Bifidobacterium longum subsp. Longum FN103 and composition thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium longum subsp. Longum as well as a composition, a pharmaceutical composition, a culture, a food product or a dietary supplement containing the bifidobacterium longum subsp. Longum and application of the bifidobacterium longum subsp. Longum. The bifidobacterium longum is preserved in the Guangdong Microbial Culture Collection Center on January 21, 2025, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) No.65836. The bifidobacterium longum subsp. Longum has relatively strong gastric acid and cholate tolerance and intestinal colonization capability; the bifidobacterium longum subsp. Longum can effectively utilize bifidobacterium bifidum to release monosaccharide or disaccharide outside cells for efficient proliferation, and the composition containing the bifidobacterium longum subsp. Longum, the bifidobacterium bifidum and breast milk oligosaccharide can release a large amount of short-chain fatty acid, so that the effects of maintaining the physiological function of an organism and improving the health of the organism are achieved.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2025101980537, filed on February 21, 2025, the entire content of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention belongs to the field of microbiological technology, and specifically relates to Bifidobacterium longum subsp. longum, as well as compositions, pharmaceutical compositions, cultures, food products or dietary supplements containing the same, and their applications. Background Art

[0003] Breast milk contains a considerable amount of bacteria and is an important source of neonatal intestinal bacteria. Bifidobacteria derived from breast milk are the dominant flora in the intestines of infants and young children. Especially in breastfed infants, Bifidobacteria can account for 60-90% of the intestinal flora. The Bifidobacteria that have been discovered include Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium longum subsp. longum.

[0004] Bifidobacteria are an important class of short-chain fatty acid-producing bacteria in the intestine. Research has shown that Bifidobacteria produce short-chain fatty acids by fermenting dietary fiber, especially human milk oligosaccharides (HMOs). Short-chain fatty acids play an important role in maintaining the physiological functions of the body, promoting intestinal health, regulating metabolism, enhancing immune function, and protecting the nervous system. Taking infant allergy as an example, multiple studies have confirmed that the numbers of Clostridium and Enterobacteriaceae bacteria in the intestines of allergic infants are increased, while the numbers of Lactobacilli and Bifidobacteria are decreased, and the short-chain fatty acids such as acetic acid, propionic acid, and butyric acid produced by the intestinal flora are also significantly lower than those of normal infants.

[0005] In addition, whether Bifidobacteria can exert the above-mentioned probiotic effects on the body also depends on whether the strains can tolerate the complexity of the body's gastrointestinal tract, such as the low pH environment in gastric juice and the high bile acid environment in the small intestine, and smoothly reach the intestine in a live state, adhere to intestinal epithelial cells, and colonize in the intestine, so as to produce short-chain fatty acids and interact with the host. Summary of the Invention

[0006] There are obvious differences among different Bifidobacterium strains in the types and yields of short-chain fatty acids produced and their specific effects on host health. It is of great significance to develop Bifidobacterium strains that can be efficiently propagated and metabolize to produce a large amount of short-chain fatty acids. Through a large number of experiments, the inventors of the present invention screened a Bifidobacterium longum subsp. longum with significant strain characteristics from more than 50 strains derived from healthy lactating mothers. The inventors of the present invention have confirmed through a large number of experiments that this Bifidobacterium longum subsp. longum has strong gastric acid and bile salt tolerance and intestinal colonization ability; this Bifidobacterium longum subsp. longum can effectively utilize the monosaccharides or disaccharides released extracellularly by Bifidobacterium bifidum for efficient propagation, and the composition containing this Bifidobacterium longum subsp. longum, Bifidobacterium bifidum and human milk oligosaccharides can release a large amount of short-chain fatty acids, thereby playing a role in maintaining the physiological functions of the body and improving the health of the body. Therefore, the applicant completed the present invention.

[0007] Bifidobacterium longum subsp. longum

[0008] In a first aspect, the present invention provides a Bifidobacterium longum subsp. longum, which is deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No. 65836.

[0009] It is easy to understand that in the growth and cultivation of microorganisms, especially in the growth and cultivation of bacteria, genetic material may undergo certain changes (for example, mutations of one or several bases), and these changes can occur spontaneously or be the result of mutagenesis by chemical and / or physical reagents (such as mutagens) and / or recombinant DNA techniques known in the art. Therefore, in this article, the Bifidobacterium longum subsp. longum of the present invention includes mutants and / or descendants of the Bifidobacterium longum subsp. longum with the deposit number GDMCC No. 65836. In certain embodiments, these mutants and / or descendants still retain the functions and / or physiological and biochemical characteristics of the Bifidobacterium longum subsp. longum with the deposit number GDMCC No. 65836 (for example, one or more of the functions and / or physiological and biochemical characteristics mentioned above).

[0010] Composition

[0011] In a second aspect, the present invention provides a composition comprising the Bifidobacterium longum subsp. longum as described in the first aspect.

[0012] In certain embodiments, the composition further comprises a probiotic selected from the group consisting of bacteria, fungi (such as yeast), or any combination thereof.

[0013] As used herein, the term "probiotic" is defined as any non-pathogenic microorganism that, when administered to a host in a live form in a sufficient amount, can have a beneficial effect on the health of the host.

[0014] In certain embodiments, the bacterium is selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus collinoides, Lactobacillus paraplantarum, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium acnes, Leuconostoc, Pediococcus, Weissella, Carnobacterium, Staphylococcus, Akkermansia, Faecalibacterium, or any combination thereof.

[0015] In certain embodiments, the probiotic of the genus Bifidobacterium is selected from: Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, or any combination thereof.

[0016] In certain embodiments, the composition comprises Bifidobacterium bifidum FN120 (Bifidobacterium bifidum with deposit number GDMCC No. 65837).

[0017] In certain embodiments, the probiotic of the genus Lactobacillus is selected from: Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens subsp. kefiranofaciens, or any combination thereof.

[0018] In certain embodiments, the probiotic bacteria of the genus Lactobacillus are selected from: Lactobacillus casei, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus rhamnosus, or any combination thereof.

[0019] In certain embodiments, the probiotic bacteria of the genus Limosilactobacillus are selected from: Limosilactobacillus fermentum, Limosilactobacillus reuteri, or a combination thereof.

[0020] In certain embodiments, the probiotic bacteria of the genus Lactiplantibacillus is Lactiplantibacillus plantarum.

[0021] In certain embodiments, the probiotic bacteria of the genus Ligilactobacillus is Ligilactobacillus salivarius.

[0022] In certain embodiments, the probiotic bacteria of the genus Streptococcus is Streptococcus salivarius subsp. thermophilus.

[0023] In certain embodiments, the probiotic bacteria of the genus Propionibacterium is Propionibacterium freudenreichii subsp. shermanii.

[0024] In certain embodiments, the probiotic bacteria of the genus Propionibacterium is Propionibacterium acidipropionici.

[0025] In certain embodiments, the probiotic bacteria of the genus Lactococcus are selected from: Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Lactis biovar diacetylactis, Lactococcus cremoris, or any combination thereof.

[0026] In certain embodiments, the probiotics of the genus Akkermansia are selected from: Akkermansia muciniphila and Akkermansia glycaniphila, or a combination thereof.

[0027] In certain embodiments, the probiotics of the genus Faecalibacterium is Faecalibacterium prausnitzii.

[0028] In certain embodiments, the composition further comprises a prebiotic selected from: human milk oligosaccharides, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, resistant dextrin, maltodextrin, polydextrose, psyllium husk, spirulina, arthrospira, polysaccharide krestin, nitrogen-containing polysaccharide of carrot, or any combination thereof.

[0029] In certain embodiments, the composition further comprises human milk oligosaccharides.

[0030] In certain embodiments, the human milk oligosaccharides are selected from: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3'-FL), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-dibiose (LNB), or any combination thereof.

[0031] In certain embodiments, the composition comprises Bifidobacterium longum subsp. longum with deposit number GDMCC No. 65836, Bifidobacterium bifidum with deposit number GDMCC No. 65837, and human milk oligosaccharide 2'-FL. In certain embodiments, the composition consists of Bifidobacterium longum subsp. longum with deposit number GDMCC No. 65836, Bifidobacterium bifidum with deposit number GDMCC No. 65837, and human milk oligosaccharide 2'-FL.

[0032] In certain embodiments, the ratio of Bifidobacterium longum subsp. longum with deposit number GDMCC No. 65836 to Bifidobacterium bifidum with deposit number GDMCC No. 65837 comprised in the composition is 1 - 100:1 - 100 (for example, 100:1, 50:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:50, 1:100), and the ratio of 2'-FL to Bifidobacterium bifidum comprised in the composition is 0.48 - 48 g:1×10 9 CFU (for example, 0.48 g:1×10 9 CFU, 1 g:1×10 9 CFU, 4.8 g:1×10 9CFU, 10 g: 1×10 9 CFU, 19.2 g: 1×10 9 CFU, 30 g: 1×10 9 CFU, 48 g: 1×10 9 CFU).

[0033] In certain embodiments, the content of Bifidobacterium bifidum with the deposit number GDMCC No. 65837 contained in the composition is higher than 1×10 6 CFU (such as 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 ), and the content of Bifidobacterium longum subsp. longum with the deposit number GDMCC No. 65836 is higher than 1×10 6 CFU (such as 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 ).

[0034] In certain embodiments, the composition has the ability to produce more than 40 μg / g of acetic acid under effective culture conditions. In certain embodiments, the composition has the ability to produce 50 μg / g to 60 μg / g, 60 μmol / l to 70 μmol / l or more acetic acid under effective culture conditions.

[0035] In certain embodiments, the composition has the ability to produce more than 8 μg / g of propionic acid under effective culture conditions. In certain embodiments, the composition has the ability to produce 8 μg / g to 10 μg / g, 10 μmol / l to 13 μmol / l or more propionic acid under effective culture conditions.

[0036] In certain embodiments, the composition has the ability to produce butyric acid greater than 15 μg / g under effective culture conditions. In certain embodiments, the composition has the ability to produce butyric acid in the range of 15 μg / g to 20 μg / g, 20 μmol / l to 30 μmol / l, 30 μmol / l to 40 μmol / l or greater under effective culture conditions.

[0037] Pharmaceutical composition

[0038] In a third aspect, the present invention provides a pharmaceutical composition comprising the Bifidobacterium longum subsp. longum as described in the first aspect or the composition as described in the second aspect.

[0039] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient, cryoprotectant, amino acid, vitamin, mineral, peptone, or any combination thereof.

[0040] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient includes: fillers, binders, lubricants, glidants, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

[0041] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient includes: sugars (such as xylose, sucrose, fructose, lactose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gums, maltodextrin), polyethers (such as polypropylene glycol, polyethylene glycol, polybutylene glycol), polyvinylpyrrolidone (such as polyvinylpyrrolidone K30, K60, K90), oils (such as rapeseed oil, sunflower oil, soybean oil, sesame oil, olive oil), surfactants (such as Tween20, Tween40, Tween60, Tween80, fatty acids, polyoxyethylene fatty alcohol ethers), inorganic salts (such as phosphates, carbonates, citrates, chlorides, sulfates, borates, citrate), talc, silica and its derivatives, hydrolysis products, or any combination thereof.

[0042] In certain embodiments, the pharmaceutical composition further comprises a cryoprotectant.

[0043] In certain embodiments, the pharmaceutical composition further comprises glycerol, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylooligosaccharide, glutathione, or any combination thereof.

[0044] In certain embodiments, the pharmaceutical composition includes amino acids.

[0045] In certain embodiments, the pharmaceutical composition comprises tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0046] In certain embodiments, the pharmaceutical composition comprises vitamins.

[0047] In certain embodiments, the pharmaceutical composition comprises vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, or any combination thereof.

[0048] In certain embodiments, the pharmaceutical composition comprises minerals.

[0049] In certain embodiments, the pharmaceutical composition comprises iron, manganese, zinc, copper, selenium, or any combination thereof.

[0050] In certain embodiments, the pharmaceutical composition comprises peptone.

[0051] In certain embodiments, the pharmaceutical composition comprises soy peptone, wheat peptone, whey peptone, or any combination thereof.

[0052] The pharmaceutical composition of the present invention can be administered to mammals including humans by various routes. The administration method can be any method commonly used in the art. For example, it can be administered orally, in vitro, intravenously, intramuscularly, subcutaneously, etc. In certain embodiments, the pharmaceutical composition is formulated for oral administration or in vitro administration.

[0053] In certain embodiments, the pharmaceutical composition comprises a preparation of Bifidobacterium bifidum.

[0054] In certain embodiments, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), oil drops, film coatings, orally soluble granules, liquids, suppositories, enemas gels, and / or creams.

[0055] In certain embodiments, the pharmaceutical composition further comprises formulation excipients (e.g., excipients for preparing powders, tablets, capsules, oil drops).

[0056] In certain embodiments, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, painkillers, anti-inflammatory agents, wound healing agents, and / or moisturizers.

[0057] In certain embodiments, Bifidobacterium longum subsp. longum in the pharmaceutical composition is present in an amount higher than 10 6 CFU / dose (e.g., 10 7 CFU / dose, 108 CFU / dose, 10 9 CFU / dose, 10 10 CFU / dose, 10 11 CFU / dose, 10 12 CFU / dose).

[0058] In certain embodiments, when the pharmaceutical composition is solid, it contains more than 10 6 CFU of Bifidobacterium longum subsp. longum per gram (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g, 10 10 CFU / g, 10 11 CFU / g, 10 12 CFU / g).

[0059] In certain embodiments, when the pharmaceutical composition is liquid, it contains more than 10 6 CFU of Bifidobacterium longum subsp. longum per milliliter (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, 10 11 CFU / mL, 10 12 CFU / mL).

[0060] Culture

[0061] In a fourth aspect, the present invention provides a culture comprising Bifidobacterium longum subsp. longum as described in the first aspect or the composition as described in the second aspect.

[0062] In certain embodiments, the culture further comprises components that provide nutrition (e.g., solid or liquid culture medium, feeder cell layer).

[0063] In certain embodiments, the components that provide nutrition are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0064] In certain embodiments, the culture further comprises a cell-free culture filtrate of Bifidobacterium longum subsp. longum.

[0065] In certain embodiments, the culture further comprises derivatives of Bifidobacterium longum subsp. longum; wherein the derivatives are selected from metabolites, enzymes, cell structure components (e.g., cell wall or its components), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0066] Food product or dietary supplement or health food

[0067] In a fifth aspect, the present invention provides a food product or a dietary supplement or a health food, which comprises the Bifidobacterium longum subsp. longum as described in the first aspect or the composition as described in the second aspect or the culture as described in the fourth aspect.

[0068] In the text, the term "food" is in a broad sense, including human food and beverages, and also covering animal food and beverages (i.e., feed). In certain embodiments, the food product is suitable for and designed for human consumption. In certain embodiments, the food product is selected from solid beverages, liquid beverages, chewable candies, puffed foods, protein bars or nuts, or alternatively, the food product is a dairy product (e.g., milk powder, milk tablets, yogurt, fermented milk with flavor, lactic acid bacteria beverage, cheese).

[0069] In the text, a "dietary supplement" refers to an edible product that can provide beneficial effects to consumers. It is also known as a nutritional supplement, nutrient, dietary supplement, etc., and is used as an auxiliary means of diet to supplement amino acids, trace elements, vitamins, minerals, etc. required by the human body.

[0070] In certain embodiments, the food product or the dietary supplement or the health food further comprises additional additives.

[0071] In certain embodiments, the additional additives are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0072] In certain embodiments, the carbohydrates are selected from monosaccharides (glucose, fructose, xylose), disaccharides (maltose, lactose, sucrose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), or any combination thereof.

[0073] In certain embodiments, the amino acids are selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0074] In certain embodiments, the vitamins are selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, or any combination thereof.

[0075] In certain embodiments, the minerals are selected from iron, manganese, zinc, copper, selenium, or any combination thereof.

[0076] In certain embodiments, Bifidobacterium longum subsp. longum is present in the food product or dietary supplement or functional food at a level higher than 10 6 CFU / dose (e.g., 10 7 CFU / dose, 10 8 CFU / dose, 10 9 CFU / dose, 10 10 CFU / dose, 10 11 CFU / dose, 10 12 CFU / dose).

[0077] In certain embodiments, when the food product or dietary supplement or functional food is solid, it contains higher than 10 6 CFU of Bifidobacterium longum subsp. longum per gram (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g, 10 10 CFU / g, 10 11 CFU / g, 10 12 CFU / g).

[0078] In certain embodiments, when the food product or dietary supplement or functional food is liquid, it contains higher than 10 6 CFU of Bifidobacterium longum subsp. longum per milliliter (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, 10 11 CFU / mL, 10 12 CFU / mL).

[0079] Use

[0080] In a sixth aspect, the present invention provides the use of Bifidobacterium longum subsp. longum as described in the first aspect or the composition as described in the second aspect or the pharmaceutical composition as described in the third aspect or the culture as described in the fourth aspect in the preparation of a drug, dietary supplement or functional food for preventing, alleviating and / or treating allergy-related diseases and / or symptoms in a subject, or for preventing, alleviating and / or treating inflammation-related diseases and / or symptoms in a subject.

[0081] In certain embodiments, the allergy-related disease is caused by an imbalance of the gut microbiota.

[0082] In certain embodiments, the allergy-related disease is associated with an increase in the levels of Clostridium and / or Enterobacteriaceae strains in the gut ecosystem.

[0083] In certain embodiments, the allergy-related disease is associated with a decrease in the levels of Lactobacillus and / or Bifidobacterium strains in the gut ecosystem.

[0084] In certain embodiments, the allergy-related disease is selected from eczema, atopic dermatitis, and food allergy.

[0085] In certain embodiments, the inflammation-related disease is selected from diseases caused by skin inflammation (e.g., dermatitis, eczema), diseases caused by respiratory inflammation (e.g., upper respiratory tract infection), and diseases caused by gastrointestinal inflammation (e.g., inflammatory bowel disease).

[0086] In certain embodiments, the drug or dietary supplement or health product is capable of activating immune cells, promoting the production of cytokines, inhibiting inflammation, or alleviating inflammatory diseases.

[0087] In certain embodiments, the cytokines are selected from IL33, IL4, IL12, IFN-γ, TSLP, or any combination thereof.

[0088] In certain embodiments, the drug or dietary supplement or health product is capable of reducing IgE levels and inhibiting IgE-mediated allergic reactions.

[0089] In certain embodiments, the drug or dietary supplement or health product is used alone or in combination with other antifungal agents, painkillers, anti-inflammatory drugs, wound healing agents, or moisturizers.

[0090] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0091] In certain embodiments, the subject is a human.

[0092] Method

[0093] In a seventh aspect, the present invention provides a method for preventing, alleviating, and / or treating allergy-related diseases and / or symptoms, or preventing, alleviating, and / or treating inflammation-related diseases and / or symptoms, the method comprising: administering to a subject in need an effective amount of Bifidobacterium longum subsp. longum as described in the first aspect, or the composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect, or the culture as described in the fourth aspect, or the food product or dietary supplement as described in the fifth aspect.

[0094] In certain embodiments, the method includes: implanting the Bifidobacterium longum subsp. longum as described in the first aspect, or the composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect, or the culture as described in the fourth aspect, or the food product or dietary supplement or health food as described in the fifth aspect into the intestine of a subject.

[0095] In certain embodiments, the method includes: administering (e.g., orally) to the subject the Bifidobacterium longum subsp. longum as described in the first aspect, or the composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect, or the culture as described in the fourth aspect, or the food product or dietary supplement or health food as described in the fifth aspect.

[0096] In certain embodiments, the Bifidobacterium longum subsp. longum is administered to the subject in an amount greater than 10 6 CFU / dose (e.g., 10 7 CFU / dose, 10 8 CFU / dose, 10 9 CFU / dose, 10 10 CFU / dose, 10 11 CFU / dose, 10 12 CFU / dose).

[0097] Term definition

[0098] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0099] As used herein, the term "probiotic" refers to live bacteria that can exert beneficial effects on the health of the host when ingested in an appropriate amount. The "probiotics" include, but are not limited to, the strains mentioned in the List of Strains Allowed to be Used in Foods and the List of Strains Allowed to be Used in Infant Foods issued by the National Health Commission.

[0100] As used herein, the term "therapeutically effective amount" or "effective amount" may refer to the amount of the strain in a composition or pharmaceutical composition that, when administered as part of a desired dosage regimen (to a human or animal, preferably a human), alleviates symptoms, improves the condition, or slows the onset of the disease condition according to clinically acceptable criteria for the condition or disease state of the subject to be treated, e.g., with a reasonable benefit-risk ratio applicable to any medical treatment. The therapeutically effective amount may vary depending on the age, weight, sex, dosage form, health status, and severity of the disease of the different subjects to be treated. In addition, the frequency may be determined by a doctor or pharmacist and administered in divided doses one to several times at fixed time intervals per day.

[0101] As used herein, the term "CFU (Colony-Forming Units)" refers to the total number of colonies of microorganisms such as bacteria, fungi, yeast, etc. in a product, and is usually used for calculating the number of viable bacteria.

[0102] As used herein, the term "CFU / dose" means the amount of bacteria present in a composition / food product or dietary supplement or functional food / drug composition provided to a subject per day or per administration. For example, in certain embodiments, the amount of Bifidobacterium longum subsp. longum in the food product or dietary supplement or functional food is higher than 10 6 CFU / dose. In such an embodiment, if Bifidobacterium bifidum is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject per day or per administration may contain higher than 10 6 CFU of Bifidobacterium longum subsp. longum. Of course, alternatively, the amount of such bacteria can be administered in multiple doses, as long as the total amount of Bifidobacterium longum subsp. longum received by the subject within any specific time period (e.g., per 24-hour period) is higher than 10 6 CFU of bacteria, i.e., it meets the requirement that the amount of Bifidobacterium longum subsp. longum in the above-mentioned food product or dietary supplement or functional food is higher than 10 6 CFU / dose.

[0103] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffer solutions; 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.

[0104] As used herein, the term "subject" is an animal. Animals can include but are not limited to primates, farm animals, sports animals, rodents and pets. More specifically, animals can include mice, rats, hamsters and guinea pigs; rabbits; dogs; cats; sheep; pigs; piglets; sows; poultry; turkeys; broiler chickens; minks; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0105] Advantageous effects of the invention

[0106] Bifidobacterium longum subsp. longum FN103 of the present invention can effectively utilize Bifidobacterium bifidum to release monosaccharides or disaccharides extracellularly, achieve efficient proliferation and produce a large amount of short-chain fatty acids. Moreover, the ability of Bifidobacterium longum subsp. longum to proliferate and produce short-chain fatty acids is stronger than that of two other strains belonging to Bifidobacterium longum subsp. longum. In addition, Bifidobacterium longum subsp. longum of the present invention, when used in combination with human milk oligosaccharides and Bifidobacterium bifidum, can inhibit the expression of inflammatory factors, improve symptoms such as skin redness and desquamation, and play a role in treating atopic dermatitis. Therefore, Bifidobacterium longum subsp. longum of the present invention has high application value in allergic diseases (especially infantile atopic dermatitis and eczema). BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Shows the colony morphology and Gram staining microscopic morphology of Bifidobacterium longum subsp. longum FN103.

[0108] Figure 2 Shows the colony morphology and Gram staining microscopic morphology of Bifidobacterium bifidum FN120.

[0109] Figure 3 Shows the phylogenetic tree of Bifidobacterium longum subsp. longum FN103 (GDMCC No. 65836) and 27 strains with completed genome sequencing submitted to the NCBI database. The retrieval date of the NCBI database was January 15, 2025. Constructed using the UBCG pipeline based on 92 single-copy core housekeeping genes, the node labels represent gene support index values, and those with the same number of common sequences are grouped into one branch. The type strain is marked with an asterisk, and the strain of this study is marked with a triangle.

[0110] Figure 4 Shows the phylogenetic tree of Bifidobacterium bifidum FN120 (GDMCC No. 65837) and 15 strains with completed genome sequencing submitted to the NCBI database. The retrieval date of the NCBI database was January 15, 2025. Constructed using the UBCG pipeline based on 92 single-copy core housekeeping genes, the node labels represent gene support index values, and those with the same number of common sequences are grouped into one branch. The type strain is marked with an asterisk, and the strain of this study is marked with a triangle.

[0111] Figure 5 Shows the experimental procedures for fecal microbiota transplantation and the establishment of an eczema model in animals.

[0112] Figure 6 Shows the effects of different intervention methods on the appearance of eczema symptoms in the right ear of mice.

[0113] Figure 7Shows the effects of different intervention methods on the eczema symptom score and ear thickness of the right ear of mice; *p<0.05, **p<0.01, two-way repeated measures analysis of variance.

[0114] Figure 8 Shows the effects of different intervention methods on the histological conditions of the right ear of mice (upper: hematoxylin & eosin staining sections; lower: toluidine blue staining sections).

[0115] Figure 9 Shows the effects of different intervention methods on the thickness of the dermis layer and the infiltration of mast cells in the right ear of mice; *p<0.05, ***p<0.0001, compared with the normal control; #p<0.05, p<0.0001, compared with the normal control; one-way analysis of variance. Specific implementation method

[0117] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0118] Unless otherwise specified, the experiments and methods described in the embodiments are basically carried out according to the conventional methods well-known in the art and described in various reference documents. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2nd Edition (1989); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., (1987)); "Methods in Enzymology" series (Academic Press): "PCR 2: A Practical Approach" (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor (1995)), and "Animal Cell Culture" (edited by R.I. Freshney (1987)).

[0119] In addition, for those conditions not specified in the examples, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially. Those skilled in the art know that the examples describe the present invention by way of illustration and are not intended to limit the scope claimed by the present invention. All the published cases and other reference materials mentioned herein are incorporated herein by reference in their entirety.

[0120] Example 1. Isolation and identification of strains

[0121] 1.1 Isolation and Identification of Bifidobacterium longum

[0122] In the present invention, more than 50 strains of bacteria were isolated from 12 breast milk samples of healthy women in Wuxi area and 35 breast milk samples of healthy women in Gansu area, and a strain of Bifidobacterium longum was screened therefrom and named Bifidobacterium longum subsp. longum FN103. Among them, the inclusion criteria for the sample donors were: (1) healthy mothers aged 20 - 45 years old; the exclusion criteria were: (1) suffering from autoimmune chronic diseases, acute and chronic infections; (3) premature birth (pregnancy duration ≤ 37 weeks, or abnormal birth weight); (4) not receiving antibiotic treatment for at least one month before sampling.

[0123] The fresh breast milk was prepared into a 10-fold serial dilution, 0.1 mL was taken from each dilution and inoculated on the PTYG agar plate, and anaerobically cultured at 36 ± 1 °C for 48 ± 6 h, and extended to 72 ± 6 h according to the colony growth on the agar plate. Single colonies were picked from the agar plate for Gram staining and microscopic examination. Monoclonal colonies of Gram-positive bacteria showing short rod-shaped, slender rod-shaped or spherical under microscopic examination were selected for further purification until pure bacteria were determined, and single clones were picked for 16S rRNA sequencing.

[0124] The sequencing results showed that, after comparison, the gene sequence of 16S rRNA of the strain isolated in the present invention had the highest similarity with Bifidobacterium longum, reaching 99.86% (the comparison strain was JCM11343), so it was initially determined to be Bifidobacterium longum, numbered Bifidobacterium longum FN103.

[0125] After sequencing analysis of this strain, the 16S rRNA gene sequence FN103-16S (SEQ ID NO.1) was:

[0126]

[0127] The whole genome extracted from this strain was sent to a professional sequencing company for whole genome sequencing. It was compared with 27 strains of *Bifidobacterium longum subsp. longum* whose complete genome sequencing maps had been submitted to the NCBI database. A phylogenetic tree was constructed based on the sequences of 92 single-copy core housekeeping genes. *Bifidobacterium longum subsp. longum* FN103 was located on an independent branch, indicating that this strain is a new strain different from these 27 strains. At the same time, *Bifidobacterium longum subsp. longum* FN103 and the type strain *Bifidobacterium* JCM1217 were in the same cluster of the three major clustering clusters, being strains with the same phenotype. This strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on January 21, 2025. The address of the depository is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 65836.

[0128] 1.2 Isolation and identification of *Bifidobacterium bifidum*

[0129] In the present invention, more than 50 strains of bacteria were isolated from 12 breast milk samples of healthy women in Wuxi area and 35 breast milk samples of healthy women in Gansu area, and a strain of *Bifidobacterium bifidum* was screened and named *Bifidobacterium bifidum* FN120. Among them, the inclusion criteria for the sample donors were the same as above.

[0130] Fresh breast milk was prepared into a 10-fold serial dilution. 0.1 mL was taken from each dilution and inoculated on PTYG agar plates, and anaerobically cultured at 36±1 °C for 48±6 h, and extended to 72±6 h according to the colony growth on the agar plates. Single colonies were picked from the agar plates for Gram staining and microscopic examination. Monoclonal colonies of Gram-positive bacteria showing short rod-shaped, slender rod-shaped or spherical under microscopic examination were selected for further purification until pure bacteria were determined, and single colonies were picked for 16S rRNA sequencing.

[0131] The sequencing results showed that after comparison, the gene sequence of 16S rRNA of the strain isolated in the present invention had the highest similarity with *Bifidobacterium bifidum*, reaching 99.79% (the comparison strain was JCM 7004). Therefore, it was preliminarily determined to be *Bifidobacterium bifidum*, numbered *Bifidobacterium bifidum* FN120.

[0132] After sequencing analysis of this strain, the 16S rRNA gene sequence FN120-16S (SEQ ID NO.2) is:

[0133]

[0134] The whole genome extracted from the strain was sent to a professional sequencing company, and the whole genome of the bacteria was sequenced using a second-generation sequencer. It was compared with the 15 bifidobacteria that had submitted genome sequencing completion maps in the NCBI database. A growth and development tree was constructed based on 92 single-copy core housekeeping gene sequences. Bifidobacterium bifidum FN120 was located in an independent branch, indicating that the strain was a new strain different from these 15 strains. At the same time, Bifidobacterium bifidum FN120 and the model strain Bifidobacterium bifidum JCM1255 were located in two cluster branches of the three major clusters, respectively, and were different phenotypic strains. The strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC, Guangdong Microbial Culture Collection Center) on January 21, 2025. The address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. GDMCC65837.

[0135] Example 2. Study on probiotic characteristics of strains

[0136] The strains used in this example are all from the probiotic strain library derived from human milk of By-Health. In addition to the Bifidobacterium longum subspecies longum FN103 mentioned in Example 1, the remaining strains tested are: Bifidobacterium longum subspecies longum FN133, FN156 (the other two strains of Bifidobacterium longum subspecies longum isolated in this separation experiment), and Bifidobacterium animalis BB12 (commercial animal Bifidobacterium).

[0137] 2.1 Gastric acid / bile salt resistance test

[0138] The human digestive tract produces a large amount of gastric acid and bile every day. Exogenously ingested probiotics need to survive in the gastrointestinal environment to perform their corresponding functions. Tolerance to gastric acid and bile salts is one of the main prerequisites for probiotic screening.

[0139] 2.1.1 Experimental methods

[0140] Prepare 900 mL of sterile PBS with pH 3.0 (experimental) and 7.0 (control) respectively, and inoculate 100 mL of Bifidobacterium liquid washed with sterile PBS (10 9 CFU / mL). After anaerobic culture at 37℃ for 4h, the mixture was fully mixed and gradient dilution was performed. 50uL of bacterial solution was evenly spread on the solid culture medium plate for each gradient. After anaerobic culture at 37℃ for 24h, the colonies were counted.

[0141] 900 mL of sterile PBS with 0.3% (w / v) bile salt added (experimental) and 900 mL of sterile PBS without bile salt added (control) were prepared, and 100 mL of bifidobacterium culture washed with sterile PBS (109 (CFU / mL). After anaerobic incubation at 37 °C for 4 h, mix well and perform serial dilutions. Take 50 mL of the bacterial suspension from each dilution and spread it evenly on the solid medium plates. After anaerobic incubation at 37 °C for 24 h, perform colony counting.

[0142] Gastric acid / bile salt tolerance (%) = CFU1 / CFU0 x 100%; CFU1: viable cell count of the experimental group, CFU / mL; CFU0: viable cell count of the control group, CFU / mL.

[0143] 2.1.2 Experimental results

[0144] To better simulate the environmental conditions of the gastrointestinal tract, considering the pH value of gastric juice and the bile salt concentration in intestinal juice, this experiment selected sterile PBS with pH = 3 and sterile PBS with 0.3% bile salt. Compared with strains of Lactobacillus (such as Lactobacillus acidophilus or Lactobacillus rhamnosus) and other genera of Lactobacillus (such as Lactobacillus casei or Lactobacillus paracasei), the tolerance of Bifidobacterium is generally weaker.

[0145] Generally speaking, Bifidobacterium longum subsp. longum FN103 has good gastric acid and bile salt tolerance; although its gastric acid and bile salt tolerance is not as good as that of the commercial strain BB-12, it is significantly better than the other two strains of the same Bifidobacterium longum subsp. longum. The specific results are shown in Table 1:

[0146] Table 1 Gastric acid tolerance and bile salt tolerance of strains

[0147] Strain Survival rate in gastric acid (%) Survival rate in bile salt (%) BB12 75.40±4.01 69.77±4.68 FN103 50.72±2.05 50.43±4.47 FN133 45.67±2.50 35.18±7.78 FN156 32.20±3.55 33.20±3.52

[0148] 2.2 Colonization ability test

[0149] Probiotics can only exert their corresponding active functions after interacting with host cells after colonizing in the gastrointestinal tract. On the one hand, compared with hydrophilic probiotics, probiotics with high hydrophobicity can adhere more effectively to colon cells; on the other hand, the auto-aggregation ability of probiotics is necessary for their adhesion to intestinal epithelial cells. Therefore, the colonization ability of probiotics can be reflected by their auto-aggregation ability and hydrophobicity.

[0150] 2.2.1 Experimental method

[0151] After activating the Bifidobacterium bacterial suspension for three generations, measure the OD600nm value A0 of the bacterial suspension. Take 2 mL of the bacterial suspension (10 9CFU / mL) and 2 mL of xylene were placed in a test tube and mixed evenly by vortex. After standing at room temperature for 30 min, the mixed solution was stratified, and the OD600 value of the aqueous phase was measured at nm to obtain A1. The surface hydrophobicity of each strain was calculated based on the OD value.

[0152] Surface hydrophobicity (%) = [1 - (A1 / A0)] × 100%.

[0153] 100 mL of the Bifidobacterium bifidum bacterial liquid after three generations of activation (10 9 CFU / mL) was placed in a 96-well plate with a pointed bottom. After thorough mixing, the OD600 value at 0 was measured to obtain A0, and then the OD600 value A1 was measured every hour for 3 consecutive hours. The auto-aggregation ability of each strain was calculated based on the OD value.

[0154] Auto-aggregation ability = [(A1 - A0) / A0] × 100%.

[0155] 2.2.2 Experimental results

[0156] Generally speaking, Bifidobacterium longum subsp. longum FN103 has good hydrophobicity and auto-aggregation ability. On the one hand, its hydrophobicity is comparable to that of the commercial strain BB-12, but stronger than the other two strains belonging to Bifidobacterium bifidum; on the other hand, its auto-aggregation ability is significantly stronger than that of the commercial strain BB-12 and the other two strains belonging to Bifidobacterium longum subsp. longum. This indicates that Bifidobacterium longum subsp. longum FN103 has good intestinal colonization ability, and the specific results are shown in Tables 2 and 3:

[0157] Table 2 Hydrophobicity of strains

[0158] Strain Hydrophobicity BB12 52.65±6.11 FN103 47.42±5.20 FN133 40.76±2.19 FN156 30.89±2.65

[0159] Table 3 Auto-aggregation activity of strains

[0160]

[0161]

[0162] Example 3 Proliferation of Bifidobacterium longum FN103 and production of short-chain fatty acids

[0163] Human milk oligosaccharides are typical natural prebiotics. Through selective fermentation, they can affect the composition or activity of intestinal microorganisms, thereby regulating mucosal immune function and being closely related to the reduction of the occurrence of allergic diseases in the body. However, there are obvious species and strain specificities in the efficiency and mechanism of Bifidobacterium using human milk oligosaccharides. Bifidobacterium bifidum can hydrolyze human milk oligosaccharides through extracellular enzymes, and Bifidobacterium longum subsp. longum can utilize monosaccharides or disaccharides released by other bacteria hydrolyzing human milk oligosaccharides.

[0164] This example studies the proliferation and short-chain fatty acid production of different Bifidobacterium longum subsp. longum during co-culture with Bifidobacterium bifidum and human milk oligosaccharides.

[0165] The strains used in this example are all from the probiotic strain library of BY-HEALTH sourced from human milk. In addition to Bifidobacterium longum subsp. longum FN103, FN133, FN156, and Bifidobacterium bifidum FN120 mentioned in Examples 1 and 2, it also includes Bifidobacterium bifidum FN121 and FN125, and these two strains of Bifidobacterium bifidum were both obtained by the methods of isolation and identification described in Example 1.

[0166] The experimental groups are set as follows:

[0167] Experimental group 1: Bifidobacterium bifidum FN120 group; Experimental group 2: Bifidobacterium bifidum FN121 group; Experimental group 3: Bifidobacterium bifidum FN125 group; Experimental group 4: Bifidobacterium longum FN103 group; Experimental group 5: Bifidobacterium longum FN133 group; Experimental group 6: Bifidobacterium longum FN156; Experimental group 7: (Bifidobacterium bifidum FN120: Bifidobacterium longum FN103 = 1:1); Experimental group 8: (Bifidobacterium bifidum FN120: Bifidobacterium longum FN133 = 1:1); Experimental group 9: (Bifidobacterium bifidum FN120: Bifidobacterium longum FN156 = 1:1); Experimental group 10: (Bifidobacterium bifidum FN121: Bifidobacterium longum FN103 = 1:1); Experimental group 11: (Bifidobacterium bifidum FN125: Bifidobacterium longum FN103 = 1:1).

[0168] Prepare 1% 2’-FL MRS liquid medium. The composition of the medium is shown in Table 4 below:

[0169] Table 4 Composition of 1% 2’-FL MRS liquid medium

[0170]

[0171]

[0172] 3.1 Growth characteristics test

[0173] After activating the strains of each above-mentioned experimental group for three generations, inoculate them into the MRS liquid medium containing 1% 2’-FL at an inoculation amount of 5%, and culture anaerobically at 37°C. Measure the OD600 value every 6 hours for a total of 48 hours, and set up three parallel experiments in the liquid medium for each experimental group.

[0174] 3.2 Short-chain fatty acid test

[0175] The short-chain fatty acid content in the culture media of experimental groups 1 to 11 was determined using gas chromatography-mass spectrometry (GC / MS). The specific method is as follows: Take 2 mL of the co-culture medium after 48 h of co-culture, centrifuge at 10,000 rpm for 10 min at 4 °C, filter the supernatant through a 0.22 μm filter membrane, take 1 mL of the filtrate, add 0.15 mL of 50% sulfuric acid and 1.6 mL of anhydrous ether, vortex and mix well, then let it stand for 30 min, centrifuge at 10,000 rpm for 10 min at 4 °C, retain the supernatant for detection, and prepare standard products of short-chain fatty acids with different gradients. The contents of acetic acid, propionic acid, and butyric acid in the supernatant were determined by gas chromatography.

[0176] 3.3 Results and Analysis

[0177] 3.3.1 Growth Characteristics

[0178] The 1% 2'-FL MRS liquid medium uses 2'-FL as the carbon source and does not contain other carbohydrate substances. Bifidobacterium longum subsp. cannot directly utilize 2'-FL, while Bifidobacterium bifidum can utilize 2'-FL and play the role of a keystone bacterium to promote the proliferation of other bifidobacteria, such as Bifidobacterium longum subsp. The growth characteristics of each experimental group are shown in Table 5. Specifically, Bifidobacterium bifidum FN120, FN121, and FN125 can all utilize 2'-FL for proliferation, while Bifidobacterium longum subsp. FN103, FN133, and FN156 cannot utilize 2'-FL for self-proliferation. Bifidobacterium longum subsp. FN103, FN133, and FN156 can all utilize the extracellular monosaccharides or disaccharides released by Bifidobacterium bifidum for proliferation, and among them, the proliferation ability of Bifidobacterium longum subsp. FN103 is significantly better than that of Bifidobacterium bifidum FN133 and FN156.

[0179] Table 5 Growth Characteristics of Single Strains and Cross-Feeding Strains in 1% 2'-FL Medium

[0180]

[0181] 3.3.2 Short-Chain Fatty Acid Production

[0182] Bifidobacterium bifidum can utilize human milk oligosaccharides and play the role of a keystone bacterium to promote the proliferation of Bifidobacterium longum subsp. When Bifidobacterium bifidum and Bifidobacterium longum subsp. are co-cultured with 2'-FL, the metabolites of Bifidobacterium bifidum can be utilized by Bifidobacterium longum subsp. to produce short-chain fatty acids. The contents of short-chain fatty acids in the culture media of experimental groups 7-11 are shown in Table 6.

[0183] As can be seen from the results in Table 6, when Bifidobacterium bifidum and Bifidobacterium longum subsp. longum were co-cultured with 2'-FL, Bifidobacterium bifidum could enzymatically hydrolyze 2'-FL to provide raw materials for the growth of Bifidobacterium longum, and simultaneously produce high yields of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. Among them, when co-cultured with Bifidobacterium bifidum FN120 and 2'-FL, Bifidobacterium longum subsp. longum FN103 had the ability to metabolize and produce higher levels of short-chain fatty acids compared to Bifidobacterium longum subsp. longum FN133 and FN156. Specifically, the contents of acetic acid, propionic acid, and butyric acid in the FN103 co-culture combination were 2.29, 1.96, and 2.63 times those in the FN156 combination, respectively.

[0184] Table 6 Contents of short-chain fatty acids of cross-feeding strains in 1% 2'-FL medium

[0185] Strain combination Acetic acid (μg / g) Propionic acid (μg / g) Butyric acid (μg / g) FN120 + FN103 70.44±4.52 13.29±2.70 40.30±3.55 FN120 + FN133 57.67±5.03 10.10±0.68 28.34±2.52 FN120 + FN156 30.78±3.53 6.78±2.05 15.31±1.78 FN121 + FN103 47.37±5.94 10.86±1.58 19.85±3.91 FN125 + FN103 32.89±2.78 8.85±1.67 12.44±2.0

[0186] Example 5: Bifidobacterium bifidum composition improves atopic dermatitis in mice

[0187] Bifidobacterium bifidum metabolizes human milk oligosaccharides to release simple sugars extracellularly, which can promote the proliferation of other microorganisms that cannot utilize human milk oligosaccharides. This example studies the improvement effect of the combination of Bifidobacterium bifidum, Bifidobacterium longum, and human milk oligosaccharides on atopic dermatitis in mice.

[0188] 5.1 Experimental protocol

[0189] 5.1.1 Formula of in vitro fermentation medium

[0190] The basal medium for simulating infant ascending colon fermentation consists of four parts (g / L). The first part is the macronutrient components, including the phosphate buffer system (KH2PO4 6.288; K2HPO4 12.852; (NH4)2SO4 2; NaCl 6), tryptone 64.8, lactose 4, bile salts 0.1, cysteine hydrochloride 2, and mucin 8. The second part is the organic micronutrients, including vitamins (inositol 0.002; calcium pantothenate 0.0012; nicotinic acid 0.0009; pyridoxal 0.0048; folic acid 0.0005595; p-aminobenzoic acid 0.00005595; biotin 0.006; thiamine 0.0005595; riboflavin 0.0009), antioxidant (glutathione 0.015), hemin chloride 0.01, potassium acetate 0.901, lipoic acid 0.001, and nucleic acids (adenine 0.01101; guanine 0.005505; uracil 0.02202; xanthine 0.00367). The third part is the minerals, including MgSO4·7H2O 1, FeSO4·7H2O 0.004, ZnSO4·7H2O 0.005, (NH4)6Mo7O2·4H2O 0.00019, MnSO4·4H2O 0.00038, CaSO4·4H2O 0.038, CoSO4·6H2O 0.000228, CuSO4·5H2O 0.00019, H3BO3 0.00076, K2SO4 0.0228, KI 0.000114. The fourth part is the chelating agents, including EDTA 0.0075 and nitrilotriacetic acid 0.0075. The media for the stationary phase and the test phase are prepared by adding lactose or 2'-fucosyllactose (1%) to the basal medium respectively. The medium is prepared fresh before use. The vitamins and antioxidants are sterilized by filtration through a 0.22 μm pore size water membrane, and the other components are sterilized at 121 °C. The trace components are first prepared in a concentrator. The newly prepared medium is placed in an anaerobic workstation and deoxygenated overnight.

[0191] 5.1.2 Recruitment of children with atopic dermatitis and collection of fecal samples

[0192] Fecal samples were collected from 6 breastfed male infants with atopic dermatitis. Only male infants were included to avoid interference of gender-related gut microbiota differences with the research results. Atopic dermatitis was diagnosed according to the Williams criteria. The average age of the children was 37 days, and they had no exposure to antibiotics or probiotics. The guardians of the children signed the informed consent form. The collected fecal samples were placed in a vacuum-sealed bag with an oxygen absorber and stored at low temperature for transportation to the laboratory. Upon arrival, the feces were homogenized under anaerobic conditions in a sterile homogenizer (LC-08, Ningbo Licheng Instrument Co., Ltd., Ningbo, China) to prepare a fecal bacterial suspension. Subsequently, these suspensions were centrifuged at 1000 rpm for 10 minutes, and then the supernatant was transferred to a sterile anaerobic tube. Then the supernatant was stored at -80 °C with the addition of 20% glycerol for subsequent in vitro fermentation experiments.

[0193] 5.1.3 Simulating continuous fermentation in the ascending colon of children

[0194] The supernatant prepared in 5.1.2 was inoculated into the bioreactor of a multifunctional gastrointestinal digestion and fermentation simulator (MGFS, Shangpin Health Technology (Qingdao) Co., Ltd.), and 80 ml (working volume) of a basal medium supplemented with 1% lactose was added to simulate the fermentation in the ascending colon of infants. Static fermentation was carried out at 37 °C for 24 hours, during which anaerobic nitrogen gas was continuously introduced and stirred to remove oxygen. The set program for continuous fermentation was as follows: pH 5.5–6.5 (adjusted with 2 mol / L NaOH); the medium flow rate was 0.0625 mL / min (residence time was 8 hours); the reactor was perfused with an external circulation water bath; the jacket temperature was controlled at 37 °C. The experiment was set up in 3 stages. The first stage was the stable period, with lactose as the carbon source (STAB, 10 days). The second stage was experimental period 1 (2'-FL intervention period), where lactose was replaced with 2'-FL (1%, continuously for 7 days, 2'-FL). The third stage was experimental period 2 (combined intervention period of Bifidobacterium and 2'-FL, 2'-FL + Bif), and continuous cultivation was continued with the 2'-FL medium, and 1 × 10 9 CFU of Bifidobacterium bifidum FN120 and Bifidobacterium longum subsp. longum FN103 (1:1) were added daily (continuously for 7 days). Fermentation broth was collected 8 hours after adding the corresponding intervention on the last day of the stable period (day 10) and the last day of experimental period 2 (day 24), and stored at -80 °C for microbiota transplantation in animal experiments.

[0195] 5.1.4 Method for animal grouping and establishing an atopic dermatitis (eczema) model

[0196] The experimental procedure was as Figure 5As shown, after one week of adaptive feeding of 4-week-old healthy BALB / C male mice, they were randomly grouped by body weight into 3 groups, with 8 mice in each group: ① healthy blank control group (without microbiota transplantation and eczema induction, normal group); ② eczema model control group (transplanting the microbiota in the stable period of in vitro fermentation, inducing eczema, model group); ③ eczema + 2’-FL + Bif (transplanting the microbiota in the experimental period 2 of in vitro fermentation, inducing eczema, intervention group). Mice in groups ② - ③ were gavaged with broad-spectrum antibiotics (ampicillin 100 mg / kg; metronidazole 100 mg / kg; vancomycin 50 mg / kg; neomycin sulfate 100 mg / kg) for 14 consecutive days to clear the intestinal microbiota, and then began to be gavaged with the fermentation broth of the microbiota in the stable / experimental period 2 collected in 5.1.3 (200 μl, 10 8 CFU / day) for 1 week. After the end of microbiota transplantation and 1 week of colonization, eczema modeling began. In groups ② - ③, the right ear was treated with 1% oxazolone every day from the 1st to the 4th day of modeling, and the right ear lesion was continuously induced chemically with 0.4% oxazolone from the 5th to the 7th day. The right ear of the healthy control mice in group ① was treated with ethanol. The healthy control mice in group ① were gavaged with an equal volume of distilled water every day from day 1 to 14 of the experiment, and an equal volume of normal saline from day 14 to 21.

[0197] 5.1.5 Data collection

[0198] Appearance of eczema status: Mainly observe the ear tissues of mice, check whether there is redness, swelling and desquamation of the ears, and at the same time pay attention to the remission of redness, swelling and desquamation. Specific evaluation requires subsequent scoring.

[0199] Eczema status scoring: During the modeling period, the thickness of the right ear was measured daily with a digital micrometer, and the skin symptoms of the right ear were recorded, and eczema scoring was performed every day. The scoring criteria are as follows:

[0200] (1) When the ear shows redness, swelling and thickening: According to the area of redness and swelling, it is divided into mild (less than 30%), moderate (30% - 60%), and severe (60% - 90%) degrees, and scored 2 points, 3 points, and 4 points respectively;

[0201] (2) When the ear shows squamous desquamation: According to the area of desquamation, it is divided into mild (less than 30%), moderate (30% - 60%), and severe (60% - 90%) degrees, and scored 2 points, 3 points, and 4 points respectively;

[0202] (3) When the ear shows ulceration and exudation: According to the area of ulceration, it is divided into mild (less than 30%), moderate (30% - 60%), and severe (60% - 90%) degrees, and scored 2 points, 3 points, and 4 points respectively; If there are no above symptoms, it is scored 0 points.

[0203] Histological examination: On the 8th day of modeling, the mice were sacrificed, the ear tissues were collected and fixed with paraformaldehyde, and tissue sections were prepared. The sections were stained with hematoxylin / eosin and toluidine blue, and the thickness of the dermis layer and the number of mast cells in the right ear were observed under the microscope.

[0204] Cytokine and immunoglobulin E detection: After sacrificing the animals, blood was quickly collected from the eye sockets, and the plasma was prepared for cytokine determination; the ears were collected and homogenates were prepared for cytokine determination; both were analyzed using the enzyme-linked immunosorbent assay (ELISA kit, Jiangsu Jingmei Biotechnology Co., Ltd.). Detection content: Immunoglobulin E (IgE), interleukin-12 (IL-12), and IFN-γ in the plasma of mice. Thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and interleukin-4 (IL-4) in ear tissues. The specific operation steps were carried out according to the kit instructions.

[0205] 5.2 Results and analysis

[0206] 5.2.1 Appearance of eczema symptoms

[0207] The results are as Figure 6 shown. On the 7th day of inducing eczema in mice, obvious swelling and desquamation appeared in the ear tissues of the model group mice, and the swelling and desquamation in the ear tissues of the intervention group mice were effectively alleviated.

[0208] 5.2.2 Eczema status score

[0209] The results are as Figure 7 shown. On the one hand, on the 5th day of inducing eczema in mice, a differential trend began to appear in the eczema scores of the mice. On the 7th day, the eczema score of the model group mice was the highest, showing significant differences from the other two groups. On the other hand, the ear thickness of the model group mice was significantly higher than that of the other two groups. Compared with the model group, the eczema score of the ear tissues of the intervention group mice decreased (P<0.05), and the ear thickness of the mice became thinner (P<0.05).

[0210] 5.2.3 Histological examination

[0211] From Figure 8 and Figure 9 it can be seen that compared with the normal group, the dermis of the ears of the model group mice showed obvious swelling, the dermis thickness increased significantly (P<0.001), there was severe infiltration of inflammatory cells, and the number of mast cells increased significantly (P<0.001). Compared with the model group, the dermal swelling of the intervention group mice was alleviated, the dermis thickness decreased (P<0.05), the infiltration of inflammatory cells was alleviated, and the number of mast cells decreased (P<0.001).

[0212] 5.2.4 Cytokine and immunoglobulin E detection

[0213] As can be seen from Table 7, compared with the normal group, the levels of the ear inflammatory factors TSLP (P<0.01), IL33 (P<0.05), IL4 (P<0.01), the plasma inflammatory factor IL12 (P<0.01) and total IgE (P<0.01) in the model group mice were significantly increased. Compared with the model group, the levels of the ear inflammatory factors TSLP (P<0.01), IL33 (P<0.01), IL4 (P<0.05), the plasma inflammatory factors IL12 (P<0.01), IFN-γ (P<0.01) and total IgE (P<0.01) in the intervention group mice were significantly decreased.

[0214] Table 7 Effects of different intervention methods on cytokines in mouse ear homogenate / plasma and plasma total IgE

[0215] Grouping Normal group Model group Intervention group Ear TSLP (pg / mg) 1.69±0.17 <![CDATA[7.09±0.76 ** > <![CDATA[3.63±0.86 *## > Ear IL33 (μg / mg) 4.23±1.04 <![CDATA[11.19±1.71 * > <![CDATA[4.76±1.29 ## > Ear IL4 (pg / mg) 7.96±0.72 <![CDATA[9.61±1.87 ** > <![CDATA[7.28±0.69 *# > Plasma total IgE (pg / mL) 78.8±7.57 <![CDATA[126.6±4.27 ** > <![CDATA[90.8±5.71 *## > Plasma IL12 (pg / mL) 170.21±30.19 <![CDATA[319.28±14.98 ** > <![CDATA[179.5±13.27 *## > Plasma IFN-γ (pg / mL) 8.04±1.41 9.85±0.86 <![CDATA[5.68±0.38 ## >

[0216] Note: The results are mean ± standard deviation, *p<0.05, **p<0.01 compared with the normal group, #p<0.05, ##p<0.01 compared with the model group, one-way ANOVA. TSLP, thymic stromal lymphopoietin; IFN-γ, interferon γ.

[0217] The results of this example show that the composition of Bifidobacterium bifidum FN120, Bifidobacterium longum subsp. longum FN103 and 2’-FL can inhibit the expression of inflammatory factors, improve symptoms such as skin redness and desquamation, and play a role in the treatment of atopic dermatitis. Therefore, this composition has high application value in allergic diseases (especially infantile atopic dermatitis and eczema).

[0218] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A Bifidobacterium longum subsp. longum, characterized in that The long bifidobacterium subspecies longum was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 21, 2025, with the storage address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No. 65836.

2. A composition, characterized in that It comprises Bifidobacterium longum subspecies longum with a deposit number of GDMCC No. 65836 as described in claim 1; Preferably, the composition further comprises a probiotic selected from the group consisting of bacteria, fungi (e.g. yeast), or any combination thereof; Preferably, the bacteria is selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosus, Lactobacillus plantarum, Lactobacillus unisomeris, Lactobacillus spp., Streptococcus, Lactococcus, Propionibacterium, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Akkermansia, Faecalibacterium prausnitzii, or any combination thereof; Preferably, the probiotic bacteria of the genus Bifidobacterium are selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, or any combination thereof; Preferably, the Bifidobacterium bifidum is Bifidobacterium bifidum FN120 having a deposit number of GDMCC No. 65837; Preferably, the composition further comprises prebiotics selected from the following: human milk oligosaccharides, fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, resistant dextrin, maltodextrin, polydextrose, psyllium husk, spirulina, arthrospira, versicolor polysaccharide, carrot nitrogen-containing polysaccharide, or any combination thereof; Preferably, the human milk oligosaccharide is selected from: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3'-FL), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), lactosyl-N-tetraose (LNT), lactosyl-N-neotetraose (LNnT), lactose-N-biose (LNB), or any combination thereof; Preferably, the composition comprises Bifidobacterium longum subspecies longum with a deposit number of GDMCC No. 65836, Bifidobacterium bifidum with a deposit number of GDMCC No. 65837, and human milk oligosaccharide 2'-FL; Preferably, the composition comprises Bifidobacterium longum subspecies longum with a preservation number of GDMCC No. 65836 and Bifidobacterium bifidum with a preservation number of GDMCC No. 65837 in a ratio of 1-10:1-10 (e.g., 100:1, 50:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:50, 1:100), and the composition comprises 2'-FL and Bifidobacterium bifidum in a ratio of 0.48-48 g:1×10 9 CFU; Preferably, the content of Bifidobacterium bifidum with the deposit number of GDMCC No.65837 contained in the composition is higher than 1×10 6 CFU (e.g. 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 ), the content of Bifidobacterium longum with the deposit number of GDMCC No.65836 is higher than 1×10 6 CFU (e.g. 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 ).

3. A pharmaceutical composition, characterized in that It comprises the Bifidobacterium longum subspecies longum of claim 1 or the composition of claim 2; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient, a cryoprotectant, an amino acid, a vitamin, a mineral, a peptone, or any combination thereof; Preferably, the pharmaceutically acceptable carrier and / or excipient comprises: a filler, a binder, a lubricant, a glidant, a thickener, a flavoring agent, an edible oil, a stabilizer, a suspending agent, a surfactant, or any combination thereof; Preferably, the pharmaceutical composition further comprises a cryoprotectant; Preferably, the pharmaceutical composition further comprises glycerol, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylo-oligosaccharide, glutathione, or any combination thereof; Preferably, the pharmaceutical composition is formulated for oral administration or in vitro administration; Preferably, the pharmaceutical composition comprises a preparation of Bifidobacterium longum subsp. longum; Preferably, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), oil drops, film-coated preparations, orally dissolving granules, liquids, suppositories, enema gels and / or creams; Preferably, the pharmaceutical composition further comprises formulation excipients (e.g., excipients for preparing powders, tablets, capsules, oil drops); Preferably, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, analgesics, anti-inflammatory drugs, healing agents and / or moisturizers; Preferably, the pharmaceutical composition contains more than 10 6 The amount of CFU / dose present (e.g., 10 7 CFU / dose, 0 8 CFU / dose, 10 9 CFU / dose); Preferably, when the pharmaceutical composition is solid, it contains more than 10 6 CFU of Bifidobacterium longum subsp. longum (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g); Preferably, when the pharmaceutical composition is a liquid, each milliliter of the composition contains more than 10 6 CFU of Bifidobacterium longum subsp. longum (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL).

4. A culture, characterized in that It comprises the Bifidobacterium longum subspecies longum of claim 1 or the composition of claim 2; Preferably, the culture further comprises components that provide nutrients (e.g., solid or liquid culture medium, feeder cell layer); Preferably, the nutrient-providing ingredients are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fibers, amino acids, or any combination thereof; Preferably, the culture further comprises Bifidobacterium longum subsp. longum and / or a cell-free culture filtrate of Bifidobacterium longum subsp. longum; Preferably, the culture further comprises Bifidobacterium longum subsp. longum and / or a derivative of Bifidobacterium longum subsp. longum; wherein the derivative is selected from metabolites, enzymes, cell structure components (e.g., cell walls or components thereof), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

5. A food product, dietary supplement or health food, characterized in that: It comprises the Bifidobacterium longum subspecies longum of claim 1 or the composition of claim 2 or the culture of claim 4; Preferably, the food product is selected from solid beverages, liquid beverages, compressed candies, puffed foods, protein bars or nuts, or the food product is a dairy product (e.g., milk powder, milk tablets, yogurt, flavored fermented milk, lactic acid bacteria beverages, cheese); Preferably, the food product or dietary supplement or nutraceutical further comprises additional additives; Preferably, the additional additive is selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fibers, amino acids, or any combination thereof; Preferably, the food product, dietary supplement or health food contains more than 10 6 The amount of CFU / dose present (e.g., 10 7 CFU / dose, 0 8 CFU / dose, 10 9 CFU / dose); Preferably, when the food product, dietary supplement or health food is solid, it contains more than 10 6 CFU of Bifidobacterium longum subsp. longum (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g); Preferably, when the food product, dietary supplement or health food is a liquid, it contains more than 10 6 CFU of Bifidobacterium longum subsp. longum (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL).

6. Use of the Bifidobacterium longum subsp. longum of claim 1, the composition of claim 2, the pharmaceutical composition of claim 3, the culture of claim 4, or the food product, dietary supplement, or health food of claim 5 in the preparation of a medicament, dietary supplement, or health food for preventing, alleviating, and / or treating allergy-related diseases and / or symptoms in a subject, or for preventing, alleviating, and / or treating inflammation-related diseases and / or symptoms in a subject; Preferably, the allergy-related disease is associated with one or more of the following conditions: (1) Increased levels of Clostridium and / or Enterobacteriaceae strains in the intestinal ecosystem; (2) It is associated with a decrease in the levels of Lactobacillus and / or Bifidobacterium strains in the intestinal ecosystem; Preferably, the allergy-related disease is selected from eczema, atopic dermatitis, and food allergy; Preselected, the inflammatory-related disease is selected from diseases caused by skin inflammation (e.g., dermatitis, eczema), diseases caused by respiratory tract inflammation (e.g., upper respiratory tract infection) and diseases caused by digestive tract inflammation (e.g., inflammatory bowel disease); Preferably, the drug or dietary supplement or health product can activate immune cells, promote the production of cytokines, inhibit inflammation or alleviate inflammatory diseases; Preferably, the cytokine is selected from IL33, IL4, IL12, IFN-γ, TSLP, or any combination thereof; Preferably, the drug or dietary supplement or health product can reduce IgE levels and inhibit IgE-mediated allergic reactions; Preferably, the drug or dietary supplement or health product is used alone or in combination with other antifungal agents, analgesics, anti-inflammatory drugs, healing agents, or moisturizers; Preferably, the subject is a mammal; Preferably, the subject is a human.

7. A method for preventing, alleviating and / or treating allergy-related diseases and / or symptoms, or preventing, alleviating and / or treating inflammation-related diseases and / or symptoms, characterized in that: The method comprises: administering an effective amount of Bifidobacterium longum subsp. longum as claimed in claim 1 or the composition as claimed in claim 2 or the pharmaceutical composition as claimed in claim 3 or the culture as claimed in claim 4 or the food product or dietary supplement or health food as claimed in claim 5 to a subject in need; Preferably, the method comprises: implanting the Bifidobacterium longum subsp. longum of claim 1 or the composition of claim 2 or the pharmaceutical composition of claim 3 or the culture of claim 4 or the food product or dietary supplement or health food of claim 5 into the intestine of a subject; Preferably, the method comprises: administering to a subject orally (e.g., orally) the Bifidobacterium longum subspecies longum as claimed in claim 1 or the composition as claimed in claim 2 or the pharmaceutical composition as claimed in claim 3 or the culture as claimed in claim 4 or the food product or dietary supplement or health food as claimed in claim 5; In certain embodiments, the Bifidobacterium longum subsp. longum is present at a concentration greater than 10 6 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the subject.

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