Isolated bifidobacterium animalis, composition containing same and application

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 microorganisms, and more particularly to isolated animal bifidobacteria, compositions containing the same, and uses thereof. Background Art

[0002] Growth refers to the growth and morphological changes of various organs and systems in the body, which is a quantitative change; development refers to the differentiation and perfection of cells, tissues and organs, the maturation of functions, and the change of production quality. The two are closely related, and growth is the material basis of development. The state of development and maturity is reflected in the change of growth quantity. Human growth and development refers to the maturation process from fertilized egg to adult. Growth and development are important characteristics that distinguish children from adults. Studies have found that intestinal bacteria can affect the growth and development of the host, and some flora can mediate the growth and development process of the host by affecting growth hormone (GH) / insulin-like growth factor-1 (IGF-1).

[0003] Immunity refers to the body's ability to resist foreign invasion and maintain the stability of the internal environment. For children, the development and protection of the immune system is particularly important because it is directly related to the healthy growth of children. Infants or children with developmental delay have insufficient immunity and low resistance due to slow development of immune organs and gastrointestinal tract, and are prone to illness. Air, food, water and other environments are full of various microorganisms: bacteria, viruses, mycoplasma, chlamydia, fungi, etc. When children have insufficient immunity, they are easily infected by harmful bacteria, and then spread to guardians and classmates, and even cause serious influenza or diseases. Although the human body will produce corresponding antibodies to different pathogens to resist reinfection, antibodies are specific and time-limited. For example, streptococcal antibodies can only protect the body from reinvasion by streptococci in a short period of time, and cannot resist infection from other viruses. The bacteria or viruses that induce influenza mutate very quickly, and infants or children with low immunity find it difficult to resist the invasion of cold viruses. This is the real reason why he often catches colds.

[0004] Probiotics are microorganisms that, when given in adequate 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. Therefore, intestinal probiotics are closely related to human health. Probiotic strains have the effects 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 improve the level of IGF-1 in mouse serum, promote the length of mouse femur, and promote the increase of adolescent height. CN114317354B discloses that animal Bifidobacterium MB-424 can promote bone cell growth and promote pituitary GH3 cells to produce growth hormone. In the prior art, the promotion of growth and development for animal Bifidobacterium or Bifidobacterium lactis is mainly focused on promoting the growth of bones and height. However, children with developmental delay have low resistance and are prone to illness, which is related to the slow development of immune organs and gastrointestinal tract. There are few studies on probiotics for promoting 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. Another study showed (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 subspecies 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 screening out new species or strains that can promote the growth and development of internal organs such as immune organs and digestive organs, and further promote growth and development, enhance immunity, and / or improve intestinal health is a huge challenge, but it also represents a huge unmet need. Summary of the invention

[0008] The present invention discloses the isolation of a novel animal Bifidobacterium ( Bifidobacterium animalis). The new strain disclosed in the present invention can 1. promote the development of major immune organs such as the thymus and spleen of developmentally retarded subjects (including increasing the weight of immune organs such as the 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 developmentally retarded subjects; 3. increase the content of serum insulin-like growth factor 1 (IGF-1) in developmentally retarded subjects, promote weight and body length growth, promote bone and muscle growth, such as promoting the growth of tibia length, femur length, soleus muscle and gastrocnemius muscle weight; 4. increase the weight of the liver, thymus, spleen, kidney, and intestinal length (large intestine length and / or small intestine length) of developmentally retarded subjects; increase food intake; increase the content of short-chain fatty acids in the intestine; improve intestinal flora; and promote intestinal development.

[0009] Therefore, the new strain disclosed in the present invention can promote the development of organs while promoting height and weight, especially promoting the development of immune organs, enhancing immunity, promoting intestinal development, improving intestinal health, etc. It can be used to improve developmental delay caused by malnutrition, and achieve the function of improving / treating / reversing developmental delay; achieving the purpose of promoting growth and development, enhancing immunity, or promoting intestinal health.

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

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

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

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

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

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

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

[0017] In a second aspect, the present disclosure provides a culture, live bacteria, freeze-dried bacteria or inactivated bacteria comprising the animal Bifidobacterium described in the first aspect, Wherein, the culture comprises any one of the following A) to D): A) fermentation broth of the animal Bifidobacterium; B) the supernatant of the fermentation broth of the animal Bifidobacterium; C) an inactivated fermentation broth of the animal Bifidobacterium; D) The concentrated or dried product of any one of A) to C) above.

[0018] In a third aspect, the present disclosure provides a composition comprising the animal Bifidobacterium described in the first aspect, or the culture, live bacteria, freeze-dried bacteria or inactivated bacteria of the animal Bifidobacterium described in the second aspect.

[0019] The present disclosure provides a composition comprising the animal Bifidobacterium, a culture thereof, and / or a metabolite thereof according to the first aspect.

[0020] In some embodiments, the culture of Bifidobacterium animalis includes a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0021] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained under anaerobic culture conditions using a liquid culture medium.

[0022] In some embodiments, the composition is provided in liquid form or in solid form.

[0023] In some embodiments, the composition comprises 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×10 12 cfu / mg of the live animal Bifidobacterium.

[0024] In some embodiments, the composition comprises 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 cfu / mg of the live animal Bifidobacterium.

[0025] In some embodiments, the composition comprises 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×10 10 cfu / mg of the live animal Bifidobacterium.

[0026] In some embodiments, the composition comprises 1×10 7 Up to 1×10 9 cfu / mL or 1×10 7Up to 1×10 9 cfu / mg of the live animal Bifidobacterium.

[0027] In some embodiments, each gram of the composition comprises 1×10 3 Up to 1×10 17 colony forming units (CFU); for example, 1×10 4 Up to 1×10 12 pcs, 1×10 5 Up to 1×10 11 or 1×10 6 Up to 1×10 10 Colony forming units (CFU), for example, 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 ,7×10 3 , 8×10 3 ,9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 ,7×10 4 , 8×10 4 ,9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 ,7×10 5 , 8×10 5 ,9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 ,7×10 6 , 8×10 6 ,9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×1013 , 8×10 13 ,9×10 13 or any value in between of colony forming units (CFU).

[0028] In some embodiments, the concentration of the animal Bifidobacterium as an active ingredient in the composition is 10 7 Up to 10 12 CFU / g.

[0029] In some embodiments, the animal Bifidobacterium in the composition is a live bacterium, an attenuated bacterium, a freeze-dried bacterium, or an inactivated bacterium, for example, a heat-inactivated bacterium, preferably a pasteurized bacterium.

[0030] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0031] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid preparation, sustained release preparation, nanoformulation, or microencapsulated capsule.

[0032] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0033] In some embodiments, the composition further comprises one or more pharmaceutically and / or food-acceptable excipients.

[0034] The pharmaceutically and / or food acceptable excipients are well known to those skilled in the art.

[0035] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0036] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0037] In some embodiments, the composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0038] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0039] In some embodiments, the organ comprises at least one of heart, thymus, liver, spleen, kidney, and intestine.

[0040] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0041] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0042] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof; In some embodiments, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus creamer.

[0043] 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, nutritional compounds, biotin, polydextrose, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof.

[0044] In some embodiments, the composition can be formulated as a frozen composition, such as a frozen composition prepared by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0045] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0046] 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 alive. In some embodiments, the strain in the composition is freeze-dried or spray-dried and can partially or completely colonize the intestine. In some embodiments, the strain is reconstituted before administration. In some cases, the reconstitution is carried out by using a diluent as described herein.

[0047] In some embodiments, the composition can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0048] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric preparation. In some embodiments, the enteric preparation is a dosage form with an enteric coating. For example, the enteric preparation 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.

[0049] In some embodiments, the composition is a medicine, or a health product or a food.

[0050] In some embodiments, the composition is in an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0051] In some embodiments, the composition is in a parenteral dosage form.

[0052] In a fourth aspect, the present disclosure provides the use of the animal Bifidobacterium described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect in the preparation of medicines, health products or foods for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0053] The present disclosure provides the use of the animal Bifidobacterium described in the first aspect, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect in promoting growth and development, enhancing immunity, or promoting intestinal health (preferably, the use does not involve the treatment of diseases).

[0054] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0055] In some embodiments, the organ comprises at least one of heart, liver, spleen, kidney, and intestine.

[0056] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0057] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0058] In some embodiments, the medicine, health product or food promotes growth and development by increasing hormone levels and / or increasing appetite.

[0059] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0060] In some embodiments, the hormone is from a body fluid; further from blood; further from serum.

[0061] In some embodiments, the medicine, health product or food enhances immunity by promoting the development of the immune system.

[0062] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0063] In some embodiments, the promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0064] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0065] In some embodiments, the medicine, health product or food promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0066] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0067] The composition comprises an isolated and purified consortium of viable microbial populations to increase body weight by at least 2%, 3%, 4%, 5%, 6%, or 7% in a subject compared to the subject's body weight prior to administration of the isolated and purified consortium of viable microbial species.

[0068] As used herein, a microbial population generally refers to a microbial population consisting essentially of a single strain, species or genus, which may be the case when a population is cultured from a subpopulation of such strain, species or genus that is separated and purified. Thus, for a given microbial population, if cultured from an isolated microbial species or strain, such a population will be referred to herein as purified or substantially pure. The resulting population may typically be at least 80% pure for the microbial species or strain, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure relative to other microbial species or strains within the particular population. In contrast, the level of non-desirable strains in any particular desired microbial population 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 population, such as other undesirable microbial strains or species, may be proportional to or less than 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 the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels for each desired population or below the above-mentioned levels. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case where the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels for each desired population or below the above-mentioned levels.

[0069] The compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by animal bifidobacteria, and the like. Cellular components or cell fractions, metabolites or secreted compounds may be obtained, for example, by recovering the supernatant of a culture of animal bifidobacteria or by extracting from a culture of animal bifidobacteria; may correspond to components in isolated form from animal bifidobacteria, or any mixture of one or more components from animal bifidobacteria.

[0070] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition described in the present disclosure can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0071] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0072] In some embodiments, the organ comprises at least one of heart, thymus, liver, spleen, kidney, and intestine.

[0073] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0074] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0075] 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.

[0076] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0077] In some embodiments, the hormone is from a body fluid; further from blood; further from serum.

[0078] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition enhances immunity by promoting the development of the immune system.

[0079] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0080] In some embodiments, the promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0081] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0082] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0083] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid.

[0084] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can promote appetite, thereby increasing food intake (preferably cumulative food intake), thereby achieving the effect of promoting growth and development.

[0085] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase the weight of the heart, liver, spleen, and / or kidney, and / or intestinal length, thereby promoting organ development.

[0086] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase body weight, body length, tibia length, femur length, soleus muscle and gastrocnemius muscle weight, thereby promoting height (body length), weight, bone, and muscle development.

[0087] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase the content of serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH), thereby promoting growth and development.

[0088] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition disclosed herein can promote the development of major immune organs such as the thymus and spleen, increase the proportion of T cells, B cells, and NK cells, thereby promoting the development of the immune system and enhancing immunity.

[0089] In some embodiments, the animal bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition of the present disclosure can increase the content of short-chain fatty acids and / or improve intestinal flora, thereby promoting intestinal health.

[0090] In some embodiments, the drug or health product or food has at least one effect selected from the following: increasing the weight of the heart, liver, spleen, and / or kidney, and / or the length of the intestine (large intestine length and / or small intestine length); increasing food intake; increasing body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight; promoting weight growth, body length growth, bone and muscle growth (wherein promoting bone growth includes the growth of bone length and density, and promoting muscle growth includes promoting specific muscle development and maintaining healthy and benign growth characteristics, such as: low body fat rate (calf muscle / body weight) and / or high muscle specific gravity (calf muscle / calf weight)); increasing serum The content of insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH); promote the development of major immune organs such as the thymus and spleen (including increasing the weight of immune organs such as the thymus and spleen, the immune organ development index (immune organ weight / body weight), etc.), increase the proportion of T cells, B cells, and NK cells; increase the content of short-chain fatty acids; improve intestinal flora; promote intestinal development; improve lesions or inflammation caused by developmental delay to achieve the function of improving / treating / reversing developmental delay; promote organ development; promote appetite; promote height (body length), weight, bone, and muscle development; promote the development of the immune system and enhance immunity; promote intestinal health; can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0091] A method for promoting growth and development, enhancing immunity, or promoting intestinal health, comprising administering an effective amount of the animal bifidobacterium described in the first aspect of the present disclosure, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria described in the second aspect, or the composition described in the third aspect to a subject in need.

[0092] In some embodiments, the growth promotion includes: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0093] In some embodiments, the organ comprises at least one of heart, liver, spleen, kidney, and intestine.

[0094] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0095] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0096] 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.

[0097] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0098] In some embodiments, the hormone is from a body fluid; further from blood; further from serum.

[0099] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition enhances immunity by promoting the development of the immune system.

[0100] In some embodiments, the promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

[0101] In some embodiments, the promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0102] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (ie, increasing the proportion of T cells, B cells, and / or NK cells).

[0103] In some embodiments, the animal Bifidobacterium, culture, live bacteria, freeze-dried bacteria or inactivated bacteria, or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0104] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, and 2-methylbutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The embodiments will be described below in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0106] Figure 1 : shows the colony morphology photograph of strain MNH17483.

[0107] Figure 2 : Gram staining photographs of strain MNH17483 are shown.

[0108] Figure 3 : shows the electron micrograph of strain MNH17483.

[0109] Figure 4 : shows the colony morphology photograph of strain MNH39288.

[0110] Figure 5 : Gram staining photographs of strain MNH39288 are shown.

[0111] Figure 6 : shows the electron micrograph of strain MNH39288.

[0112] Figure 7 : Shows the results of the tolerance of strain MNH17483 to different pH.

[0113] Figure 8 : Shows the tolerance results of strain MNH17483 to different concentrations of NaCl.

[0114] Fig. 9 : Shows the tolerance results of strain MNH17483 to different concentrations of bile salts.

[0115] Fig.10 : Shows the results of the tolerance of strain MNH39288 to different pH.

[0116] Fig.11 : Shows the tolerance results of strain MNH39288 to different concentrations of NaCl.

[0117] Fig.12 : Shows the tolerance results of strain MNH39288 to different concentrations of bile salts.

[0118] Fig.13 : The phylogenetic tree of strain MNH17483 is shown.

[0119] Fig.14 : The phylogenetic tree of strain MNH39288 is shown.

[0120] Fig.15 :Shows that strains MNH17483 and MNH39288 increased cumulative food intake (cumulative food intake line graph). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's ttest; *, p < 0.05 compared with the LFD-Control group.

[0121] Fig.16 :Shows that strains MNH17483 and MNH39288 increased cumulative food intake (cumulative food intake bar graph). Data are presented as mean ± SD. Statistical analysis was performed using the Student's ttest; *, p < 0.05 compared with the LFD-Control group.

[0122] Fig.17:Shows that strains MNH17483 and MNH39288 can promote organ development (liver weight). Data are shown 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.

[0123] Fig.18 :Shows that strains MNH17483 and MNH39288 can promote organ development (spleen weight). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0124] Fig.19 :Shows that strains MNH17483 and MNH39288 can promote organ development (kidney weight). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; **, p < 0.01 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0125] Fig. 20 :Shows that strains MNH17483 and MNH39288 can promote organ development (intestinal length). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; **, p < 0.01 compared with the LFD-Control group.

[0126] Fig.21 :Shows that strains MNH17483 and MNH39288 promote body weight development (weight gain). Data are shown 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] Fig. 22:Shows that strains MNH17483 and MNH39288 promote the development of height (body length) (body length increment). Data are shown as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; ****, p < 0.0001 compared with the LFD-Control group.

[0128] Fig.23 :Shows that strains MNH17483 and MNH39288 promote bone development (femur 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 < 0.001 compared with the LFD-Control group.

[0129] Fig.24 :Shows that strains MNH17483 and MNH39288 promote bone development (tibia length). Data are shown 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.

[0130] Fig.25 :Shows that strains MNH17483 and MNH39288 promote muscle development (soleus 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.

[0131] Fig.26 :Shows that strains MNH17483 and MNH39288 promote muscle development (gastrocnemius weight). Data are shown 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 < 0.001 compared with the LFD-Control group.

[0132] Fig. 27:Shows that strain MNH39288 increases serum insulin-like growth factor 1 (IGF-1) content. Data are shown 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 < 0.0001 compared with the LFD-Control group.

[0133] Fig.28 :Shows that strains MNH17483 and MNH39288 promote immune system development and enhance immunity (thymus 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.

[0134] Fig.29 :Shows that strains MNH17483 and MNH39288 promote the development of the immune system and enhance immunity (spleen weight). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0135] Fig.30 :Shows that strains MNH17483 and MNH39288 promote the development of the immune system and enhance immunity (immune organ weight). Data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's ttest; *, p < 0.05 compared with the LFD-Control group.

[0136] Fig.31 :Shows that strain MNH39288 promotes immune system development and enhances immunity (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.

[0137] Fig.32 :Shows that strain MNH17483 promotes the production of short-chain fatty acids (butyrate content) in cecal contents. 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.

[0138] Fig.33 :Shows that strain MNH39288 promotes the production of short-chain fatty acids (isovaleric acid content) in cecal contents. Data are shown 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 < 0.001 compared with the LFD-Control group.

[0139] Fig.34 :Shows that strain MNH39288 promotes the production of short-chain fatty acids in cecal contents (2-methylbutyrate ratio). 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.

[0140] Deposit of strains

[0141] strain Bifidobacterium animalis ( Bifidobacterium animalis )MNH17483, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65553, deposited on November 28, 2024, at the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit name Bifidobacterium animalis MNH17483, taxonomic name: Bifidobacterium animalis .

[0142] strain Bifidobacterium animalis ( Bifidobacterium animalis ) MNH39288, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65555, the deposit date is November 28, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, the deposit name is Bifidobacterium animalis MNH39288, taxonomic name: Bifidobacterium animalis . DETAILED DESCRIPTION

[0143] The present invention separates two new strains of Bifidobacterium animalis, with the deposit numbers 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 are new strains of Bifidobacterium animalis. Furthermore, the present invention studies the biochemical properties and therapeutic uses of the two strains.

[0144] It is known in the art that bacterial species can be classified and identified by traditional classification methods and molecular biological methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biological methods include, but are not limited to, ribosomal RNA sequence determination methods, determination methods based on whole genome sequencing, etc.

[0145] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0146] The terms "subject," "subject," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including: humans, mammals, laboratory animals, livestock, and domestic pets.

[0147] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical foods. In some cases, the composition is applied in the form of a pharmaceutical preparation. In some cases, the composition is applied in the form of a nutritional supplement. In some cases, the composition is applied in the form of a dietary supplement. In some cases, the composition is applied in the form of a medical food. In some cases, the composition is applied in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplements, nutritional supplements, medical probiotics or medical foods) can be orally administered, for example as a capsule, pill or tablet.

[0148] 16S rRNA is a ribosomal RNA of prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is common to all bacteria, while the variable region has different degrees of difference between different bacteria. By comparing the 16S rRNA gene sequences of bacteria, according to the base number of sequence differences and their evolutionary distance, an evolutionary tree can be drawn. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence 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).

[0149] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG program package, BLASTN or FASTA. Commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

[0150] The second generation sequencing technology can also be used to identify bacterial species based on whole genome sequencing, making the identification results more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated by methods such as BLAST. 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 species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9 (1): 5114.).

[0151] Various existing mature ANI value calculation tools can be used, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0152] By using the above method, a person skilled in the art can determine whether an isolated strain belongs to the animal Bifidobacterium species identified by the inventors. For example, when compared with the animal Bifidobacterium with the deposit number GDMCC No: 65553 or GDMCC No: 65555 ( Bifidobacterium animalis ) has an average nucleotide identity ANI value of at least 95%, for example at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.1%, 99.2%, 99.3%, 9 ... .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that they belong to the same species.

[0153] For another example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO: 3 or 4, for example, at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity, it can be determined to belong to the same bacterial species.

[0154] A "strain" refers to a member of a bacterial species that has a genetic characteristic that allows it to be distinguished from closely related members of the same bacterial species. The genetic characteristic can be the total or partial absence of at least one gene, the total or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosomal binding site), the absence ("cure" of at least one native plasmid), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosomal binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case of a strain that gains or loses antibiotic resistance (compared to another strain of the same species) or gains or loses a biosynthetic ability (e.g., an auxotrophic strain), the strains can be distinguished by selection or counter-selection using antibiotics or nutrients / metabolites.

[0155] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0156] Compositions can be prepared using the animal bifidobacterium described herein, for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the animal bifidobacterium, for example, the animal bifidobacterium with a deposit number of GDMCC No: 65553 or GDMCC No: 65555. Similarly, the animal bifidobacterium with a deposit number of GDMCC No: 65553 or GDMCC No: 65555 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, which comprises a pharmaceutically effective amount of the animal bifidobacterium.

[0157] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0158] The composition herein can be formulated into any form suitable for enhancing the abundance of animal bifidobacteria in a subject. The composition can be administered orally (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, intragastrointestinal, bile infusion, cardiac infusion, anterior anus, rectum, spinal subcutaneous, sublingual, topical, intravaginal, transdermal, ureteral or urethral.

[0159] Examples of dosage forms suitable for the composition herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0160] In some embodiments, the composition is a sugar-coated tablet, a gel capsule, a gel, an emulsion, a tablet, a sheet capsule, a hydrogel, a nanofiber gel, an electrospun fiber, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder or a toothpaste, etc.

[0161] In some embodiments, administration can also be by inclusion in a subject's diet, such as inclusion in a functional food for humans or companion animals.

[0162] The compositions provided herein may include a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier is well known in the art.

[0163] In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is spray-dried. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried or spray-dried and is alive. In some embodiments, the animal bifidobacterium in the composition of the present disclosure is freeze-dried or spray-dried and can be partially or completely colonized in the intestine. In some embodiments, the freeze-dried animal bifidobacterium is reconstituted before administration. In some embodiments, the reconstitution is carried out using a diluent as described herein.

[0164] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0165] In some embodiments, the composition is prepared by freeze drying or spray drying.

[0166] The composition disclosed herein includes medicine, health care product or food.

[0167] The subject of the present disclosure may be a human or an animal, and the animal includes but is not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0168] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature of the art or in accordance with the product or instrument instructions are used. All reagents or instruments are indicated by the manufacturer and can be purchased commercially.

[0169] Example

[0170] The anaerobic blood agar plate involved in the embodiment was purchased from Huankai Microorganisms, with a formula of 10 g / L casein pancreatic digest, 3 g / L heart pancreatic digest, 1 g / L corn starch, 5 g / L meat gastric digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50 mL / L sterile defibrinated sheep blood, pH 7.3±0.2.

[0171] The PYG liquid culture medium involved in the embodiment, each 1 L PYG liquid culture medium contains: Trypticase peptone (trypticase peptone) 5.0 g, Peptone (peptone) 5.0 g, Yeast extract (yeast extract powder) 10.0 g, Beef extract (beef extract powder) 5.0 g, Glucose (glucose) 5.0 g, K2HPO4 2.0 g, Tween 80 (Tween 80) 1.0 ml, Resazurin (resazurin) 1.0 mg, Salt solution (salt solution) 40 ml (its components are as follows: CaCl2·2H2O0.01 g, MgSO4·7H2O 0.02 g, K2HPO4 0.04 g, NaHCO3 0.4 g, NaCl 0.08 g, balance water), balance water, pH 7.2±0.2 (25°C).

[0172] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0173] Example 1. Isolation and identification of strains

[0174] 1.1 Isolation and purification of strains MNH17483 and MNH39288 Two intestinal strains of Bifidobacterium animalis ( Bifidobacterium animalis MNH17483 and Bifidobacterium animalisMNH39288) were isolated from fecal samples of healthy volunteers. The isolation method adopted the conventional strain isolation method, using gradient dilution, and then picking a single colony for isolation and culture, and then purifying the strain and anaerobic culture at 37°C. The pure culture strain was prepared into 20% glycerol / water-bacteria solution and stored at -80°C.

[0175] Specifically, the separation and purification methods of the strains are as follows: The donor takes 2-5 g of fresh feces and puts it into a sample collection and storage tube. After shaking and homogenization, the processed feces sample is placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0176] Dispense physiological saline in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation culture medium, and transfer them to the anaerobic workstation 24 hours in advance, marking the sample information, culture medium type, isolation date, etc.

[0177] Take a fresh stool sample and place it in an anaerobic workstation and shake it on a vortex shaker for 1 min to mix it. Pipette 1 mL of the sample into 9 mL of normal saline and mix it for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution for later use.

[0178] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a drop size of 100 μL / plate. Spread evenly. After the surface of the plate is dry, invert the plate and culture at 37°C for 3-5 days.

[0179] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterilized toothpick for strain purification. The purified strain is placed at 37°C for anaerobically cultured. The pure culture strain is prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0180] 1.2 Morphological characteristics of strains MNH17483 and MNH39288 1.2.1 Morphological characteristics of strain MNH17483 The strain MNH17483 was inoculated on a PYG plate medium and cultured anaerobically at 37°C for 48 hours. Visible colonies were formed on the PYG plate medium. The colonies were round, with regular and smooth edges, about 0.5-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic morphological observation revealed that it had no flagella, was non-motile, rod-shaped, and about 0.5-1µm×1.5-3 µm in size. The colony morphology of the strain MNH 17483 cultured on a PYG plate for 48 hours is shown in Figure 1 The Gram staining photos of strain MNH17483 are shown in Figure 2 See the electron microscope photos for Figure 3 .

[0181] 1.2.2 Morphological characteristics of strain MNH39288 The strain MNH39288 was inoculated on a PYG plate medium and cultured anaerobically at 37°C for 48 hours. Visible colonies were formed on the PYG plate medium. The colonies were round, with regular and smooth edges, about 0.5-2 mm in diameter, white, and opaque. The strain was Gram-positive. Microscopic morphological observation revealed that it had no flagella, was non-motile, rod-shaped, and about 0.5-1µm×1.5-3 µm in size. The colony morphology of the strain MNHMNH39288 cultured on a PYG plate for 48 hours is shown in the figure. Figure 4 The Gram staining photos of strain MNH39288 are shown in Figure 5 See the electron microscope photos for Figure 6 .

[0182] 1.3 Physiological and biochemical characteristics of strains MNH17483 and MNH39288 1.3.1 Physiological and biochemical characteristics of strain MNH17483 The strain MNH17483 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 5.0 to 10.0, with the optimal growth pH being 7.0-9.0 (see the results of strain tolerance to different pH values ​​for details). Figure 7 ); it does not grow in a medium with a NaCl content of more than 3% (the results of the strain's tolerance to different concentrations of NaCl are shown in Figure 8 ); strain MNH17483 can survive and grow in the bile salt concentration range of 0%-0.15%, and cannot grow when the bile salt concentration is greater than or equal to 0.2% (the strain's tolerance to different concentrations of bile salts is shown in Fig. 9 ).

[0183] 1.3.2 Physiological and biochemical characteristics of strain MNH39288 The strain MNH39288 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 6.0 to 9.0, with the optimal growth pH being 7.0 (see the results of strain tolerance to different pH values ​​for details). Fig.10 ); can still grow in medium with NaCl content exceeding 6% (the results of strain tolerance to different concentrations of NaCl are shown in Fig.11 ); strain MNH39288 can survive and grow in the bile salt concentration range of 0%-0.1%, and cannot grow when the bile salt concentration is greater than or equal to 0.15% (the strain's tolerance to different concentrations of bile salts is shown in Fig.12 ).

[0184] 1.4 Biochemical identification results of strains MNH17483 and MNH39288 1.4.1 Results of biochemical identification of strain MNH17483 using API 50CHL API 50CHL (Mérieux, CN5041010) was used. For specific experimental operations, please refer to the conventional API reagent operation guide. Culture conditions: 37°C, anaerobic. The experimental results are shown in Table 1.

[0185] MNH17483 can utilize L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, α-methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, and D-turanose TUR to ferment and produce acid. Therefore, in the process of fermentation or cultivation of strain MNH17483, L-arabinose LARA, ribose RIB, D-xylose DXYL, glucose GLU, α-methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, and their derivatives can be used as carbon sources.

[0186]

[0187] 1.4.2 Results of biochemical identification of strain MNH39288 by API 50CHL API 50CHL (Mérieux, CN5041010) was used. For specific experimental operations, please refer to the conventional API reagent operation guide. Culture conditions: 37°C, anaerobic. The experimental results are shown in Table 2.

[0188] MNH39288 can utilize L-arabinose LARA, ribose RIB, D-xylose DXYL, Galactose GAL, Glucose GLU, Fructose FRU, α-methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR are fermented to produce acid. Therefore, during the fermentation or cultivation of strain MNH39288, L-arabinose LARA, ribose RIB, D-xylose DXYL, Galactose GAL, Glucose GLU, Fructose FRU,α-Methyl-D-glucoside MDG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, raffinose RAF, starch, gentiobiose GEN, D-turanose TUR, and their derivatives can be used as carbon sources.

[0189]

[0190] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strains MNH17483 and MNH39288 1.5.1 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH17483 The minimum inhibitory concentration of antibiotics for strain MNH17483 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 3.

[0191]

[0192] The results showed that MNH17483 was 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 antibiotic resistance in subjects is low.

[0193] 1.5.2 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH39288 The minimum inhibitory concentration of antibiotics for strain MNH39288 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 4.

[0194]

[0195] The results showed that MNH39288 was 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 antibiotic resistance in subjects is low.

[0196] 1.6 Identification of strains MNH17483 and MNH39288 1.6.1 Amplification of 16S rRNA genes of strains MNH17483 and MNH39288 Fresh cultures of strains MNH17483 and MNH39288 were taken to extract genomic DNA of the strains, and the extracted genomic DNA of the strains was used as a template for 16S rRNA gene amplification.

[0197] The primer pairs used for PCR of 16S rRNA gene are: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1); 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0198] The PCR reaction procedure is as follows: Pre-denaturation: 94℃, 4 min; (denaturation: 94℃, 50 sec, annealing: 52℃, 40 sec; extension: 72℃, 70sec), 36 cycles; final extension: 72℃, 10 min.

[0199] 1.6.2 16S rRNA gene sequencing of strains MNH17483 and MNH39288 The PCR product was purified and sequenced by Bioengineering to obtain the 16S rRNA gene.

[0200]

[0201] 1.6.3 Identification results of strains MNH17483 and MNH39288 The 16S rRNA gene sequences measured above, such as those shown in SEQ ID NO: 3 and SEQ ID NO: 4, were used to perform strain 16S rRNA gene analysis using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0202] The measured sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH17483 was Bifidobacterium animalis subsp. lactis , the similarity is 100%, and the whole genome ANI analysis shows Bifidobacterium animalis (GCF_000260715.1) has a similarity of 95.88% and an alignment score (AF) of 92%, so strain MNH17483 is Bifidobacterium animalis subsp. lactis strains planted; the strain with the highest similarity to MNH39288 is Bifidobacterium animalis subsp. lactis , the similarity is 100%, and the whole genome ANI analysis shows Bifidobacterium animalis (GCF_000260715.1) has a similarity of 95.83% and an alignment score (AF) of 92%, so strain MNH39288 is Bifidobacterium animalis subsp. lactis Planted strains.

[0203] MNH17483 and MNH39288 were compared with the Bifidobacterium animalis The 16S rRNA gene sequences of related strains of the genus were compared, and phylogenetic trees were constructed respectively.

[0204] The sequences of MNH17483 and MNH39288 were aligned with those of the model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method). Fig.13 , 14 Only the Bootstrap values ​​greater than 50% are displayed in the developmental tree nodes.

[0205] From the phylogenetic tree ( Fig.13 , 14 ) It can be seen that strains MNH17483 and MNH39288 Bifidobacterium animalis Get together for Bifidobacterium animalis New plants planted.

[0206] 1.7 Genome analysis of strains MNH17483 and MNH39288 1.7.1 Genome analysis of strain MNH17483 The genome of strain MNH17483 was fragmented by ultrasound, with a fragment length range of ~350bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. The sequencing obtained 1.11Gbp of data, of which Q20 accounted for: 96.25%.

[0207] The raw sequencing data of the genome were 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 were assembled using SPAdes (version: v3.14.0) with assembly parameters "--isolate --cov-cutoff 10". The total gene length of the genome assembly was 1.92 Mbp, the N50 length was 182.2 kbp, and the GC content was 60.49%.

[0208] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version: 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 1554 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0209] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), where the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 5.

[0210]

[0211] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed comparison results are shown in Table 6.

[0212]

[0213] The analysis of potential secondary metabolism gene clusters in the genome was performed using antiSMASH6 (version: 6.0.1). No secondary metabolism gene clusters were found.

[0214] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 7.

[0215]

[0216] 1.7.2 Genome analysis of strain MNH39288 The genome of strain MNH39288 was fragmented by ultrasound, with a fragment length range of ~350bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. 1.33Gbp of data was obtained by sequencing, of which Q20 accounted for 96.34%.

[0217] The raw sequencing data of the genome were 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 were assembled using SPAdes (version: v3.14.0) with assembly parameters "--isolate --cov-cutoff 10". The total gene length of the genome assembly was 1.92 Mbp, the N50 length was 182.4 kbp, and the GC content was 60.49%.

[0218] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version: 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 1556 CDS sequences were predicted, with an average CDS sequence length of 1077 bp.

[0219] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), where the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 8.

[0220]

[0221] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare the virulence factor database VFDB (virulence factor database, 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.

[0222]

[0223] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 10.

[0224]

[0225] Example 2: Strains MNH17483 and MNH39288 promote growth and development in a low-fat, low-protein diet-induced growth retardation mouse model The growth retardation mouse model induced by low-fat and low-protein diet was used to conduct experiments on the improvement of growth and development indicators by MNH17483 and MNH39288. This invention has passed the ethical review of the Animal Ethics Committee of Muen Biological.

[0226] 2.1 Experimental methods 1) Experimental animals: The experimental mice were C57BL / 6J mice, 3 weeks old, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0227] 2) Preparation of test materials of strains MNH17483 and MNH39288: After thawing the glycerol cryovials of MNH17483 and MNH39288 strains at 37°C, they were inoculated into PYG plate medium in an anaerobic workstation for activation. The activated strains were inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0228] 3) Negative control: Use PBS containing 25% glycerol and 0.05% L-Cys HCl as the negative control.

[0229] 4) Positive control: Bifidobacterium lactis CGMCC No.20847. After thawing the glycerol cryopreservation tube of Bifidobacterium lactis CGMCC No.20847 (disclosed in patent CN112980725B) at 37°C, inoculate it into PYG medium in an anaerobic workstation for activation. Inoculate the activated strain into PYG liquid medium and culture it anaerobically to obtain a sufficient amount of culture. After centrifuging and concentrating the cultured bacterial solution, resuspend the bacterial strain in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0230] 5) Experimental process: After the quarantine period, 40 C57BL / 6J male mice weighing 13g-16g were selected for 3-week-old mice. They were randomly divided into 5 groups according to their weight, 8 mice / group, namely NCD-Control group, LFD-Control group, MNH17483 group, MNH39288 group, and positive control group. The NCD-Control group was fed with maintenance feed (Cat. No.: PD24043001, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), and the other four groups were fed with low-fat and low-protein feed (Cat. No.: PD24043003, Changzhou Shuyi Shuer Biotechnology Co., Ltd.). After grouping (D1), medication began. The NCD-Control group and the LFD-Control group were given negative control products. The positive control group was gavaged with Bifidobacterium lactis CGMCC No.20847, the MNH17483 group was gavaged with MNH17483, and the MNH39288 group was gavaged with MNH39288. The medication was given once a day, 200 μL each time, for a total of 35 days. During the experiment, mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the experiment, a general clinical observation was performed after each medication. The endpoint of this experiment was the day after the end of medication (D36). The end point of the experiment was anatomical sampling according to the protocol, and the results of each anatomical data and serum test data were summarized and analyzed. All data were expressed in the form of Mean±SD, and GraphPadPrism software was used for drawing and statistical analysis. For pairwise comparisons, the t test (Student's t test) analysis method was used. No significance is indicated; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0231] Food intake was determined by weighing the remaining amount of feed.

[0232] The weight of internal organs such as the liver and spleen, the length of the large intestine, and the length and weight of various bones and muscles are measured through dissection.

[0233] Method for detecting serum insulin-like growth factor-1: Mouse serum was collected and detected using Mouse IGF-1 ELISA Development Kit (PeproTech).

[0234] The immune cell detection method is to take 0.1g of mouse spleen tissue, wash the surface with PBS and then crush the tissue, suspend the crushed spleen tissue in PBS, filter through a 70μm filter membrane, and prepare a mouse spleen single cell suspension. Take a small amount of the mouse spleen single cell suspension prepared above, stain it, and complete the machine detection in the flow cytometer to accurately count the number of mouse spleen immune cells.

[0235] The method for determining the content of short-chain fatty acids in cecal contents is as follows: weigh about 100 mg of cecal contents and place it in a 2 mL EP tube, add 1 ml of ether, vortex mix immediately, extract short-chain fatty acids by ultrasonication for 10 min, centrifuge for 10 min to remove precipitation, pass the supernatant through a 0.22 μm organic filter membrane, transfer the supernatant to a gas phase vial, wrap it with a sealing film and store it in a -20 ° C refrigerator, waiting for determination. Use a DB-FFAP column and a mass spectrometer to separate and detect each short-chain fatty acid.

[0236] 2.2 Experimental Results See also Figure 15-16 The results showed that MNH17483 and MNH39288 could significantly increase the food intake of the growth retardation mouse model induced by a low-fat and low-protein diet, indicating that MNH17483 and MNH39288 have the function of promoting appetite.

[0237] See also Figure 17-20 The results showed that MNH17483 and MNH39288 could significantly increase the weight of liver, spleen and kidney and the length of intestine in developmentally retarded mice, indicating that MNH17483 and MNH39288 can promote the development of liver, spleen, kidney and / or intestine, and have the use of promoting organ development. As a control, there was no significant difference in the weight of liver, spleen and kidney between the positive control group and the developmentally retarded mice.

[0238] See also Figure 21-26 The results showed that MNH17483 and MNH39288 could significantly promote the increase in body weight, body length, tibia length, femur length, and soleus and / or gastrocnemius muscle weight in the growth retardation mouse model induced by a low-fat and low-protein diet, indicating that MNH17483 and MNH39288 have the use of promoting height (body length), weight, bone, and muscle development.

[0239] See also Fig. 27The results showed that MNH39288 could significantly increase the serum insulin-like growth factor 1 (IGF-1) content in the growth retardation mouse model induced by a low-fat and low-protein diet, indicating that MNH39288 has the use of promoting growth and development.

[0240] See also Figure 28-31 The results showed that MNH17483 and MNH39288 can significantly promote the development of major immune organs such as the thymus and spleen in the low-fat and low-protein diet-induced growth retardation mouse model; MNH39288 can increase the proportion of T cells, indicating that MNH17483 and MNH39288 have the function of promoting the development of the immune system and enhancing immunity.

[0241] See also Figure 32-34 The results showed that MN17483 could significantly promote the content of butyric acid in the cecum of a mouse model of growth retardation induced by a low-fat and low-protein diet; MNH39288 could significantly increase the production of isovaleric acid and the proportion of 2-methylbutyric acid in total short-chain fatty acids. This indicates that MNH17483 and MNH39288 can promote the production of short-chain fatty acids and have the effect of promoting intestinal health.

[0242] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations may be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples disclosed illustrate embodiments of the present invention, they are provided only as non-limiting examples and therefore should not be considered as limiting the various aspects of the present invention thus described. The present invention is intended to have the full scope defined by the language of the present disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered illustrative rather than restrictive.

Claims

1. An isolated animal Bifidobacterium ( Bifidobacterium animalis ), which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 65553 or GDMCC No: 65555.

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

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

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

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

6. The composition according to any one of claims 3 to 5, which is any one of a medicine, a health product or a food.

7. The composition according to claim 6, wherein the health product or food is any one of a nutritional composition, a food, a candy, a food bar, a food additive, a drink additive, and a dietary supplement.

8. Use of the animal bifidobacterium according to claim 1, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria according to claim 2, or the composition according to any one of claims 3 to 7 in promoting growth and development, enhancing immunity, or promoting intestinal health.

9. Use of the animal bifidobacterium according to claim 1, the culture, live bacteria, freeze-dried bacteria or inactivated bacteria according to claim 2, or the composition according to any one of claims 3 to 7 in the preparation of medicines, health products or foods for promoting growth and development, enhancing immunity, or promoting intestinal health.

10. According to the use according to any one of claims 8-9, the growth and development promotion includes: promoting at least one of the development of organs, height, weight, bones, and muscles.

11. The use according to claim 10, wherein the organ comprises at least one of liver, thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle includes at least one of the soleus muscle and the gastrocnemius muscle.

12. The use according to any one of claims 8 to 9, wherein the medicine, health product or food promotes growth and development by increasing hormone content and / or increasing appetite; or The medicine, health product or food enhances immunity by promoting the development of the immune system; or The medicine, health product or food promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

13. The use according to claim 12, wherein the hormone comprises insulin-like growth factor 1 and / or growth hormone; or The promoting the development of the immune system comprises at least one of promoting the development of immune organs and promoting the generation of immune cells.

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