Lactobacillus paracasei strain capable of promoting growth and development and enhancing immunity, prebiotics containing lactobacillus paracasei strain and application of lactobacillus paracasei strain

Abstract

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

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of microbiology, more particularly to an isolated Lactobacillaceae strain, compositions comprising the same and uses thereof. BACKGROUND

[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 of the body, which is a quantitative change; development refers to the differentiation and maturation of cells, tissues and organs, and functional maturation, which is a qualitative change. The two are closely related, and growth is the material basis of development. The maturation status of development is also reflected in the quantitative changes of growth. Studies have found that intestinal bacteria can affect the growth and development of the host, and some bacterial flora can mediate the growth and development of the host by affecting growth hormone (GH) / insulin-like growth factor-1 (IGF-1).

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

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

[0005] Lactobacillus crispatus and Lactobacillus paracasei are common Lactobacillus probiotics, and their safety has been widely verified. Lactobacillus crispatus is mainly studied in promoting the production performance of poultry and pigs, including increasing average daily gain, average daily feed intake, feed utilization rate, and improving the absorption and utilization rate of calcium and phosphorus in feed. For example, CN114891657B discloses that Lactobacillus crispatus DC529 significantly improves the average daily gain and average daily feed intake of broilers. CN108546663B discloses that Lactobacillus crispatus ZLC020 can increase the final weight of the pig group by 5.52% (P<0.05), increase the average daily gain by 7.32%, and reduce the feed-to-weight ratio by 5.68 (P<0.05) compared with the control group, indicating that Lactobacillus crispatus from pigs has a positive effect on improving the production performance of growing pigs. There are few reports on Lactobacillus crispatus promoting the growth and development of mice or infants.

[0006] For Lactobacillus paracasei, CN116875515A discloses that Lactobacillus paracasei GF027 promotes the growth of rat height, bone weight and bone density, and increases insulin-like growth factor 1 in serum. The prior art for Lactobacillus paracasei to promote growth and development mainly focuses on promoting the growth of bones and height. However, children with delayed development have low resistance and are prone to illness, which is related to the slow development of immune organs and the gastrointestinal tract. Few studies have targeted 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 it is a great challenge to screen new strains or species from them 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, but also represents a great unmet need. SUMMARY

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

[0009] Therefore, the new strain of the present disclosure can have the purposes of promoting the development of height and weight, promoting the development of organs, especially promoting the development of immune organs, enhancing immunity, promoting the development of the intestinal tract, improving intestinal health, etc. It can be used to improve the growth retardation caused by malnutrition or weak absorption and digestion of children, realize the functions of improving / treating / reversing growth retardation; realize the purposes of promoting growth and development, enhancing immunity, or promoting intestinal health, etc.

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

[0011] In a first aspect, the present disclosure provides an isolated Lactobacillaceae strain having an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, or at least 97.5% to MNH06277 with the accession number GDMCC No: 65552; and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence as shown in SEQ ID NO: 3; or

[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 as shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 4.

[0013] In some embodiments, the Lactobacillaceae strain is two new strains of Lactobacillaceae.

[0014] In some embodiments, the Lactobacillaceae strain has the name: Lacticaseibacillus paracasei MNH06277, is preserved in Guangdong Microbial Culture Collection Center (GDMCC), has the preservation number of GDMCC No: 65552, is preserved on November 28, 2024, is located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, Guangdong Microbiology Institute of Guangdong Academy of Sciences, and has the preservation name of Lacticaseibacillus paracasei MNH06277.

[0015] In some embodiments, the Lactobacillaceae strain has the name: Lactobacillus crispatus MNH45330, is preserved in Guangdong Microbial Culture Collection Center (GDMCC), has the preservation number of GDMCC No: 65556, is preserved on November 28, 2024, is located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, Guangdong Microbiology Institute of Guangdong Academy of Sciences, and has the preservation name of Lactobacillus crispatus MNH45330.

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

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

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

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

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

[0021] C) an inactivated product of the fermentation broth of the Lacticaseibacillus paracasei MNH06277 and / or Lactobacillus crispatus MNH45330;

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

[0024] In some embodiments, the culture of the Lactobacillaceae strain comprises a solid culture, a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof of the Lactobacillaceae strain.

[0025] In some embodiments, the fermentation culture or fermentation culture supernatant is obtained using a liquid culture medium under anaerobic culture conditions.

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

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

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

[0029] In some embodiments, the composition comprises 1 x 10 6 to 1 x 10 10 cfu / mL or 1 x 10 6 to 1 x 10 101 x 10

[0029] In some embodiments, the composition comprises 1 x 10 7 to 1 x 10 9 cfu / mL or 1 x 10 7 to 1 x 10 9 cfu / mL of viable bacteria of the Lactobacillaceae strain.

[0030] In some embodiments, the composition comprises 1 x 10 3 to 1 x 10 17 colony forming units (CFU) of bacteria per g of the composition; for example, 1 x 10 4 to 1 x 10 12 , 1 x 10 5 to 1 x 10 11 , or 1 x 10 6 to 1 x 10 10 colony forming units (CFU) of bacteria, specifically, for example, 1 x 10 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 , 1 x 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , 1 x 10 5 , 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 , 1 x 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 106 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12, 1 x 10 13 , 2 x 10 13 , 3 x 10 13 , 4 x 10 13 , 5 x 10 13 , 6 x 10 13 , 7 x 10 13 , 8 x 10 13 , 9 x 10 13 or any value therebetween, of colony forming units (CFU) of bacteria.

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

[0032] In some embodiments, the Lactobacillaceae strain in the composition is a live, attenuated, lyophilized or inactivated bacteria, for example can be heat-inactivated, preferably pasteurized.

[0033] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (e.g. 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 preparation, sustained release preparation, nano-preparation or micro-encapsulated capsule.

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

[0036] In some embodiments, the composition further comprises one or more pharmaceutical and / or food acceptable excipients.

[0037] The pharmaceutical and / or food acceptable excipients are well known to those skilled in the art.

[0038] In some embodiments, the excipient can be at least one selected from the group consisting of carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavorings.

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

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

[0041] In some embodiments, the promoting growth development comprises: promoting at least one of organ, height / length, weight, bone, muscle development.

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

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

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

[0045] In some embodiments, the other active agent can be one or more or a combination of probiotic, prebiotic;

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

[0047] In some embodiments, the prebiotic is selected from inulin, mulberry leaf extract, berberine, ganoderma, green coffee bean extract, oat, pectin, potato or extract thereof, citrus polyphenol, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidin, resistant dextrin, yeast beta-glucan, ginseng or extract thereof, nutritional compound, biotin, polydextrose, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, mannan-oligosaccharide, mannan-oligosaccharide (MOS), inulin rich in fructo-oligosaccharide, gluco-oligosaccharide, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS), and any combination thereof.

[0048] In some embodiments, the composition can be formulated into a frozen composition, such as a frozen composition obtained by flash 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 strains in the composition are freeze-dried or spray-dried. In some embodiments, the strains in the composition are electrostatic spray-dried. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are viable. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are capable of partial or complete colonization of the intestine. In some embodiments, the strains are reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

[0051] In some embodiments, the composition is 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 formulation. In some embodiments, the enteric formulation is a dosage form with an enteric coating. For example, the enteric formulation can be an enteric granule, an enteric tablet, or an enteric capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained release capsule, a controlled release capsule, or an enteric capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0053] In some embodiments, the composition is a medicament, or a health product, or a food product.

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

[0055] In some embodiments, the composition is a dosage form for gastrointestinal administration or a dosage form for non-gastrointestinal administration.

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

[0057] The present disclosure provides use of the composition of the second aspect in a medicament, a health product, or a food product for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0058] The present disclosure provides use of the composition of the second aspect in the preparation of a medicament, a health product, or a food product for promoting growth and development, enhancing immunity, or promoting intestinal health.

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

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

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

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

[0063] In some embodiments, the drug, nutraceutical, or food promotes growth and development by increasing hormone levels, and / or facilitating digestive absorption.

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

[0065] In some embodiments, the hormone is from a bodily fluid; further from blood; still further from serum.

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

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

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

[0069] In some embodiments, the promoting generation of immune cells comprises promoting generation of T cells, B cells, and / or NK cells (i.e., increasing the proportion of T cells, B cells, and / or NK cells; further comprises promoting generation of T cells.

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

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

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

[0073] As used herein, a microbiota generally refers to a population of microorganisms that consists essentially of a single strain, species, or genus, which can be the case when a population is cultured from a subpopulation of an isolated and purified strain, species, or genus. Thus, for a given population of microorganisms, if cultured from an isolated microorganism species or strain, such population will be referred to herein as purified or substantially pure. The resulting population can be at least 80% pure for the microorganism species or strain, at least 90% pure relative to other microorganism species or strains within that particular population, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure. Conversely, the level of non-desired strains in any particular desired population of microorganisms will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate for the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate for the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population.

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

[0075] In some embodiments, the Lactobacillaceae strain or composition of the present disclosure can be used to promote growth and development, enhance immunity, or promote gut health.

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

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

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

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

[0080] In some embodiments, the Lachnospiraceae strain or composition promotes growth and development by increasing hormone levels, and / or facilitating digestive absorption.

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

[0082] In some embodiments, the hormone is from a bodily fluid; further from blood; still further from serum.

[0083] In some embodiments, the Lachnospiraceae strain or composition enhances immunity by promoting immune system development.

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

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

[0086] In some embodiments, the promoting generation of immune cells comprises promoting generation of (i.e., increasing the proportion of) T cells, B cells, and / or NK cells; further comprises promoting generation of T cells.

[0087] In some embodiments, the Lachnospiraceae strain or composition promotes gut health by increasing short-chain fatty acids, and / or improving gut microbiota.

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

[0089] In some embodiments, the Lachnospiraceae strain or composition of the present disclosure can increase heart, liver, spleen, and / or kidney weight, and / or intestinal length, thereby promoting organ development.

[0090] In some embodiments, the Lactobacillaceae strains or compositions 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), body weight, bone, and muscle development.

[0091] In some embodiments, the Lactobacillaceae strains or compositions of the present disclosure can increase serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH) content, thereby promoting growth and development.

[0092] In some embodiments, the Lactobacillaceae strains or compositions of the present disclosure can promote digestive absorption, thereby promoting growth and development without increasing cumulative food intake.

[0093] In some embodiments, the Lactobacillaceae strains or compositions of the present disclosure can promote the development of major immune organs such as the thymus and spleen, and / or increase the proportion of T cells, thereby promoting immune system development and enhancing immunity.

[0094] In some embodiments, the Lactobacillaceae strains or compositions of the present disclosure can increase short-chain fatty acid content and / or improve gut flora, thereby promoting intestinal health.

[0095] In some embodiments, the drug or health product or food has at least one effect selected from the group consisting of: increasing heart, liver, spleen, and / or kidney weight, and / or intestinal length (large intestine length and / or small intestine length); not increasing food intake; increasing body weight, body length, tibia length, femur length, soleus muscle and / or gastrocnemius muscle weight; promoting body weight and body length, promoting bone and muscle growth (where promoting bone growth includes increasing 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 ratio (calf muscle / body weight) and / or high muscle ratio (calf muscle / calf weight)); increasing serum insulin-like growth factor 1 (IGF-1) and / or growth hormone (GH) content; promoting the development of major immune organs such as the thymus and spleen (including increasing immune organ weight, immune organ development index (immune organ weight / body weight), etc.), increasing the proportion of T cells; increasing short-chain fatty acid content; improving gut flora; promoting intestinal development; improving pathologies or inflammation caused by developmental retardation to achieve the function of improving / treating / reversing developmental retardation; promoting organ development; promoting digestive absorption; promoting height (body length), body weight, bone, and muscle development; promoting immune system development and enhancing immunity; promoting intestinal health; and being useful for promoting growth and development, enhancing immunity, or promoting intestinal health.

[0096] A method of promoting growth and development, enhancing immunity, or promoting gut health, administering an effective amount of the Lactobacillaceae strain of the first aspect or the composition of the second aspect to a subject in need thereof.

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

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

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

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

[0101] In some embodiments, the Lactobacillaceae strain or composition promotes growth and development by increasing hormone level, and / or promoting digestive absorption.

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

[0103] In some embodiments, the hormone is from a body fluid; further from blood; further from serum.

[0104] In some embodiments, the Lactobacillaceae strain or composition enhances immunity by promoting immune system development.

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

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

[0107] In some embodiments, the promoting generation of immune cells comprises promoting generation of T cells, B cells, and / or NK cells (i.e. increasing the proportion of T cells, B cells, and / or NK cells); further comprises promoting generation of T cells.

[0108] In some embodiments, the Lactobacillaceae strain or composition promotes gut health by increasing short-chain fatty acids, and / or improving gut microbiota.

[0109] In some embodiments, the short-chain fatty acid comprises at least one of propionic acid, butyric acid, isovaleric acid, isobutyric acid, 2-methylbutyric acid. BRIEF DESCRIPTION OF DRAWINGS

[0110] The above and other aspects and advantages of the present application will become more apparent by describing in detail the embodiments thereof, with reference made to the accompanying drawings, in which:

[0111] Figure 1 : shows a photograph of colony morphology of strain MNH06277.

[0112] Figure 2 : shows a photograph of Gram staining of strain MNH06277.

[0113] Figure 3 : shows a photograph of electron microscopy of strain MNH06277.

[0114] Figure 4 : shows a photograph of colony morphology of strain MNH45330.

[0115] Figure 5 : shows a photograph of Gram staining of strain MNH45330.

[0116] Figure 6 : shows a photograph of electron microscopy of strain MNH45330.

[0117] Figure 7 : shows results of tolerance of strain MNH06277 to different pH.

[0118] Figure 8 : shows results of tolerance of strain MNH06277 to different concentrations of NaCl.

[0119] Figure 9 : shows results of tolerance of strain MNH06277 to different concentrations of bile salts.

[0120] Figure 10 : shows results of tolerance of strain MNH45330 to different pH.

[0121] Figure 11 : shows results of tolerance of strain MNH45330 to different concentrations of NaCl.

[0122] Figure 12 : shows results of tolerance of strain MNH45330 to different concentrations of bile salts.

[0123] Figure 13 : shows a phylogenetic tree of strain MNH06277.

[0124] Figure 14 : shows a phylogenetic tree of strain MNH45330.

[0125] Figure 15: Shows that strains MNH06277 and MNH45330 promote the development of organs (spleen weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control group; **, p < 0.01 compared to LFD-Control group.

[0126] Figure 16 : Shows that strains MNH06277 and MNH45330 promote the development of organs (kidney weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; **, p < 0.01 compared to LFD-Control group; ****, p < 0.0001 compared to LFD-Control group.

[0127] Figure 17 : Shows that strains MNH06277 and MNH45330 promote the development of organs (intestine length). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; **, p < 0.01 compared to LFD-Control group.

[0128] Figure 18 : Shows that strains MNH06277 and MNH45330 promote the development of body weight (body weight gain). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control group; ****, p < 0.0001 compared to LFD-Control group.

[0129] Figure 19 : Shows that strains MNH06277 and MNH45330 promote the development of body height (body length) (body length gain). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; **, p < 0.01 compared to LFD-Control group; ****, p < 0.0001 compared to LFD-Control group.

[0130] Figure 20: Shows that strains MNH06277 and MNH45330 promote bone development (femur length). Data are shown as Mean ± SD. Statistical analysis was performed using 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.

[0131] Figure 21 : Shows that strains MNH06277 and MNH45330 promote bone development (tibia length). Data are shown as Mean ± SD. Statistical analysis was performed using 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.

[0132] Figure 22 : Shows that strains MNH06277 and MNH45330 promote muscle development (soleus weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared with the LFD-Control group.

[0133] Figure 23 : Shows that strains MNH06277 and MNH45330 promote muscle development (gastrocnemius weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared with the LFD-Control group; **, p < 0.01 compared with the LFD-Control group.

[0134] Figure 24 : Shows that strain MNH06277 increases serum insulin-like growth factor 1 (IGF-1) content. Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared with the LFD-Control group; ****, p < 0.0001 compared with the LFD-Control group.

[0135] Figure 25: Shows that strains MNH06277 and MNH45330 did not increase cumulative food intake (Cumulative food intake line graph). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test.

[0136] Figure 26 : Shows that strains MNH06277 and MNH45330 did not increase cumulative food intake (Cumulative food intake bar graph). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test.

[0137] Figure 27 : Shows that strains MNH06277 and MNH45330 promoted immune system development and enhanced immunity (Thymus weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control; **, p < 0.01 compared to LFD-Control.

[0138] Figure 28 : Shows that strains MNH06277 and MNH45330 promoted immune system development and enhanced immunity (Spleen weight). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control; **, p < 0.01 compared to LFD-Control.

[0139] Figure 29 : Shows that strain MNH45330 promoted immune system development and enhanced immunity (T cell percentage). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control.

[0140] Figure 30 : Shows that strain MNH45330 promoted the production of short chain fatty acids in cecal content (Propionic acid content). Data are shown as Mean ± SD. Statistical analysis was performed using Student's t test; *, p < 0.05 compared to LFD-Control.

[0141] Figure 31: Shows that strain MNH45330 promotes the production of short-chain fatty acids in cecal contents (isobutyric acid content). Data are shown as Mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared with the LFD-Control group, **, p < 0.01 compared with the LFD-Control group.

[0142] Figure 32 : Shows that strain MNH45330 promotes the production of short-chain fatty acids in cecal contents (2-methylbutyric acid content). Data are shown as Mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared with the LFD-Control group, **, p < 0.01 compared with the LFD-Control group.

[0143] Figure 33 : Shows that strain MNH45330 promotes the production of short-chain fatty acids in cecal contents (isovaleric acid content). Data are shown as Mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared with the LFD-Control group, **, p < 0.01 compared with the LFD-Control group.

[0144] Figure 34 : Shows that strain MNH06277 promotes the production of short-chain fatty acids in cecal contents (butyric acid content). Data are shown as Mean ± SD. Statistical analysis was performed using Student’s t test; *, p < 0.05 compared with the LFD-Control group.

[0145] Preservation of strains

[0146] Strain Lacticaseibacillus paracasei MNH06277, preserved in the Guangdong Microbial Culture Collection Center (GDMCC), with the preservation number GDMCC No: 65552, preserved on November 28, 2024, at address No. 59, Building 5, 100, Martyrs' Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the preservation name is Lacticaseibacillus paracasei MNH06277, the taxonomic name is Lacticaseibacillus paracasei.

[0147] Lactobacillus crispatus MNH45330, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC No: 65556, deposited on November 28, 2024, at 5th Floor, Building 59, No. 100, Martyrs' Avenue, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposited name of Lactobacillus crispatus MNH45330, taxonomic name of Lactobacillus crispatus. DETAILED DESCRIPTION

[0148] The present disclosure isolates two new strains of the Lactobacillaceae family, with accession numbers of GDMCC No: 65552 and GDMCC No: 65556, and identifies them using traditional classification methods and molecular biology methods. The identification results show that the strain with the accession number of GDMCC No: 65552 belongs to a new strain under the Paracasei lactis species, and the strain with the accession number of GDMCC No: 65556 belongs to a new strain under the Lactobacillus crispatus species. Further, the present disclosure studies the biochemical properties and therapeutic uses of the two strains.

[0149] It is known in the art that the classification and identification of bacterial species can be performed by traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, for example, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biology methods include, but are not limited to, ribosomal RNA sequence determination method, whole genome sequencing-based determination method, etc.

[0150] The term "prebiotic" as used herein can be a general term referring to a chemical substance and / or component that can affect the growth and / or activity of microorganisms in a host (e.g., can allow specific changes in the composition and / or activity of the microbiome).

[0151] The terms "subject", "object", "individual", "host", and "patient" are used interchangeably herein to refer to any animal subject, including: human, mammal, experimental animal, livestock, and domestic pet.

[0152] The compositions or formulations of the present disclosure can be administered as a pharmaceutical formulation, a therapeutic composition, a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food. In some cases, the composition is administered as a pharmaceutical formulation. In some cases, the composition is administered as a nutritional supplement. In some cases, the composition is administered as a dietary supplement. In some cases, the composition is administered as a medical food. In some cases, the composition is administered as a medical probiotic. In some cases, the composition (e.g., a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food) can be administered orally, for example, as a capsule, pill, or tablet.

[0153] 16S rRNA is a ribosomal RNA of prokaryotes, and the 16S rRNA gene is composed of variable regions and conserved regions, with the conserved regions being shared by all bacteria and the variable regions differing to varying degrees among different bacteria. By comparing the 16S rRNA gene sequences of bacteria, an evolutionary tree can be drawn according to the evolutionary distance based on the number of sequence differences. When the sequence identity between the 16S rRNA genes of two strains is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 2), 346-351, and Liu, C., Du, M.-X., Abuduaini, R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), p23).

[0154] “Identity” between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the “FASTA” program, the GCG program package, BLASTN, or FASTA. Commercially or publicly available programs can also be, for example, the DNAStar “MegAlign” program.

[0155] The second-generation sequencing technology can also be used for bacterial strain identification based on whole genome sequencing, which makes the bacterial strain identification result more accurate. The average nucleotide identity (ANI) of bacterial genome refers to the similarity of homologous genes between two bacterial genomes. The ANI value can be calculated by BLAST and other methods. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach more than 95% to be identified as belonging to the same bacterial strain (Jain C, Rodriguez-R L M, Phillippy A M, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries [J]. Nature Communications, 2018, 9(1): 5114.).

[0156] The existing various mature ANI value calculation tools can be used, such as the local operation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), the online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0157] Using the above methods, one skilled in the art can determine whether an isolated strain belongs to the Lacticaseibacillus paracasei species or the Lactobacillus crispatus species identified by the present inventors. For example, when the average nucleotide identity ANI value with Lacticaseibacillus paracasei having the accession number GDMCC No: 65552 is at least 95%, such as at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, it can be determined to belong to the same species (i.e. to the Lacticaseibacillus paracasei species); or when the average nucleotide identity ANI value with Lactobacillus crispatus having the accession number GDMCC No: 65556 is at least 95%, such as at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, it can be determined to belong to the same species (i.e. to the Lactobacillus crispatus species).

[0158] For example, when its 16S rRNA sequence has at least 98.65% identity, for example at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or is 100% to the sequence set forth in SEQ ID NO: 3, it can be determined to belong to the same bacterial species (i.e., to belong to the species Paracaseicivorax casei); when its 16S rRNA sequence has at least 98.65% identity, for example at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or is 100% to the sequence set forth in SEQ ID NO: 4, it can be determined to belong to the same bacterial species (i.e., to belong to the species Lactobacillus crispatus).

[0159] A "strain" refers to 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. The genetic characteristics can be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence of at least one native plasmid ("cure"), 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., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. The genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case where a strain (as compared to another strain of the same species) gains or loses antibiotic resistance or gains or loses biosynthetic capabilities (e.g., an auxotrophic strain), the strain or the nutrient / metabolite can be distinguished by selection or counter-selection using the antibiotic.

[0160] "Supernatant" or "supernatant fluid" within the meaning herein refers to the culture supernatant of a bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0161] Compositions can be prepared using the Lactobacillaceae strains described herein, for example by using pharmaceutically acceptable excipients. The pharmaceutical compositions comprise a pharmaceutically effective amount of the Lactobacillaceae strain, for example Lacticaseibacillus paracasei having accession number GDMCC No: 65552, or Lactobacillus crispatus having accession number GDMCC No: 65556. Likewise, Lacticaseibacillus paracasei having accession number GDMCC No: 65552, or Lactobacillus crispatus having accession number GDMCC No: 65556 can be prepared into pharmaceutical compositions, for example by using pharmaceutically acceptable excipients, comprising a pharmaceutically effective amount of the Lactobacillaceae strain.

[0162] Suitable pharmaceutically acceptable excipients that can be used are, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0163] The compositions herein can be formulated in any form suitable for enhancing the abundance of Lactobacillaceae strains in a subject. The compositions can be administered by oral administration (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, gastrointestinal, biliary perfusion, cardiac perfusion, pre-anal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transdermal, ureteral, or urethral routes, among others.

[0164] Examples of dosage forms in which the compositions herein are suitable include, but are not limited to, tablets, aerosols, chewable bars, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, concentrates.

[0165] In some embodiments, the composition is a sugar-coated tablet, a gel capsule, a gel, an emulsion, a tablet, a tablet 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, among others.

[0166] In some embodiments, administration can also be by inclusion in the subject’s diet, for example in a functional food for a human or a companion animal.

[0167] The compositions provided herein can comprise a pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

[0168] In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is lyophilized. 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 lyophilized or spray-dried and is viable. In some embodiments, the Lactobacillaceae strain in the composition of the present disclosure is lyophilized or spray-dried and is able to partially or completely colonize the intestine. In some embodiments, the lyophilized Lactobacillaceae strain is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0169] In some embodiments, the composition of the present disclosure is administered orally. Oral administration can involve swallowing, so that the composition enters the gastrointestinal tract, and / or administration through the buccal cavity, tongue or sublingually.

[0170] In some embodiments, the composition is prepared by freeze-drying, or spray-drying.

[0171] The composition of the present disclosure includes a pharmaceutical, nutraceutical, or food product.

[0172] The subject of the present disclosure can be a human or an animal, including but not limited to a cow, a sheep, a cat, a dog, a horse, a rabbit, a monkey, a mouse, a rat, an alpaca, a camel, etc.

[0173] In 2020, Zheng et al. proposed to reclassify the genus Lactobacillus into 25 genera, including Lactobacillus and 23 new genera, based on phylogenomic analysis of the core genes of 261 Lactobacillus species, average nucleotide identity (ANI) comparison between species, analysis of characteristic genes, and physiological criteria and ecological analysis of species. They also suggested that one genus in the family Leuconostocaceae should be divided into the family Lactobacillaceae. According to this study, Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. tolerans, Lactobacillus rhamnosus, and Lactobacillus chiayiensis in the Lactobacillus casei group were renamed as Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lacticaseibacillus paracasei subsp. tolerans, Lacticaseibacillus rhamnosus, and Lacticaseibacillus chiayiensis, respectively. The genus Lactobacillus contains many important probiotic resources, and the change in its classification may affect the update of regulations related to microorganisms in various industries and the application of species.

[0174] The schemes of the present application will be explained below in connection with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the application. If specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the field or according to the product or instrument instructions. If the manufacturer of a reagent or instrument is specified, it can be purchased on the market.

[0175] Examples

[0176] The anaerobic blood agar plates involved in the examples were purchased from HuanKai Microbiology, and the formula was as follows: casein enzymatic digest 10 g / L, heart-pancreatic enzymatic digest 3 g / L, corn starch 1 g / L, meat-peptone enzymatic digest 5 g / L, yeast extract powder 5 g / L, sodium chloride 5 g / L, agar 15 g / L, sterile defibrinated sheep blood 50-100 mL / L, pH 7.3±0.2.

[0177] The PYG liquid culture medium involved in the examples contains the following per 1 LPYG: 5.0 g Trypticasepeptone, 5.0 g Peptone, 10.0 g Yeast extract, 5.0 g Beef extract, 5.0 g Glucose, 2.0 g K2HPO4, 1.0 ml Tween 80, 1.0 mg Resazurin, 40 ml Salt solution (composition as follows: 0.01 g CaCl2·2H2O, 0.02 g MgSO4·7H2O, 0.04 g K2HPO4, 0.4 g NaHCO3, 0.08 g NaCl, water balance), pH 7.2 ± 0.2 (25°C).

[0178] The MRS plate culture medium mentioned in the examples contains, per 1 LMRS plate: 10.0 g peptone, beef extract powder

[0179] 10.0g yeast extract, 5.0g glucose, 20.0g Tween-80, 1.0mL dipotassium hydrogen phosphate, 2.0g sodium acetate, 2.0g triammonium citrate, 0.1g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), 15.0g agar, water balance.

[0180] The MRS liquid culture medium mentioned in the examples contains, per 1 LMRS liquid culture medium: 10.0 g peptone, beef extract powder

[0181] 10.0g yeast extract powder, 5.0g glucose, 20.0g Tween-80, 1.0mL dipotassium hydrogen phosphate, 2.0g sodium acetate, 2.0g triammonium citrate, 0.1g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), water balance.

[0182] The above-mentioned culture medium can be prepared using 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 strains of intestinal bacteria, Lacticaseibacillus paracasei MNH06277 and Lactobacillus crispatus MNH45330, were isolated from the fecal samples of healthy volunteers. The isolation method used a conventional strain isolation method using gradient dilution, followed by single colony picking after strain purification and anaerobic culture at 37°C. The pure culture strain was prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0186] Specifically, the isolation and purification method of the strain is as follows:

[0187] The donor took 2-5g of fresh feces, placed it in a sample collection and preservation tube, and homogenized it. The treated fecal sample was then placed in an ice box and delivered to the laboratory within 24 hours for strain isolation.

[0188] Physiological saline was dispensed in a biological safety cabinet, 9mL / tube; strain isolation medium anaerobic blood agar plates were prepared and transferred into the anaerobic workstation 24h in advance, labeled with sample information, medium type, isolation date, etc.

[0189] Fresh fecal samples were taken and placed in an anaerobic workstation using a vortex shaker for 1min, mixed, and 1mL of sample was taken into 9mL of physiological saline, mixed to 10 -1 dilution, then gradient diluted to 10 -6 dilution, ready for use.

[0190] Take 10 -6 dilution and drop it on the anaerobic blood agar plate, drop the amount of 100μL / dish, evenly spread, after the surface of the plate is dry, the plate is inverted and cultured at 37°C for 3-5 days.

[0191] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick single colonies with a sterile toothpick for strain purification. The purified strain was cultured anaerobically at 37°C. The pure culture strain was prepared into a 20% glycerol / water-bacteria solution and stored 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 on MRS plate medium, and visible colonies were formed on MRS plate medium after anaerobic incubation at 37°C for 48 h. The colonies were round, regular and smooth in edge, white and opaque, with a diameter of about 1-2 mm. The strain was gram-positive. Microscopic observation showed that the strain was non-motile, non-flagellated, and rod-shaped, with a size of about 0.5-1 μm x 1.5-3 μm. The colony morphology of strain MNH 06277 on MRS plate medium after incubation for 48 h is shown in Figure 1 . The gram staining and electron microscopic photographs of strain MNH06277 are shown in Figure 2 and Figure 3 , respectively.

[0195] 1.2.2 Morphological characteristics of strain MNH45330

[0196] Strain MNH45330 was inoculated on MRS plate medium, and visible colonies were formed on MRS plate medium after anaerobic incubation at 37°C for 48 h. The colonies were round, regular and smooth in edge, white and opaque, with a diameter of about 0.5-2 mm. The strain was gram-positive. Microscopic observation showed that the strain was non-motile, non-flagellated, and rod-shaped, with a size of about 0.5-1 μm x 1.5-4 μm. The colony morphology of strain MNH 45330 on MRS plate medium after incubation for 48 h is shown in Figure 4 . The gram staining and electron microscopic photographs of strain MNH45330 are shown in Figure 5 and Figure 6 , respectively.

[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 grew under aerobic and anaerobic conditions. It could grow in a pH range of 4.0 to 10.0, with an optimal growth pH of 6.0 (the results of strain tolerance to different pH are shown in Figure 7 ). It could grow in a medium with a NaCl content of more than 6% (the results of strain tolerance to different concentrations of NaCl are shown in Figure 8 ). Strain MNH06277 could not grow in a medium with a bile salt concentration of more than 0.1% (the results of strain tolerance to different concentrations of bile salt are shown in Figure 9 ).

[0200] 1.3.2 Physiological and biochemical characteristics of strain MNH45330

[0201] Strain MNH45330 could grow in a pH range of 4.0 to 10.0, with an optimal growth pH of 8.0 (the results of strain tolerance to different pH are shown in Figure 10) ; still grow on medium with NaCl content over 4% (see results of tolerance of strains to different concentrations of NaCl in Table 1 Figure 11 ) ; strain MNH45330 is unable to grow on medium with bile salt concentration over 0.1% (see results of tolerance of strains to different concentrations of bile salt in Table 1 Figure 12 ).

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

[0203] 1.4.1 Biochemical identification results of strain MNH06277 by API 50CHL

[0204] API 50CHL (Merieux, CN5041010) was used, and the specific experimental operation was according to the routine API reagent operation instruction. The culture condition was 37°C, anaerobic. The experimental results are shown in Table 1.

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

[0206] Table 1. Test results of strain MNH06277 MNH06277

[0207] 0 1 2 3 4 5 6 7 8 9 CTRL GLY ERY DARA LARA RIB DXYL LXYL ADO MDX - - - - - + - - - - 10 11 12 13 14 15 16 17 18 19 GAL GLU FRU MNE SBE RHA DUL INO MAN SOR + + + + + - - - + + 20 21 22 23 24 25 26 27 28 29 MDM MDG NAG AMY ARB ESC SAL CEL MAL LAC - - + + + + + + + + 30 31 32 33 34 35 36 37 38 39 MEL SAC TRE INU MLZ RAF AMD GLYG XLT GEN - + + + + - - - - + 40 41 42 43 44 45 46 47 48 49 TUR LYX TAG DFUC LFUC DARL LARL GNT 2KG 5KG + - + - - - + + - -

[0208] Note: "+" represents positive, "-" represents negative.

[0209] 1.4.2 Biochemical identification results of strain MNH45330 by API 50CHL

[0210] API 50CHL (bioMerieux, CN5041010) was used according to the manufacturer's instructions. Incubation conditions: 37°C, anaerobic. The results are shown in Table 2.

[0211] MNH45330 is capable of fermenting galactose GAL, glucose GLU, fructose FRU, mannose MNE, mannitol MAN, a-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, aesculin ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melanzoese MLZ, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, D-lyxose LYX to produce acid. Thus, during fermentation or cultivation of the strain MNH45330, galactose GAL, glucose GLU, fructose FRU, mannose MNE, mannitol MAN, a-methyl-D-glucoside MDG, N-acetyl-glucosamine NAG, amygdalin AMY, arbutin ARB, aesculin ESC, salicin SAL, cellobiose CEL, maltose MAL, lactose LAC, melibiose MEL, sucrose SAC, trehalose TRE, inulin INU, melanzoese MLZ, raffinose RAF, starch AMD, gentiobiose GEN, D-turanose TUR, D-lyxose LYX, and derivatives thereof can be used as carbon sources.

[0212] Table 2. Test results for strain MNH45330 MNH45330

[0213] 0 1 2 3 4 5 6 7 8 9 CTRL GLY ERY DARA LARA RIB DXYL LXYL ADO MDX - - - - - - - - - - 10 11 12 13 14 15 16 17 18 19 GAL GLU FRU MNE SBE RHA DUL INO MAN SOR + + + + - - - - + - 20 21 22 23 24 25 26 27 28 29 MDM MDG NAG AMY ARB ESC SAL CEL MAL LAC - + + + + + + + + + 30 31 32 33 34 35 36 37 38 39 MEL SAC TRE INU MLZ RAF AMD GLYG XLT GEN + + + + + + + - - + 40 41 42 43 44 45 46 47 48 49 TUR LYX TAG DFUC LFUC DARL LARL GNT 2KG 5KG + + - - - - - - - -

[0214] Note: "+" means positive, "-" means 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 antibiotic minimum inhibitory concentration of strain MNH06277 was determined using E-test (Liofilchem) strips. The results are shown in Table 3.

[0218] Table 3. Antibiotic minimum inhibitory concentration test results for strain MNH06277

[0219] Antibiotic Minimum inhibitory concentration (mg / L) Imipenem (I / R) 3.30 Ampicillin Sulbactam (SAM) 1.50 Doripenem (DOR) 3.50 Amoxicillin (AMC) 1.00 Rifampicin (RD) 0.25 Meropenem (MRP) 12.00 Cefquinome (CZX) 24.00 Ceftriaxone (CRO) 0.35 Imipenem (I / R) 3.30 Ampicillin Sulbactam (SAM) 1.50

[0220] The results show that MNH06277 is resistant to imipenem (I / R), ampicillin sulbactam (SAM), doripenem (DOR)

[0221] Amoxicillin (AMC), Rifampicin (RD), Ceftriaxone (CRO) sensitive. It can be seen that MNH06277 is sensitive to most types of antibiotics, and the risk of long-term use of MNH06277 leading to the subject developing antibiotic resistance is low.

[0222] 1.5.2 Antibiotic minimum inhibitory concentration test of strain MNH45330

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

[0224] Table 4. Test results of antibiotic minimum inhibitory concentration of strain MNH45330

[0225]

[0226]

[0227] The results show that MNH45330 is sensitive to imipenem, ampicillin sulbactam, doripenem, ertapenem, meropenem, piperacillin-tazobactam, and ceftriaxone. It can be seen that MNH45330 is sensitive to most types of antibiotics, and the risk of long-term use of MNH45330 leading to the subject developing antibiotic resistance is low.

[0228] 1.6 Identification of strains MNH06277 and MNH45330

[0229] 1.6.1 16S rRNA gene amplification of strains MNH06277 and MNH45330

[0230] Fresh cultures of strains MNH06277 and MNH45330 were taken for extraction of strain genomic DNA. The extracted strain genomic DNA was used as a template for 16S rRNA gene amplification.

[0231] The primer pair used for 16S rRNA gene PCR was:

[0232] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0233] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0234] The PCR reaction program was as follows:

[0235] 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.

[0236] 1.6.2 16S rRNA gene sequencing of strains MNH06277 and MNH45330

[0237] The PCR products were purified and 16S rRNA gene sequencing was performed by Shenguo Company, and the 16S rRNA gene was obtained.

[0238]

[0239]

[0240] 1.6.3 Identification results of strains MNH06277 and MNH45330

[0241] The measured 16S rRNA gene sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4 was analyzed by NCBI Basic Local Alignment Search Tool for strain 16S rRNA gene analysis to confirm the strain classification information.

[0242] The measured sequence was analyzed by BLAST with the data in GenBank, and the alignment result showed that the strain with the highest similarity to MNH06277 was Lacticaseibacillus paracasei with a similarity of 100%, so the strain MNH06277 was a strain under the species of Lacticaseibacillus paracasei; the strain with the highest similarity to MNH45330 was Lactobacillus crispatus with a similarity of 100%, so the strain MNH45330 was a strain under the species of Lactobacillus crispatus.

[0243] MNH06277 and MNH45330 were compared with the 16S rRNA gene sequences of related strains of Lacticaseibacillus paracasei and Lactobacillus crispatus retrieved from databases such as GenBank to construct phylogenetic trees.

[0244] MNH06277 and MNH45330 and the sequences of the 16S rRNA gene sequence of the strain with high similarity in the NCBI database were subjected to multiple sequence alignment, and then the software MEGA 5 was used to construct a phylogenetic tree (the maximum likelihood method was used to construct a phylogenetic tree), Figure 13 、 14 The phylogenetic tree node only shows the Bootstrap value greater than 50% value.

[0245] From the phylogenetic tree, Figure 13 、 14)It can be seen that strain MNH06277 is clustered with Lacticaseibacillus paracasei of the genus Lacticaseibacillus, and is a new strain under the species Lacticaseibacillus paracasei. Strain MNH45330 is clustered with Lactobacillus crispatus of the genus Lactobacillus, and is a new strain under the species Lactobacillus crispatus.

[0246] The genomes of MNH06277 and MNH45330 were extracted, sequenced, and then detected by the gtdb method. The strain with the highest similarity to the genome of MNH06277 is Lacticaseibacillus paracasei (GCF_000829035.1), with an average nucleotide similarity (ANI) of 98.2% and an alignment fraction (AF) of 88%. Therefore, MNH06277 can be identified as Lacticaseibacillus paracasei. The strain with the highest similarity to the genome of MNH45330 is Lactobacillus crispatus (GCF_002088015.1), with an average nucleotide similarity (ANI) of 97.4% and an alignment fraction (AF) of 86%. Therefore, MNH45330 can be identified as Lactobacillus crispatus.

[0247] 1.7 Genome analysis of strains MNH06277 and MNH45330

[0248] 1.7.1 Genome analysis of strain MNH06277

[0249] The genome of strain MNH06277 was fragmented by ultrasonic method, with a fragmentation length range of ~ 350 bp, and then an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was subjected to double-end 150 bp sequencing using NovaSeq (Illumina). The sequencing obtained 0.89 Gbp data, of which the Q20 accounted for 94.82%.

[0250] The raw sequencing data of the genome was 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 was used for genome assembly using SPAdes (version: v3.14.0) with the assembly parameters “--isolate --cov-cutoff 10”. The genome assembly resulted in a total length of 2.90Mbp of genes, with an N50 length of 196.8kbp, and a GC content of 46.43%.

[0251] The genome genes were analyzed for genome gene prediction 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 909bp.

[0252] The potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2) with the antibiotic resistance gene database CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found.

[0253] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to align 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 alignment results are shown in Table 5.

[0254] Table 5. List of potential virulence genes of MNH06277

[0255] Strain gene VFDB gene Gene name Alignment identity (%) MNH06277_00263 VFG037100 pilB 60.145 MNH06277_00317 VFG002190 cpsA 63.71 MNH06277_00416 VFG048830 gnd 67.308 MNH06277_00497 VFG000080 clpE 64.364 MNH06277_00709 VFG000077 clpP 69.744 MNH06277_00840 VFG000964 hasC 70.134 MNH06277_01569 VFG006717 lap 61.124 MNH06277_01630 VFG000079 clpC 61.063 MNH06277_01753 VFG002165 efaA 60.993

[0256] 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 6.

[0257] Table 6. List of potential primary metabolic gene clusters of MNH06277

[0258]

[0259] 1.7.2 Genome analysis of strain MNH 45330

[0260] The genome of strain MNH 45330 was sequenced by ultrasonic fragmentation, with a fragment length range of ~350 bp, and then an Illumina sequencing library was constructed using a standard DNA library kit (NEB UltraTM). The constructed sequencing library was sequenced by NovaSeq (Illumina) with a double-end 150 bp sequencing. The sequencing obtained 1.43 Gbp data, of which Q20 accounted for 95.33%.

[0261] The raw genome sequencing data was filtered using fastp (version: 0.20.0), with filtering parameters: “--poly_g_min_len 10--poly_x_min_len 10-q 15-u 40-n 5-l 50”. The filtered raw data was used for genome assembly using SPAdes (version: v3.14.0), with assembly parameters “--isolate--cov-cutoff10”. The genome assembly obtained a total gene length of 2.45 Mbp, with an N50 length of 43.3 kbp and a GC content of 36.51%.

[0262] The genome genes were analyzed for genome gene prediction using the prokka (version: 1.14.5) of prokaryotic analysis software, with parameters “--gcode 11--evalue 1e-09”. A total of 2468 CDS sequences were predicted, with an average CDS sequence length of 870 bp.

[0263] The potential antibiotic resistance genes in the genome were analyzed using the RGI process (version: 4.2.2), with the antibiotic resistance gene database being CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). No resistance genes were found.

[0264] The analysis of potential virulence factors and related genes in the genome used NCBI blastp (version: 2.7.1+) to align 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 alignment results are shown in Table 7.

[0265] Table 7. List of potential virulence genes of MNH45330

[0266] Strain gene VFDB gene Gene name Alignment identity (%) MNH45330_00056 VFG000964 hasC 67.458 MNH45330_00120 VFG000077 clpP 69.634 MNH45330_01304 VFG002182 cpsI 65.312 MNH45330_01839 VFG037100 pilB 61.268

[0267] The analysis of potential primary metabolic gene clusters in the genome used gutSMASH5 (version: 1.0.0). The detailed comparison results are shown in Table 8.

[0268] Table 8. MNH45330 potential primary metabolic gene cluster list

[0269]

[0270] Example 2: Strains MNH06277 and MNH45330 promote growth and development in a low-fat low-protein diet-induced growth and development retardation mouse model

[0271] MNH06277 and MNH45330 were used to improve the growth and development index in a low-fat low-protein diet-induced growth and development retardation mouse model. The present application has been ethically reviewed by the Muen Biological Animal Ethics Committee.

[0272] 2.1 Experimental method

[0273] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 3 weeks old, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.

[0274] 2) Preparation of MNH06277 and MNH45330 test substances: After thawing the glycerol stock tube of MNH06277 and MNH45330 strains at 37°C, inoculate in MRS plate culture medium in an anaerobic workstation, activate, inoculate the activated strain in MRS liquid culture medium, anaerobically culture, obtain sufficient amount of culture, centrifuge the cultured bacterial liquid, concentrate, resuspend the bacterial body with PBS containing 25% glycerol and 0.05% L-Cys HCl, obtain the test substance with purity and viable bacterial count (2×10 9 CFU / mL) meeting the requirements of animal experiment.

[0275] 3) Negative control: PBS containing 25% glycerol and 0.05% L-Cys HCl was used as the negative control.

[0276] 4) Positive control: Bifidobacterium lactis CGMCC No. 20847, after thawing the glycerol stock tube of Bifidobacterium lactis CGMCC No. 20847 (which has been disclosed in patent CN112980725B) at 37°C, inoculate in PYG culture medium in an anaerobic workstation, activate, inoculate the activated strain in PYG liquid culture medium, anaerobically culture, obtain sufficient amount of culture, centrifuge the cultured bacterial liquid, concentrate, resuspend the bacterial body with PBS containing 25% glycerol and 0.05% L-Cys HCl, obtain the positive control with purity and viable bacterial count (2×10 9 CFU / mL) meeting the requirements of animal experiment.

[0277] 5) Experimental Procedure: After the quarantine period, 40 male C57BL / 6J mice weighing between 13g and 16g were selected at 3 weeks of age and randomly stratified into 5 groups of 8 mice each: NCD-Control group, LFD-Control group, MNH06277 group, MNH45330 group, and positive control group. The NCD-Control group was fed a maintenance diet (product number: PD24043001, Changzhou Shuyi Shuer Biotechnology Co., Ltd.), while the other four groups were fed a low-fat, low-protein diet (product number: PD24043003, Changzhou Shuyi Shuer Biotechnology Co., Ltd.). After grouping (D1), drug administration began. The NCD-Control and LFD-Control groups received negative controls, the positive control group received positive controls via gavage, the MNH06277 group received MNH06277 via gavage, and the MNH45330 group received MNH45330 via gavage. Drug administration was once daily, 200 μL each time, for a total of 35 days. Mice had free access to water and food during the experiment, using a 12h / 12h diurnal cycle. A routine clinical observation was performed after each administration period. The endpoint of this experiment was the day after the end of administration (D36). Dissection and tissue sampling were performed according to the protocol to reach the endpoint. Data from each dissection and serum testing were summarized and analyzed. All data are expressed as Mean ± SD and plotted and statistically analyzed using GraphPad Prism software. Student's t-test was used for pairwise comparisons. No significance indicates no difference; significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0278] Food intake is determined by weighing the remaining amount of feed.

[0279] The weight of internal organs such as the liver and spleen, the length of the large intestine, and the length and weight of various bones and muscles are measured through anatomical sampling.

[0280] Method for detecting serum insulin-like growth factor 1: Mouse serum was collected and detected using the Mouse IGF-1 ELISA Development Kit (PeproTech).

[0281] For the immune cell detection method, 0.1g of mouse spleen tissue was taken, washed with PBS, and then the tissue was pulverized. The pulverized spleen tissue was suspended in PBS and filtered through a 70μm filter membrane to prepare a mouse spleen single-cell suspension. A small amount of the prepared mouse spleen single-cell suspension was stained and then analyzed by flow cytometry to accurately count the number of mouse spleen immune cells.

[0282] The method for determining the content of short-chain fatty acids in cecal contents is as follows: about 100 mg of cecal content sample is weighed and placed in a 2 ml EP tube, 1 ml of ether is added, and vortexed immediately, ultrasonic extraction is performed for 10 min, and the precipitate is removed by centrifugation for 10 min. The supernatant is passed through a 0.22 micron organic filter membrane, and the supernatant is transferred to a gas phase vial, wrapped with a sealing film and stored in a -20°C refrigerator, and then determined. DB-FFAP column and mass spectrometry detector are used for separation and detection of each short-chain fatty acid.

[0283] 2.2 Experimental results

[0284] See Figure 15-17 , the results show that MNH06277 and MNH45330 can significantly increase the length of the large intestine, the weight of the spleen and the kidney of the growth retardation mice, indicating that MNH06277 and MNH45330 can promote the development of the spleen, kidney and / or intestine, and have the use of promoting the development of organs.

[0285] See Figure 18-23 , the results show that MNH06277 and MNH45330 can significantly promote the weight of the body, the length of the body, the length of the tibia, the length of the femur, the weight of the soleus muscle and / or the gastrocnemius muscle of the low-fat low-protein diet-induced growth retardation mouse model, indicating that MNH06277 and MNH45330 have the use of promoting the development of height (body length), body weight, bone and / or muscle.

[0286] See Figure 24 , the results show that MNH06277 can significantly increase the content of serum insulin-like growth factor 1 (IGF-1) of the low-fat low-protein diet-induced growth retardation mouse model, indicating that MNH06277 has the use of promoting growth and development.

[0287] See Figure 25-26 , the results show that MNH06277 and MNH45330 do not increase food intake, indicating that MNH06277 and MNH45330 have the use of promoting digestion and absorption when promoting growth and development.

[0288] See Figure 27-29 , the results show that MNH06277 and MNH45330 can significantly promote the development of the thymus, spleen and other major immune organs of the low-fat low-protein diet-induced growth retardation mouse model; MNH45330 can increase the proportion of T cells; indicating that MNH06277 and MNH45330 have the use of promoting the development of the immune system and enhancing immunity.

[0289] See Figure 30-34, the results show that MNH45330 can promote the production of short-chain fatty acids in the cecum of the low-fat low-protein diet-induced growth and development retardation mouse model, significantly promote the production of propionic acid, isobutyric acid, 2-methylbutyric acid, and isovaleric acid; MNH06277 can increase the proportion of butyric acid in total short-chain fatty acids. It is shown that MNH06277 and / or MNH45330 can promote the production of short-chain fatty acids and have the effect of promoting intestinal health.

[0290] While the application has been disclosed with reference to certain implementations, it is apparent that modifications and changes can be made without departing from the spirit and scope of the application as disclosed herein and as provided in the following claims. Further, it is to be understood that even though embodiments have been disclosed in the context of certain implementations, other implementations will be apparent to those skilled in the art and are intended to be within the scope of the application, which is to be limited solely by the language of the appended claims. The drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

Claims

1. A type of *Lactobacillus paracasei*, characterized in that, The preservation number of Lacticaseibacillus paracasei MNH06277 is GDMCC No: 65552.

2. A probiotic or prebiotic containing Lacticaseibacillus paracasei, wherein the Lacticaseibacillus paracasei MNH06277 has the accession number GDMCC No: 65552.

3. The probiotics or prebiotics as described in claim 2, characterized in that, The probiotics or prebiotics may also contain one or more other active agents that promote growth and development, enhance immunity, or promote gut health.

4. The probiotics or prebiotics as described in claim 3, characterized in that, The probiotics or prebiotics also include any one of the following: food additives, beverage additives, and dietary supplements.

5. The use of Lactobacillus paracasei as described in claim 1 in the preparation of health products that enhance immunity.

6. The use of Lactobacillus paracasei as described in claim 1 in the preparation of probiotics or prebiotics that promote growth and development or enhance immunity.

7. The use of any one of the probiotics or prebiotics according to claims 2-4 in the preparation of products that promote growth and development or products that enhance immunity.

8. The application according to claim 6 or 7, characterized in that, The growth and development promotion includes promoting at least one of the following: organ, height, weight, bone, and muscle development; wherein, the organ development promotion includes at least one of the thymus, spleen, kidney, and intestine; the bone includes at least one of the femur and tibia; and the muscle includes at least one of the soleus and gastrocnemius muscles.

9. The application according to claim 6 or 7, characterized in that, The growth and development promotion is selected from at least one of the following: (1) promoting growth and development by improving digestion and absorption; (2) enhancing immunity by promoting immune system development; (3) promoting intestinal health by increasing short-chain fatty acids; (4) promoting growth and development by increasing hormone content.

10. The application according to claim 9, characterized in that, The promotion of immune system development includes at least one of promoting the development of immune organs and promoting the generation of immune cells, wherein the immune cells include T cells; and the hormone includes insulin-like growth factor 1.

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