Bifidobacterium animalis for enhancing immunity and application thereof

Abstract

The present disclosure provides isolated Bifidobacterium animalis, a composition comprising the same, and uses thereof, which can be used for promoting growth and development, enhancing immunity, or promoting intestinal health, etc.

Description

Technical Field

[0001] This disclosure relates to the field of microbiology, and more specifically to isolated Bifidobacterium animalis, compositions comprising thereof, and uses thereof. Background Technology

[0002] Growth refers to the increase in size and morphological changes of various organs and systems in the body; it is a quantitative change. Development refers to the differentiation, maturation, and functional development of cells, tissues, and organs; it is a qualitative change. The two are closely related; growth is the material basis of development. The state of development and maturation is reflected in the quantitative changes in growth. Human growth and development refers to the maturation process from a fertilized egg to an adult. Growth and development are important characteristics that distinguish children from adults. Studies have found that gut bacteria can affect the host's growth and development; some bacterial groups can mediate the host's growth and development process by influencing growth hormone (GH) / insulin-like growth factor-1 (IGF-1).

[0003] Immunity refers to the body's ability to resist external invasion and maintain the stability of its internal environment. For children, the development and protection of the immune system are particularly important, as they directly relate to their healthy growth. Infants and children with developmental delays often have insufficient immunity and low resistance due to slow development of their immune organs and gastrointestinal tract, making them prone to illness. The environment, including air, food, and water, is teeming with various microorganisms: bacteria, viruses, mycoplasma, chlamydia, fungi, etc. When children have insufficient immunity, they are easily infected by harmful pathogens and can then transmit them to their caregivers and classmates, even leading to severe influenza or other illnesses. Although the body produces corresponding antibodies against different pathogens to resist reinfection, antibodies are specific and time-limited. For example, streptococcal antibodies only protect the body from reinfection by streptococci for a short period and cannot protect against other viral infections. The bacteria or viruses that cause influenza mutate extremely rapidly, making it difficult for infants and children with low immunity to resist cold viruses; this is the real reason why they frequently catch colds.

[0004] Probiotics are microorganisms that, when given in sufficient quantities, provide health benefits to the host. Studies have shown that probiotics can maintain the balance of gut microbiota and inhibit the growth of harmful bacteria through their own metabolism. Therefore, gut probiotics are closely related to human health, and probiotic strains have the effects of regulating gut health and enhancing 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 femoral length in mice, and promote height increase in adolescents. CN114317354B discloses that Bifidobacterium animalis MB-424 can promote bone cell growth and promote the production of growth hormone by pituitary GH3 cells. Existing technologies for promoting growth and development with Bifidobacterium animalis or Bifidobacterium lactis mainly focus on promoting bone and height growth. However, children with developmental delays have low immunity and are prone to illness, which is related to the slow development of immune organs and the gastrointestinal tract. Few studies target 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 did not affect the weight of the spleen / thymus in mice. Another study (Effect of probiotics on thymus size and markers of infection inlate infancy: a randomized controlled trial) involved 186 healthy Danish infants with *Lactobacillus rhamnosus* LGG and *Bifidobacterium animalis* subsp. *lactobacter* BB-12 for 6 months. There was no significant difference in thymus size between the probiotic group and the placebo group (p≥0.248). The probiotics had no effect on CRP (C-reactive protein) (p=0.331).

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

[0008] This disclosure discloses the isolation of a novel *Bifidobacterium animalis*. This novel strain can: 1. Promote the development of major immune organs such as the thymus and spleen in growth-retarded individuals (including increasing the weight of immune organs such as the thymus and spleen, and the immune organ development index (immune organ weight / body weight)); 2. Promote the development and proliferation of immune cells such as T cells, thereby enhancing the immunity of growth-retarded individuals; 3. Increase the serum insulin-like growth factor 1 (IGF-1) levels in growth-retarded individuals, promoting weight and body length growth, and promoting bone and muscle growth, such as promoting tibia length growth, femur length growth, and increasing the weight of the soleus and gastrocnemius muscles; 4. Increase the weight of the liver, thymus, spleen, and kidneys, and the length of the intestines (large intestine length and / or small intestine length) in growth-retarded individuals; increase food intake; increase the content of short-chain fatty acids in the intestines; improve the gut microbiota; and promote intestinal development.

[0009] Therefore, the novel strain disclosed herein can promote height and weight while simultaneously promoting organ development, particularly the development of immune organs, enhancing immunity, promoting intestinal development, and improving intestinal health. It can be used to improve developmental delays caused by malnutrition, achieving the functions of improving / treating / reversing developmental delays; and to achieve the purposes of promoting growth and development, enhancing immunity, or promoting intestinal health.

[0010] In a first aspect, this disclosure provides isolated Bifidobacterium animalis with 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 respect to strains with accession numbers GDMCC No: 65553 or GDMCC No: 65555; and / or having a 16S rRNA sequence with at least 98.65% identity to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0011] In some embodiments, the Bifidobacterium animalis has an average nucleotide identity (ANI) 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 respect to the sequence shown in SEQ ID NO:3; and / or has a 16S rRNA sequence with at least 98.65% identity to the sequence shown in SEQ ID NO:3.

[0012] In some embodiments, the Bifidobacterium animalis has an average nucleotide identity (ANI) 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 respect to the sequence shown in SEQ ID NO:4; and / or has a 16S rRNA sequence with at least 98.65% identity to the sequence shown in SEQ ID NO:4.

[0013] In some embodiments, the animal Bifidobacterium has a 16S rRNA sequence having 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% identity with the sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0014] In some embodiments, the animal bifidobacterium is two novel strains of Bifidobacterium animalis.

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

[0016] In some embodiments, the name of the Bifidobacterium animalis is: Bifidobacterium animalis MNH39288, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 65555, deposited on November 28, 2024, at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0017] Secondly, this disclosure provides cultures, live bacteria, lyophilized bacteria, or inactivated bacterial cells comprising the Bifidobacterium animalis described in the first aspect, wherein the cultures comprise any one of the following A) to D):

[0018] A) The fermentation broth of the animal Bifidobacterium;

[0019] B) The supernatant of the fermentation broth of the animal Bifidobacterium;

[0020] C) The inactivated fermentation broth of the animal Bifidobacterium;

[0021] D) The concentrated or dried product of any one of A)-C).

[0022] Thirdly, this disclosure provides a composition comprising the Bifidobacterium animalis described in the first aspect, or a culture, live bacteria, freeze-dried bacteria, or inactivated bacteria of the Bifidobacterium animalis described in the second aspect.

[0023] This disclosure provides compositions comprising Bifidobacterium animalis, its cultures, and / or its metabolites as described in the first aspect.

[0024] In some embodiments, the culture of Bifidobacterium animalis includes a solid culture of Bifidobacterium animalis, a fermentation culture, the supernatant of a fermentation culture, or a dried product thereof.

[0025] 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.

[0026] In some embodiments, the composition is provided in liquid or solid form.

[0027] In some embodiments, the composition contains 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×10 12 The live animal Bifidobacterium cfu / mg.

[0028] In some embodiments, the composition contains 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 The live animal Bifidobacterium cfu / mg.

[0029] In some embodiments, the composition contains 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×1010 The live animal Bifidobacterium cfu / mg.

[0030] In some embodiments, the composition contains 1×10 7 Up to 1×10 9 cfu / mL or 1×10 7 Up to 1×10 9 The live animal Bifidobacterium cfu / mg.

[0031] In some embodiments, each gram of the composition contains 1 × 10⁻⁶ 3 Up to 1×10 17 Bacteria with colony-forming units (CFU); for example, 1 × 10⁶. 4 Up to 1×10 12 1×10 5 Up to 1×10 11 One or 1×10 6 Up to 1×10 10 A colony-forming unit (CFU) of bacteria, specifically, for example, 1 × 10⁶. 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 7×106 、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 、8×10 129×10 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 A number of colony-forming units (CFU) or any number of colonies forming units (CFU) of bacteria.

[0032] In some embodiments, the concentration of the animal Bifidobacterium as the active ingredient in the composition is 10. 7 Up to 10 12 CFU / g.

[0033] In some embodiments, the Bifidobacterium animalis in the composition is a live, attenuated, freeze-dried, or inactivated bacterium, for example, it can be a heat-inactivated bacterium, preferably pasteurized.

[0034] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (e.g., lyophilized powder), or gel.

[0035] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0036] In some embodiments, the composition is in the form of an oral or injectable dosage form.

[0037] In some embodiments, the composition further comprises one or more pharmaceutical and / or food-acceptable excipients.

[0038] The excipients used in the pharmaceutical and / or food products are well known to those skilled in the art.

[0039] In some embodiments, the excipient may be at least one selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0040] In some embodiments, the composition comprises one or more of the following: a buffer (e.g., sodium bicarbonate, infant formula or sterile human milk or other agents that allow bacteria to survive and grow (e.g., to survive in the acidic environment of the stomach and to grow in the intestinal environment)), a lyophilization protectant, a preservative, a stabilizer, a binder, a compactor, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0041] In some embodiments, the composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting gut health.

[0042] In some implementations, the growth and development promotion includes promoting at least one of the following: organ, height / body length, weight, bone, and muscle development.

[0043] In some embodiments, the organ includes at least one of the heart, thymus, liver, spleen, kidney, and intestines.

[0044] In some embodiments, the bone comprises at least one of the femur and tibia.

[0045] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0046] In some embodiments, the other active agents may be one or more of probiotics and prebiotics, or a combination thereof.

[0047] In some embodiments, the probiotics are selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus curvatureii, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus mare's milk, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatureii, Lactobacillus sakei, Lactococcus lactis, and Lactococcus fatii.

[0048] In some embodiments, the prebiotic is selected from 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 β-glucan, ginseng or its extract, nutrient compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannooligosaccharides (MOS), inulin rich in fructooligosaccharides, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0049] In some embodiments, the composition may be formulated as a frozen composition, such as a frozen composition prepared by quick-freezing and drying, or freeze-drying, for storage and / or transport.

[0050] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0051] 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 the diluent described herein.

[0052] In some embodiments, the composition may be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0053] 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 with an enteric coating. For example, the enteric-coated formulation may be enteric-coated granules, enteric-coated tablets, or enteric-coated capsules. 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 may be a microencapsulated capsule or microcapsule.

[0054] In some embodiments, the composition is a medicine, or a health product or food.

[0055] In some embodiments, the composition is an infant-specific dosage form, a pediatric-specific dosage form, or an adult-specific dosage form.

[0056] In some embodiments, the composition is a gastrointestinal or non-gastrointestinal dosage form.

[0057] Fourthly, this 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 in the preparation of a medicament, health product or food for promoting growth and development, enhancing immunity or promoting intestinal health.

[0058] This 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 in promoting growth and development, enhancing immunity, or promoting intestinal health (preferably, the use does not involve the treatment of disease).

[0059] In some implementations, the growth and development promotion includes promoting at least one of the following: organ, height / body length, weight, bone, and muscle development.

[0060] In some implementations, the organ includes at least one of the heart, liver, spleen, kidney, and intestines.

[0061] In some embodiments, the bone comprises at least one of the femur and tibia.

[0062] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0063] In some implementations, the drug, health product, or food promotes growth and development by increasing hormone levels and / or increasing appetite.

[0064] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0065] In some implementations, the hormone is derived from bodily fluids; further, from blood; and even further, from serum.

[0066] In some implementations, the drug, health product, or food enhances immunity by promoting the development of the immune system.

[0067] In some implementations, the promotion of immune system development includes at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0068] In some embodiments, promoting the development of immune organs includes promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; further includes promoting the development of the thymus and / or spleen.

[0069] In some implementations, the promotion of immune cell production includes promoting the production of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells).

[0070] In some implementations, the drug, health product, or food promotes gut health by increasing short-chain fatty acids and / or improving gut microbiota.

[0071] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0072] The composition comprises aggregates of isolated and purified live microbial communities to increase body weight by at least 2%, 3%, 4%, 5%, 6%, or 7% compared to the subject's body weight before administration of the aggregates of isolated and purified microbial species.

[0073] As used herein, a microbiome generally refers to a group of microorganisms that consists essentially of a single strain, species, or genus, as may be the case when a subgroup of such strain, species, or genus is isolated and purified. Therefore, for a given microbial community, if cultured from an isolated microbial species or strain, such a community will be referred to herein as purified or substantially pure. The resulting community will typically be at least 80% pure with respect to the said microbial species or strain, and at least 90%, 95%, 98%, 99%, 99.5%, or 99.9% pure relative to other microbial species or strains within that particular community. Conversely, the level of undesired strains in any particular desired microbial community 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 in the purified community aggregates, such as other undesirable microbial strains or species, may be proportional to or below the aforementioned levels for each desired community. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where the composition comprises an aggregate of multiple microbial populations, each population may have the above-mentioned purity, either before its incorporation into the composition or when polymerization measurements are performed on the aggregate. For example, the level of impurities in the purified aggregate, such as other undesirable microbial strains or species, may be proportionally at or below the above-mentioned level for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where the composition comprises an aggregate of multiple microbial populations, each population may have the above-mentioned purity, either before its incorporation into the composition or when polymerization measurements are performed on the aggregate. For example, the level of impurities in the purified aggregate, such as other undesirable microbial strains or species, may be proportionally at or below the above-mentioned level for each desired population.

[0074] The compositions disclosed herein may also include cellular components, metabolites, secreted molecules and compounds of Bifidobacterium animalis, and the like. This can be achieved, for example, by recovering the supernatant of a Bifidobacterium animalis culture or by extracting cellular components or cell fractions, metabolites, or secreted compounds from a Bifidobacterium animalis culture; these may correspond to components in isolated forms from Bifidobacterium animalis, or any mixture of one or more components from Bifidobacterium animalis.

[0075] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or compositions described in this disclosure can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0076] In some implementations, the growth and development promotion includes promoting at least one of the following: organ, height / body length, weight, bone, and muscle development.

[0077] In some embodiments, the organ includes at least one of the heart, thymus, liver, spleen, kidney, and intestines.

[0078] In some embodiments, the bone comprises at least one of the femur and tibia.

[0079] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0080] 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.

[0081] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0082] In some implementations, the hormone is derived from bodily fluids; further, from blood; and even further, from serum.

[0083] In some embodiments, the animal bifidobacteria, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or compositions enhance immunity by promoting the development of the immune system.

[0084] In some implementations, the promotion of immune system development includes at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0085] In some embodiments, promoting the development of immune organs includes promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; further includes promoting the development of the thymus and / or spleen.

[0086] In some implementations, the promotion of immune cell production includes promoting the production of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells).

[0087] In some embodiments, the animal Bifidobacterium, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition promotes gut health by increasing short-chain fatty acids and / or improving gut microbiota.

[0088] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0089] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or combinations thereof disclosed herein can stimulate appetite, thereby increasing food intake (preferably cumulative food intake) and achieving the effect of promoting growth and development.

[0090] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, lyophilized bacteria or inactivated bacteria, or compositions thereof disclosed herein can increase the weight of the heart, liver, spleen, and / or kidneys, and / or intestinal length, thereby promoting organ development.

[0091] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or compositions disclosed herein can increase body weight, increase body length, increase tibial length, increase femur length, increase the weight of soleus and gastrocnemius muscles, thereby promoting height (body length), weight, bone and muscle development.

[0092] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, lyophilized bacteria or inactivated bacteria, or compositions disclosed herein can increase serum levels of insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH), thereby promoting growth and development.

[0093] In some embodiments, the animal bifidobacteria, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or combinations thereof 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.

[0094] In some embodiments, the animal Bifidobacterium, cultures, live bacteria, lyophilized bacteria or inactivated bacteria, or compositions disclosed herein can increase the content of short-chain fatty acids and / or improve the gut microbiota, thereby promoting gut health.

[0095] In some embodiments, the drug, health product, or food has at least one effect selected from the following: increasing the weight of the heart, liver, spleen, and / or kidneys, and / or intestinal length (large intestine length and / or small intestine length); increasing food intake; causing weight gain, body length increase, tibia length increase, femur length increase, soleus muscle and / or gastrocnemius muscle weight increase; promoting weight gain, body length increase, promoting bone and muscle growth (wherein, promoting bone growth includes increasing bone length and density, and promoting muscle growth includes promoting the development of specific muscles and maintaining healthy growth characteristics, such as: low body fat percentage (calf muscle / body weight) and / or high muscle mass (calf muscle / calf weight)); increasing serum... Increases the levels of insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH); promotes 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, and the immune organ development index (immune organ weight / body weight), etc.), increases the proportion of T cells, B cells, and NK cells; increases the content of short-chain fatty acids; improves the gut microbiota; promotes intestinal development; improves lesions or inflammation caused by developmental delay, thereby achieving the function of improving / treating / reversing developmental delay; promotes organ development; promotes appetite; promotes height (body length), weight, bone, and muscle development; promotes the development of the immune system and enhances immunity; promotes intestinal health; can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0096] A method for promoting growth and development, enhancing immunity, or promoting gut health involves applying an effective amount of the animal Bifidobacterium as described in the first aspect of this disclosure, the culture as described in the second aspect, live bacteria, freeze-dried bacteria or inactivated bacteria, or the composition as described in the third aspect, to a person in need.

[0097] In some implementations, the growth and development promotion includes promoting at least one of the following: organ, height / body length, weight, bone, and muscle development.

[0098] In some implementations, the organ includes at least one of the heart, liver, spleen, kidney, and intestines.

[0099] In some embodiments, the bone comprises at least one of the femur and tibia.

[0100] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0101] 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.

[0102] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0103] In some implementations, the hormone is derived from bodily fluids; further, from blood; and even further, from serum.

[0104] In some embodiments, the animal bifidobacteria, cultures, live bacteria, freeze-dried bacteria or inactivated bacteria, or compositions enhance immunity by promoting the development of the immune system.

[0105] In some implementations, the promotion of immune system development includes at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0106] In some embodiments, promoting the development of immune organs includes promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; further includes promoting the development of the thymus and / or spleen.

[0107] In some implementations, the promotion of immune cell production includes promoting the production of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells).

[0108] In some embodiments, the animal Bifidobacterium, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition promotes gut health by increasing short-chain fatty acids and / or improving gut microbiota.

[0109] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid. Attached Figure Description

[0110] The embodiments will now be described in conjunction with the accompanying drawings, thereby making the above and other aspects and advantages of the present invention apparent and readily understood.

[0111] Figure 1 The image shows a photograph of the colony morphology of strain MNH17483.

[0112] Figure 2 The image shows a Gram staining photograph of strain MNH17483.

[0113] Figure 3 The image shows an electron microscope image of strain MNH17483.

[0114] Figure 4 The image shows a photograph of the colony morphology of strain MNH39288.

[0115] Figure 5 The image shows a Gram staining photograph of strain MNH39288.

[0116] Figure 6 The image shows an electron microscope image of strain MNH39288.

[0117] Figure 7 The results show the tolerance of strain MNH17483 to different pH values.

[0118] Figure 8 The results show the tolerance of strain MNH17483 to different concentrations of NaCl.

[0119] Figure 9 The results show the tolerance of strain MNH17483 to different concentrations of bile salts.

[0120] Figure 10 The results show the tolerance of strain MNH39288 to different pH values.

[0121] Figure 11 The results show the tolerance of strain MNH39288 to different concentrations of NaCl.

[0122] Figure 12 The results show the tolerance of strain MNH39288 to different concentrations of bile salts.

[0123] Figure 13 The phylogenetic tree of strain MNH17483 is shown.

[0124] Figure 14 The phylogenetic tree of strain MNH39288 is shown.

[0125] Figure 15-16 The chart shows the increased cumulative food intake of strains MNH17483 and MNH39288 (15: line graph of cumulative food intake; 16: bar graph of cumulative food intake). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t-test; *, p < 0.05 compared to the LFD-Control group.

[0126] Figure 17-20 The results showed that strains MNH17483 and MNH39288 promoted organ development (17: liver weight; 18: spleen weight; 19: kidney weight; 20: intestinal length). 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; ****, p < 0.0001 compared with the LFD-Control group.

[0127] Figure 21-26The results showed that strains MNH17483 and MNH39288 promoted the development of height (body length), weight, bone, and muscle (21: weight gain; 22: body length gain; 23: femur length; 24: tibia length; 25: soleus muscle weight; 26: gastrocnemius muscle 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; ***, p < 0.001 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0128] Figure 27 The results showed that strain MNH39288 increased serum insulin-like growth factor 1 (IGF-1) levels. Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t-test; * indicates p < 0.05 compared to the LFD-Control group; **** indicates p < 0.0001 compared to the LFD-Control group.

[0129] Figure 28-31 The results showed that strains MNH17483 and MNH39288 promoted immune system development and enhanced immunity (28: thymus weight; 29: spleen weight; 30: immune organ weight; 31: T cell percentage). 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.

[0130] Figures 32-34 The results showed that strains MNH17483 and MNH39288 promoted the production of short-chain fatty acids in cecal contents (32: butyric acid content; 33: isovaleric acid content; 34: 2-methylbutyric acid percentage). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t-test; * indicates p < 0.05 compared to the LFD-Control group, *** indicates p < 0.001 compared to the LFD-Control group.

[0131] Preservation of strains

[0132] The strain *Bifidobacterium animalis* MNH17483 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 65553, on November 28, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. The deposit name is *Bifidobacterium animalis* MNH17483, and the taxonomic name is *Bifidobacterium animalis*.

[0133] The strain *Bifidobacterium animalis* MNH39288 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 65555, on 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, and the taxonomic name is *Bifidobacterium animalis*. Detailed Implementation

[0134] This disclosure discloses the isolation of two novel strains of *Bifidobacterium animalis*, with accession numbers GDMCC No: 65553 and GDMCC No: 65555, respectively, and their identification using traditional classification and molecular biological methods. The identification results indicate that these two strains are novel strains belonging to the *Bifidobacterium animalis* species. Furthermore, this disclosure investigates the biochemical properties and therapeutic uses of these two strains.

[0135] It is known in the art that bacterial species can be classified and identified using both traditional classification methods and molecular biological methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic experiments. Molecular biological methods include, but are not limited to, ribosomal RNA sequencing and whole-genome sequencing-based methods.

[0136] As used in this article, the term "prebiotic" can be a general term referring to chemical substances and / or components that can affect the growth and / or activity of microorganisms in a host (e.g., that can allow specific changes in the composition and / or activity of the microbiome).

[0137] The terms “subject,” “object,” “individual,” “host,” and “patient” are used interchangeably in this document to refer to any animal subject, including: humans, mammals, laboratory animals, livestock, and domestic pets.

[0138] The compositions or formulations disclosed herein can be administered as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics, or medical foods. In some cases, the compositions are administered as pharmaceutical preparations. In some cases, the compositions are administered as nutritional supplements. In some cases, the compositions are administered as dietary supplements. In some cases, the compositions are administered as medical foods. In some cases, the compositions are administered as medical probiotics. In some cases, the compositions (e.g., dietary supplements, nutritional supplements, medical probiotics, or medical foods) can be administered orally, for example, as capsules, pills, or tablets.

[0139] 16S rRNA is a type of ribosomal RNA found in prokaryotes. The 16S rRNA gene consists of variable and conserved regions. The conserved regions are common to all bacteria, while the variable regions vary to varying degrees among different bacteria. By comparing the 16S rRNA gene sequences of bacteria and analyzing their evolutionary distances based on sequence differences, an evolutionary tree can be constructed. When the 16S rRNA gene sequence identity between two strains is less than 98.65%, they can be identified as belonging to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary 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 the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), p. 23).

[0140] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG package, BLASTN, or FASTA. Commercially or publicly available programs can also include, for example, the DNAStar "MegAlign" program.

[0141] Second-generation sequencing technology can also be used to identify bacterial species based on whole-genome sequencing, making the identification results more accurate. Average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. ANI values ​​can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that an ANI value of more than 95% is required to identify them as belonging to the same bacterial species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear speciesboundaries[J]. Nature Communications, 2018, 9(1): 5114.).

[0142] You can use various existing mature ANI value calculation tools, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), and online calculation tools ANI caculator (http: / / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0143] Using the methods described above, those skilled in the art can determine whether an isolated strain belongs to the *Bifidobacterium animalis* species identified by the inventors. For example, when the average nucleotide identity (ANI) value with *Bifidobacterium animalis* with accession numbers GDMCC No: 65553 or GDMCC No: 65555 is 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%, the ANI value is considered to be at least 95%. When the percentages are 97.4%, 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 bacterial species.

[0144] For example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO: 3 or 4, such as 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%, it can be determined that it belongs to the same bacterial species.

[0145] A “strain” refers to a member of a bacterial species that possesses genetic characteristics that distinguish it from closely related members of the same bacterial species. These genetic characteristics 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), absence (“cure” of at least one natural plasmid), the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one exogenous gene (a gene from another species), at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-natural 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 a strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic capacity (e.g., auxotrophic strains), the strain or nutrient / metabolite can be distinguished by selection or anti-selection using antibiotics.

[0146] As used herein, “supernatant” or “supernatant” means the culture supernatant of a bacterial strain according to the present disclosure, optionally containing compounds and / or cell debris of the strain, and / or metabolites and / or molecules secreted by the strain.

[0147] Compositions can be prepared using the *Bifidobacterium animalis* described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical composition contains a pharmaceutically effective amount of the *Bifidobacterium animalis*, such as *Bifidobacterium animalis* with accession numbers GDMCC No: 65553 or GDMCC No: 65555. Similarly, *Bifidobacterium animalis* with accession numbers GDMCC No: 65553 or GDMCC No: 65555 can also be prepared into pharmaceutical compositions, for example, by using pharmaceutically acceptable excipients, containing a pharmaceutically effective amount of the *Bifidobacterium animalis*.

[0148] Suitable pharmaceutically acceptable excipients include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0149] The compositions described herein can be formulated in any form suitable for enhancing the abundance of Bifidobacterium in the animal body. The compositions can be administered orally (e.g., via oral gavage), intramuscularly, by inhalation, intracranially, intralymphaticly, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrically, intra-gastrointestinally, via bile infusion, via cardiac infusion, via preanal, rectal, subcutaneous, sublingual, local, intravaginal, percutaneously, via the ureter, or via the urethra.

[0150] Examples of dosage forms to which the compositions described herein are applicable include, but are not limited to, tablets, aerosols, chewing sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0151] In some embodiments, the composition is a sugar-coated tablet, gel capsule, gel, emulsion, tablet, sheet capsule, hydrogel, nanofiber gel, electrospun fiber, food bar, candy, fermented milk, fermented cheese, chewing gum, powder or toothpaste, etc.

[0152] In some implementations, administration may also be carried out by including the subject in their diet, for example, in a functional food intended for humans or companion animals.

[0153] The compositions provided herein may contain pharmaceutically acceptable excipients, diluents, or carriers. Such pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

[0154] In some embodiments, the *Bifidobacterium animalis* in the compositions of this disclosure is lyophilized. In some embodiments, the *Bifidobacterium animalis* in the compositions of this disclosure is spray-dried. In some embodiments, the *Bifidobacterium animalis* in the compositions of this disclosure is lyophilized or spray-dried and is viable. In some embodiments, the *Bifidobacterium animalis* in the compositions of this disclosure is lyophilized or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the lyophilized *Bifidobacterium animalis* is reconstituted prior to administration. In some embodiments, the reconstitution is performed using the diluents described herein.

[0155] In some embodiments, the compositions of this disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration via the mouth, tongue, or sublingual route.

[0156] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0157] The compositions disclosed herein include pharmaceuticals, health products, or food.

[0158] The subject of this 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.

[0159] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are merely illustrative and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature or the product / instrument instruction manual. All reagents or instruments whose manufacturers are specified are commercially available.

[0160] Example

[0161] The anaerobic blood agar plates used in the examples were purchased from Huankai Microbiology and had the following formula: 10 g / L casein pancreatic enzyme digest, 3 g / L cardiac pancreatic enzyme digest, 1 g / L corn starch, 5 g / L vesicular enzyme digest, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar, 50 mL / L sterile defibrinated sheep blood, and pH 7.3 ± 0.2.

[0162] The PYG liquid culture medium involved in the examples contains the following per 1 LPYG: 5.0 g Trypticasepeptone, 5.0 g Peptone, 10.0 g Yeast extract, 5.0 g Beef extract, 5.0 g Glucose, 2.0 g K2HPO4, 1.0 ml Tween 80, 1.0 mg Resazurin, 40 ml Salt solution (composition as follows: 0.01 g CaCl2·2H2O, 0.02 g MgSO4·7H2O, 0.04 g K2HPO4, 0.4 g NaHCO3, 0.08 g NaCl, water balance), water balance, pH 7.2 ± 0.2 (25°C).

[0163] The above-mentioned culture medium can be prepared using conventional preparation methods and sterilization methods.

[0164] Example 1. Isolation and Identification of Strains

[0165] 1.1 Isolation and purification of strains MNH17483 and MNH39288

[0166] Two intestinal strains of Bifidobacterium animalis (Bifidobacterium animalis MNH17483 and Bifidobacterium animalis MNH39288) were isolated from fecal samples of healthy volunteers. The isolation method employed standard bacterial isolation techniques, including serial dilution, followed by isolation culture to collect single colonies. The strains were then purified and anaerobically cultured at 37°C. The purified cultures were prepared into 20% glycerol / water-bacterial suspensions and stored at -80°C.

[0167] Specifically, the methods for isolating and purifying the strain are as follows:

[0168] Donors collect 2-5g of fresh feces, place it in a sample collection and preservation tube, shake to homogenize, and then place the processed fecal sample in an ice box and deliver it to the laboratory within 24 hours for bacterial isolation.

[0169] Dispense physiological saline into tubes of 9 mL each in a biosafety cabinet; prepare anaerobic blood agar plates for bacterial isolation, and transfer them to the anaerobic workstation 24 hours in advance, labeling them with sample information, culture medium type, isolation date, etc.

[0170] Take a fresh stool sample and place it in an anaerobic workstation. Vortex the sample for 1 minute to mix thoroughly. Transfer 1 mL of the sample to 9 mL of physiological saline and mix well to a final volume of 10 mL. -1 Diluent, then serially diluted to 10. -6 Diluent, for later use.

[0171] Take 10 -6 Add 100 μL of the diluent to an anaerobic blood agar plate and spread it evenly. After the plate surface dries, invert the plate and incubate at 37°C for 3-5 days.

[0172] Observe the growth of the strains on the isolation medium (anaerobic blood agar plates) and pick single colonies with sterile toothpicks for strain purification. The purified strains are then cultured anaerobically at 37°C. The pure culture strains are prepared into 20% glycerol / water-bacterial suspensions and stored at -80°C.

[0173] 1.2 Morphological characteristics of strains MNH17483 and MNH39288

[0174] 1.2.1 Morphological characteristics of strain MNH17483

[0175] Strain MNH17483 was inoculated onto PYG agar plates and anaerobically cultured at 37°C for 48 hours. Visible colonies formed on the PYG plates; the colonies were round, with regular and smooth edges, approximately 0.5-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic observation revealed that it was non-motile, rod-shaped, and lacked flagella, measuring approximately 0.5-1 μm × 1.5-3 μm. A photograph of the colony morphology of strain MNH 17483 after 48 hours of culture on PYG plates is shown below. Figure 1 Gram staining images of strain MNH17483 can be found here. Figure 2 See electron microscope images Figure 3 .

[0176] 1.2.2 Morphological characteristics of strain MNH39288

[0177] Strain MNH39288 was inoculated onto PYG agar plates and anaerobically cultured at 37°C for 48 hours. Visible colonies formed on the PYG plates; the colonies were round, with regular and smooth edges, approximately 0.5-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic observation revealed that it was non-motile, rod-shaped, and non-flagellated, measuring approximately 0.5-1 μm × 1.5-3 μm. A photograph of the colony morphology of strain MNH39288 after 48 hours of culture on PYG plates is shown below. Figure 4 Gram staining images of strain MNH39288 can be found here. Figure 5 See electron microscope images Figure 6 .

[0178] 1.3 Physiological and biochemical characteristics of strains MNH17483 and MNH39288

[0179] 1.3.1 Physiological and biochemical characteristics of strain MNH17483

[0180] Strain MNH17483 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in a pH range of 5.0 to 10.0, with the optimal growth pH being 7.0–9.0 (see [link to results on strain tolerance to different pH values]). Figure 7 It does not grow in media containing more than 3% NaCl (see results for strain tolerance to different NaCl concentrations). Figure 8 Strain MNH17483 can survive and grow in a bile salt concentration range of 0%-0.15%, but cannot grow at a bile salt concentration greater than or equal to 0.2% (see [link to strain's tolerance to different bile salt concentrations]). Figure 9 ).

[0181] 1.3.2 Physiological and biochemical characteristics of strain MNH39288

[0182] Strain MNH39288 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in a pH range of 6.0 to 9.0, with the optimal growth pH being 7.0 (see [link to results on strain tolerance to different pH values]). Figure 10 It can still grow in media with NaCl concentrations exceeding 6% (see results for strain tolerance to different NaCl concentrations). Figure 11 Strain MNH39288 can survive and grow in a bile salt concentration range of 0%-0.1%, but cannot grow at a bile salt concentration greater than or equal to 0.15% (see [link to strain's tolerance to different bile salt concentrations]). Figure 12 ).

[0183] 1.4 Biochemical identification results of strains MNH17483 and MNH39288

[0184] 1.4.1 Results of biochemical identification of strain MNH17483 using API 50 CHL

[0185] API 50 CHL (bioMérieux, CN5041010) was used; specific experimental procedures are detailed in the standard API reagent handling guidelines. Culture conditions: 37℃, anaerobic. Experimental results are shown in Table 1.

[0186] MNH17483 can produce acid through fermentation using L-arabinose (LARA), ribose (RIB), D-xylose (DXYL), glucose (GLU), α-methyl-D-glucoside (MDG), amygdalin (AMY), arbutin (ARB), aesculin (ESC), salicylic acid (SAL), cellobiose (CEL), maltose (MAL), lactose (LAC), melibiose (MEL), sucrose (SAC), raffinose (RAF), starch (AMD), gentiobiose (GEN), and D-minobiose (TUR). Therefore, during the 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), aesculin (ESC), salicylic acid (SAL), cellobiose (CEL), maltose (MAL), lactose (LAC), melibiose (MEL), sucrose (SAC), raffinose (RAF), starch (AMD), gentiobiose (GEN), D-minobiose (TUR), and their derivatives can serve as carbon sources.

[0187] Table 1. Test results of strain MNH17483

[0188] 0 1 2 3 4 5 6 7 8 9 CTRL GLY ERY DARA LARA RIB DXYL LXYL ADO MDX - - - - + + + - - - 10 11 12 13 14 15 16 17 18 19 GAL GLU FRU MNE SBE RHA DUL INO MAN SOR - + - - - - - - - - 20 21 22 23 24 25 26 27 28 29 MDM MDG NAG AMY ARB ESC SAL CEL MAL LAC - + - + + + + + + + 30 31 32 33 34 35 36 37 38 39 MEL SAC TRE INU MLZ RAF AMD GLYG XLT GEN + + - - - + + - - + 40 41 42 43 44 45 46 47 48 49 TUR LYX TAG DFUC LFUC DARL LARL GNT 2KG 5KG + - - - - - - - - -

[0189] Note: "+" indicates positive, and "-" indicates negative.

[0190] 1.4.2 Results of biochemical identification of strain MNH39288 using API 50 CHL

[0191] API 50 CHL (bioMérieux, CN5041010) was used; specific experimental procedures are detailed in the standard API reagent handling guidelines. Culture conditions: 37℃, anaerobic. Experimental results are shown in Table 2.

[0192] MNH39288 can produce acid through fermentation using L-arabinose (LARA), ribose (RIB), D-xylose (DXYL), galactose (GAL), glucose (GLU), fructose (FRU), α-methyl-D-glucoside (MDG), amygdalin (AMY), arbutin (ARB), aesculin (ESC), salicylic acid (SAL), cellobiose (CEL), maltose (MAL), lactose (LAC), melibiose (MEL), sucrose (SAC), raffinose (RAF), starch, gentiobiose (GEN), and D-minobiose (TUR). 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), aesculin (ESC), salicylic acid (SAL), cellobiose (CEL), maltose (MAL), lactose (LAC), melibiose (MEL), sucrose (SAC), raffinose (RAF), starch, gentiobiose (GEN), D-minobiose (TUR), and their derivatives can serve as carbon sources.

[0193] Table 2. Test results of strain MNH39288

[0194]

[0195]

[0196] Note: "+" indicates positive, and "-" indicates negative.

[0197] 1.5 Minimum inhibitory concentration (MIC) test of antibiotics for strains MNH17483 and MNH39288

[0198] 1.5.1 Minimum inhibitory concentration test of antibiotics for strain MNH17483

[0199] The minimum inhibitory concentration of antibiotics for strain MNH17483 was determined using E-test (purchased from Liofilchem) discs, and the test results are shown in Table 3.

[0200] Table 3. Results of antibiotic minimum inhibitory concentration tests for strain MNH17483

[0201] 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 Cefquinoxime (CZX) 6.00 Tetracycline (TE) 5.00 Ceftriaxone (CRO) 1.90

[0202] The results showed that MNH17483 was sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampin, penicillin, cefquinoxime, tetracycline, and ceftriaxone. This indicates that MNH17483 is sensitive to most types of antibiotics, and the risk of developing antibiotic resistance with long-term use of MNH17483 is low.

[0203] 1.5.2 Minimum inhibitory concentration test of antibiotics for strain MNH39288

[0204] The minimum inhibitory concentration of antibiotics for strain MNH39288 was determined using E-test (purchased from Liofilchem) discs, and the test results are shown in Table 4.

[0205] Table 4. Results of antibiotic minimum inhibitory concentration tests for strain MNH39288

[0206] 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 Cefquinoxime (CZX) 8.00 Tetracycline (TE) 6.00 Ceftriaxone (CRO) 1.80

[0207] The results showed that MNH39288 was sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampin, penicillin, cefquinoxime, tetracycline, and ceftriaxone. This indicates that MNH39288 is sensitive to most types of antibiotics, and the risk of antibiotic resistance developing with long-term use of MNH39288 is low.

[0208] 1.6 Identification of strains MNH17483 and MNH39288

[0209] 1.6.1 Amplification of the 16S rRNA gene in strains MNH17483 and MNH39288

[0210] Fresh cultures of strains MNH17483 and MNH39288 were collected, and genomic DNA was extracted from them. The extracted genomic DNA was used as a template for 16S rRNA gene amplification.

[0211] The primer pair used for PCR of the 16S rRNA gene is:

[0212] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0213] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0214] The PCR reaction procedure is as follows:

[0215] Pre-denaturation: 94℃, 4min; (denaturation: 94℃, 50sec; annealing: 52℃, 40sec; extension: 72℃, 70sec), 36 cycles; final extension: 72℃, 10min.

[0216] 1.6.2 Sequencing of 16S rRNA genes of strains MNH17483 and MNH39288

[0217] The PCR product was purified and the 16S rRNA gene was sequenced by Sangon Biotech to obtain the 16S rRNA gene.

[0218]

[0219]

[0220] 1.6.3 Identification results of strains MNH17483 and MNH39288

[0221] The 16S rRNA gene sequences obtained above, such as SEQ ID NO:3 and SEQ ID NO:4, were analyzed using the NCBIB Basic Local Alignment Search Tool to confirm the strain's classification information.

[0222] The sequenced data were analyzed using BLAST with data from GenBank. The alignment results showed that the strain with the highest similarity to MNH17483 was *Bifidobacterium animalis* subsp. *lactis*, with a similarity of 100%. Whole-genome ANI analysis showed that *Bifidobacterium animalis* (GCF_000260715.1) had a similarity of 95.88% and an alignment score (AF) of 92%. Therefore, strain MNH17483 is a strain of *Bifidobacterium animalis* subsp. *lactis*. Similarly, the strain with the highest similarity to MNH39288 was also *Bifidobacterium animalis* subsp. *lactis*, with a similarity of 100%. Whole-genome ANI analysis showed that *Bifidobacterium animalis* (GCF_000260715.1) had a similarity of 95.83% and an alignment score (AF) of 92%. Therefore, strain MNH39288 is a strain of *Bifidobacterium animalis* subsp. *lactis*. The strain of animalissubsp.lactis.

[0223] Phylogenetic trees were constructed by comparing the 16S rRNA gene sequences of MNH17483 and MNH39288 with those of related strains of the genus *Bifidobacterium animalis* retrieved from databases such as GenBank.

[0224] Multiple sequence alignment was performed on the sequences of MNH17483 and MNH39288 and type strains with high 16S rRNA gene sequence similarity in the NCBI database. Then, a phylogenetic tree was constructed using MEGA 5 software (the phylogenetic tree was constructed using the maximum likelihood method). Figure 13 , 14 The growth tree nodes only display values ​​where the Bootstrap value is greater than 50%.

[0225] From the phylogenetic tree ( Figure 13 , 14It can be seen that strains MNH17483 and MNH39288 clustered together with Bifidobacterium animalis, representing new strains of Bifidobacterium animalis.

[0226] 1.7 Genome analysis of strains MNH17483 and MNH39288

[0227] 1.7.1 Genome analysis of strain MNH17483

[0228] The genome of strain MNH17483 was fragmented using ultrasound, with fragment lengths ranging from ~350 bp. An Illumina sequencing library was then constructed using a standard DNA library preparation kit (NEB Ultra™). The constructed sequencing library was then sequenced at 150 bp paired ends using NovaSeq (Illumina). Sequencing yielded 1.11 Gbp of data, with Q20 accounting for 96.25%.

[0229] Raw genome 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 was then assembled using SPAdes (version v3.14.0) with the following assembly parameters: "--isolate--cov-cutoff 10". The assembled genome yielded a total length of 1.92 Mbp, an N50 length of 182.2 kbp, and a GC content of 60.49%.

[0230] Genomic gene prediction analysis was performed using the prokaryotic analysis software genome annotation workflow Prokka (version 1.14.5) with parameters "--gcode 11--evalue 1e-09". A total of 1554 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0231] Potential antibiotic resistance genes in the genome were analyzed using the RGI workflow (version 4.2.2), with the antibiotic resistance gene database being CARD (version 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 5.

[0232] Table 5. List of MNH17483 drug resistance genes

[0233]

[0234] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version 2.7.1+) to align with the virulence factor database VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed alignment results are shown in Table 6.

[0235] Table 6. List of Potential Toxicity Genes in MNH17483

[0236] strain genes VFDB gene Gene name Comparison consistency (%) MNH17483_00919 VFG048797 ugd 62.26

[0237] The analysis of potential secondary metabolic gene clusters in the genome was performed using antiSMASH6 (version 6.0.1). No secondary metabolic gene clusters were found.

[0238] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version 1.0.0). Detailed alignment results are shown in Table 7.

[0239] Table 7. List of potential primary metabolic gene clusters of MNH17483

[0240]

[0241]

[0242] 1.7.2 Genome analysis of strain MNH39288

[0243] The genome of strain MNH39288 was fragmented using ultrasound, with fragment lengths ranging from ~350 bp. An Illumina sequencing library was then constructed using a standard DNA library preparation kit (NEB Ultra™). The constructed sequencing library was then sequenced at 150 bp paired ends using NovaSeq (Illumina). Sequencing yielded 1.33 Gbp of data, with Q20 accounting for 96.34%.

[0244] Raw genome 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 was then assembled using SPAdes (version v3.14.0) with the following assembly parameters: "--isolate--cov-cutoff 10". The assembled genome yielded a total length of 1.92 Mbp, an N50 length of 182.4 kbp, and a GC content of 60.49%.

[0245] Genomic gene prediction analysis was performed using the prokaryotic analysis software genome annotation workflow Prokka (version 1.14.5) with parameters "--gcode 11--evalue 1e-09". A total of 1556 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0246] Potential antibiotic resistance genes in the genome were analyzed using the RGI workflow (version 4.2.2), with the antibiotic resistance gene database being CARD (version 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 8.

[0247] Table 8. List of MNH39288 drug resistance genes

[0248]

[0249] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version 2.7.1+) to align with the virulence factor database VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed alignment results are shown in Table 9.

[0250] Table 9. List of Potential Toxicity Genes in MNH39288

[0251] strain genes VFDB gene Gene name Consistency (%) MNH39288_00707 VFG048797 ugd 62.26

[0252] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version 1.0.0). Detailed alignment results are shown in Table 10.

[0253] Table 10. List of potential primary metabolic gene clusters of MNH39288

[0254]

[0255] Example 2: Strains MNH17483 and MNH39288 promote growth and development in a mouse model of growth retardation induced by a low-fat, low-protein diet.

[0256] This invention utilizes a low-fat, low-protein diet to induce a growth-retarded mouse model, and investigates the effects of MNH17483 and MNH39288 on growth and development indicators. This invention has passed the ethical review of the Muen Bio Animal Ethics Committee.

[0257] 2.1 Experimental Methods

[0258] 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 3 weeks old, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0259] 2) Preparation of test samples from strains MNH17483 and MNH39288: Glycerol cryovials of MNH17483 and MNH39288 were thawed at 37°C and activated by inoculation into PYG agar plates in an anaerobic workstation. The activated strains were then inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain purity and viable count (2 × 10⁻⁶). 9 Test substances (CFU / mL) that meet the requirements for animal experiments.

[0260] 3) Negative control: PBS containing 25% glycerol and 0.05% L-Cys HCl was used as the negative control.

[0261] 4) Positive control: Bifidobacterium lactis CGMCC No. 20847. The Bifidobacterium lactis CGMCC No. 20847 strain (disclosed in patent CN112980725B) was thawed in glycerol cryovials at 37°C and activated in PYG medium in an anaerobic workstation. The activated strain was then inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was centrifuged and concentrated, and the bacterial resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain purity and viable count (2×10⁻⁶). 9 A positive control (CFU / mL) that meets the requirements for animal experiments.

[0262] 5) Experimental Procedure: After the quarantine period, 40 male C57BL / 6J mice weighing between 13g and 16g were selected at 3 weeks of age and randomly stratified into 5 groups of 8 mice each: NCD-Control group, LFD-Control group, MNH17483 group, MNH39288 group, and positive control group. The NCD-Control group was fed a maintenance diet (product number: PD24043001, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), while the other four groups were fed a low-fat, low-protein diet (product number: PD24043003, Changzhou Shuyi Shuer Biotechnology Co., Ltd.). After grouping (D1), drug administration began. The NCD-Control and LFD-Control groups received negative controls, while the positive control group received Bifidobacterium lactis CGMCC No. 20847 via gavage. The MNH17483 group received MNH17483 via gavage, and the MNH39288 group received MNH39288 via gavage. Drugs were administered once daily at 200 μL each time for 35 days. Mice had free access to water and food during the experiment, following a 12h / 12h diurnal cycle. A routine clinical observation was performed after each administration period. The endpoint of this experiment was the day after the end of administration (D36). Dissection and tissue sampling were performed according to the protocol to reach the endpoint. Data were collected and analyzed from each dissection and serum assay. All data are expressed as Mean ± SD and plotted and statistically analyzed using GraphPadPrism software. Student's t-test was used for pairwise comparisons. No significance indicates no difference; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0263] Food intake is determined by weighing the remaining amount of feed.

[0264] 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 anatomical sampling.

[0265] Method for detecting serum insulin-like growth factor 1: Mouse serum was collected and detected using the Mouse IGF-1 ELISA Development Kit (PeproTech).

[0266] For the immune cell detection method, 0.1g of mouse spleen tissue was taken, washed with PBS, and then the tissue was pulverized. The pulverized spleen tissue was suspended in PBS and filtered through a 70μm filter membrane to prepare a mouse spleen single-cell suspension. A small amount of the prepared mouse spleen single-cell suspension was stained and then analyzed by flow cytometry to accurately count the number of mouse spleen immune cells.

[0267] The method for determining the short-chain fatty acid content in cecal contents is as follows: Weigh approximately 100 mg of cecal contents sample and place it in a 2 mL EP tube. Add 1 mL of diethyl ether, immediately vortex to mix, and sonicate for 10 min to extract the short-chain fatty acids. Centrifuge for 10 min to remove the precipitate. Pass the supernatant through a 0.22 μm organic filter membrane, transfer the supernatant to a gas chromatography vial, seal it with a sealing film, and store it at -20°C for analysis. The short-chain fatty acids are separated and detected using a DB-FFAP column and a mass spectrometer.

[0268] 2.2 Experimental Results

[0269] See Figure 15-16 The results showed that MNH17483 and MNH39288 could significantly increase the food intake of a low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH17483 and MNH39288 have the potential to promote appetite.

[0270] See Figure 17-20 The results showed that MNH17483 and MNH39288 significantly increased the weight of the liver, spleen, and kidneys, as well as the intestinal length, in growth-retarded mice, indicating that MNH17483 and MNH39288 can promote the development of the liver, spleen, kidneys, and / or intestines, and have the potential to promote organ development. As a control, the weight of the liver, spleen, and kidneys in the positive control group showed no significant difference compared to the growth-retarded mice.

[0271] See Figure 21-26 The results showed that MNH17483 and MNH39288 could significantly promote the increase of body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight in a low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH17483 and MNH39288 have the potential to promote height (body length), weight, bone and muscle development.

[0272] See 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 potential to promote growth and development.

[0273] See Figure 28-31 The results showed that MNH17483 and MNH39288 significantly promoted the development of major immune organs such as the thymus and spleen in a low-fat, low-protein diet-induced growth-retarded mouse model; MNH39288 increased the proportion of T cells, indicating ....

[0274] MNH39288 has the function of promoting the development of the immune system and enhancing immunity.

[0275] See Figures 32-34 The results showed that MN17483 significantly increased butyrate levels in the cecum of a low-fat, low-protein diet-induced growth-retarded mouse model; MNH39288 significantly increased isovaleric acid production and the proportion of 2-methylbutyrate in total short-chain fatty acids. These results indicate that MNH17483 and MNH39288 can promote the production of short-chain fatty acids and thus contribute to gut health.

[0276] Although the invention has been disclosed with reference to certain embodiments, it will be apparent that modifications and variations can be made without departing from the spirit and scope of the invention as disclosed herein and as set forth in the appended claims. Furthermore, it should be understood that while all embodiments disclosed illustrate implementations of the invention, they are provided only as non-limiting examples and should not be construed as limiting the various aspects of the invention thus illustrated. The invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed descriptions should be considered illustrative rather than restrictive.

Claims

1. A probiotic, prebiotic, or pharmaceutical composition comprising Bifidobacterium animalis with accession number GDMCC No: 65555 and / or accession number GDMCC No: 65553.

2. The composition of claim 1, further comprising at least one of the following characteristics: (1) Contains one or more pharmaceutical and / or food-acceptable excipients; (2) The composition further comprises other active agents for enhancing immunity, preferably, other active agents include one or more of other probiotics, prebiotics, nutritional supplements, and dietary supplements; (3) The Bifidobacterium animalis in the composition is a live bacterium, a virus-attenuated bacterium, a freeze-dried bacterium, or an inactivated bacterium; (4) The composition is an infant-suitable dosage form, a pediatric-suitable dosage form, or an adult-suitable dosage form; (5) The composition is provided in liquid or solid form, preferably in the form of a liquid, foam, cream, spray, powder or gel; or the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid preparation, sustained-release preparation, nano preparation or microencapsulated capsule.

3. The application of Bifidobacterium animalis in the preparation of probiotics or prebiotics for enhancing immunity, wherein the Bifidobacterium animalis is Bifidobacterium animalis with accession number GDMCC No: 65555 and / or accession number GDMCC No: 65553.

4. The use of the composition according to claim 1 or 2 in the preparation of a product for enhancing immunity, preferably, the product being a pharmaceutical or health product.

5. The use of the composition in the preparation of products that enhance the immunity of infants, children, or individuals with malnutrition or developmental delays, characterized in that, The composition is the composition according to claim 1 or 2.

6. The application according to any one of claims 3-5, wherein the enhanced immunity includes at least one of the following: (1) promoting the development of the immune system; (2) increasing short-chain fatty acids and / or improving gut microbiota to promote gut health.

7. The application according to claim 6, wherein promoting immune system development includes promoting the development of immune organs and / or promoting the generation of immune cells; preferably, promoting the development of immune organs includes promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; preferably, promoting the development of the thymus and / or spleen.

8. The application according to claim 6, wherein promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells; preferably, increasing the proportion of T cells, B cells, and / or NK cells.

9. The application according to claim 5, wherein the short-chain fatty acid comprises at least one selected from propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

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