Isolated lactobacilliaceae strains, compositions containing the same and uses thereof

Abstract

The present disclosure provides isolated Lactobacillus strains, compositions comprising the same, and uses thereof, which can be used to promote growth and development, enhance immunity, or promote intestinal health, etc.

Description

Technical Field

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

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

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

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

[0005] Lactobacillus crispatus and Lactobacillus paracasei are common Lactobacillus probiotics, and their safety has been widely verified. Research on Lactobacillus crispatus for promoting animal growth and development has primarily focused on improving the production performance of poultry and pigs, including increasing average daily gain, average daily feed intake, and feed utilization, and improving the absorption and utilization of calcium and phosphorus in feed. For example, CN114891657B discloses that Lactobacillus crispatus DC529 significantly improved the average daily gain and average daily feed intake of broiler chickens. CN108546663B discloses that Lactobacillus crispatus ZLC020 increased pig weight by 5.52% (P < 0.05), average daily gain by 7.32%, and reduced feed-to-weight ratio by 5.68 (P < 0.05) compared to the control group, demonstrating that porcine Lactobacillus crispatus has a positive effect on improving the production performance of growing pigs. However, there are few reports that Lactobacillus crispatus can promote the growth and development of mice or infants.

[0006] For Lactobacillus paracasei, CN116875515A discloses Lactobacillus paracasei GF027, which promotes rat height, bone weight and bone density growth, and increases the insulin-like growth factor 1 in serum. Prior art mainly focuses on promoting the growth of bone and height for Lactobacillus paracasei. However, developmentally retarded children have low resistance and are prone to illness, which is relevant to the slow development of immune organs and gastrointestinal tract. There is little research on the probiotics that promote the growth and development of internal organs such as immune organs and digestive organs.

[0007] 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 disclosure discloses the isolation of a new strain of the family Lactobacillaceae. The new strain disclosed herein can 1. promote the development of major immune organs such as the thymus and spleen in developmentally delayed subjects (including increasing the weight of immune organs such as the thymus and spleen, and the immune organ development index (immune organ weight / body weight), etc.); 2. promote the development and proliferation of immune cells such as T cells, thereby boosting the immunity of developmentally delayed subjects; 3. increase serum insulin-like growth factor 1 (IGF-1) levels in developmentally delayed subjects, promoting weight and length growth, and promoting bone and muscle growth, such as increasing tibia length, femur length, and soleus and gastrocnemius muscle weight; 4. increase the weight of the thymus, spleen, and kidneys, and intestinal length (large intestine length and / or small intestine length) in developmentally delayed subjects; increase the content of short-chain fatty acids in the intestine; improve intestinal flora; and promote intestinal development.

[0009] Therefore, the new strain disclosed herein can promote growth and weight while also promoting organ development, particularly immune organ development, enhancing immunity, promoting intestinal development, and improving intestinal health. It can be used to improve developmental delays caused by malnutrition or poor digestion and absorption in children, thereby improving, treating, or reversing developmental delays; achieving goals such as promoting growth and development, enhancing immunity, or promoting intestinal health.

[0010] In a first aspect, the present disclosure provides an isolated Lactobacillaceae strain having an average nucleotide identity (ANI) of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% to MNH06277 deposited with GDMCC No: 65552; ​​and / or having a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO: 3; or

[0011] The average nucleotide identity (ANI) value with MNH45330 deposited with GDMCC No: 65556 is 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%; and / or, the 16S rRNA sequence has at least 98.65% identity to the sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the Lactobacillaceae strain 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 set forth in SEQ ID NO:3, or SEQ ID NO:4.

[0013] In some embodiments, the Lactobacillaceae strains are two novel strains of the Lactobacillaceae family.

[0014] In some embodiments, the name of the Lactobacillaceae strain is: Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65552, 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 Lacticaseibacillus paracasei MNH06277.

[0015] In some embodiments, the name of the Lactobacillaceae strain is: Lactobacillus crispatus ( Lactobacillus crispatus ) MNH45330, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65556, 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 Lactobacillus crispatus MNH45330.

[0016] In a second aspect, the present disclosure provides a composition comprising the Lactobacillaceae strain, a culture thereof, and / or a metabolite thereof according to the first aspect.

[0017] The present disclosure provides a composition for promoting growth and development, which comprises Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277 and / or Lactobacillus crispatus ( Lactobacillus crispatus ) culture, live bacteria, freeze-dried bacteria or inactivated bacteria of MNH45330, wherein the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The deposit number of MNH06277 is GDMCC No: 65552; ​​the Lactobacillus crispatus ( Lactobacillus crispatus ) The deposit number of MNH45330 is GDMCC No: 65556.

[0018] In some embodiments, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277 and / or Lactobacillus crispatus ( Lactobacillus crispatus ) The culture of MNH45330 includes any of the following A) to D):

[0019] A) the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277 and / or Lactobacillus crispatus ( Lactobacillus crispatus ) fermentation broth of MNH45330;

[0020] B) the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277 and / or Lactobacillus crispatus ( Lactobacillus crispatus ) fermentation supernatant of MNH45330;

[0021] C) the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277 and / or Lactobacillus crispatus ( Lactobacillus crispatus ) Inactivated fermentation broth of MNH45330;

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

[0023] In some embodiments, the culture of the Lactobacillaceae strain includes a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0024] 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.

[0025] In some embodiments, the composition is provided in liquid form or solid form.

[0026] In some embodiments, the composition comprises 1×10 4 to 1×10 12 cfu / mL or 1×10 4 to 1×10 12 cfu / mg of live bacteria of the Lactobacillus family strain.

[0027] In some embodiments, the composition comprises 1×10 5 to 1×10 11 cfu / mL or 1×10 5 to 1×10 11 cfu / mg of live bacteria of the Lactobacillus family strain.

[0028] In some embodiments, the composition comprises 1×10 6 to 1×10 10 cfu / mL or 1×10 6 to 1×10 10 cfu / mg of live bacteria of the Lactobacillus family strain.

[0029] In some embodiments, the composition comprises 1×10 7 to 1×10 9 cfu / mL or 1×10 7 to 1×10 9 cfu / mg of live bacteria of the Lactobacillus family strain.

[0030] In some embodiments, each gram of the composition comprises 1×10 3 to 1×10 17 colony-forming units (CFU) of bacteria; for example, 1×10 4 to 1×10 12 1×10 5 to 1×10 11 or 1×10 6 to 1×10 10colony forming units (CFU), specifically, for example, 1 × 10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 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 , 1×109 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 or any value in between of colony forming units (CFU).

[0031] In some embodiments, the concentration of the Lactobacillusaceae strain as an active ingredient in the composition is 10 7 to 10 12 CFU / g.

[0032] In some embodiments, the Lactobacillus strain 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.

[0033] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0034] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0035] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0036] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients.

[0037] The pharmaceutically acceptable excipients are well known to those skilled in the art.

[0038] 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.

[0039] In some embodiments, the composition comprises one or more of a buffering agent (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0040] In some embodiments, the composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0041] In some embodiments, the growth promotion comprises: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0042] In some embodiments, the organ comprises at least one of the heart, liver, spleen, kidney, and intestine.

[0043] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0044] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0045] In some embodiments, the other active agent may be one or more of probiotics, prebiotics, or a combination thereof.

[0046] In some embodiments, the probiotic is selected from at least one of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus krumelans, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentans, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus cremoris.

[0047] In some embodiments, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast beta-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0048] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0049] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0050] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0051] 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.

[0052] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form having an enteric coating. For example, the enteric-coated formulation can be an enteric granule, an enteric-coated tablet, or an enteric-coated capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained-release capsule, a controlled-release capsule, or an enteric-coated capsule, or the capsule can be a microencapsulated capsule or a microcapsule.

[0053] In some embodiments, the composition is a medicine, or a health product for enhancing immunity.

[0054] In some embodiments, the composition is in an infant-appropriate dosage form, a child-appropriate dosage form, or an adult-appropriate dosage form.

[0055] In some embodiments, the composition is in a parenteral or parenteral dosage form.

[0056] In a third aspect, the present disclosure provides the use of the Lactobacillus strain described in the first aspect or the composition described in the second aspect in the preparation of a medicine for promoting growth and development, enhancing immunity, or promoting intestinal health, or a health product for enhancing immunity.

[0057] Use of the Lactobacillaceae strain of the first aspect (the strain is Lacticaseibacillus paracasei MNH06277) or the composition of the second aspect (the strain is Lacticaseibacillus paracasei MNH06277) in the preparation of a health product for enhancing immunity.

[0058] The present disclosure provides use of the composition of the second aspect (the strain is Lacticaseibacillus paracasei MNH06277) in enhancing immunity.

[0059] The present disclosure provides use of the composition described in the second aspect in preparing a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0060] The present disclosure provides use of the composition of the second aspect (the strain is Lacticaseibacillus paracasei MNH06277) in preparing a health product for enhancing immunity.

[0061] In some embodiments, the growth promotion comprises: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0062] In some embodiments, the organ comprises at least one of the heart, liver, spleen, kidney, and intestine.

[0063] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0064] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0065] In some embodiments, the drug promotes growth and development by increasing hormone levels and / or promoting digestion and absorption.

[0066] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0067] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0068] In some embodiments, the medicine or health product can enhance immunity.

[0069] 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.

[0070] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0071] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells; further comprising promoting the generation of T cells.

[0072] In some embodiments, the drug promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0073] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, isobutyric acid, and 2-methylbutyric acid.

[0074] The composition comprises a consortium of isolated and purified viable microbial populations to increase body weight by at least 2%, 3%, 4%, 5%, 6%, or 7% in a subject compared to the subject's body weight before administration of the isolated and purified consortium of viable microbial species.

[0075] As used herein, a microbial population generally refers to a population of microorganisms consisting essentially of a single strain, species, or genus, as may be the case when the population is cultured from an isolated and purified subpopulation of such a strain, species, or genus. Thus, for a given population of microorganisms, if such population is cultured from an isolated species or strain of microorganisms, such population will be referred to herein as purified or substantially pure. The resulting population will typically be at least 80% pure with respect to the species or strain of microorganism, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure relative to other species or strains of microorganisms within that particular population. Conversely, the level of undesirable strains in any particular desired population of microorganisms will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. For example, the level of impurities, such as other undesirable strains or species, in a purified population of microorganisms can be at or below the aforementioned levels for each desired population, proportionally. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case of a composition comprising a consortium of multiple populations of microorganisms, each population may have the above-mentioned purities, either prior to its incorporation into the composition, or when measured in aggregate for the consortium. For example, the level of impurities, such as other undesirable strains or species of microorganisms, in the purified population consortium may be proportional to or below the above-mentioned levels for each desired population.

[0076] The compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by the Lactobacillaceae strain, and the like. This can be obtained, for example, by recovering the supernatant of a culture of the Lactobacillaceae strain or by extracting cellular components or cell fractions, metabolites or secreted compounds from a culture of the Lactobacillaceae strain; this can correspond to a component in isolated form from the Lactobacillaceae strain, or any mixture of one or more components from the Lactobacillaceae strain.

[0077] In some embodiments, the Lactobacillus strains or compositions described herein can be used to promote growth and development, enhance immunity, or promote intestinal health.

[0078] In some embodiments, the growth promotion comprises: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0079] In some embodiments, the organ comprises at least one of a thymus, a heart, a liver, a spleen, a kidney, and an intestine.

[0080] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0081] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0082] In some embodiments, the Lactobacillusaceae strain or composition promotes growth and development by increasing hormone content and / or promoting digestion and absorption.

[0083] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0084] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0085] In some embodiments, the Lactobacillusaceae strain or composition enhances immunity by promoting immune system development.

[0086] 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.

[0087] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0088] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells); and further comprises promoting the generation of T cells.

[0089] In some embodiments, the Lactobacillus strain or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0090] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, isobutyric acid, and 2-methylbutyric acid.

[0091] In some embodiments, the Lactobacillus strain or composition of the present disclosure can increase heart, liver, spleen, and / or kidney weight, and / or intestinal length, thereby promoting organ development.

[0092] In some embodiments, the Lactobacillus strain or composition of the present disclosure can increase body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight, thereby promoting height (body length), weight, bone, and muscle development.

[0093] In some embodiments, the Lactobacillus strain or composition of the present disclosure can increase the levels of serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH), thereby promoting growth and development.

[0094] In some embodiments, the Lactobacillus strains of the present disclosure or compositions comprising the same can promote digestion and absorption, thereby promoting growth and development without increasing cumulative food intake.

[0095] In some embodiments, the Lactobacillaceae strain or composition disclosed herein can promote the development of major immune organs such as the thymus and spleen, and / or increase the proportion of T cells, thereby promoting the development of the immune system and enhancing immunity.

[0096] In some embodiments, the Lactobacillus strain or composition of the present disclosure can increase the content of short-chain fatty acids and / or improve intestinal flora, thereby promoting intestinal health.

[0097] In some embodiments, the drug has at least one effect selected from the following: increasing the weight of the heart, liver, spleen, and / or kidney, and / or intestinal length (large intestine length and / or small intestine length); not increasing food intake; increasing body weight, body length, tibia length, femur length, soleus and / or gastrocnemius muscle weight; promoting weight and body length growth, promoting bone and muscle growth (wherein, promoting bone growth includes growth in bone length and density, and promoting muscle growth includes promoting specific muscle development and maintaining healthy and benign growth characteristics, such as: low body fat rate (calf muscle / weight) and / or high muscle mass ratio (calf muscle / calf weight)); increasing blood Clear 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; 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 digestion and absorption; 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.

[0098] In some embodiments, the health product has the following effects: enhancing immunity.

[0099] A method for promoting growth and development, enhancing immunity, or promoting intestinal health, comprising administering an effective amount of the Lactobacillusaceae strain described in the first aspect of the present disclosure or the composition described in the second aspect to a subject in need.

[0100] In some embodiments, the growth promotion comprises: promoting at least one of the development of organs, height / body length, weight, bones, and muscles.

[0101] In some embodiments, the organ comprises at least one of a thymus, a heart, a liver, a spleen, a kidney, and an intestine.

[0102] In some embodiments, the bone comprises at least one of a femur and a tibia.

[0103] In some embodiments, the muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

[0104] In some embodiments, the Lactobacillusaceae strain or composition promotes growth and development by increasing hormone content and / or promoting digestion and absorption.

[0105] In some embodiments, the hormone comprises insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH).

[0106] In some embodiments, the hormone is derived from body fluids; further from blood; still further from serum.

[0107] In some embodiments, the Lactobacillusaceae strain or composition enhances immunity by promoting immune system development.

[0108] 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.

[0109] In some embodiments, promoting the development of immune organs comprises promoting the development of the thymus, spleen, bone marrow, and / or lymph nodes; and further comprises promoting the development of the thymus and / or spleen.

[0110] In some embodiments, the promoting the generation of immune cells comprises promoting the generation of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells); and further comprises promoting the generation of T cells.

[0111] In some embodiments, the Lactobacillus strain or composition promotes intestinal health by increasing short-chain fatty acids and / or improving intestinal flora.

[0112] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, isobutyric acid, and 2-methylbutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] The following description of the embodiments will be made in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0114] Figure 1 : shows the colony morphology photograph of strain MNH06277.

[0115] Figure 2 : shows Gram staining photographs of strain MNH06277.

[0116] Figure 3 : shows an electron micrograph of strain MNH06277.

[0117] Figure 4 : shows a photograph of the colony morphology of strain MNH45330.

[0118] Figure 5 : Gram-stained photographs of strain MNH45330 are shown.

[0119] Figure 6 : shows an electron micrograph of strain MNH45330.

[0120] Figure 7 : Shows the results of the tolerance of strain MNH06277 to different pH.

[0121] Figure 8 : Shows the tolerance results of strain MNH06277 to different concentrations of NaCl.

[0122] Figure 9 : Shows the tolerance results of strain MNH06277 to different concentrations of bile salts.

[0123] Figure 10 : Shows the results of the tolerance of strain MNH45330 to different pH.

[0124] Figure 11 : Shows the tolerance results of strain MNH45330 to different concentrations of NaCl.

[0125] Figure 12 : Shows the results of the tolerance of strain MNH45330 to different concentrations of bile salts.

[0126] Figure 13 : shows the phylogenetic tree of strain MNH06277.

[0127] Figure 14: shows the phylogenetic tree of strain MNH45330.

[0128] Figure 15 : Shows that strains MNH06277 and MNH45330 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.

[0129] Figure 16 : Shows that strains MNH06277 and MNH45330 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.

[0130] Figure 17 : This study demonstrates that strains MNH06277 and MNH45330 promote organ development (intestinal length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. **, p < 0.01 compared with the LFD-Control group.

[0131] Figure 18 Figure 2: Strains MNH06277 and MNH45330 promote weight gain. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0132] Figure 19 Figure 2: Strains MNH06277 and MNH45330 promote height (length increase). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. **, p < 0.01 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0133] Figure 20Figure 2: MNH06277 and MNH45330 strains promote bone development (femur length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0134] Figure 21 Figure 2: MNH06277 and MNH45330 strains promote bone development (tibia length). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group; ***, p < 0.001 compared with the LFD-Control group.

[0135] Figure 22 Figure 3: Muscle development (soleus muscle weight) of strains MNH06277 and MNH45330. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0136] Figure 23 Figure 2: MNH06277 and MNH45330 strains promote muscle development (gastrocnemius muscle weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0137] Figure 24 Figure 2: Strain MNH06277 increases serum insulin-like growth factor 1 (IGF-1) levels. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0138] Figure 25Strains MNH06277 and MNH45330 did not increase cumulative food intake (line graph of cumulative food intake). Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using the Student's t test.

[0139] Figure 26 Strains MNH06277 and MNH45330 did not increase cumulative food intake (cumulative food intake bar graph). Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using the Student's t test.

[0140] Figure 27 The results show that strains MNH06277 and MNH45330 promote immune system development and enhance immunity (thymus weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0141] Figure 28 The results show that strains MNH06277 and MNH45330 promote immune system development and enhance immunity (spleen weight). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0142] Figure 29 This study demonstrates that strain MNH45330 promotes immune system development and enhances immunity (T cell percentage). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0143] Figure 30 This figure shows that strain MNH45330 promotes the production of short-chain fatty acids (propionic acid content) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0144] Figure 31This figure shows that strain MNH45330 promotes the production of short-chain fatty acids (isobutyric acid content) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0145] Figure 32 Figure 3: Strain MNH45330 promotes the production of short-chain fatty acids (2-methylbutyrate) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0146] Figure 33 Figure 2: Strain MNH45330 promotes the production of short-chain fatty acids (isovalerate) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0147] Figure 34 Figure 2: Strain MNH06277 promotes the production of short-chain fatty acids (butyrate as a percentage) in cecal contents. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t test. *, p < 0.05 compared with the LFD-Control group.

[0148] Strain preservation

[0149] Lactobacillus paracasei ( Lacticaseibacillus paracasei ) MNH06277, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65552, 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 Lacticaseibacillus paracasei MNH06277, taxonomic name Lacticaseibacillus paracasei .

[0150] strain Lactobacillus crispatus ( Lactobacillus crispatus) MNH45330, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65556, 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 Lactobacillus crispatus MNH45330, taxonomic name Lactobacillus crispatus . DETAILED DESCRIPTION

[0151] The present invention discloses the isolation of two new strains of the Lactobacillaceae family, with deposit numbers GDMCC No. 65552 and GDMCC No. 65556, respectively. These strains were identified using traditional taxonomic and molecular biological methods. The identification results indicate that strain GDMCC No. 65552 belongs to the species Lactobacillus paracasei, while strain GDMCC No. 65556 belongs to the species Lactobacillus crispatus. Furthermore, the present invention investigates the biochemical properties and therapeutic uses of these two strains.

[0152] As is known in the art, bacterial species can be classified and identified using traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic assays. Molecular biology methods include, but are not limited to, ribosomal RNA sequencing and whole genome sequencing-based methods.

[0153] 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).

[0154] 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.

[0155] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, nutritional supplements, or medical probiotics. In some cases, the composition is used in the form of a pharmaceutical preparation. In some cases, the composition is used in the form of a nutritional supplement. In some cases, the composition is used in the form of a medical probiotic. In some cases, the composition (e.g., nutritional supplements, medical probiotics) can be administered orally, for example, as a capsule, pill, or tablet.

[0156] 16S rRNA is a type of ribosomal RNA in prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is shared by all bacteria, while the variable region varies to varying degrees among different bacteria. By comparing bacterial 16S rRNA gene sequences and basing the number of sequence differences and their evolutionary distance, an evolutionary tree can be constructed. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence betweenaverage nucleotide identity and 16S rRNA gene sequence similarity for speciesdemarcation of prokaryotes. International Journal of Systematic andEvolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini,R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening thetaxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), p23).

[0157] 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.

[0158] Second-generation sequencing technology can also be used to identify bacterial species based on whole-genome sequencing, making the identification results more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value must reach above 95% to be considered as belonging to the same species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9(1):5114.).

[0159] Various existing mature ANI value calculation tools can be used, such as the local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0160] Utilize the above method, those skilled in the art can judge whether a bacterial strain isolate belongs to the lactobacillus paraceasi kind or the lactobacillus crispatus kind that the inventor has identified.For example, when being the lactobacillus paraceasi kind of GDMCC No: 65552 ( Lacticaseibacillus paracasei) 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%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98 ...6%, 98.7%, 98.8%, 98.9%, 98. 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 (i.e., they belong to the species Lactobacillus paracasei); or when they are combined with Lactobacillus crispatus ( Lactobacillus crispatus ), an average nucleotide identity ANI value of at least 95%, for example at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9 ... .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 (i.e., the Lactobacillus crispatus species).

[0161] For another example, when its 16S rRNA sequence is at least 98.65% identical to the sequence shown in SEQ ID NO: 3, 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%, it can be determined to belong to the same species (i.e., the species Lactobacillus paracasei); when its 16S rRNA sequence is at least 98.65% identical to the sequence shown in SEQ ID NO: 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%, it can be determined to belong to the same species (i.e., the species Lactobacillus crispatus).

[0162] A "strain" is a member of a bacterial species that has genetic characteristics that allow it to be distinguished from closely related members of the same bacterial species. A genetic characteristic can be the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence ("cure") of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In cases where one strain (compared to another strain of the same species) acquires or loses antibiotic resistance or a biosynthetic capacity (e.g., an auxotrophic strain), the strains can be distinguished by selection or counterselection using antibiotics or nutrients / metabolites.

[0163] "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.

[0164] The composition can be prepared using the Lactobacillus strains described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical composition comprises a pharmaceutically effective amount of the Lactobacillus strain, for example, Lactobacillus paracasei (GDMCC No: 65552). Lacticaseibacillus paracasei ), or Lactobacillus crispatus with a deposit number of GDMCC No: 65556 ( Lactobacillus crispatus). Similarly, the Lactobacillus paracasei with a deposit number of GDMCC No: 65552 ( Lacticaseibacillus paracasei ), or Lactobacillus crispatus with a deposit number of GDMCC No: 65556 ( Lactobacillus crispatus ) can also be prepared into a pharmaceutical composition, for example, by using pharmaceutically acceptable excipients, which contains a pharmaceutically effective amount of the Lactobacillus family strain.

[0165] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

[0166] The compositions herein can be formulated into any form suitable for enhancing the abundance of the Lactobacillusaceae strain in a subject. The compositions can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by bile infusion, by cardiac infusion, pre-anally, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, or by ureteral or urethral administration.

[0167] Examples of dosage forms suitable for the compositions herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0168] In some embodiments, the immunity-enhancing health product is a sugar-coated tablet, a gel capsule, a tablet, a sheet capsule, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder, and the like.

[0169] In some embodiments, administration can also be by inclusion in the subject's diet.

[0170] The compositions provided herein may comprise a pharmaceutically acceptable excipient, diluent or carrier. Such pharmaceutically acceptable excipients, diluents or carriers are well known in the art.

[0171] In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is freeze-dried. In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is spray-dried. In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is freeze-dried or spray-dried and is alive. In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is freeze-dried or spray-dried and can partially or completely colonize in the intestine. In some embodiments, the freeze-dried Lactobacillaceae strain is reconstructed before administration. In some embodiments, the reconstruction is carried out using a diluent as described herein.

[0172] 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.

[0173] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0174] The compositions disclosed herein include medicines or health products for enhancing immunity.

[0175] The subject of the present disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0176] In 2020, ZHENG et al. conducted a study on Lactobacillus spp. Lactobacillus ) of 261 bacterial species, the comparison of average nucleotide indentity (ANI) between species, the analysis of characteristic genes, the physiological standards and the ecological analysis of species, proposed that the genus Lactobacillus be redivided into 25 genera, including Lactobacillus and 23 new genera, and suggested that the family Leuconostocaceae ( Leuconostocaceae ) is assigned to the Lactobacillaceae family ( Lactobacillaceae ). According to this study, the Lactobacillus casei group Lactobacillus casei 、 Lactobacillus paracasei subsp. paracasei 、 Lactobacillus paracasei subsp. tolerans 、 Lactobacillus rhamnosus 、 Lactobacillus chiayiensis Renamed to Lacticaseibacillus casei 、 Lacticaseibacillus paracasei subsp. paracasei 、 Lacticaseibacillus paracasei subsp. tolerans 、 Lacticaseibacillus rhamnosus 、 Lacticaseibacillus chiayiensis Among them, the Lactobacillus genus contains many important probiotic resources, and changes in its classification may affect the update of microbial-related industry regulations and the application of bacterial strains in various countries.

[0177] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature of the art or in accordance with the product or instrument specifications are used. All reagents or instruments are commercially available if their manufacturers are specified. Example

[0178] The anaerobic blood agar plates involved in the examples were purchased from Huankai Microorganisms, with the following formula: 10 g / L casein pancreatic digest, 3 g / L cardiac pancreatic digest, 1 g / L corn starch, 5 g / L meat peptic digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50-100 mL / L sterile defibrinated sheep blood, pH 7.3±0.2.

[0179] The PYG liquid culture medium involved in the examples, each 1 L PYG liquid culture medium contains: Trypticase peptone 5.0 g, Peptone 5.0 g, Yeast extract 10.0 g, Beef extract 5.0 g, Glucose 5.0 g, K2HPO4 2.0 g, Tween 80 1.0 ml, Resazurin 1.0 mg, 40 ml 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, the balance being water), the balance being water, pH 7.2±0.2 (25°C).

[0180] The MRS plate medium involved in the embodiment, each 1LMRS plate medium contains: peptone 10.0 g, beef extract powder 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, Tween-80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, ammonium citrate tribasic 2.0 g, magnesium sulfate (MgSO4·7H2O) 0.1 g, manganese sulfate (MnSO4·4H2O) 0.05 g, agar 15.0 g, and water balance.

[0181] The MRS liquid culture medium involved in the embodiment, each 1LMRS liquid culture medium contains: peptone 10.0 g, beef extract powder 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, Tween-80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, triammonium citrate 2.0 g, magnesium sulfate (MgSO4·7H2O) 0.1 g, manganese sulfate (MnSO4·4H2O) 0.05 g, and water balance.

[0182] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0183] Example 1. Isolation and identification of strains

[0184] 1.1 Isolation and purification of strains MNH06277 and MNH45330

[0185] Two intestinal bacterial strains of the family Lactobacillaceae (Lactobacillus paracasei Lacticaseibacillus paracasei MNH06277 and Lactobacillus crispatus Lactobacillus crispatus MNH45330) were isolated from fecal samples of healthy volunteers. Conventional strain isolation methods were used, using gradient dilution, followed by isolation and culture of single colonies. Purification was performed and anaerobically cultured at 37°C. Pure cultures were prepared into a 20% glycerol / water suspension and stored at -80°C.

[0186] Specifically, the isolation and purification methods of the strains are as follows:

[0187] The donor collects 2-5 g of fresh feces, places it in a sample collection and storage tube, shakes it to homogenize it, and places the processed fecal sample in an ice box. It is then sent to the laboratory for strain isolation within 24 hours.

[0188] Aliquot physiological saline solution in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation and transfer them to the anaerobic workstation 24 h in advance. Label the plates with sample information, culture medium type, isolation date, etc.

[0189] Take fresh fecal sample and place it in anaerobic workstation and shake it with vortex shaker for 1 min to mix it. Pipette 1 mL of sample into 9 mL of normal saline and mix it for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution, set aside.

[0190] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the plate surface is dry, invert the plate and culture at 37°C for 3-5 days.

[0191] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterile toothpick for strain purification. Incubate the purified strain anaerobically at 37°C. Prepare a 20% glycerol / water solution of the pure culture and store it at -80°C.

[0192] 1.2 Morphological characteristics of strains MNH06277 and MNH45330

[0193] 1.2.1 Morphological characteristics of strain MNH06277

[0194] Strain MNH06277 was inoculated onto MRS plates and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the MRS plates. These colonies were round, with regular, smooth edges, approximately 1-2 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1.5-3 µm in size. A photograph of the colony morphology of strain MNH06277 after 48 hours of MRS plate culture is shown in the accompanying image. Figure 1 Gram staining photos of strain MNH06277 are shown in Figure 2 See the electron microscope photos for Figure 3 .

[0195] 1.2.2 Morphological characteristics of strain MNH45330

[0196] Strain MNH45330 was inoculated onto MRS plates and incubated anaerobically at 37°C for 48 hours. Visible colonies formed on the MRS plates. These colonies were round, with regular, smooth edges, approximately 0.5-2 mm in diameter, and white, opaque. The strain was Gram-positive. Microscopic observation revealed that the strain lacked flagella, was nonmotile, and had a rod-shaped shape, approximately 0.5-1 µm × 1.5-4 µm in size. A photograph of the colony morphology of strain MNH 45330 after 48 hours of MRS plate culture is shown in the accompanying image. Figure 4 The Gram staining photos of strain MNH45330 are shown in Figure 5 See the electron microscope photos for Figure 6 .

[0197] 1.3 Physiological and biochemical characteristics of strains MNH06277 and MNH45330

[0198] 1.3.1 Physiological and biochemical characteristics of strain MNH06277

[0199] Strain MNH06277 grows under both aerobic and anaerobic conditions. It can grow in the pH range of 4.0 to 10.0, with the optimal growth pH being 6.0 (see the results of the strain's tolerance to different pH values ​​for details). Figure 7 ); can still grow on medium containing more than 6% NaCl (the results of strain tolerance to different concentrations of NaCl are shown in Figure 8 ); strain MNH06277 cannot grow in culture medium with bile salt concentrations above 0.1% (the results of strain tolerance to different concentrations of bile salts are shown in Figure 9 ).

[0200] 1.3.2 Physiological and biochemical characteristics of strain MNH45330

[0201] The strain MNH45330 can grow in the pH range of 4.0 to 10.0, with the optimal growth pH being 8.0 (see the results of the strain's tolerance to different pH values ​​for details). Figure 10); can still grow on medium containing more than 4% NaCl (the results of strain tolerance to different concentrations of NaCl are shown in Figure 11 ); strain MNH45330 cannot grow in culture medium with bile salt concentration above 0.1% (the results of strain tolerance to different concentrations of bile salts are shown in Figure 12 ).

[0202] 1.4 Biochemical identification results of strains MNH06277 and MNH45330

[0203] 1.4.1 Results of biochemical identification of strain MNH06277 using API 50CHL

[0204] API 50CHL (Mérieux, CN5041010) was used. Specific experimental procedures are described in the general API reagent operating instructions. Incubation conditions: 37°C, anaerobic. Results are shown in Table 1.

[0205] MNH06277 can utilize ribose RIB, galactose GAL, glucose GLU, fructose FRU, mannose MNE, sorbitol SBE, mannitol MAN, sorbitol SOR, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, gentiobiose GEN, D-turanose TUR, D-tagatose TAG, L-arabinitol LARL, and gluconate GNT to ferment and produce acid. Therefore, during the fermentation or cultivation of strain MNH06277, ribose RIB, galactose GAL, glucose GLU, fructose FRU, mannose MNE, sorbitol SBE, mannitol MAN, sorbitol SOR, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, gentiobiose GEN, D-turanose TUR, D-tagatose TAG, L-arabinitol LARL, gluconate GNT, and their derivatives can be used as carbon sources.

[0206] Table 1. Test results of strain MNH06277

[0207]

[0208] Note: “+” indicates positive, “-” indicates negative.

[0209] 1.4.2 Results of biochemical identification of strain MNH45330 using API 50CHL

[0210] API 50CHL (Mérieux, CN5041010) was used. Specific experimental procedures are described in the standard API reagent operating instructions. Incubation conditions: 37°C, anaerobic. Results are shown in Table 2.

[0211] MNH45330 can ferment and produce acid by using galactose GAL, glucose GLU, fructose FRU, mannose MNE, mannitol MAN, α-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, and D-lyxose LYX. Therefore, during the fermentation or cultivation of strain MNH45330, galactose GAL, glucose GLU, fructose FRU, mannose MNE, mannitol MAN, α-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, esculin ESC, salicylate SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melezitose MLZ, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, D-lyxose LYX, and their derivatives can be used as carbon sources.

[0212] Table 2. Test results of strain MNH45330

[0213]

[0214] Note: “+” indicates positive, “-” indicates negative.

[0215] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strains MNH06277 and MNH45330

[0216] 1.5.1 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH06277

[0217] The minimum inhibitory concentration of antibiotics for strain MNH06277 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 3.

[0218] Table 3. Minimum inhibitory concentration test results of antibiotics for strain MNH06277

[0219]

[0220] The results showed that MNH06277 was effective in treating imipenem (I / R), ampicillin-sulbactam (SAM), and dopenem (DOR).

[0221] MNH06277 is sensitive to amoxicillin (AMC), rifampicin (RD), and ceftriaxone (CRO). Therefore, MNH06277 is sensitive to most types of antibiotics, and the risk of long-term use of MNH06277 leading to antibiotic resistance in subjects is low.

[0222] 1.5.2 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH45330

[0223] The minimum inhibitory concentration of antibiotics for strain MNH45330 was determined using E-test (purchased from Liofilchem) paper. The test results are shown in Table 4.

[0224] Table 4. Minimum inhibitory concentration test results of antibiotics for strain MNH45330

[0225]

[0226] The results showed that MNH45330 was sensitive to imipenem, ampicillin-sulbactam, dopenem, ertapenem, meropenem, piperacillin-tazobactam, and ceftriaxone. This suggests that MNH45330 is sensitive to most antibiotics, and the risk of developing antibiotic resistance in subjects with long-term MNH45330 use is low.

[0227] 1.6 Identification of strains MNH06277 and MNH45330

[0228] 1.6.1 16S rRNA gene amplification of strains MNH06277 and MNH45330

[0229] Fresh cultures of strains MNH06277 and MNH45330 were used to extract genomic DNA, which was then used as a template for 16S rRNA gene amplification.

[0230] The primer pairs used for PCR of the 16S rRNA gene are:

[0231] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0232] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0233] The PCR reaction procedure is as follows:

[0234] 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.

[0235] 1.6.2 16S rRNA gene sequencing of strains MNH06277 and MNH45330

[0236] The PCR product was purified and sequenced by Sangon to obtain the 16S rRNA gene.

[0237]

[0238]

[0239] 1.6.3 Identification results of strains MNH06277 and MNH45330

[0240] The 16S rRNA gene sequences measured above, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, were analyzed for the strain 16S rRNA gene using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0241] The obtained sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH06277 was Lacticaseibacillus paracasei , the similarity is 100%, so the strain MNH06277 is Lacticaseibacillus paracasei strains planted; the strain with the highest similarity to MNH45330 is Lactobacillus crispatus The similarity is 100%, so strain MNH45330 is Lactobacillus crispatus Planted strains.

[0242] MNH06277 and MNH45330 were compared with the Lacticaseibacillus paracasei and Lactobacillus crispatus The 16S rRNA gene sequences of related strains of the genus were compared and phylogenetic trees were constructed.

[0243] The sequences of MNH06277 and MNH45330 were aligned with those of model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method). Figure 13 、 14 The nodes in the developmental tree only display the values ​​whose Bootstrap values ​​are greater than 50%.

[0244] From the phylogenetic tree ( Figure 13 、 14 ) It can be seen that strain MNH06277 and Lacticaseibacillus Fungus Lacticaseibacillus paracasei Get together for Lacticaseibacillus paracasei The new strain MNH45330 and Lactobacillus Fungus Lactobacillus crispatus Get together for Lactobacillus crispatus New plants planted.

[0245] The genomes of MNH06277 and MNH45330 were extracted and sequenced, and then tested using the GTDB method. The strain with the highest similarity to the MNH06277 genome was:Lacticaseibacillus paracasei (GCF_000829035.1), with an average nucleotide similarity (ANI) of 98.2% and an alignment score (AF) of 88%. Therefore, MNH06277 can be identified as Lacticaseibacillus paracasei The strain with the highest genome similarity to MNH45330 is: Lactobacillus crispatus (GCF_002088015.1), with an average nucleotide similarity (ANI) of 97.4% and an alignment score (AF) of 86%. Therefore, MNH45330 can be identified as Lactobacillus crispatus .

[0246] 1.7 Genome analysis of strains MNH06277 and MNH45330

[0247] 1.7.1 Genome analysis of strain MNH06277

[0248] The genome of strain MNH06277 was fragmented using ultrasound to fragments ranging from 350 bp in length. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) with paired-end 150-bp sequencing. The resulting data yielded 0.89 Gbp of data, of which Q20 accounted for 94.82%.

[0249] The raw sequencing data were filtered using 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 were assembled using SPAdes (version v3.14.0) with the assembly parameters "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 2.90 Mbp, an N50 length of 196.8 kbp, and a GC content of 46.43%.

[0250] Genomic gene prediction analysis was performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 2728 CDS sequences were predicted, with an average CDS sequence length of 909 bp.

[0251] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), using the CARD antibiotic resistance gene database (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found.

[0252] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed comparison results are shown in Table 5.

[0253] Table 5. List of potential toxic genes of MNH06277

[0254]

[0255] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version 1.0.0). Detailed alignment results are shown in 6.

[0256] Table 6. List of potential primary metabolic gene clusters in MNH06277

[0257]

[0258] 1.7.2 Genome analysis of strain MNH 45330

[0259] The genome of strain MNH 45330 was fragmented by ultrasonication to fragments ranging from 350 bp in length. An Illumina sequencing library was then constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) with paired-end 150-bp sequencing. The resulting data was 1.43 Gbp, of which Q20 accounted for 95.33%.

[0260] The raw sequencing data were filtered using 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 were assembled using SPAdes (version v3.14.0) with the assembly parameters "--isolate --cov-cutoff 10". The assembled genome yielded a total gene length of 2.45 Mbp, an N50 length of 43.3 kbp, and a GC content of 36.51%.

[0261] Genomic gene prediction analysis was performed using the prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) with the parameters "--gcode 11 --evalue 1e-09". A total of 2468 CDS sequences were predicted, with an average CDS sequence length of 870 bp.

[0262] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), using the CARD antibiotic resistance gene database (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found.

[0263] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). Detailed comparison results are shown in Table 7.

[0264] Table 7. List of potential toxic genes of MNH45330

[0265]

[0266] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 8.

[0267] Table 8. List of potential primary metabolic gene clusters in MNH45330

[0268]

[0269] Example 2: Strains MNH06277 and MNH45330 promote growth and development in a low-fat, low-protein diet-induced growth retardation mouse model

[0270] A low-fat, low-protein diet was used to induce growth retardation in mice to test the effects of MNH06277 and MNH45330 on growth and development indicators. This invention has passed the ethical review of the Muen Biotechnology Animal Ethics Committee.

[0271] 2.1 Experimental methods

[0272] 1) Experimental Animals: 3-week-old C57BL / 6J mice were purchased from Guangdong Weitonglihua Laboratory Animal Technology Co., Ltd.

[0273] 2) Preparation of test articles for strains MNH06277 and MNH45330: Glycerol cryovials of MNH06277 and MNH45330 were thawed at 37°C and activated in MRS plate culture medium in an anaerobic workstation. The activated strains were inoculated into MRS liquid culture medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured liquid was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable cell count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0274] 3) Negative control: Use PBS containing 25% glycerol and 0.05% L-Cys HCl as the negative control.

[0275] 4) Positive control: Bifidobacterium lactis CGMCC No. 20847. After thawing the glycerol cryopreserved tube of Bifidobacterium lactis CGMCC No. 20847 (disclosed in patent CN112980725B) at 37°C, the strain was inoculated into PYG medium in an anaerobic workstation for activation. The activated strain was inoculated into PYG liquid medium and cultured anaerobically to obtain a sufficient amount of culture. The cultured liquid was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable cell count (2×10 9 CFU / mL) is a positive control substance that meets the requirements of animal experiments.

[0276] 5) Experimental Procedure: After the quarantine period, 40 3-week-old C57BL / 6J male mice weighing 13-16 g were randomly stratified according to body weight and divided into five groups, 8 mice per group: NCD-Control, LFD-Control, MNH06277, MNH45330, and a positive control. The NCD-Control group was fed a maintenance diet (Cat. No. PD24043001, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), while the other four groups were fed a low-fat, low-protein diet (Cat. No. PD24043003, Changzhou Shuyi Shuer Biotechnology Co., Ltd.). Drug administration began after grouping (D1). The NCD-Control and LFD-Control groups were administered a negative control. The positive control group received a positive control by gavage. The MNH06277 group received MNH06277, and the MNH45330 group received MNH45330 by gavage. 200 μL of drug was administered daily for 35 days. Mice had free access to water and food during the experiment, and a 12-hour day / night cycle was maintained. General clinical observations were conducted after each dose. The endpoint was the day after the end of drug administration (D36). Dissections were performed according to the protocol, and data were summarized and analyzed for individual dissection data and serum test results. All data are presented as mean ± SD and analyzed using GraphPad Prism software. Pairwise comparisons were analyzed using the Student's t test. No significance is indicated; significant differences are indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0277] Food intake was determined by weighing the remaining amount of feed.

[0278] 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.

[0279] Method for detecting serum insulin-like growth factor-1: Mouse serum was collected and detected using the Mouse IGF-1 ELISA Development Kit (PeproTech).

[0280] To detect immune cells, 0.1 g of mouse spleen tissue was collected, rinsed with PBS, and then pulverized. The pulverized spleen tissue was suspended in PBS and filtered through a 70 μm filter to prepare a mouse spleen single-cell suspension. A small amount of this suspension was stained and analyzed on a flow cytometer to accurately count the number of immune cells in the mouse spleen.

[0281] The short-chain fatty acid content of cecal contents was determined as follows: approximately 100 mg of cecal content was weighed and placed in a 2 mL EP tube. 1 mL of ether was added and immediately vortexed to mix thoroughly. Short-chain fatty acids were extracted by sonication for 10 minutes. The precipitate was removed by centrifugation for 10 minutes. The supernatant was passed through a 0.22 μm organic filter membrane and transferred to a gas phase vial. The vial was sealed with parafilm and stored at -20°C until assayed. Short-chain fatty acids were separated and detected using a DB-FFAP column and mass spectrometer.

[0282] 2.2 Experimental Results

[0283] See also Figures 15 - 17 The results showed that MNH06277 and MNH45330 could significantly increase the length of the large intestine, spleen and kidney weight of developmentally retarded mice, indicating that MNH06277 and MNH45330 can promote the development of the spleen, kidney, and / or intestine, and have the purpose of promoting organ development.

[0284] See also Figures 18 - 23 The results showed that MNH06277 and MNH45330 could significantly promote the body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight of the low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH06277 and MNH45330 have the use of promoting height (body length), weight, bone, and / or muscle development.

[0285] See also Figure 24 The results showed that MNH06277 could significantly increase the serum insulin-like growth factor 1 (IGF-1) content in a low-fat, low-protein diet-induced growth retardation mouse model, indicating that MNH06277 has the use of promoting growth and development.

[0286] See also Figures 25 - 26 The results showed that MNH06277 and MNH45330 did not increase food intake, indicating that MNH06277 and MNH45330 have the function of promoting digestion and absorption when promoting growth and development.

[0287] See also Figures 27 - 29 The results showed that MNH06277 and MNH45330 can significantly promote the development of major immune organs such as the thymus and spleen in a mouse model of growth retardation induced by a low-fat and low-protein diet; MNH45330 can increase the proportion of T cells, indicating that MNH06277 and MNH45330 have the purpose of promoting the development of the immune system and enhancing immunity.

[0288] See also Figures 30 - 34Results showed that MNH45330 promoted the production of short-chain fatty acids in the cecum of a mouse model of growth retardation induced by a low-fat, low-protein diet, significantly boosting the production of propionic acid, isobutyric acid, 2-methylbutyric acid, and isovaleric acid. MNH06277 also increased the proportion of butyric acid in the total short-chain fatty acids. This suggests that MNH06277 and / or MNH45330 can promote the production of short-chain fatty acids and promote intestinal health.

[0289] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations can be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples in the disclosure illustrate embodiments of the present invention, they are provided as non-limiting examples only and, therefore, should not be construed as limiting the various aspects of the invention thus described. The present invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Accordingly, the drawings and detailed description should be regarded as illustrative rather than restrictive.

Claims

1. A composition for promoting growth and development, enhancing immunity, or promoting intestinal health, characterized in that: The composition comprises live bacteria or freeze-dried bacteria of Lactobacillus crispatus MNH45330, wherein the preservation number of the Lactobacillus crispatus MNH45330 is GDMCC No: 65556; and the composition is a medicine.

2. The composition according to claim 1, wherein The composition further comprises one or more pharmaceutically acceptable carriers and / or excipients; and / or The composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

3. The composition according to claim 2, wherein The other active agents include one or more of probiotics and prebiotics.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

5. The use according to claim 4, characterized in that The growth promotion includes: promoting at least one of the development of organs, height, weight, bones, and muscles.

6. The use according to claim 5, characterized in that The organ includes at least one of the thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

7. The use according to any one of claims 4 to 6, characterized in that The drug promotes growth and development by improving digestion and absorption; or The drug enhances immunity by promoting the development of the immune system; or The drug promotes intestinal health by increasing short-chain fatty acids.

8. The use according to claim 7, characterized in that 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, wherein the immune cells include T cells.

9. A composition for promoting growth and development, enhancing immunity, or promoting intestinal health, characterized in that: The composition comprises live bacteria or freeze-dried bacteria of Lactobacillus crispatus MNH45330 and Lactobacillus paracasei MNH06277, wherein the preservation number of Lactobacillus paracasei MNH06277 is GDMCC No: 65552; ​​the preservation number of Lactobacillus crispatus MNH45330 is GDMCC No: 65556; and the composition is a medicine.

10. The composition according to claim 9, wherein The composition further comprises one or more pharmaceutically acceptable carriers and / or excipients; and / or The composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

11. The composition according to claim 10, wherein The other active agents include one or more of probiotics and prebiotics.

12. Use of the composition according to any one of claims 9 to 11 in the preparation of a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

13. The use according to claim 12, characterized in that The growth promotion includes: promoting at least one of the development of organs, height, weight, bones, and muscles.

14. The use according to claim 13, characterized in that The organ includes at least one of the thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

15. The use according to any one of claims 12 to 14, characterized in that: The drug promotes growth and development by increasing hormone levels and / or facilitating digestion and absorption; or The drug enhances immunity by promoting the development of the immune system; or The drug promotes intestinal health by increasing short-chain fatty acids.

16. The use according to claim 15, characterized in that The hormone comprises insulin-like growth factor 1; 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, wherein the immune cells include T cells.

17. A composition for promoting growth and development, enhancing immunity, or promoting intestinal health, characterized in that: The composition comprises live bacteria or freeze-dried bacteria of Lacticaseibacillus paracasei MNH06277, wherein the preservation number of the Lacticaseibacillus paracasei MNH06277 is GDMCC No: 65552; ​​and the composition is a medicine.

18. The composition according to claim 17, wherein The composition further comprises one or more pharmaceutically acceptable carriers and / or excipients; and / or The composition further comprises one or more other active agents for promoting growth and development, enhancing immunity, or promoting intestinal health.

19. The composition according to claim 18, wherein The other active agents include one or more of probiotics and prebiotics.

20. Use of the composition according to any one of claims 17 to 19 in the preparation of a medicament for promoting growth and development, enhancing immunity, or promoting intestinal health.

21. The use according to claim 20, characterized in that The growth promotion includes: promoting at least one of the development of organs, height, weight, bones, and muscles.

22. The use according to claim 21, characterized in that The organ includes at least one of the thymus, spleen, kidney, and intestine; or The bone comprises at least one of a femur and a tibia; or The muscle comprises at least one of the soleus muscle and the gastrocnemius muscle.

23. The use according to any one of claims 20 to 22, characterized in that The drug promotes growth and development by increasing hormone levels and / or facilitating digestion and absorption; or The drug enhances immunity by promoting the development of the immune system; or The drug promotes intestinal health by increasing short-chain fatty acids.

24. The use according to claim 23, characterized in that The hormone comprises insulin-like growth factor 1; or The promoting the development of the immune system includes promoting the development of immune organs.

25. A composition for enhancing immunity, characterized in that The composition comprises live bacteria or freeze-dried bacteria of Lacticaseibacillus paracasei MNH06277, wherein the preservation number of the Lacticaseibacillus paracasei MNH06277 is GDMCC No: 65552; ​​and the composition is a health product.

26. The composition of claim 25, wherein The composition further comprises one or more food-acceptable carriers and / or excipients; and / or The composition may also contain one or more other active agents for enhancing immunity.

27. The composition of claim 26, wherein The other active agents include one or more of probiotics and prebiotics.

28. The composition according to any one of claims 25 to 27, wherein The health care product is a nutritional composition.

29. The composition according to any one of claims 25 to 27, wherein The health care product is food.

30. The composition according to any one of claims 25 to 27, wherein The health care product is candy.

31. The composition according to any one of claims 25 to 27, wherein The health product is a food bar.

32. The composition according to any one of claims 25 to 27, wherein The health product is a dietary supplement.

33. Use of the composition according to any one of claims 25 to 32 for enhancing immunity for non-therapeutic purposes.

34. Use of the composition according to any one of claims 25 to 32 in the preparation of a health product for enhancing immunity.

35. The use according to claim 34, characterized in that The health supplement enhances immunity by promoting the development of the immune system.

36. The use according to claim 35, characterized in that The promoting the development of the immune system includes promoting the development of immune organs.

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