Bifidobacterium animalis for promoting growth and development and intestinal health and application of bifidobacterium animalis

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] The present disclosure relates to the field of microbiology, more particularly to an isolated animal Bifidobacterium, compositions comprising the same and uses thereof. BACKGROUND

[0002] Growth refers to the size and shape changes of various organs, systems, and is a quantitative change; development refers to the differentiation and maturation of cells, tissues and organs, and is a qualitative change. The two are closely related, and growth is the material basis of development. The maturation status of development is also reflected in the quantitative change of growth. Human growth and development refers to the maturing process from a fertilized egg to an adult. Growth and development are important characteristics of children that distinguish them from adults. Studies have found that intestinal bacteria can affect the growth and development of the host, and some bacterial flora can mediate the growth and development of the host by affecting growth hormone (GH) / insulin-like growth factor-1 (IGF-1).

[0003] Immunity refers to the ability of the body to resist external invasion and maintain the stability of the internal environment. For children, the development and protection of the immune system are particularly important, as it is directly related to the healthy growth of children. Infants or children with delayed development have slow development of immune organs and gastrointestinal tracts, insufficient immunity, low resistance, and are prone to illness. The environment, such as air, food, and water, is full of various microorganisms: bacteria, viruses, mycoplasma, chlamydia, fungi, etc. In the case of insufficient immunity in children, they are easily infected by harmful bacteria, and then transmitted to guardians and classmates, and even cause serious influenza or diseases. Although the human body produces corresponding antibodies to different pathogens to resist reinfection, antibodies have specificity and time limit, such as streptococcal antibodies that can only protect the body from streptococcal infection for a short period of time, and cannot resist infection by other viruses. The bacteria or viruses that induce influenza vary rapidly, and infants or children with low immunity are difficult to resist the invasion of cold viruses, which is the real reason for their frequent colds.

[0004] Probiotics are microorganisms that, when given in sufficient amounts, bring health benefits to the host. Studies have shown that probiotics can maintain the balance of intestinal flora through their own metabolism and inhibit the growth of harmful bacteria, so that intestinal probiotics are closely related to human health, and probiotic strains have the functions of regulating intestinal health and enhancing immunity.

[0005] Bifidobacterium lactis CGMCC No.20847 is a strain with excellent growth-promoting activity on the market. CN112980725B discloses that Bifidobacterium lactis CGMCC No.20847 can significantly increase the level of IGF-1 in the serum of mice, promote the length of the femur of mice, and promote the increase of height of adolescents. CN114317354B discloses that Bifidobacterium animalis MB-424 can promote the growth of bone cells and the production of growth hormone by pituitary GH3 cells. In the prior art, the growth-promoting activity of Bifidobacterium animalis or Bifidobacterium lactis is mainly focused on promoting the growth of bones and height. However, children with developmental retardation have low resistance and are prone to illness, which is related to the slow development of immune organs and gastrointestinal tract. Few studies have focused 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 have no effect on the weight of the spleen / thymus of mice. Studies have shown (Effect of probiotics on thymus size and markers of infection in late infancy: a randomized controlled trial) that 186 healthy Danish infants were intervened for 6 months using Lactobacillus rhamnosus LGG + Bifidobacterium animalis subsp. lactis BB-12. There was no significant difference in thymus size between the probiotic group and the placebo group (p≥0.248). Probiotics 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 it is a great challenge to screen new strains 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. However, it also represents a huge unmet demand. SUMMARY

[0008] The present disclosure isolates a new animal Bifidobacterium (Bifidobacterium animalis). The new strain of the present disclosure can 1, promote the development of major immune organs such as thymus and spleen of the developmentally delayed subject (including increasing the weight of immune organs such as thymus and spleen, immune organ development index (immune organ weight / body weight), etc.); 2, promote the development and proliferation of immune cells such as T cells, and promote the immunity of the developmentally delayed subject; 3, increase the content of serum insulin-like growth factor 1 (IGF-1) of the developmentally delayed subject, promote the increase of body weight and body length, and promote the growth of bone and muscle, for example, promote the increase of tibial length, femoral length, soleus muscle and gastrocnemius muscle weight; 4, increase the weight of liver, thymus, spleen and kidney of the developmentally delayed subject, and the length of intestinal tract (length of large intestine and / or length of small intestine); increase food intake; increase the content of short-chain fatty acids in the intestinal tract; improve intestinal flora; and promote the development of intestinal tract.

[0009] Therefore, the new strain of the present disclosure can have the use of promoting the development of organs while promoting the increase of body height and weight, especially promoting the development of immune organs, enhancing immunity, promoting the development of intestinal tract, and improving intestinal health. It can be used to improve the developmentally delayed caused by malnutrition, achieve the function of improving / treating / reversing the developmentally delayed; achieve the purpose of promoting growth and development, enhancing immunity, or promoting intestinal health, etc.

[0010] In the first aspect, the present disclosure provides an isolated animal Bifidobacterium (Bifidobacterium animalis) having 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% to the strain with the accession number of GDMCC No: 65553, or GDMCC No: 65555; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence as shown in SEQ ID NO: 3, or as shown in 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% to the strain MNH17483 with the accession number of GDMCC No: 65553; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence as shown in SEQ ID NO: 3.

[0012] In some embodiments, the Bifidobacterium animalis 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% to the strain MNH39288 with the accession number GDMCC No: 65555; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence as set forth 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 as set forth in SEQ ID NO: 3, or as set forth in SEQ ID NO: 4.

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

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

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

[0017] In a second aspect, the present disclosure provides a composition comprising the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the animal Bifidobacterium of the first aspect, wherein the culture comprises any one of the following A) to D):

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

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

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

[0021] D) a concentrated or dried product of any one of A) to C).

[0022] In a third aspect, the present disclosure provides a composition comprising the animal Bifidobacterium of the first aspect, or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the animal Bifidobacterium of the second aspect.

[0023] The present disclosure provides a composition comprising the animal Bifidobacterium of the first aspect, a culture thereof, and / or a metabolite thereof.

[0024] In some embodiments, the culture of the animal Bifidobacterium comprises a solid culture, a fermentation culture, a fermentation culture supernatant of the animal Bifidobacterium, 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 a liquid form or a solid form.

[0027] In some embodiments, the composition comprises 1 x 10 4 to 1 x 10 12 cfu / mL or 1 x 10 4 to 1 x 10 12 cfu / mg of the live animal Bifidobacterium.

[0028] In some embodiments, the composition comprises 1 x 10 5 to 1 x 10 11 cfu / mL or 1 x 10 5 to 1 x 10 11 cfu / mg of the live animal Bifidobacterium.

[0029] In some embodiments, the composition comprises 1 x 10 6 to 1 x 10 10 cfu / mL or 1 x 10 6 to 1 x 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 12, 9 x 10 12 , 1 x 10 13 , 2 x 10 13 , 3 x 10 13 , 4 x 10 13 , 5 x 10 13 , 6 x 10 13 , 7 x 10 13 , 8 x 10 13 , 9 x 10 13 or any value therebetween, of colony forming units (CFU) of bacteria.

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

[0033] In some embodiments, the Bifidobacterium animalis in the composition is a live, attenuated, lyophilized or inactivated bacteria, for example can be heat inactivated, 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 preparation, sustained release preparation, nano-preparation or microencapsulated capsule.

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

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

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

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

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

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

[0042] In some embodiments, the promoting growth development comprises: promoting at least one of organ, height / length, weight, bone, muscle development.

[0043] In some embodiments, the organ comprises at least one of heart, thymus, liver, spleen, kidney, intestine.

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

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

[0046] In some embodiments, the additional active agent can be one or more than one or a combination of probiotics, prebiotics;

[0047] 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 kefiranofaciens, Lactobacillus kefiri, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sake, Lactococcus lactis, Lactococcus cremoris.

[0048] In some embodiments, the prebiotic is selected from inulin, mulberry leaf extract, berberine, ganoderma, green coffee bean extract, oat, pectin, potato or extract thereof, citrus polyphenol, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidin, resistant dextrin, yeast beta-glucan, ginseng or extract thereof, nutritional compound, biotin, polydextrose, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, mannan-oligosaccharide, mannan-oligosaccharide (MOS), inulin rich in fructo-oligosaccharide, gluco-oligosaccharide, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS), and any combination thereof.

[0049] In some embodiments, the composition can be formulated into a frozen composition, such as a frozen composition prepared by flash freezing and drying, or lyophilization, for storage and / or transportation.

[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 strains in the composition are freeze-dried or spray-dried. In some embodiments, the strains in the composition are electrostatic spray-dried. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are viable. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are capable of partial or complete colonization of the intestine. In some embodiments, the strains are reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

[0052] In some embodiments, the composition is 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 formulation. In some embodiments, the enteric formulation is a dosage form with an enteric coating. For example, the enteric formulation can be an enteric granule, an enteric tablet, or an enteric 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 capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

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

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

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

[0057] In a fourth aspect, the present disclosure provides use of the animal bifidobacterium of the first aspect, the culture, live bacteria, freeze-dried bacteria, or inactivated bacteria of the second aspect, or the composition of the third aspect in the preparation of a medicament, a health product, or a food product for promoting growth and development, enhancing immunity, or promoting gut health.

[0058] The present disclosure provides use of the animal bifidobacterium of the first aspect, the culture, live bacteria, freeze-dried bacteria, or inactivated bacteria of the second aspect, or the composition of the third aspect in promoting growth and development, enhancing immunity, or promoting gut health (preferably, the use does not involve the treatment of a disease).

[0059] In some embodiments, the promoting growth and development comprises: promoting at least one of organ, height / length, weight, bone, muscle development.

[0060] In some embodiments, the organ comprises at least one of a heart, a liver, a spleen, a kidney, an intestine.

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

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

[0063] In some embodiments, the drug, nutraceutical, 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 embodiments, the hormone is from a bodily fluid; further from blood; still further from serum.

[0066] In some embodiments, the drug, nutraceutical, or food enhances immunity by promoting immune system development.

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

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

[0069] In some embodiments, the promoting generation of immune cells comprises promoting generation 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 embodiments, the drug, nutraceutical, 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, 2-methylbutyric acid.

[0072] The composition comprises a population of isolated and purified live microorganisms in a consortium to increase body weight by at least 2%, 3%, 4%, 5%, 6%, or 7% in a subject as compared to the body weight of the subject prior to administration of the population of isolated and purified microorganism species in a consortium.

[0073] As used herein, a microbiota generally refers to a population of microorganisms that consists essentially of a single strain, species, or genus, which can be the case when a population is cultured from a subpopulation of an isolated and purified strain, species, or genus. Thus, for a given population of microorganisms, if cultured from an isolated microorganism species or strain, such population will be referred to herein as purified or substantially pure. The resulting population can be at least 80% pure for the microorganism species or strain, at least 90% pure relative to other microorganism species or strains within that particular population, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure. Conversely, the level of non-desired 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 in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate for the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate for the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population.

[0074] The compositions of the present disclosure can also include cell components, metabolites, secreted molecules and compounds, and the like, metabolized by the animal Bifidobacterium. These can be recovered, for example, by recovering the supernatant of an animal Bifidobacterium culture or by extracting cell components or cell fractions, metabolites or secreted compounds from an animal Bifidobacterium culture; can correspond to components in isolated form from the animal Bifidobacterium, or any mixture of one or more components from the animal Bifidobacterium.

[0075] In some embodiments, the animal Bifidobacterium, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition of the present disclosure can be used to promote growth and development, enhance immunity, or promote gut health.

[0076] In some embodiments, the promoting growth and development comprises: promoting at least one of organ, height / length, weight, bone, muscle development.

[0077] In some embodiments, the organ comprises at least one of a heart, a thymus, a liver, a spleen, a kidney, an intestine.

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

[0079] In some embodiments, the muscle comprises at least one of a soleus muscle, a 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 levels, 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 embodiments, the hormone is from a bodily fluid; further from blood; further from serum.

[0083] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition enhances immunity by promoting immune system development.

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

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

[0086] In some embodiments, the promoting generation of immune cells comprises promoting generation 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, freeze-dried 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, 2-methylbutyric acid.

[0089] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can promote appetite, and in turn increase food intake (preferably cumulative food intake), to achieve the effect of promoting growth and development.

[0090] In some embodiments, the Bifidobacterium animalis of the present disclosure, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition can increase heart, liver, spleen, and / or kidney weight, and / or intestinal length, thereby promoting organ development.

[0091] In some embodiments, the Bifidobacterium animalis of the present disclosure, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition can increase body weight, body length, tibia length, femur length, gastrocnemius and gastrocnemius muscle weight, thereby promoting height (body length), body weight, bone, muscle development.

[0092] In some embodiments, the Bifidobacterium animalis of the present disclosure, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition can increase serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH) content, thereby promoting growth development.

[0093] In some embodiments, the Bifidobacterium animalis of the present disclosure, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition can promote the development of major immune organs such as thymus, spleen, increase the proportion of T cells, B cells, NK cells, thereby promoting immune system development, enhancing immunity.

[0094] In some embodiments, the Bifidobacterium animalis of the present disclosure, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition can increase short-chain fatty acid content, and / or improve intestinal flora, thereby promoting intestinal health.

[0095] In some embodiments, the drug or health product or food product has at least one effect selected from the group consisting of: increasing heart, liver, spleen, and / or kidney weight, 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 body weight, body length growth, promoting bone and muscle growth (wherein promoting bone growth comprises bone length and density growth, and promoting muscle growth comprises promoting specific muscle development and maintaining healthy and benign growth characteristics, such as: low body fat ratio (calf muscle / body weight) and / or high muscle ratio (calf muscle / calf weight)); increasing serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH) content; promoting the development of major immune organs such as thymus, spleen, etc. (including increasing thymus, spleen, etc. immune organ weight, immune organ development index (immune organ weight / body weight), etc.), increasing T cell, B cell, NK cell proportion; increasing short-chain fatty acid content; improving intestinal flora; promoting intestinal development; improving pathologies or inflammation caused by developmental retardation to achieve the function of improving / treating / reversing developmental retardation; promoting organ development; promoting appetite; promoting height (body length), body weight, bone, muscle development; promoting immune system development, enhancing immunity; promoting intestinal health; can be used for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0096] A method for promoting growth and development, enhancing immunity, or promoting intestinal health, administering an effective amount of the animal bifidobacterium of the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the second aspect, or the composition of the third aspect to a subject in need thereof.

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

[0098] In some embodiments, the organ comprises at least one of heart, liver, spleen, kidney, intestine.

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

[0100] In some embodiments, the muscle comprises at least one of soleus muscle, 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 embodiments, the hormone is from a bodily fluid; further from blood; still further from serum.

[0104] In some embodiments, the animal bifidobacteria, culture, live bacteria, lyophilized bacteria or inactivated bacteria, or composition enhances immunity by promoting immune system development.

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

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

[0107] In some embodiments, the promoting generation of immune cells comprises promoting generation 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 bifidobacteria, 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 acids comprise at least one of propionic acid, butyric acid, isovaleric acid, 2-methylbutyric acid. BRIEF DESCRIPTION OF DRAWINGS

[0110] The above and other aspects and advantages of the present application will become apparent and easier to understand from the following description, taken in conjunction with the accompanying drawings.

[0111] Figure 1 : shows a photograph of colony morphology of strain MNH17483.

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

[0113] Figure 3 : shows a photograph of electron microscopy of strain MNH17483.

[0114] Figure 4 : shows a photograph of colony morphology of strain MNH39288.

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

[0116] Figure 6 : shows a photograph of electron microscopy of strain MNH39288.

[0117] Figure 7 Figure 2: shows the results of the tolerance of strain MNH17483 to different pH.

[0118] Figure 8 Figure 3: shows the results of the tolerance of strain MNH17483 to different concentrations of NaCl.

[0119] Figure 9 Figure 4: shows the results of the tolerance of strain MNH17483 to different concentrations of bile salts.

[0120] Figure 10 Figure 5: shows the results of the tolerance of strain MNH39288 to different pH.

[0121] Figure 11 Figure 6: shows the results of the tolerance of strain MNH39288 to different concentrations of NaCl.

[0122] Figure 12 Figure 7: shows the results of the tolerance of strain MNH39288 to different concentrations of bile salts.

[0123] Figure 13 Figure 8: shows the phylogenetic tree of strain MNH17483.

[0124] Figure 14 Figure 9: shows the phylogenetic tree of strain MNH39288.

[0125] Figures 15-16 Figure 10: shows that strains MNH17483 and MNH39288 increase cumulative food intake (15: cumulative food intake line graph; 16: cumulative food intake bar graph). Data are shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared to the LFD-Control group.

[0126] Figures 17-20 Figure 11: shows that strains MNH17483 and MNH39288 promote organ development (17: liver weight; 18: spleen weight; 19: kidney weight; 20: intestinal length). Data are shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared to the LFD-Control group; **, p < 0.01 compared to the LFD-Control group; ****, p < 0.0001 compared to the LFD-Control group.

[0127] Figures 21-26: Shows that strains MNH17483 and MNH39288 promote 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 shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with the LFD-Control group.

[0128] Figure 27 : Shows that strain MNH39288 increases the serum insulin-like growth factor 1 (IGF-1) content. Data are shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05; ****, p < 0.0001 compared with the LFD-Control group.

[0129] Figures 28-31 : Shows that strains MNH17483 and MNH39288 promote the development of the immune system and enhance immunity (28: thymus weight; 29: spleen weight; 30: immune organ weight; 31: T cell percentage). Data are shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05; **, p < 0.01 compared with the LFD-Control group.

[0130] Figures 32-34 : Shows that strains MNH17483 and MNH39288 promote the production of short-chain fatty acids in cecal contents (32: butyric acid content; 33: isovaleric acid content; 34: 2-methylbutyric acid ratio). Data are shown as mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05; ***, p < 0.001 compared with the LFD-Control group.

[0131] Preservation of strains

[0132] Bifidobacterium animalis MNH17483, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC No: 65553, on November 28, 2024, at 5th floor, Building 59, 100, Jiefang Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposited name of Bifidobacterium animalis MNH17483, and the taxonomic name of: Bifidobacterium animalis.

[0133] Bifidobacterium animalis MNH39288, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC No: 65555, on November 28, 2024, at 5th floor, Building 59, 100, Jiefang Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposited name of Bifidobacterium animalis MNH39288, and the taxonomic name of: Bifidobacterium animalis. DETAILED DESCRIPTION

[0134] The present disclosure isolates two new strains of Bifidobacterium animalis species, with accession numbers of GDMCC No: 65553 and GDMCC No: 65555, respectively, and identifies them using traditional classification methods and molecular biology methods. The identification results show that the two strains belong to new strains under the Bifidobacterium animalis species. Further, the present disclosure studies the biochemical properties and therapeutic uses of the two strains.

[0135] It is known in the art that a strain can be classified and identified by traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, for example, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biology methods include, but are not limited to, ribosomal RNA sequence determination method, whole genome sequencing-based determination method, etc.

[0136] The term "prebiotic" as used herein can be a general term referring to a chemical substance and / or component that can affect the growth and / or activity of microorganisms in a host (e.g., 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 herein to refer to any animal subject, including: human, mammal, experimental animal, livestock, and domestic pet.

[0138] The compositions or formulations of the present disclosure can be administered as a pharmaceutical formulation, a therapeutic composition, a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food. In some cases, the composition is administered as a pharmaceutical formulation. In some cases, the composition is administered as a nutritional supplement. In some cases, the composition is administered as a dietary supplement. In some cases, the composition is administered as a medical food. In some cases, the composition is administered as a medical probiotic. In some cases, the composition (e.g., a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food) can be administered orally, for example, as a capsule, pill, or tablet.

[0139] 16S rRNA is a ribosomal RNA of prokaryotes, and the 16S rRNA gene is composed of variable regions and conserved regions, with the conserved regions being shared by all bacteria and the variable regions differing to varying degrees among different bacteria. By comparing the 16S rRNA gene sequences of bacteria, an evolutionary tree can be drawn according to the evolutionary distance based on the number of sequence differences. When the sequence identity between the 16S rRNA genes 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 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), p23).

[0140] “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. Commercially or publicly available programs can also be, for example, the DNAStar “MegAlign” program.

[0141] The second-generation sequencing technology can also be used for bacterial strain identification based on whole genome sequencing, which makes the bacterial strain identification result more accurate. The average nucleotide identity (ANI) of bacterial genome refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated by BLAST and other methods. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach more than 95% to be identified as belonging to the same bacterial species (Jain C, Rodriguez-R L M, Phillippy A M, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries [J]. Nature Communications, 2018, 9(1): 5114.).

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

[0143] Using the above method, a person skilled in the art can determine whether a separated strain belongs to the animal bifidobacterium species identified by the present inventors. For example, when the average nucleotide identity ANI value with the animal bifidobacterium (Bifidobacterium animalis) with the preservation number of GDMCC No: 65553 or GDMCC No: 65555 is at least 95%, such as 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%, 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 it belongs to the same bacterial species.

[0144] For another example, when its 16S rRNA sequence has at least 98.65% identity, 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 is 100% to the sequence set forth in SEQ ID NO: 3 or 4, it can be determined to belong to the same species.

[0145] A "strain" refers to a member of a bacterial species that has genetic characteristics that distinguish it from closely related members of the same bacterial species. The genetic characteristics can be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence of at least one native plasmid ("curing"), 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), at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a 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. The 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 the case where a strain gains or loses antibiotic resistance or gains or loses biosynthetic capabilities (e.g., an auxotrophic strain) compared to another strain of the same species, the strains or nutrients / metabolites can be distinguished by selection or counter-selection using antibiotics.

[0146] "Supernatant" or "supernate" within the meaning herein refers to a culture supernatant of a 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.

[0147] Compositions can be prepared using the animal Bifidobacterium described herein, for example by using pharmaceutically acceptable excipients. The pharmaceutical compositions comprise a pharmaceutically effective amount of the animal Bifidobacterium, for example the animal Bifidobacterium having the accession number GDMCC No: 65553, or GDMCC No: 65555. Likewise, the animal Bifidobacterium having the accession number GDMCC No: 65553, or GDMCC No: 65555 can also be prepared into a pharmaceutical composition, for example by using pharmaceutically acceptable excipients, comprising a pharmaceutically effective amount of the animal Bifidobacterium.

[0148] Suitable pharmaceutically acceptable excipients that can be used are, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweetening agents, and flavoring agents.

[0149] The compositions herein can be formulated in any form suitable for enhancing the abundance of animal bifidobacteria in a subject. The compositions can be administered by oral administration (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, gastrointestinal, biliary perfusion, cardiac perfusion, pre-anal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transdermal, ureteral, or urethral routes, among others.

[0150] Examples of suitable dosage forms for the compositions herein include, but are not limited to, tablets, aerosols, chewable bars, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, concentrates.

[0151] In some embodiments, the compositions are sugar-coated tablets, gel capsules, gels, emulsions, tablets, tablet-like capsules, hydrogels, nanofiber gels, electrospun fibers, food bars, candies, fermented milk, fermented cheese, chewing gum, powders, or toothpaste, among others.

[0152] In some embodiments, administration can also be by inclusion in the subject’s diet, for example, in a functional food for a human or companion animal.

[0153] The compositions provided herein can include a pharmaceutically acceptable excipient, diluent, or carrier. Such pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

[0154] In some embodiments, the animal bifidobacteria in the compositions of the present disclosure are lyophilized. In some embodiments, the animal bifidobacteria in the compositions of the present disclosure are spray-dried. In some embodiments, the animal bifidobacteria in the compositions of the present disclosure are lyophilized or spray-dried and are viable. In some embodiments, the animal bifidobacteria in the compositions of the present disclosure are lyophilized or spray-dried and are capable of partial or complete colonization of the intestine. In some embodiments, the lyophilized animal bifidobacteria are reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0155] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration can involve swallowing, so that the composition enters the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

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

[0157] The compositions of the present disclosure include a pharmaceutical, nutraceutical, or food product.

[0158] The subject of the present disclosure can be a human or an animal, including but not limited to a cow, a sheep, a cat, a dog, a horse, a rabbit, a monkey, a mouse, a rat, an alpaca, a camel, etc.

[0159] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the examples below are merely for illustration of the present application and should not be considered as limiting the scope of the application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature of the art or according to the product or instrument manual is used. If the manufacturer of all reagents or instruments is specified, they can be purchased commercially.

[0160] Example

[0161] The anaerobic blood agar plates involved in the examples are purchased from HuanKai Microbiology, and the formula is: casein enzymatic digest 10 g / L, heart pancreatic enzymatic digest 3 g / L, corn starch 1 g / L, meat pancreatic enzymatic digest 5 g / L, yeast extract powder 5 g / L, sodium chloride 5 g / L, agar 15 g / L, sterile defibrillated sheep blood 50 mL / L, pH 7.3±0.2.

[0162] The PYG liquid medium involved in the examples contains, per 1 L of PYG liquid medium: 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, water to volume, water to volume), water to volume, pH 7.2±0.2 (25℃).

[0163] The above-mentioned culture medium can be prepared by 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 strains of intestinal bacteria, Bifidobacterium animalis MNH17483 and Bifidobacterium animalis MNH39288, are isolated from the fecal samples of healthy volunteers. The isolation method uses conventional strain isolation method, gradient dilution, and then single colony is picked up by isolation culture, strain purification, and anaerobic culture at 37°C. The pure culture strain is prepared into 20% glycerol / water-bacteria liquid, and stored at -80°C.

[0167] Specifically, the isolation and purification method of the strain is as follows:

[0168] The donor takes 2-5g of fresh feces, puts it into a sample collection and preservation tube, shakes it evenly, and then puts the treated fecal sample into an ice box, and delivers it to the laboratory within 24 hours for strain isolation.

[0169] Physiological saline is dispensed in a biological safety cabinet, 9mL / tube; strain isolation medium anaerobic blood agar plate is prepared, and it is transferred into the anaerobic workstation 24h in advance, and the sample information, medium type, isolation date, etc. are marked.

[0170] Take fresh fecal sample and place it in an anaerobic workstation, use vortex shaker to shake for 1min, mix evenly, take 1mL sample into 9mL physiological saline, mix evenly to 10 -1 dilution, then gradient dilution to 10 -6 dilution, standby.

[0171] Take 10 -6 dilution drops into anaerobic blood agar plate, drop amount is 100μL / dish, evenly spread, after the surface of the plate is dry, the plate is inverted, and cultured at 37°C for 3-5 days.

[0172] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick up single colony with sterilized toothpick for strain purification, and the purified strain is cultured anaerobically at 37°C. The pure culture strain is prepared into 20% glycerol / water-bacteria liquid, 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 into PYG plate medium, and after anaerobic culture at 37°C for 48 h, visible colonies were formed on the PYG plate medium, which were round, regular and smooth in edge, about 0.5-2 mm in diameter, white and opaque; the strain was gram-positive; microscopic observation found that it was non-flagellated, non-motile and rod-shaped, about 0.5-1 μm x 1.5-3 μm in size. The colony morphology photo of strain MNH17483 cultured on PYG plate for 48 h is shown in Figure 1 . The gram staining photo of strain MNH17483 is shown in Figure 2 and the electron microscope photo is shown in Figure 3 .

[0176] 1.2.2 Morphological characteristics of strain MNH39288

[0177] Strain MNH39288 was inoculated into PYG plate medium, and after anaerobic culture at 37°C for 48 h, visible colonies were formed on the PYG plate medium, which were round, regular and smooth in edge, about 0.5-2 mm in diameter, white and opaque; the strain was gram-positive; microscopic observation found that it was non-flagellated, non-motile and rod-shaped, about 0.5-1 μm x 1.5-3 μm in size. The colony morphology photo of strain MNH MNH39288 cultured on PYG plate for 48 h is shown in Figure 4 . The gram staining photo of strain MNH39288 is shown in Figure 5 and the electron microscope photo is shown in 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 did not grow under aerobic conditions, and only grew under anaerobic conditions. It could grow at pH 5.0 to 10.0, and the optimal growth pH was 7.0-9.0 (the results of strain tolerance to different pH are shown in Figure 7 ); it did not grow in the medium with NaCl content exceeding 3% (the results of strain tolerance to different concentrations of NaCl are shown in Figure 8 ); strain MNH17483 could survive and grow at a bile salt concentration of 0%-0.15%, and could not grow at a bile salt concentration of 0.2% or more (the results of strain tolerance to different concentrations of bile salt are shown in 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 at pH 6.0 to 9.0, with the optimum pH being 7.0 (see Table 2 for the results of the strain's tolerance to different pH values); it can grow in medium with NaCl content of more than 6% (see Table 3 for the results of the strain's tolerance to different concentrations of NaCl); and it can survive and grow in the presence of bile salt at a concentration of 0% to 0.1%, but cannot grow in the presence of bile salt at a concentration of 0.15% or more (see Table 4 for the results of the strain's tolerance to different concentrations of bile salt). Figure 10 Figure 11 Figure 12

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

[0184] 1.4.1 Biochemical identification results of strain MNH17483 by API 50CHL

[0185] API 50CHL (Merieux, CN5041010) was used, and the specific experimental operation was in accordance with the routine API reagent operation guide. The culture conditions were 37°C and anaerobic. The experimental results are shown in Table 1.

[0186] MNH17483 can ferment L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, alpha-methyl-D-glucoside MDG, amygdaloside AMY, arbutin ARB, escine ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, and D-turanose TUR to produce acid. Therefore, L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, alpha-methyl-D-glucoside MDG, amygdaloside AMY, arbutin ARB, escine ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, and D-turanose TUR, and their derivatives can be used as carbon sources in the fermentation or culture of strain MNH17483.

[0187] Table 1. Test results of strain MNH17483

[0188] MNH17483

[0189]

[0190] Note: "+" indicates positive, and "-" indicates negative.

[0191] ​​​1.4.2 Results of biochemical identification of strain MNH39288 by API 50CHL

[0192] API 50CHL (bioMerieux, CN5041010) was used according to the manufacturer's instructions. The incubation conditions were 37°C, anaerobiosis. The results are shown in Table 2.

[0193] MNH39288 is able to ferment L-arabinose LARA, ribose RIB, D-xylose DXYL, galactose GAL, glucose GLU, fructose FRU, alpha-methyl-D-glucoside MDG, amygdalose AMY, arbutin ARB, escine ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR to produce acid. Thus, during the fermentation or cultivation of strain MNH39288, L-arabinose LARA, ribose RIB, D-xylose DXYL, galactose GAL, glucose GLU, fructose FRU, alpha-methyl-D-glucoside MDG, amygdalose AMY, arbutin ARB, escine ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR, and derivatives thereof can be used as carbon sources.

[0194] Table 2. Test results for strain MNH39288 MNH39288

[0195]

[0196]

[0197] Note: "+" means positive, "-" means negative.

[0198] 1.5 Antibiotic minimal inhibitory concentration tests for strains MNH17483 and MNH39288

[0199] 1.5.1 Antibiotic minimal inhibitory concentration test for strain MNH17483

[0200] The antibiotic minimal inhibitory concentration of strain MNH17483 was determined using E-test (Liofilchem) strips. The results are shown in Table 3.

[0201] Table 3. Antibiotic minimal inhibitory concentration test results for strain MNH17483

[0202] Antibiotic MIC (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

[0203] The results show that MNH17483 is sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, penicillin, cefquinome, tetracycline, and ceftriaxone. It can be seen that MNH17483 is sensitive to most types of antibiotics, and the risk of long-term use of MNH17483 leading to the subject developing antibiotic resistance is low.

[0204] 1.5.2 Antibiotic minimum inhibitory concentration test of strain MNH39288

[0205] The antibiotic minimum inhibitory concentration of strain MNH39288 was determined using E-test (purchased from Liofilchem) strips, and the test results are shown in Table 4.

[0206] Table 4. Antibiotic minimum inhibitory concentration test results of strain MNH39288

[0207] Antibiotic MIC (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

[0208] The results show that MNH39288 is sensitive to moxifloxacin, ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, penicillin, cefquinome, tetracycline, and ceftriaxone. It can be seen that MNH39288 is sensitive to most types of antibiotics, and the risk of long-term use of MNH39288 leading to the subject developing antibiotic resistance is low.

[0209] 1.6 Identification of strains MNH17483 and MNH39288

[0210] 1.6.1 16S rRNA gene amplification of strains MNH17483 and MNH39288

[0211] Fresh cultures of strains MNH17483 and MNH39288 were taken for extraction of strain genomic DNA. The extracted strain genomic DNA was used as a template for 16S rRNA gene amplification.

[0212] The primer pair used for 16S rRNA gene PCR was:

[0213] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0214] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0215] The PCR reaction program was as follows:

[0216] 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.

[0217] 1.6.2 16S rRNA gene sequencing of strains MNH17483 and MNH39288

[0218] The PCR products were purified and 16S rRNA gene sequencing was performed by Shenguo Company, and the 16S rRNA gene was obtained.

[0219]

[0220]

[0221] 1.6.3 Identification results of strains MNH17483 and MNH39288

[0222] The measured 16S rRNA gene sequence shown as SEQ ID NO: 3, SEQ ID NO: 4 was used to analyze the 16S rRNA gene of the strain by NCBI Basic Local Alignment Search Tool, and the strain classification information was confirmed.

[0223] The measured sequence was analyzed by BLAST with the data in GenBank, and the comparison results showed that the strain with the highest similarity to MNH17483 was Bifidobacterium animalis subsp. lactis, with a similarity of 100%, and the whole genome ANI analysis showed that the similarity of Bifidobacterium animalis (GCF_000260715.1) was 95.88%, and the alignment score (AF) was 92%, so the strain MNH17483 was a strain under the species of Bifidobacterium animalis subsp. lactis; the strain with the highest similarity to MNH39288 was Bifidobacterium animalis subsp. lactis, with a similarity of 100%, and the whole genome ANI analysis showed that the similarity of Bifidobacterium animalis (GCF_000260715.1) was 95.83%, and the alignment score (AF) was 92%, so the strain MNH39288 was a strain under the species of Bifidobacterium animalis subsp. lactis.

[0224] MNH17483 and MNH39288 were compared with the 16S rRNA gene sequences of related strains of Bifidobacterium animalis genus retrieved from databases such as GenBank, and phylogenetic trees were constructed.

[0225] MNH17483 and MNH39288 and the sequences of the strains with high similarity to the 16S rRNA gene sequence in the NCBI database were subjected to multiple sequence alignment, and then the software MEGA 5 was used to construct a phylogenetic tree (the maximum likelihood method was used to construct the phylogenetic tree), Figure 13 、 14 Only Bootstrap values greater than 50% were displayed on the phylogenetic tree nodes.

[0226] From the phylogenetic tree, Figure 13 、 14)It can be seen that strains MNH17483 and MNH39288 are clustered together with Bifidobacterium animalis, which is a new strain under the species Bifidobacterium animalis.

[0227] 1.7 Genome analysis of strains MNH17483 and MNH39288

[0228] 1.7.1 Genome analysis of strain MNH17483

[0229] The genome of strain MNH17483 was sequenced by ultrasonic fragmentation, with a fragmentation length range of ~ 350 bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced by NovaSeq (Illumina) with a double-end 150 bp. The sequencing obtained 1.11 Gbp data, of which the Q20 accounted for 96.25%.

[0230] The raw genome sequencing data was filtered using fastp (version: 0.20.0), with filtering parameters: “--poly_g_min_len 10--poly_x_min_len 10-q 15-u 40-n 5-l 50”. The filtered raw data was assembled into a genome using SPAdes (version: v3.14.0), with assembly parameters “--isolate--cov-cutoff 10”. The genome assembly obtained a total length of 1.92 Mbp, with an N50 length of 182.2 kbp and a GC content of 60.49%.

[0231] The genome genes were analyzed for genome gene prediction using the prokka (version: 1.14.5) software for prokaryotic analysis, with parameters “--gcode 11--evalue 1e-09”. A total of 1554 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0232] The potential antibiotic resistance genes in the genome were analyzed using the RGI process (version: 4.2.2), with the antibiotic resistance gene database being CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). For detailed comparison information, refer to Table 5.

[0233] Table 5. MNH17483 drug resistance gene information list

[0234]

[0235] The potential virulence factors and related genes in the genome were analyzed by NCBI blastp (version: 2.7.1+) against the virulence factor database VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed alignment results are shown in Table 6.

[0236] Table 6. List of potential virulence genes of MNH17483

[0237] Strain gene VFDB gene Gene name Alignment identity (%) MNH17483_00919 VFG048797 ugd 62.26

[0238] The potential secondary metabolism gene clusters in the genome were analyzed by antiSMASH6 (version: 6.0.1). No secondary metabolism gene cluster was found.

[0239] The potential primary metabolism gene clusters in the genome were analyzed by gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 7.

[0240] Table 7. List of potential primary metabolism gene clusters of MNH17483

[0241]

[0242]

[0243] 1.7.2 Genome analysis of strain MNH39288

[0244] The genome of strain MNH39288 was fragmented by ultrasonic method, with a fragmentation length of ~ 350 bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced by NovaSeq (Illumina) with a double-end 150 bp. The sequencing obtained 1.33 Gbp data, of which the Q20 accounted for 96.34%.

[0245] The raw sequencing data of the genome was filtered by fastp (version: 0.20.0) with the filtering parameters: “--poly_g_min_len 10--poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50”. The filtered raw data was used for genome assembly by SPAdes (version: v3.14.0) with the assembly parameters “--isolate--cov-cutoff 10”. The genome assembly obtained a total gene length of 1.92 Mbp, with an N50 length of 182.4 kbp and a GC content of 60.49%.

[0246] Genome genes were analyzed by 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.

[0247] The potential antibiotic resistance genes in the genome were analyzed by RGI (version: 4.2.2) with the antibiotic resistance gene database CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). The detailed comparison information is shown in Table 8.

[0248] Table 8. MNH39288 drug resistance gene information list

[0249]

[0250] The potential virulence factors and related genes in the genome were analyzed by NCBI blastp (version: 2.7.1+) against the virulence factor database VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed comparison results are shown in Table 9.

[0251] Table 9. MNH39288 potential virulence gene list

[0252] Strain gene VFDB gene Gene name Alignment identity (%) MNH39288_00707 VFG048797 ugd 62.26

[0253] The potential primary metabolic gene clusters in the genome were analyzed by gutSMASH5 (version: 1.0.0). The detailed comparison results are shown in Table 10.

[0254] Table 10. MNH39288 potential primary metabolic gene cluster list

[0255]

[0256] Example 2: Strains MNH17483 and MNH39288 promote growth and development in a low-fat low-protein diet-induced growth and development retardation mouse model

[0257] The low-fat low-protein diet-induced growth and development retardation mouse model was used to improve the growth and development indicators of MNH17483 and MNH39288. The present application has been ethically reviewed by the Muen Biological Animal Ethics Committee.

[0258] 2.1 Experimental method

[0259] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 3 weeks old, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.

[0260] 2) Preparation of test substances of strains MNH17483 and MNH39288: After the glycerol frozen tubes of MNH17483 and MNH39288 strains were thawed at 37°C, they were inoculated into PYG plate culture medium in an anaerobic workstation for activation. The activated strains were inoculated into PYG liquid culture medium and anaerobically cultured to obtain a sufficient amount of culture. After the cultured bacterial liquid was centrifuged and concentrated, the bacterial bodies were resuspended with PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain test substances with a purity and viable bacterial count (2×10 9 CFU / mL) meeting the requirements of animal experiments.

[0261] 3) Negative control: PBS containing 25% glycerol and 0.05% L-Cys HCl was used as the negative control.

[0262] 4) Positive control: Bifidobacterium lactis CGMCC No. 20847. After the glycerol frozen tube of Bifidobacterium lactis CGMCC No. 20847 strain (which has been disclosed in patent CN112980725B) was thawed at 37°C, it was inoculated into PYG culture medium in an anaerobic workstation for activation. The activated strain was inoculated into PYG liquid culture medium and anaerobically cultured to obtain a sufficient amount of culture. After the cultured bacterial liquid was centrifuged and concentrated, the bacterial bodies were resuspended with PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain a positive control with a purity and viable bacterial count (2×10 9 CFU / mL) meeting the requirements of animal experiments.

[0263] 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.

[0264] Food intake is determined by weighing the remaining amount of feed.

[0265] 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.

[0266] Method for detecting serum insulin-like growth factor 1: Mouse serum was collected and detected using the Mouse IGF-1 ELISA Development Kit (PeproTech).

[0267] 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.

[0268] The method for determining the content of short-chain fatty acids in cecal contents is as follows: about 100 mg of cecal content sample is placed in a 2 ml EP tube, 1 ml of ether is added, and vortexed immediately, ultrasonic extraction is performed for 10 min, and the supernatant is separated by centrifugation for 10 min to remove the precipitate. The supernatant is transferred to a gas phase vial through a 0.22 micron organic filter membrane, wrapped with a sealing film and stored in a -20°C refrigerator, and then determined. DB-FFAP column and mass spectrometry detector are used for separation and detection of each short-chain fatty acid.

[0269] 2.2 Experimental results

[0270] See Figure 15 a-15b, the results show that MNH17483 and MNH39288 can significantly increase the food intake of low-fat low-protein diet-induced growth and development retardation mouse models, indicating that MNH17483 and MNH39288 have the purpose of promoting appetite.

[0271] See Figure 16 a-16d, the results show that MNH17483 and MNH39288 can significantly increase the liver, spleen and kidney weights and intestinal length of growth and development retardation mice, indicating that MNH17483 and MNH39288 can promote the development of liver, spleen, kidney and / or intestine, and have the purpose of promoting the development of organs. As a control, there is no significant difference in the liver, spleen and kidney weights of the positive control group and the growth and development retardation mice.

[0272] See Figure 17 a-17f, the results show that MNH17483 and MNH39288 can significantly promote the weight gain, length increase, tibia length increase, femur length increase, soleus muscle and / or gastrocnemius muscle weight increase of low-fat low-protein diet-induced growth and development retardation mouse models, indicating that MNH17483 and MNH39288 have the purpose of promoting the development of height (length), weight, bone and muscle.

[0273] See Figure 18 , the results show that MNH39288 can significantly increase the content of serum insulin-like growth factor 1 (IGF-1) of low-fat low-protein diet-induced growth and development retardation mouse models, indicating that MNH39288 has the purpose of promoting growth and development.

[0274] See Figure 19 a-19d, the results show that MNH17483 and MNH39288 can significantly promote the development of major immune organs such as thymus and spleen of low-fat low-protein diet-induced growth and development retardation mouse models; MNH39288 can increase the proportion of T cells, indicating that MNH17483 and MNH39288 have the function of promoting the development of immune system and enhancing immunity.

[0275] See Figure 20 a-20c, the results showed that MN17483 can promote the content of butyric acid in the cecum of the low-fat low-protein diet-induced growth and development retardation mouse model; MNH39288 can significantly increase the production of isovaleric acid and significantly increase the proportion of 2-methylbutyric acid in total short-chain fatty acids. It shows that MNH17483 and MNH39288 can promote the production of short-chain fatty acids and have the effect of promoting intestinal health.

[0276] While the application has been disclosed with reference to certain implementations, it will be understood by those skilled in the art that various modifications and changes can be made without departing from the spirit and scope of the application as disclosed herein and as provided by the appended claims. Moreover, it should be understood that the disclosure, in its broadest form, is to be taken as illustrative and not in a limiting sense. The application is intended and adapted to encompass all modifications and variations of the implementations described herein that are within the scope of the claims.

Claims

1. A probiotic, prebiotic or pharmaceutical composition comprising Bifidobacterium animalis deposited under GDMCC No: 65555 and / or GDMCC No: 65553.

2. The composition of claim 1, further comprising at least one of the following features: (1) comprising one or more pharmaceuticals, and / or food acceptable excipients; (2) further comprising other active agents for enhancing immunity, preferably, the other active agents comprise one or more of other probiotics, prebiotics, nutritional supplements, dietary supplements; (3) the Bifidobacterium animalis in the composition is live, attenuated, lyophilized or inactivated; (4) the composition is in a dosage form suitable for infants, children or adults; (5) the composition is provided in a liquid form or a solid form, preferably, the composition is 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 micro-encapsulated capsule.

3. Use of Bifidobacterium animalis deposited under GDMCC No: 65555 and / or GDMCC No: 65553 in the manufacture of a probiotic or prebiotic for promoting growth and development, promoting gut health and / or modulating gut microbiota.

4. Use of the composition of claim 1 or 2 in the manufacture of a product for promoting growth and development, promoting gut health and / or modulating gut microbiota.

5. Use of the composition of claim 1 or 2 in the manufacture of a health product for promoting growth and development, promoting gut health and / or modulating gut microbiota.

6. The use of any one of claims 3-5, wherein the promoting growth and development comprises promoting growth and development of an infant or a child, or ameliorating, treating or reversing stunting caused by malnutrition.

7. The use of any one of claims 3-6, wherein the promoting growth and development comprises at least one of: (1) promoting development of at least one of height, weight, bone, muscle, kidney; (2) promoting development of immune system; (3) promoting development of digestive organs; (4) increasing hormone level; (5) increasing appetite to promote growth and development.

8. The use of claim 7, wherein the digestive organs comprise at least one of liver and gut; and / or the bone comprises at least one of femur and tibia; and / or the muscle comprises at least one of soleus and gastrocnemius; and / or the hormone comprises at least one of insulin-like growth factor 1 and growth hormone.

9. The use of claim 7, wherein the promoting development of immune system comprises at least one of promoting development of immune organs and promoting generation of immune cells; preferably, the immune system comprises thymus and / or spleen, and the immune cells comprise T cells. ​ ​ ​ ​ ​ 10. Use according to any one of claims 3-5, wherein said promoting of intestinal health comprises promoting intestinal health by increasing short chain fatty acids; preferably, said short chain fatty acids comprise at least one of propionic acid, butyric acid, isovaleric acid, 2-methylbutyric acid.

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