Isolated animal bifidobacterium, compositions containing same and uses thereof

Abstract

The present disclosure provides isolated animal bifidobacteria, compositions containing the same, and uses thereof, which can be used to promote growth and development, enhance immunity, or promote intestinal health.

Description

Technical Field

[0001] The present disclosure relates to the field of microorganisms, and more particularly to isolated animal Bifidobacterium, compositions containing the same, and uses thereof. Background Art

[0002] Growth refers to the enlargement and morphological changes of the body's organs and systems, representing quantitative changes; development refers to the differentiation, perfection, and functional maturation of cells, tissues, and organs, representing changes in their quality. The two are closely related, with growth providing the material foundation for development. Developmental maturity, in turn, is reflected in quantitative changes in growth. Human growth and development refers to the maturation process from fertilized egg to adult. Growth and development are key characteristics that distinguish children from adults. Studies have found that intestinal bacteria can influence host growth and development, with some microbiota mediating growth and development by influencing growth hormone (GH) / insulin-like growth factor-1 (IGF-1).

[0003] Immunity refers to the body's ability to resist foreign invasion and maintain a stable internal environment. For children, the development and protection of the immune system is particularly important, as it is directly related to their healthy growth. Infants and children with developmental delays, due to slower development of their immune organs and gastrointestinal tract, have weak immunity and low resistance, making them more susceptible to illness. Air, food, water, and other environmental sources are filled with a wide variety of microorganisms, including bacteria, viruses, mycoplasmas, chlamydia, and fungi. Children with weakened immunity are easily infected by harmful pathogens, which can then spread to their caregivers and classmates, potentially leading to severe influenza or other illnesses. While the human body produces antibodies to protect against reinfection with different pathogens, these antibodies are specific and short-lived. For example, antibodies against streptococci only provide short-term protection against reinfection and do not protect against infection with other viruses. The bacteria and viruses that cause influenza mutate rapidly, making it difficult for infants and children with weakened immunity to resist the cold virus. This is the real reason for their frequent colds.

[0004] Probiotics are microorganisms that, when administered in adequate amounts, confer health benefits on the host. Studies have shown that probiotics can maintain a balanced intestinal flora through their own metabolism and inhibit the growth of harmful bacteria. Therefore, intestinal probiotics are closely related to human health. Probiotic strains have been shown to regulate intestinal health and enhance immunity.

[0005] Bifidobacterium lactis CGMCC No. 20847 is a commercially available strain with excellent growth-promoting activity. CN112980725B discloses that Bifidobacterium lactis CGMCC No. 20847 can significantly increase the level of IGF-1 in mouse serum, promote mouse femur length, and promote the increase of adolescent height. CN114317354B discloses that Bifidobacterium animalis MB-424 can promote bone cell growth and promote pituitary GH3 cells to produce growth hormone. In the prior art, the promotion of growth and development for Bifidobacterium animalis or Bifidobacterium lactis mainly focuses on promoting the growth of bones and height. However, children with developmental delay have low resistance and are prone to illness, which is related to the slow development of immune organs and gastrointestinal tract. Few studies have been conducted on probiotics that promote the growth and development of internal organs such as immune organs and digestive organs.

[0006] CN110964657B discloses that Bifidobacterium lactis BL-99 and BB-12 had no effect on spleen and thymus weight in mice. Another study (Effect of probiotics on thymus size and markers of infection in late infancy: a randomized controlled trial) showed that 186 healthy Danish infants were treated with Lactobacillus rhamnosus LGG and Bifidobacterium animalis subsp. lactis BB-12 for six months. There was no significant difference in thymus size between the probiotic and placebo groups (p ≥ 0.248). Probiotics also had no effect on CRP (C-reactive protein) (p = 0.331).

[0007] It can be seen that the number of microbial resources is extremely large, and screening out new species or strains that can promote the growth and development of internal organs such as immune organs and digestive organs, and further promote growth and development, enhance immunity, and / or improve intestinal health is a huge challenge, but it also represents a huge unmet need. Summary of the Invention

[0008] The present invention discloses the isolation and preparation of a novel animal Bifidobacterium ( Bifidobacterium animalisThe new strain disclosed herein can 1. promote the development of major immune organs such as the thymus and spleen in subjects with developmental delay (including increasing the weight of immune organs such as the thymus and spleen, and the immune organ development index (immune organ weight / body weight), etc.); 2. promote the development and proliferation of immune cells such as T cells, thereby improving the immunity of subjects with developmental delay; 3. increase the serum insulin-like growth factor 1 (IGF-1) content in subjects with developmental delay, promote weight and body length growth, and promote bone and muscle growth, such as promoting the growth of tibia length, femur length, and soleus and gastrocnemius muscle weight; 4. increase the weight of the liver, thymus, spleen, kidney, and intestinal length (large intestine length and / or small intestine length) in subjects with developmental delay; increase food intake; increase the content of short-chain fatty acids in the intestine; improve intestinal flora; and promote intestinal development.

[0009] Therefore, the new strain disclosed herein can promote growth and weight while also promoting organ development, particularly immune organ development, enhancing immunity, promoting intestinal development, and improving intestinal health. It can be used to improve, treat, or reverse developmental delay caused by malnutrition, achieving goals such as promoting growth and development, enhancing immunity, or promoting intestinal health.

[0010] In a first aspect, the present disclosure provides isolated Bifidobacterium animalis ( Bifidobacterium animalis ), which has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with the strain deposited with GDMCC No: 65553 or GDMCC No: 65555; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0011] In some embodiments, the animal bifidobacterium has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with strain MNH17483 deposited with GDMCC No: 65553; and / or has a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 3.

[0012] In some embodiments, the animal bifidobacterium has an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with strain MNH39288 deposited with GDMCC No: 65555; and / or has a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 4.

[0013] In some embodiments, the Bifidobacterium animalis has a 16S rRNA sequence that is at least 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the sequence shown in SEQ ID NO: 3, or SEQ ID NO: 4.

[0014] In some embodiments, the Bifidobacterium animalis is Bifidobacterium animalis ( Bifidobacterium animalis ) two new strains.

[0015] In some embodiments, the name of the animal Bifidobacterium is: Animal Bifidobacterium ( Bifidobacterium animalis ) MNH17483, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65553, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Bifidobacterium animalis MNH17483.

[0016] In some embodiments, the name of the animal Bifidobacterium is: Animal Bifidobacterium ( Bifidobacterium animalis ) MNH39288, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65555, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Bifidobacterium animalis MNH39288.

[0017] In a second aspect, the present disclosure provides a culture, live bacteria, freeze-dried bacteria or inactivated bacteria comprising the animal Bifidobacterium described in the first aspect,

[0018] Wherein, the culture comprises any one of the following A) to D):

[0019] A) fermentation broth of the animal Bifidobacterium;

[0020] B) the supernatant of the fermentation broth of Bifidobacterium animalis;

[0021] C) an inactivated fermentation broth of the animal Bifidobacterium;

[0022] D) The concentrated or dried product of any one of A) to C) above.

[0023] In a third aspect, the present disclosure provides a composition comprising the animal Bifidobacterium described in the first aspect, or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the animal Bifidobacterium described in the second aspect, wherein the composition is a medicine.

[0024] The present disclosure provides a composition comprising the animal Bifidobacterium, a culture thereof, and / or a metabolite thereof according to the first aspect, wherein the composition is a medicine.

[0025] In some embodiments, the culture of Bifidobacterium animalis includes a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0026] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained using a liquid culture medium under anaerobic culture conditions.

[0027] In some embodiments, the composition is provided in liquid form or solid form.

[0028] In some embodiments, the composition comprises 1×10 4 to 1×10 12 cfu / mL or 1×10 4 to 1×10 12 cfu / mg of the live animal Bifidobacterium.

[0029] In some embodiments, the composition comprises 1×10 5 to 1×10 11 cfu / mL or 1×10 5 to 1×10 11 cfu / mg of the live animal Bifidobacterium.

[0030] In some embodiments, the composition comprises 1×10 6 to 1×10 10 cfu / mL or 1×10 6 to 1×10 10cfu / mg of the live animal Bifidobacterium.

[0031] In some embodiments, the composition comprises 1×10 7 to 1×10 9 cfu / mL or 1×10 7 to 1×10 9 cfu / mg of the live animal Bifidobacterium.

[0032] In some embodiments, each gram of the composition comprises 1×10 3 to 1×10 17 colony-forming units (CFU) of bacteria; for example, 1×10 4 to 1×10 12 1×10 5 to 1×10 11 or 1×10 6 to 1×10 10 colony forming units (CFU), specifically, for example, 1 × 10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×106 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12, 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 or any value in between of colony forming units (CFU).

[0033] In some embodiments, the concentration of the Bifidobacterium animalis as an active ingredient in the composition is 10 7 to 10 12 CFU / g.

[0034] In some embodiments, the animal Bifidobacterium in the composition is a live bacterium, an attenuated bacterium, a freeze-dried bacterium, or an inactivated bacterium, for example, a heat-inactivated bacterium, preferably pasteurized.

[0035] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0036] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0037] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0038] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients.

[0039] The pharmaceutically acceptable excipients are well known to those skilled in the art.

[0040] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0041] In some embodiments, the composition comprises one or more of a buffering agent (e.g., sodium bicarbonate or other agent that allows bacterial survival and growth (e.g., survival in the acidic environment of the stomach and growth in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0042] In some embodiments, the composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0043] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0044] In some embodiments, the organ comprises at least one of the heart, thymus, liver, spleen, kidney, and intestine.

[0045] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0046] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0047] In some embodiments, the other active agent may be one or more of probiotics, prebiotics, or a combination thereof.

[0048] In some embodiments, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentans, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus cremoris.

[0049] In some embodiments, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast beta-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0050] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0051] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0052] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0053] In some embodiments, the composition can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0054] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form having an enteric coating. For example, the enteric-coated formulation can be an enteric granule, an enteric-coated tablet, or an enteric-coated capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained-release capsule, a controlled-release capsule, or an enteric-coated capsule, or the capsule can be a microencapsulated capsule or a microcapsule.

[0055] In some embodiments, the composition is a medicament.

[0056] In some embodiments, the composition is in an infant-appropriate dosage form, a child-appropriate dosage form, or an adult-appropriate dosage form.

[0057] In some embodiments, the composition is in a parenteral or parenteral dosage form.

[0058] A composition for enhancing immunity, comprising the animal Bifidobacterium described in the first aspect or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the animal Bifidobacterium described in the second aspect, wherein the composition is a health product.

[0059] The present disclosure provides an immunity-enhancing composition comprising the animal Bifidobacterium, a culture thereof, and / or a metabolite thereof according to the first aspect, wherein the composition is a health product.

[0060] In some embodiments, the culture of Bifidobacterium animalis includes a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0061] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained using a liquid culture medium under anaerobic culture conditions.

[0062] In some embodiments, the composition is provided in liquid form or solid form.

[0063] In some embodiments, the composition comprises 1×10 4 to 1×10 12 cfu / mL or 1×10 4 to 1×10 12 cfu / mg of the live animal Bifidobacterium.

[0064] In some embodiments, the composition comprises 1×10 5 to 1×10 11 cfu / mL or 1×10 5 to 1×10 11 cfu / mg of the live animal Bifidobacterium.

[0065] In some embodiments, the composition comprises 1×10 6 to 1×10 10 cfu / mL or 1×10 6 to 1×10 10 cfu / mg of the live animal Bifidobacterium.

[0066] In some embodiments, the composition comprises 1×10 7 to 1×10 9 cfu / mL or 1×10 7 to 1×10 9 cfu / mg of the live animal Bifidobacterium.

[0067] In some embodiments, each gram of the composition comprises 1×10 3 to 1×10 17 colony-forming units (CFU) of bacteria; for example, 1×10 4 to 1×10 12 1×10 5 to 1×10 11 or 1×10 6 to 1×10 10 colony forming units (CFU), specifically, for example, 1 × 10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×104 、3×10 4 、4×10 4 、5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10, 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 or any value in between of colony forming units (CFU).

[0068] In some embodiments, the concentration of the Bifidobacterium animalis as an active ingredient in the composition is 10 7 to 10 12 CFU / g.

[0069] In some embodiments, the animal Bifidobacterium in the composition is a live bacterium, an attenuated bacterium, a freeze-dried bacterium, or an inactivated bacterium, for example, a heat-inactivated bacterium, preferably pasteurized.

[0070] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0071] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0072] In some embodiments, the composition is in the form of an oral dosage.

[0073] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0074] In some embodiments, the composition comprises one or more of a buffering agent (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0075] In some embodiments, the composition further comprises one or more other active agents for enhancing immunity.

[0076] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof;

[0077] In some embodiments, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentans, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus cremoris.

[0078] In some embodiments, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast beta-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0079] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0080] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0081] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0082] In a fourth aspect, the present disclosure provides the use of the animal bifidobacterium described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect (the composition is a drug) in the preparation of a drug for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0083] Use of the animal bifidobacterium described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect (the composition is a health product) in the preparation of a health product for enhancing immunity.

[0084] The present disclosure provides uses of the animal Bifidobacterium described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect in promoting growth and development, enhancing immunity, or promoting intestinal health (preferably, the uses do not involve the treatment of diseases).

[0085] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0086] In some embodiments, the organ comprises at least one of the heart, liver, spleen, kidney, and intestine.

[0087] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0088] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0089] In some embodiments, the drug promotes growth and development by increasing hormone levels, and / or increasing appetite.

[0090] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0091] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0092] In some embodiments, the drug or health product enhances immunity by promoting the development of the immune system.

[0093] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0094] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0095] In some embodiments, promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0096] In some embodiments, the drug promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0097] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0098] The composition comprises a consortium of isolated and purified viable microbial populations to increase body weight by at least 2%, 3%, 4%, 5%, 6%, or 7% in a subject compared to the subject's body weight before administration of the isolated and purified consortium of viable microbial species.

[0099] As used herein, a microbial population generally refers to a population of microorganisms consisting essentially of a single strain, species, or genus, as may be the case when the population is cultured from an isolated and purified subpopulation of such a strain, species, or genus. Thus, for a given population of microorganisms, if such population is cultured from an isolated species or strain of microorganisms, such population will be referred to herein as purified or substantially pure. The resulting population will typically be at least 80% pure with respect to the species or strain of microorganism, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure relative to other species or strains of microorganisms within that particular population. Conversely, the level of undesirable strains in any particular desired population of microorganisms will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. For example, the level of impurities, such as other undesirable strains or species, in a purified population of microorganisms can be at or below the aforementioned levels for each desired population, proportionally. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population.

[0100] The compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by Bifidobacterium animalis, and the like. This can be obtained, for example, by recovering the supernatant of a culture of Bifidobacterium animalis or by extracting cellular components or cell fractions, metabolites or secreted compounds from a culture of Bifidobacterium animalis; this can correspond to components in isolated form from Bifidobacterium animalis, or any mixture of one or more components from Bifidobacterium animalis.

[0101] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition described in the present disclosure can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0102] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0103] In some embodiments, the organ comprises at least one of the heart, thymus, liver, spleen, kidney, and intestine.

[0104] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0105] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0106] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes growth and development by increasing hormone content and / or increasing appetite.

[0107] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0108] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0109] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition enhances immunity by promoting immune system development.

[0110] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0111] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0112] In some embodiments, promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0113] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0114] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0115] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can promote appetite, thereby increasing food intake (preferably cumulative food intake), thereby achieving the effect of promoting growth and development.

[0116] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase heart, liver, spleen, and / or kidney weight, and / or intestinal length, thereby promoting organ development.

[0117] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase body weight, body length, tibia length, femur length, soleus muscle and gastrocnemius muscle weight, thereby promoting height (body length), weight, bone, and muscle development.

[0118] In some embodiments, the animal Bifidobacterium, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition of the present disclosure can increase the levels of serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH), thereby promoting growth and development.

[0119] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can promote the development of major immune organs such as the thymus and spleen, increase the proportion of T cells, B cells, and NK cells, thereby promoting the development of the immune system and enhancing immunity.

[0120] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase the content of short-chain fatty acids and / or improve intestinal flora, thereby promoting intestinal health.

[0121] In some embodiments, the drug has at least one effect selected from the following: increasing the weight of the heart, liver, spleen, and / or kidney, and / or intestinal length (large intestine length and / or small intestine length); increasing food intake; increasing body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight; promoting weight growth, body length growth, bone and muscle growth (wherein promoting bone growth includes growth in bone length and density, and promoting muscle growth includes promoting specific muscle development and maintaining healthy and benign growth characteristics, such as: low body fat rate (calf muscle / body weight) and / or high muscle mass ratio (calf muscle / calf weight)); increasing serum insulin The invention relates to an enzyme that is present in the gut and is used to increase the content of IGF-1 and / or growth hormone (GH); promote the development of major immune organs such as the thymus and spleen (including increasing the weight of immune organs such as the thymus and spleen, the immune organ development index (immune organ weight / body weight), etc.), increase the proportion of T cells, B cells, and NK cells; increase the content of short-chain fatty acids; improve intestinal flora; promote intestinal development; improve lesions or inflammation caused by developmental delay to achieve the function of improving / treating / reversing developmental delay; promote organ development; promote appetite; promote height (body length), weight, bone, and muscle development; promote the development of the immune system and enhance immunity; promote intestinal health; can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0122] In some embodiments, the health product has the following effects: enhancing immunity.

[0123] A method for promoting growth and development, enhancing immunity, or promoting intestinal health, comprising administering an effective amount of the animal bifidobacterium described in the first aspect of the present disclosure, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect to a subject in need.

[0124] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0125] In some embodiments, the organ comprises at least one of the heart, liver, spleen, kidney, and intestine.

[0126] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0127] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0128] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes growth and development by increasing hormone content and / or increasing appetite.

[0129] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0130] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0131] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition enhances immunity by promoting immune system development.

[0132] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0133] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0134] In some embodiments, promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0135] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0136] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] The following description of the embodiments will be made in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0138] Figure 1 : shows a photograph of the colony morphology of strain MNH17483.

[0139] Figure 2 : Gram-stained photographs of strain MNH17483 are shown.

[0140] Figure 3 : shows an electron micrograph of strain MNH17483.

[0141] Figure 4 : shows a photograph of the colony morphology of strain MNH39288.

[0142] Figure 5 : Gram-stained photographs of strain MNH39288 are shown.

[0143] Figure 6 : shows an electron micrograph of strain MNH39288.

[0144] Figure 7 : Shows the results of the tolerance of strain MNH17483 to different pH.

[0145] Figure 8 : Shows the tolerance results of strain MNH17483 to different concentrations of NaCl.

[0146] Figure 9 : Shows the results of the tolerance of strain MNH17483 to different concentrations of bile salts.

[0147] Figure 10 : Shows the results of the tolerance of strain MNH39288 to different pH.

[0148] Figure 11 : Shows the results of the tolerance of strain MNH39288 to different concentrations of NaCl.

[0149] Figure 12 : Shows the results of the tolerance of strain MNH39288 to different concentrations of bile salts.

[0150] Figure 13 : shows the phylogenetic tree of strain MNH17483.

[0151] Figure 14 : shows the phylogenetic tree of strain MNH39288.

[0152] Figure 15 Figure 2: Strains MNH17483 and MNH39288 increase cumulative food intake (line graph). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0153] Figure 16 Figure 2: MNH17483 and MNH39288 strains increased cumulative food intake (cumulative food intake bar graph). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0154] Figure 17: This study demonstrates that strains MNH17483 and MNH39288 promote organ development (liver weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0155] Figure 18 : Shows that strains MNH17483 and MNH39288 promote organ development (spleen weight). Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0156] Figure 19 : Shows that strains MNH17483 and MNH39288 promote organ development (kidney weight). Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using the Student's t test; **, p < 0.01 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0157] Figure 20 : This study demonstrates that strains MNH17483 and MNH39288 promote organ development (intestinal length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. **, p < 0.01 compared with the LFD-Control group.

[0158] Figure 21 Figure 2: Strains MNH17483 and MNH39288 promote weight gain. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0159] Figure 22: Shows that strains MNH17483 and MNH39288 promote height (length increase). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; ****, p < 0.0001 compared with the LFD-Control group.

[0160] Figure 23 Figure 3: MNH17483 and MNH39288 strains promote bone development (femur length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0161] Figure 24 Figure 3: MNH17483 and MNH39288 strains promote bone development (tibia length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0162] Figure 25 Figure 2: MNH17483 and MNH39288 strains promote muscle development (soleus muscle weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0163] Figure 26 Figure 3: MNH17483 and MNH39288 strains promote muscle development (gastrocnemius muscle weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0164] Figure 27Figure 3: MNH39288 strain increases serum insulin-like growth factor-1 (IGF-1) levels. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0165] Figure 28 The results show that strains MNH17483 and MNH39288 promote immune system development and enhance immunity (thymus weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0166] Figure 29 The MNH17483 and MNH39288 strains promote immune system development and enhance immunity (spleen weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0167] Figure 30 The results show that strains MNH17483 and MNH39288 promote immune system development and enhance immunity (immune organ weights). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0168] Figure 31 This study demonstrates that strain MNH39288 promotes immune system development and enhances immunity (T cell percentage). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0169] Figure 32 This figure shows that strain MNH17483 promotes the production of short-chain fatty acids (butyrate content) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0170] Figure 33 Figure 3: Strain MNH39288 promotes the production of short-chain fatty acids (isovalerate) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0171] Figure 34 Figure 3: Strain MNH39288 promotes the production of short-chain fatty acids (2-methylbutyrate as a percentage) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0172] Strain preservation

[0173] strain Bifidobacterium animalis ( Bifidobacterium animalis ) MNH17483, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65553, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the deposit name is Bifidobacterium animalis MNH17483, taxonomic name: Bifidobacterium animalis .

[0174] strain Bifidobacterium animalis ( Bifidobacterium animalis ) MNH39288, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65555, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, the deposit name is Bifidobacterium animalis MNH39288, taxonomic name: Bifidobacterium animalis . DETAILED DESCRIPTION

[0175] The present invention isolated two new strains of the species Bifidobacterium animalis, deposited with GDMCC No. 65553 and GDMCC No. 65555, respectively. These strains were identified using traditional taxonomic and molecular biological methods. The results indicate that these two strains are new strains of the species Bifidobacterium animalis. Furthermore, the present invention investigates the biochemical properties and therapeutic uses of these two strains.

[0176] As is known in the art, bacterial species can be classified and identified using traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic assays. Molecular biology methods include, but are not limited to, ribosomal RNA sequencing and whole genome sequencing-based methods.

[0177] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0178] The terms "subject," "subject," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including humans, mammals, laboratory animals, livestock, and domestic pets.

[0179] The compositions (which are medicaments) or formulations disclosed herein can be administered as pharmaceutical preparations, therapeutic compositions, or medical probiotics. In some cases, the compositions are administered in the form of pharmaceutical preparations. In some cases, the compositions are administered in the form of medical probiotics. In some cases, the compositions (e.g., medical probiotics) can be administered orally, for example, as capsules, pills, or tablets.

[0180] 16S rRNA is a type of ribosomal RNA in prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is shared by all bacteria, while the variable region varies to varying degrees among different bacteria. By comparing bacterial 16S rRNA gene sequences and basing the number of sequence differences and their evolutionary distance, an evolutionary tree can be constructed. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence betweenaverage nucleotide identity and 16S rRNA gene sequence similarity for speciesdemarcation of prokaryotes. International Journal of Systematic andEvolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini,R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening thetaxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), p23).

[0181] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG program package, BLASTN or FASTA. Commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

[0182] Second-generation sequencing technology can also be used to identify bacterial species based on whole-genome sequencing, making the identification results more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value must reach above 95% to be considered as belonging to the same species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9(1):5114.).

[0183] Various existing mature ANI value calculation tools can be used, such as the local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0184] Using the above method, a person skilled in the art can determine whether an isolated strain belongs to the animal Bifidobacterium species identified by the present inventors. For example, when compared with the animal Bifidobacterium with the deposit number GDMCC No: 65553 or GDMCC No: 65555 ( Bifidobacterium animalis ), an average nucleotide identity ANI value of at least 95%, for example at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98 ... .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that they belong to the same species.

[0185] For another example, when its 16S rRNA sequence is at least 98.65% identical to the sequence shown in SEQ ID NO: 3 or 4, for example, at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the sequence shown in SEQ ID NO: 3 or 4, it can be determined to belong to the same bacterial species.

[0186] A "strain" is a member of a bacterial species that has genetic characteristics that allow it to be distinguished from closely related members of the same bacterial species. A genetic characteristic can be the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence ("cure") of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In cases where one strain (compared to another strain of the same species) acquires or loses antibiotic resistance or a biosynthetic capacity (e.g., an auxotrophic strain), the strains can be distinguished by selection or counterselection using antibiotics or nutrients / metabolites.

[0187] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0188] The animal Bifidobacterium described herein can be used to prepare a composition (a pharmaceutical composition), for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the animal Bifidobacterium, for example, the animal Bifidobacterium with a deposit number of GDMCC No. 65553 or GDMCC No. 65555. Similarly, the animal Bifidobacterium with a deposit number of GDMCC No. 65553 or GDMCC No. 65555 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, comprising a pharmaceutically effective amount of the animal Bifidobacterium.

[0189] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

[0190] The composition herein (the composition being a medicament) can be formulated into any form suitable for enhancing the abundance of animal Bifidobacterium in a subject. The composition can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by bile infusion, by cardiac infusion, pre-anally, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, or by ureteral or urethral administration.

[0191] Examples of dosage forms suitable for the compositions herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0192] In some embodiments, the immunity-enhancing health product is a sugar-coated tablet, a gel capsule, a tablet, a sheet capsule, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder, and the like.

[0193] The compositions provided herein may comprise a pharmaceutically acceptable excipient, diluent or carrier. Such pharmaceutically acceptable excipients, diluents or carriers are well known in the art.

[0194] In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is spray-dried. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried or spray-dried and is alive. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried or spray-dried and can partially or completely colonize the intestine. In some embodiments, the freeze-dried animal bifidobacterium is reconstituted before administration. In some embodiments, the reconstitution is performed using a diluent as described herein.

[0195] In some embodiments, the compositions of the present disclosure (which are pharmaceuticals) are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0196] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0197] The composition of the present disclosure (the composition is a drug) is a pharmaceutical product.

[0198] The immunity-enhancing composition disclosed herein is a health product.

[0199] The subject of the present disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0200] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature of the art or in accordance with the product or instrument specifications are used. All reagents or instruments are commercially available if their manufacturers are specified.

[0201] Example

[0202] The anaerobic blood agar plates involved in the examples were purchased from Huankai Microorganisms, with the following formula: 10 g / L casein pancreatic digest, 3 g / L cardiac pancreatic digest, 1 g / L corn starch, 5 g / L meat peptic digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50 mL / L sterile defibrinated sheep blood, pH 7.3 ± 0.2.

[0203] The PYG liquid medium involved in the examples, each 1 L of PYG liquid medium contains: Trypticase peptone 5.0 g, Peptone 5.0 g, Yeast extract 10.0 g, Beef extract 5.0 g, Glucose 5.0 g, K2HPO4 2.0 g, Tween 80 1.0 ml, Resazurin 1.0 mg, Salt solution 40 ml (its components are as follows: CaCl2·2H2O 0.01 g, MgSO4·7H2O 0.02 g, K2HPO4 0.04 g, NaHCO3 0.4 g, NaCl 0.08 g, balance water), balance water, pH 7.2±0.2 (25°C).

[0204] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0205] Example 1. Isolation and identification of strains

[0206] 1.1 Isolation and purification of strains MNH17483 and MNH39288

[0207] Two intestinal strains of Bifidobacterium animalis ( Bifidobacterium animalis MNH17483 and Bifidobacterium animalisMNH39288) were isolated from fecal samples of healthy volunteers. Conventional strain isolation methods were used, using gradient dilution, followed by isolation and culture of single colonies. Purification was performed and anaerobically cultured at 37°C. Pure cultures were prepared into a 20% glycerol / water suspension and stored at -80°C.

[0208] Specifically, the isolation and purification methods of the strains are as follows:

[0209] The donor collects 2-5 g of fresh feces, places it in a sample collection and storage tube, shakes it to homogenize it, and places the processed fecal sample in an ice box. It is then sent to the laboratory for strain isolation within 24 hours.

[0210] Aliquot physiological saline solution in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation and transfer them to the anaerobic workstation 24 h in advance. Label the plates with sample information, culture medium type, isolation date, etc.

[0211] Take fresh fecal sample and place it in anaerobic workstation and shake it with vortex shaker for 1 min to mix it. Pipette 1 mL of sample into 9 mL of normal saline and mix it for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution, set aside.

[0212] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the plate surface is dry, invert the plate and culture at 37°C for 3-5 days.

[0213] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterile toothpick for strain purification. Incubate the purified strain anaerobically at 37°C. Prepare a 20% glycerol / water solution of the pure culture and store it at -80°C.

[0214] 1.2 Morphological characteristics of strains MNH17483 and MNH39288

[0215] 1.2.1 Morphological characteristics of strain MNH17483

[0216] Strain MNH17483 was inoculated onto PYG plates and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the PYG plates. These colonies were round, with regular, smooth edges, approximately 0.5-2 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1.5-3 µm in size. A photograph of the colony morphology of strain MNH17483 after 48 hours of culture on PYG plates is shown in the accompanying image. Figure 1 The Gram staining photos of strain MNH17483 are shown in Figure 2 See the electron microscope photos for Figure 3 .

[0217] 1.2.2 Morphological characteristics of strain MNH39288

[0218] Strain MNH39288 was inoculated onto PYG plates and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the PYG plates. These colonies were round, with regular, smooth edges, approximately 0.5-2 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1.5-3 µm in size. A photograph of the colony morphology of strain MNHMNH39288 after 48 hours of culture on PYG plates is shown in the figure. Figure 4 The Gram staining photos of strain MNH39288 are shown in Figure 5 See the electron microscope photos for Figure 6 .

[0219] 1.3 Physiological and biochemical characteristics of strains MNH17483 and MNH39288

[0220] 1.3.1 Physiological and biochemical characteristics of strain MNH17483

[0221] Strain MNH17483 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 5.0 to 10.0, with the optimal growth pH being 7.0-9.0 (see the results of the strain's tolerance to different pH values ​​for details). Figure 7 ); it does not grow in a culture medium with a NaCl content exceeding 3% (the results of the strain's tolerance to different concentrations of NaCl are shown in Figure 8 ); strain MNH17483 can survive and grow in the bile salt concentration range of 0%-0.15%, and cannot grow when the bile salt concentration is greater than or equal to 0.2% (the results of the strain's tolerance to different bile salt concentrations are shown in Figure 9 ).

[0222] 1.3.2 Physiological and biochemical characteristics of strain MNH39288

[0223] The strain MNH39288 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 6.0 to 9.0, with the optimal growth pH being 7.0 (see the results of the strain's tolerance to different pH values ​​for details). Figure 10 ); can still grow in culture medium with NaCl content exceeding 6% (the results of strain tolerance to different concentrations of NaCl are shown in Figure 11 ); strain MNH39288 can survive and grow in the bile salt concentration range of 0%-0.1%, and cannot grow when the bile salt concentration is greater than or equal to 0.15% (the results of the strain's tolerance to different concentrations of bile salts are shown in Figure 12 ).

[0224] 1.4 Biochemical identification results of strains MNH17483 and MNH39288

[0225] 1.4.1 Results of biochemical identification of strain MNH17483 using API 50CHL

[0226] API 50CHL (Mérieux, CN5041010) was used. Specific experimental procedures are described in the general API reagent operating instructions. Incubation conditions: 37°C, anaerobic. Results are shown in Table 1.

[0227] MNH17483 can ferment and produce acid by using L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, α-methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, and D-turanose TUR. Therefore, in the process of fermentation or cultivation of strain MNH17483, L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, α-methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, and their derivatives can be used as carbon sources.

[0228] Table 1. Test results of strain MNH17483

[0229] MNH17483 0 1 2 3 4 5 6 7 8 9 - - - - + + + - - - 10 11 12 13 14 15 16 17 18 19 - + - - - - - - - - 20 21 22 23 24 25 26 27 28 29 - + - + + + + + + + 30 31 32 33 34 35 36 37 38 39 + + - - - + + - - + 40 41 42 43 44 45 46 47 48 49 + - - - - - - - - -

[0230] Note: “+” indicates positive, “-” indicates negative.

[0231] 1.4.2 Results of biochemical identification of strain MNH39288 using API 50CHL

[0232] API 50CHL (Mérieux, CN5041010) was used. Specific experimental procedures are described in the standard API reagent operating instructions. Incubation conditions: 37°C, anaerobic. Results are shown in Table 2.

[0233] MNH39288 can utilize L-arabinose LARA, ribose RIB, D-xylose DXYL, Galactose GAL, Glucose GLU, Fructose FRU,α-Methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR are fermented to produce acid. Therefore, during the fermentation or cultivation of strain MNH39288, L-arabinose LARA, ribose RIB, D-xylose DXYL, Galactose GAL, Glucose GLU, Fructose FRU, α-Methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salvinol SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR, and their derivatives can be used as carbon sources.

[0234] Table 2. Test results of strain MNH39288

[0235] MNH39288 0 1 2 3 4 5 6 7 8 9 - - - - + + + - - - 10 11 12 13 14 15 16 17 18 19 + + + - - - - - - - 20 21 22 23 24 25 26 27 28 29 - + - + + + + + + + 30 31 32 33 34 35 36 37 38 39 + + - - - + - - - + 40 41 42 43 44 45 46 47 48 49 + - - - - - - - - -

[0236] Note: “+” indicates positive, “-” indicates negative.

[0237] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strains MNH17483 and MNH39288

[0238] 1.5.1 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH17483

[0239] The minimum inhibitory concentration of antibiotics for strain MNH17483 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 3.

[0240] Table 3. Minimum inhibitory concentration test results of antibiotics for strain MNH17483

[0241] antibiotic Antibacterial concentration (mg / L) Moxifloxacin (MXF) 5.50 Ampicillin (AMP) 0.75 Chloramphenicol (C) 1.80 Clindamycin (CD) 0.96 Amoxicillin (AMC) 0.42 Rifampicin (RD) 0.38 Penicillin (P) 0.5 Cefquinome (CZX) 6.00 Tetracycline (TE) 5.00 Ceftriaxone (CRO) 1.90

[0242] The results showed that MNH17483 was sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, penicillin, cefquinome, tetracycline, and ceftriaxone. This suggests that MNH17483 is sensitive to most antibiotics, and the risk of developing antibiotic resistance in subjects with long-term MNH17483 use is low.

[0243] 1.5.2 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH39288

[0244] The minimum inhibitory concentration of antibiotics for strain MNH39288 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 4.

[0245] Table 4. Minimum inhibitory concentration test results of antibiotics for strain MNH39288

[0246] antibiotic Antibacterial concentration (mg / L) Moxifloxacin (MXF) 4.00 Ampicillin (AMP) 2.00 Chloramphenicol (C) 1.20 Clindamycin (CD) 0.070 Amoxicillin (AMC) 0.40 Rifampicin (RD) 0.27 Penicillin (P) 0.45 Cefquinome (CZX) 8.00 Tetracycline (TE) 6.00 Ceftriaxone (CRO) 1.80

[0247] The results showed that MNH39288 was sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, penicillin, cefquinome, tetracycline, and ceftriaxone. This suggests that MNH39288 is sensitive to most antibiotics, and the risk of developing antibiotic resistance in subjects with long-term MNH39288 use is low.

[0248] 1.6 Identification of strains MNH17483 and MNH39288

[0249] 1.6.1 16S rRNA gene amplification of strains MNH17483 and MNH39288

[0250] Fresh cultures of strains MNH17483 and MNH39288 were used to extract genomic DNA, which was then used as a template for 16S rRNA gene amplification.

[0251] The primer pairs used for PCR of the 16S rRNA gene are:

[0252] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0253] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0254] The PCR reaction procedure is as follows:

[0255] Pre-denaturation: 94°C, 4 min; (denaturation: 94°C, 50 sec, annealing: 52°C, 40 sec; extension: 72°C, 70 sec), 36 cycles; final extension: 72°C, 10 min.

[0256] 1.6.2 16S rRNA gene sequencing of strains MNH17483 and MNH39288

[0257] The PCR product was purified and sequenced by Sangon to obtain the 16S rRNA gene.

[0258]

[0259]

[0260] 1.6.3 Identification results of strains MNH17483 and MNH39288

[0261] The 16S rRNA gene sequences obtained above, such as those shown in SEQ ID NO: 3 and SEQ ID NO: 4, were analyzed using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0262] The obtained sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH17483 was Bifidobacterium animalis subsp. lactis , the similarity is 100%, and the whole genome ANI analysis shows Bifidobacterium animalis (GCF_000260715.1) with a similarity of 95.88% and an alignment score (AF) of 92%, so strain MNH17483 is Bifidobacterium animalis subsp. lactis strains planted; the strain with the highest similarity to MNH39288 is Bifidobacterium animalis subsp. lactis , the similarity is 100%, and the whole genome ANI analysis shows Bifidobacterium animalis (GCF_000260715.1) has a similarity of 95.83% and an alignment score (AF) of 92%, so strain MNH39288 is Bifidobacterium animalis subsp. lactis Planted strains.

[0263] MNH17483 and MNH39288 were compared with the Bifidobacterium animalis The 16S rRNA gene sequences of related strains of the genus were compared and phylogenetic trees were constructed.

[0264] The sequences of MNH17483 and MNH39288 were aligned with the sequences of model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method). Figure 13 、 14 The nodes in the developmental tree only display the values ​​whose Bootstrap values ​​are greater than 50%.

[0265] From the phylogenetic tree ( Figure 13 、 14 ) It can be seen that strains MNH17483 and MNH39288 Bifidobacterium animalis Get together for Bifidobacterium animalis New plants planted.

[0266] 1.7 Genome analysis of strains MNH17483 and MNH39288

[0267] 1.7.1 Genome analysis of strain MNH17483

[0268] The genome of strain MNH17483 was fragmented using ultrasound to fragments of approximately 350 bp. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) with paired-end 150-bp sequencing. The resulting data was 1.11 Gbp, of which Q20 accounted for 96.25%.

[0269] The raw sequencing data were filtered using fastp (version 0.20.0) with the following parameters: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50". The filtered raw data were then assembled using SPAdes (version 3.14.0) with the following parameters: "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 1.92 Mbp, an N50 length of 182.2 kbp, and a GC content of 60.49%.

[0270] Genomic gene prediction and analysis were performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 1554 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0271] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), using the CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi) database. Detailed comparison information is shown in Table 5.

[0272] Table 5. MNH17483 drug resistance gene information list

[0273] strain genes resistance genes Gene name Comparison consistency (%) MNH17483_00301 ARO:3000194 tet(W) 96.87 MNH17483_00874 ARO:3004480 Bifidobacterium adolescentis rpoB mutants conferring resistance to rifampicin 92.66

[0274] The genome was analyzed for potential virulence factors and related genes using NCBI blastp (version 2.7.1+) against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated September 19, 2019). Detailed comparison results are shown in Table 6.

[0275] Table 6. List of potential toxic genes in MNH17483

[0276] strain genes VFDB gene Gene name Comparison consistency (%) MNH17483_00919 VFG048797 ugd 62.26

[0277] AntiSMASH6 (version 6.0.1) was used to analyze potential secondary metabolism gene clusters in the genome. No secondary metabolism gene clusters were found.

[0278] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 7.

[0279] Table 7. List of potential primary metabolic gene clusters in MNH17483

[0280] Gene cluster range type from arrive Most similar known gene cluster abbreviation Similarity Region 2.1 Fumarate → succinate metabolic pathway, pyruvate → acetate-formate metabolic pathway (Fumarate2succinate, Pyruvate2acetate-formate) 164242 202462 Pyruvate to acetate-formate pathway in E. coli PFL_acetate 100%

[0281] 1.7.2 Genome analysis of strain MNH39288

[0282] The genome of strain MNH39288 was fragmented using ultrasound to fragments of approximately 350 bp. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) system for paired-end 150-bp sequencing. The resulting data was 1.33 Gbp, of which Q20 accounted for 96.34%.

[0283] The raw sequencing data were filtered using fastp (version 0.20.0) with the following parameters: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50". The filtered raw data were assembled using SPAdes (version v3.14.0) with the following parameters: "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 1.92 Mbp, an N50 length of 182.4 kbp, and a GC content of 60.49%.

[0284] Genomic gene prediction and analysis were performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 1556 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0285] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version 4.2.2), using the CARD (version 3.0.0, https: / / card.mcmaster.ca / analyze / rgi) database. Detailed comparison information is shown in Table 8.

[0286] Table 8. MNH39288 drug resistance gene information list

[0287] strain genes resistance genes Gene name Comparison consistency (%) MNH39288_00354 ARO:3000194 tet(W) 96.87 MNH39288_00752 ARO:3004480 Bifidobacterium adolescentis rpoB mutants conferring resistance to rifampicin 92.66

[0288] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed comparison results are shown in Table 9.

[0289] Table 9. List of potential toxic genes of MNH39288

[0290] strain genes VFDB gene Gene name Comparison consistency (%) MNH39288_00707 VFG048797 ugd 62.26

[0291] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 10.

[0292] Table 10. List of potential primary metabolic gene clusters in MNH39288

[0293] Gene cluster range type from arrive Most similar known gene cluster abbreviation Similarity Region 2.1 Fumarate → succinate metabolic pathway, pyruvate → acetate-formate metabolic pathway (Fumarate2succinate, Pyruvate2acetate-formate) 164040 202260 Pyruvate toacetate-formate metabolic pathway in E. coli PFL_acetate 100% .

[0294] Example 2: Strains MNH17483 and MNH39288 promote growth and development in a low-fat, low-protein diet-induced growth retardation mouse model

[0295] MNH17483 and MNH39288 were used to improve growth and development indicators in a mouse model induced by a low-fat, low-protein diet. This invention has passed the ethical review of the Animal Ethics Committee of Muen Biotechnology.

[0296] 2.1 Experimental methods

[0297] 1) Experimental Animals: 3-week-old C57BL / 6J mice were purchased from Guangdong Weitonglihua Laboratory Animal Technology Co., Ltd.

[0298] 2) Preparation of test articles for strains MNH17483 and MNH39288: Glycerol cryovials of MNH17483 and MNH39288 strains were thawed at 37°C and inoculated onto PYG plate medium in an anaerobic workstation for activation. The activated strains were inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured liquid was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable cell count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0299] 3) Negative control: Use PBS containing 25% glycerol and 0.05% L-Cys HCl as the negative control.

[0300] 4) Positive control: Bifidobacterium lactis CGMCC No. 20847. After thawing the glycerol cryopreserved tube of Bifidobacterium lactis CGMCC No. 20847 (disclosed in patent CN112980725B) at 37°C, the strain was inoculated into PYG medium in an anaerobic workstation for activation. The activated strain was inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured liquid was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable cell count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0301] 5) Experimental Procedure: After the quarantine period, 40 3-week-old C57BL / 6J male mice weighing 13-16 g were randomly stratified according to body weight and divided into five groups, 8 mice per group: NCD-Control, LFD-Control, MNH17483, MNH39288, and a positive control. The NCD-Control group was fed a maintenance diet (Cat. No. PD24043001, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), while the other four groups were fed a low-fat, low-protein diet (Cat. No. PD24043003, Changzhou Shuyi Shuer Biotechnology Co., Ltd.). Drug administration began after grouping (D1). The NCD-Control and LFD-Control groups were given a negative control. The positive control group received oral gavage with Bifidobacterium lactis CGMCC No. 20847. The MNH17483 group received oral gavage with MNH17483, and the MNH39288 group received oral gavage with MNH39288. Dosing was once daily, 200 μL per dose, for 35 days. Mice had free access to water and food during the experiment, and a 12-hour day / night cycle was maintained. General clinical observations were conducted after each dose. The endpoint was the day after the end of dosing (D36). Dissections were performed according to the protocol, and data were summarized and analyzed for individual dissection data and serum test results. All data are presented as mean ± SD and analyzed using GraphPad Prism software. Pairwise comparisons were analyzed using the Student's t test. No significance is indicated; significant differences are indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0302] Food intake was determined by weighing the remaining amount of feed.

[0303] The weight of internal organs such as the liver and spleen, the length of the large intestine, and the length and weight of various bones and muscles are measured through dissection.

[0304] Method for detecting serum insulin-like growth factor-1: Mouse serum was collected and detected using the Mouse IGF-1 ELISA Development Kit (PeproTech).

[0305] To detect immune cells, 0.1 g of mouse spleen tissue was collected, rinsed with PBS, and then pulverized. The pulverized spleen tissue was suspended in PBS and filtered through a 70 μm filter to prepare a mouse spleen single-cell suspension. A small amount of this suspension was stained and analyzed on a flow cytometer to accurately count the number of immune cells in the mouse spleen.

[0306] The short-chain fatty acid content of cecal contents was determined as follows: approximately 100 mg of cecal content was weighed and placed in a 2 mL EP tube. 1 mL of ether was added and immediately vortexed to mix thoroughly. Short-chain fatty acids were extracted by sonication for 10 minutes. The precipitate was removed by centrifugation for 10 minutes. The supernatant was passed through a 0.22 μm organic filter membrane and transferred to a gas phase vial. The vial was sealed with parafilm and stored at -20°C until assayed. Short-chain fatty acids were separated and detected using a DB-FFAP column and mass spectrometer.

[0307] 2.2 Experimental Results

[0308] See also Figure 15-16 The results showed that MNH17483 and MNH39288 could significantly increase the food intake of growth retardation mice induced by a low-fat, low-protein diet, indicating that MNH17483 and MNH39288 have the function of promoting appetite.

[0309] See also Figure 17-20 The results showed that MNH17483 and MNH39288 significantly increased liver, spleen, and kidney weights, as well as intestinal length, in developmentally retarded mice. This suggests that MNH17483 and MNH39288 can promote liver, spleen, kidney, and / or intestinal development and have potential uses in promoting organ development. As a control, liver, spleen, and kidney weights in the positive control group were not significantly different from those in the developmentally retarded mice.

[0310] See also Figure 21-26 The results showed that MNH17483 and MNH39288 could significantly promote the increase in body weight, length, tibia length, femur length, and soleus and / or gastrocnemius muscle weight in a low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH17483 and MNH39288 have the use of promoting height (body length), weight, bone, and muscle development.

[0311] See also Figure 27 The results showed that MNH39288 could significantly increase the serum insulin-like growth factor 1 (IGF-1) content in a low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH39288 has the function of promoting growth and development.

[0312] See also Figure 28-31 The results showed that MNH17483 and MNH39288 can significantly promote the development of major immune organs such as the thymus and spleen in a mouse model of growth retardation induced by a low-fat and low-protein diet; MNH39288 can increase the proportion of T cells, indicating that MNH17483 and MNH39288 have the function of promoting the development of the immune system and enhancing immunity.

[0313] See also Figures 32-34Results showed that MN17483 significantly increased butyrate content in the cecum of a mouse model of growth retardation induced by a low-fat, low-protein diet; MNH39288 significantly increased the production of isovaleric acid and the proportion of 2-methylbutyrate in total short-chain fatty acids. This suggests that MNH17483 and MNH39288 can promote the production of short-chain fatty acids and promote intestinal health.

[0314] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations can be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples in the disclosure illustrate embodiments of the present invention, they are provided as non-limiting examples only and, therefore, should not be construed as limiting the various aspects of the invention thus described. The present invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Accordingly, the drawings and detailed description should be regarded as illustrative rather than restrictive.

Claims

1. An isolated animal Bifidobacterium ( Bifidobacterium animalis ), which is deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65555.

2. The culture, live bacteria or freeze-dried bacteria of animal Bifidobacterium according to claim 1, in, The culture includes any one of the following A) to D): A) fermentation broth of the animal Bifidobacterium; B) the fermentation broth of the animal Bifidobacterium and the supernatant of the fermentation broth of the animal Bifidobacterium; C) the fermentation broth of the animal Bifidobacterium and the inactivated fermentation broth of the animal Bifidobacterium; D) The concentrated or dried product of any one of A) to C) above.

3. A composition comprising the animal Bifidobacterium according to claim 1 or the culture, live bacteria or freeze-dried bacteria of the animal Bifidobacterium according to claim 2, wherein the composition is a medicine.

4. The composition according to claim 3, further comprising one or more pharmaceutically acceptable excipients; and / or The composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

5. The composition of claim 4, wherein the other active agent comprises one or more of probiotics and prebiotics.

6. A composition for enhancing immunity, comprising the animal Bifidobacterium according to claim 1 or the culture, live bacteria or freeze-dried bacteria of the animal Bifidobacterium according to claim 2, wherein the composition is a health product.

7. The composition of claim 6, further comprising one or more food-acceptable excipients; and / or The composition may also contain one or more other active agents for enhancing immunity.

8. The composition of claim 7, wherein the other active agent comprises one or more of probiotics and prebiotics.

9. The composition according to any one of claims 6 to 8, wherein the health product is a nutritional composition.

10. The composition according to any one of claims 6 to 8, wherein the health product is a food.

11. The composition according to any one of claims 6 to 8, wherein the health product is candy.

12. The composition according to any one of claims 6 to 8, wherein the health product is a food bar.

13. The composition according to any one of claims 6 to 8, wherein the health product is a dietary supplement.

14. Use of the animal Bifidobacterium according to claim 1, the culture, live bacteria or freeze-dried bacteria according to claim 2, or the composition according to any one of claims 6 to 13 for enhancing immunity for non-therapeutic purposes.

15. Use of the animal Bifidobacterium according to claim 1, the culture, live bacteria or freeze-dried bacteria according to claim 2, or the composition according to any one of claims 3 to 5 in the preparation of a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

16. The use according to claim 15, wherein the growth promotion comprises: promoting at least one of the development of organs, height, weight, bones, and muscles.

17. The use according to claim 16, wherein the organ comprises at least one of the liver, thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

18. The use according to any one of claims 15 to 17, wherein the drug promotes growth and development by increasing hormone content and / or increasing appetite, and the hormone comprises insulin-like growth factor 1; or The drug promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

19. The use according to any one of claims 15 to 17, wherein the drug enhances immunity by promoting the development of the immune system.

20. The use according to claim 19, wherein the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells, and the immune cells comprise T cells.

21. Use of the animal Bifidobacterium according to claim 1, the culture, live bacteria or freeze-dried bacteria according to claim 2, or the composition according to any one of claims 6 to 13 in the preparation of a health product for enhancing immunity.

22. The use according to claim 21, wherein the health product enhances immunity by promoting the development of the immune system.

23. The use according to claim 22, wherein the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells, and the immune cells comprise T cells.

24. An isolated animal Bifidobacterium ( Bifidobacterium animalis ), which is deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65553.

25. The culture, live bacteria or freeze-dried bacteria of animal Bifidobacterium according to claim 24, in, The culture includes any one of the following A) to D): A) fermentation broth of the animal Bifidobacterium; B) the fermentation broth of the animal Bifidobacterium and the supernatant of the fermentation broth of the animal Bifidobacterium; C) the fermentation broth of the animal Bifidobacterium and the inactivated fermentation broth of the animal Bifidobacterium; D) The concentrated or dried product of any one of A) to C) above. 26 . A composition comprising the animal Bifidobacterium according to claim 24 or the culture, live bacteria or freeze-dried bacteria of the animal Bifidobacterium according to claim 25 , wherein the composition is a medicine.

27. The composition of claim 26, further comprising one or more pharmaceutically acceptable excipients; and / or The composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

28. The composition of claim 27, wherein the other active agent comprises one or more of a probiotic and a prebiotic.

29. A composition for enhancing immunity, comprising the animal Bifidobacterium according to claim 24 or the culture, live bacteria or freeze-dried bacteria of the animal Bifidobacterium according to claim 25, wherein the composition is a health product.

30. The composition of claim 29, further comprising one or more food-acceptable excipients; and / or The composition may also contain one or more other active agents for enhancing immunity.

31. The composition of claim 30, wherein the other active agent comprises one or more of a probiotic and a prebiotic.

32. The composition according to any one of claims 29 to 31, wherein the health product is a nutritional composition.

33. The composition according to any one of claims 29 to 31, wherein the health product is a food.

34. The composition according to any one of claims 29 to 31, wherein the health product is candy.

35. The composition of any one of claims 29-31, wherein the health product is a food bar.

36. The composition of any one of claims 29-31, wherein the health product is a dietary supplement.

37. Use of the animal Bifidobacterium according to claim 24, the culture, live bacteria or freeze-dried bacteria according to claim 25, or the composition according to any one of claims 29 to 36 for enhancing immunity for non-therapeutic purposes.

38. Use of the animal Bifidobacterium according to claim 24, the culture, live bacteria or freeze-dried bacteria according to claim 25, or the composition according to any one of claims 26 to 28 in the preparation of a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

39. The use according to claim 38, wherein the growth promotion comprises: promoting at least one of the development of organs, height, weight, bones, and muscles.

40. The use according to claim 39, wherein the organ comprises at least one of the liver, thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

41. The use according to any one of claims 38 to 40, wherein the drug promotes growth and development by increasing appetite; or The drug promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

42. The use according to any one of claims 38 to 40, wherein the drug enhances immunity by promoting the development of the immune system. The use according to claim 42, wherein the promoting the development of the immune system is promoting the development of immune organs.

44. Use of the animal Bifidobacterium according to claim 24, the culture, live bacteria or freeze-dried bacteria according to claim 25, or the composition according to any one of claims 29 to 36 in the preparation of a health product for enhancing immunity. The use according to claim 44 , wherein the health product enhances immunity by promoting the development of the immune system. The use according to claim 45 , wherein the promoting the development of the immune system is promoting the development of immune organs.

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