Composition with functions of improving spleen deficiency and promoting growth of children and application

Abstract

The invention provides a composition with functions of improving spleen deficiency and promoting growth of children and application, the composition comprises 5-20 parts of bovine spleen peptide, 5-20 parts of hawthorn fruit powder, 5-15 parts of Chinese yam and 1-10 parts of radish seed powder, and the composition is prepared by fermentation of plant lactobacillus PPL19. The composition has the characteristic of improving the intestinal absorption rate by improving the permeability of the fluorescein sodium; secondly, the intestinal mucosa permeability problem of the mouse with spleen deficiency is improved by adjusting expression of an ERK / MLCK signal channel mediated intestinal tight junction gene; in addition, by improving MTL and SP levels and reducing VIP level, gastric emptying and gastrointestinal motility are promoted so as to improve spleen and stomach functions; finally, the composition can efficiently retain a receptor agonist similar to GPR109A, improve the level of IGF-1 and OCN, improve the weight, body length, femoral length, bone weight and bone density of the spleen-deficiency mice, and effectively promote the growth and development of the spleen-deficiency mice. The composition is simple in preparation method and can be produced on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of probiotics, specifically relating to compositions and applications that improve spleen deficiency and promote children's growth. Background Technology

[0002] The incidence of picky eating among children in my country exceeds 30%. Long-term picky eating weakens the spleen and stomach's digestive function, leading to an imbalance in the intestinal microecology. This causes a sharp drop in the absorption efficiency of key nutrients for bones, forming a vicious cycle of "picky eating → weak spleen and stomach → poor intestinal absorption → slow bone development." It can even lead to serious bone development problems such as growth retardation (height 5cm or more shorter than peers), frequent growing pains (night awakenings or bone pain after exercise ≥3 times per week), and premature closure of the epiphyseal cartilage, causing irreversible effects on children's lifelong growth.

[0003] Current technological solutions for children's bone development problems have significant limitations, making it difficult to break the vicious cycle described above. In the field of gastrointestinal digestion, existing products mostly rely on single food-medicine homologous ingredients such as hawthorn and chicken gizzard lining. While these can promote digestive enzyme secretion to some extent, they lack systematic regulation of the spleen and stomach's digestive function. Furthermore, due to the lack of biotransformation processing, the utilization rate of active ingredients (such as hawthorn flavonoids and chicken gizzard lining protease) is insufficient, and they also suffer from problems such as a sour and astringent taste and poor child compliance. In the field of bone nutrition supplementation, products mostly focus on single-pathway interventions, such as simply supplementing calcium or vitamin D, but neglect the synergistic needs of "nutrient absorption-transportation-bone deposition"—for example, effective calcium deposition depends on the guidance of vitamin K2, and existing products often lack such synergistic ingredients, resulting in supplementation without absorption or absorption without utilization. In addition, although probiotic preparations can regulate the intestinal flora, they only remain at the level of improving the intestinal microenvironment and cannot connect the enhancement of spleen and stomach function with the regulation of bone synthesis, making it difficult to overcome the deep barrier of "intestinal and spleen deficiency leading to nutrient conversion disorders and insufficient bone cell activity."

[0004] Therefore, it is urgent to develop an innovative technical solution with multi-component synergy and high utilization rate of active ingredients. This solution needs to simultaneously achieve gastrointestinal health to enhance nutrient conversion capacity, regulate intestinal microecology to improve absorption efficiency, and activate bone metabolic pathways to promote osteoblast function. Through multi-dimensional synergistic regulation of "spleen-stomach-intestine-bone", it breaks through the limitations of single intervention in traditional technologies and achieves integrated regulation of height development and gastrointestinal health from the root.

[0005] The invention patent with publication number CN 119999917 A discloses a composition and preparation method for promoting children's bone growth and physical development. The composition contains a variety of nutrients and traditional Chinese medicine ingredients, including fructooligosaccharides, concentrated whey protein, bovine bone collagen peptides, hawthorn, malt, astragalus, red dates, oysters, jujube seeds, γ-aminobutyric acid, calcium citrate, calcium gluconate, and citric acid. These ingredients have the effects of promoting bone health, enhancing immunity, improving nutrient absorption and regulating metabolism, improving sleep quality, and promoting growth hormone secretion, which together promote healthy bone growth and overall physical development.

[0006] Chinese patent application CN 118077899 A discloses a composition, a solid beverage, and a preparation method for regulating children's spleen and stomach and / or improving their intestinal flora. This invention selects hawthorn, malt, chicken gizzard lining, yam, tangerine peel, white hyacinth bean, and poria cocos, all listed by the National Health Commission as having medicinal and edible properties, as the main raw materials. The combined use of these ingredients can strengthen the spleen and stomach, promote digestion, and replenish vital energy. Probiotics can regulate and maintain the balance of intestinal flora and promote gastrointestinal motility, thereby promoting digestion. Through the internal regulation of both, the digestive organs of children are fundamentally regulated, ensuring their normal function and stabilizing children's growth and development. Summary of the Invention

[0007] Based on the above background, the first objective of this invention is to provide a composition that improves spleen deficiency and promotes children's growth. The second objective of this invention is to provide a composition that improves spleen deficiency and promotes children's growth, and its application and preparation method.

[0008] The third objective of this invention is to provide an application of a composition that improves spleen deficiency and promotes children's growth.

[0009] This invention is achieved through the following technical solution:

[0010] A composition that improves spleen deficiency and promotes children's growth is obtained by fermentation with Lactobacillus plantarum PPL19, comprising 5-20 parts by weight of bovine spleen peptide, 5-20 parts by weight of hawthorn powder, 5-15 parts by weight of yam and 1-10 parts by weight of radish seed powder.

[0011] The molecular weight of the bovine spleen peptide is 3000-8000 Da.

[0012] The preparation method of the composition for improving spleen deficiency and promoting children's growth includes the following steps:

[0013] Bovine spleen peptides, hawthorn powder, yam and radish seed powder are mixed, water is added, and the mixture is sterilized to obtain fermentation raw materials;

[0014] Inoculate the fermentation raw material with a bacterial culture of *Lactobacillus plantarum* PPL19, centrifuge after fermentation, and collect the supernatant.

[0015] The ratio of the fermentation raw material to water is 1g:(5-10mL);

[0016] The sterilization temperature is 115℃ and the sterilization time is 20 minutes.

[0017] The viable count of the bacterial solution is 10. 7 CFU / mL;

[0018] The fermentation temperature is 32-37℃, the fermentation time is 12-24 hours, and the rotation speed is 50-100 r / min.

[0019] The centrifuge speed is 3000-5000 r / min;

[0020] Centrifugation time is 5 to 10 minutes.

[0021] The application of the composition having the functions of improving spleen deficiency and promoting children's growth is in the preparation of food or medicine that improves intestinal absorption; or

[0022] Foods or medicines used to improve intestinal mucosal permeability in spleen-deficient mice; or

[0023] It can be applied to the preparation of food or medicine that improves intestinal mucosal permeability in spleen-deficient mice by regulating the expression of intestinal tight junction genes mediated by the ERK / MLCK signaling pathway.

[0024] It can be used to prepare food or medicine that enhances the body weight, body length, femur length, bone weight, and bone density of mice with spleen deficiency;

[0025] It can be used to prepare foods or medicines that promote bone growth by increasing the levels of IGF-1 and OCN.

[0026] This invention can be applied to the preparation of foods or pharmaceuticals that promote growth and development by efficiently retaining the effective components of GPR109A receptor agonists; compared to existing technologies, the advantages of this invention are as follows:

[0027] The composition provided by this invention can increase the expression levels of Occludin and ZO-1 genes, repair the tight junction structure of intestinal epithelial cells, and maintain the integrity of the intestinal mucosal barrier.

[0028] The composition provided by this invention can improve intestinal mucosal permeability in spleen-deficient mice by regulating the expression of intestinal tight junction genes mediated by the ERK / MLCK signaling pathway. This invention also provides a fermentation composition similar to a GPR109A receptor agonist, which can significantly increase the levels of bone growth regulator factor-1 (IGF-1) and osteocalcin (OCN) in mice, promoting growth and development. The fermentation technology significantly retains the effective components of the GPR109A receptor agonist in the composition. Attached Figure Description

[0029] Figure 1 Effects of different intervention groups on the expression of tight junction-related genes in rat intestinal tissue.

[0030] Figure 2 Effects of different intervention groups on the expression of ERK / MLCK pathway-related genes. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments, and the experimental methods for which specific conditions are not specified in the embodiments, are generally performed under conventional conditions. The materials and reagents used in the embodiments are commercially available unless otherwise specified.

[0032] This invention provides a *Lactobacillus plantarum* PPL19, which was screened from children's saliva and deposited on September 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32110. This bacterium is disclosed in Chinese invention patent application CN119752727A, entitled "*Lactobacillus plantarum* and its fermentation products, probiotic preparations for preventing dental caries".

[0033] Example 1

[0034] Raw material screening

[0035] Caco-2 cell monolayer model: Transmembrane resistance (TEER) analysis, alkaline phosphatase (AKP) analysis, and fluorescent yellow permeability analysis were used; when the TEER value was greater than 350 Ω·cm 2When the ratio of AKP activity in the upper chamber to that in the lower chamber of the Transwell cell is greater than 2 (i.e., APAKP / BLAKP>2) and the permeability of fluorescein is <3%, Caco-2 cells can be considered to have formed a dense, differentiated monolayer cell membrane with characteristics of intestinal epithelial cells. The monolayer model was used to assess whether the raw materials listed in Table 1 could promote intestinal absorption. The experimental group used the raw materials described in Table 1, and the control group used no raw materials. Hawthorn and other plant-based raw materials, as well as peptide raw materials, were all purchased commercially. The bacterial powder raw material was from Hunan Nutrition Tree Biotechnology Co., Ltd., with a viable bacteria count of 600 billion / g. Evaluation indicators: Caco-2 cell survival rate was used to assess the safety of the raw materials, and sodium fluorescein permeability was used to assess the absorption and utilization rate of the raw materials.

[0036] Table 1. Effects of different raw materials on Caco-2 cell viability

[0037]

[0038] According to Table 1, the ingredients that promote the proliferation of Caco-2 cells are bovine spleen peptide, hawthorn, radish seed, polygonatum, and Lactobacillus plantarum PPL19. Therefore, these ingredients were selected to evaluate the permeability of sodium fluorescein.

[0039] Sodium fluorescein permeability (P) app In Caco-2 cells, P primarily represents the ability of drugs or substances to cross the intestinal epithelial cell barrier. app The size of P is mainly related to the absorption rate in the body. app Less than 2×10 -6 cm / s represents low permeability, with an absorption rate between 0-20%; P app Greater than 2×10 -6 cm / s, less than 10 -5 At a speed of cm / s, it indicates moderate permeability, with an absorption rate between 20-70%. P app Greater than 10 -5 A value of cm / s indicates high permeability, with an absorption rate between 70-100%.

[0040] Table 2. Permeability of sodium fluorescein to different raw materials (P) app The impact of

[0041]

[0042] As shown in Table 2, the absorption rates of all raw materials are between 20% and 70%. In order to enhance the synergistic effect of the raw materials and improve their absorption rate, bio-fermentation technology is now widely used to improve their bioavailability. Therefore, the raw materials are combined and fermented.

[0043] Example 2

[0044] This embodiment provides a composition comprising, by weight, 5-20 parts of bovine spleen peptide, 5-20 parts of hawthorn powder, 5-15 parts of yam, and 1-10 parts of radish seed powder. The composition is obtained by fermenting the above raw materials with *Lactobacillus plantarum* PPL19.

[0045] The specific characteristics of the fermentation raw materials are as follows:

[0046] The bovine spleen peptide, with a molecular weight of 3000-8000 Da, is filtered through a 0.22 μm microporous membrane to remove large molecular impurities and is ready for use; the hawthorn is dried hawthorn that has been pitted, pulverized, and then filtered through a 60-80 mesh screen and is ready for use.

[0047] The radish seeds mentioned are dried radish seeds that have been baked at a low temperature of 60℃±2℃ for 2 hours (moisture content ≤8%), then pulverized to 80 mesh and set aside.

[0048] The yam mentioned is freeze-dried yam that has been directly pulverized and passed through a 60-80 mesh filter for later use;

[0049] Compound fermentation strain: Lactobacillus plantarum PPL19 is an internally produced bacterial powder with a live bacteria count of 1 billion CFU / g.

[0050] Bovine spleen peptides have the effect of nourishing the spleen and stomach and improving nutrient absorption. Hawthorn has the effect of promoting digestion and eliminating food stagnation. Yam has the effect of nourishing the spleen and stomach. Radish seed powder has the effect of regulating qi and relieving stagnation, and reducing gastrointestinal irritation.

[0051] The specific preparation method of the fermentation composition is as follows:

[0052] S1: Mixing and sterilization

[0053] Mixing: Weigh the pretreatment raw materials according to the composition formula and mix them evenly; add deionized water at a material-to-liquid ratio of 1:(5-10)(g:mL) and stir to form a homogeneous suspension;

[0054] Sterilization: Autoclave at 115℃ for 20 minutes, then cool to room temperature.

[0055] S2: Inoculation and Fermentation

[0056] Inoculation: The viable bacterial count in the inoculation solution should be 10. 7 The fermentation parameters were set as follows: CFU / mL, temperature 32-37℃, fermentation time 12-24h, and rotation speed 50-100r / min.

[0057] S3: Post-processing

[0058] The fermentation broth was centrifuged at 3000-5000 r / min for 5-10 minutes to obtain the supernatant, which is the desired substance.

[0059] To verify whether it has a synergistic effect, the active ingredients in its fermentation broth were determined, and the permeability of sodium fluorescein was evaluated using a Caco-2 monolayer cell model to assess the absorption rate of the fermentation composition.

[0060] Example 3

[0061] 1. Detection of active ingredient content

[0062] Amino acid composition analysis:

[0063] The raw materials before and after fermentation were prepared according to Example 2, wherein the raw materials before fermentation were uninoculated with microorganisms; the raw materials after fermentation were the fermentation supernatant. The above raw materials were analyzed using an amino acid analyzer, and the amino acid ratio was calculated.

[0064] Polyphenol determination: The total polyphenol content was determined by the Folin-Ciocalteu colorimetric method.

[0065] Flavonoid determination: The total flavonoid content was determined by the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method.

[0066] Polysaccharide determination: The polysaccharide content was determined using the sulfuric acid-phenol method.

[0067] Vitexin Determination: Liquid Chromatography

[0068] Groups: To compare the synergistic effect of the compositions, two comparative groups were set up.

[0069] Comparative Example 1: Following the method in Example 2, bovine spleen peptides were fermented at a material-to-liquid ratio of 1:(5-10)(g:mL);

[0070] Comparative Example 2: Following the method of Example 2, except for bovine spleen peptides, the other raw materials were fermented in the same proportion and with a material-to-liquid ratio of 1:(5-10)(g:mL);

[0071] Table 3. Determination of active ingredient content before and after fermentation

[0072]

[0073] Compared with the raw materials before fermentation, the composition obtained after fermentation showed a 50% increase in polyphenols, a 70.79% increase in flavonoids, a 107.20% increase in polysaccharides, and a 30.39% increase in vitexin. Furthermore, compared with comparative groups 1 and 2, the composition obtained after fermentation showed a significant increase in active ingredients. Literature reports that vitexin and flavonoids can regulate ion channels (such as Ca2+) on cell membranes. 2 It can enhance the activity of channels and nutrient transport proteins (such as glucose transporters), optimize the efficiency of transmembrane transport of substances, and improve the ability to absorb substances.

[0074] Compared to the raw materials before fermentation, the composition obtained after fermentation showed a significant increase in leucine and lysine content, with leucine increasing by 24.07% and lysine by 65.79%. Furthermore, compared to comparative groups 1 and 2, the composition obtained after fermentation exhibited a marked increase in lysine and leucine content. Literature reports that leucine and lysine, as amino acids, are raw materials for the synthesis of cell membrane proteins (including transport proteins), promoting the synthesis and repair of membrane transport proteins and indirectly enhancing the cell membrane's permeability and transport capacity for nutrients.

[0075] 2. Enzyme activity assay

[0076] The enzyme activity of each group was detected using protease, amylase, and cellulase activity kits.

[0077] Groups: Fermented composition, Comparative Example 1, Comparative Example 2

[0078] Table 4 Enzyme activity detection in different groups

[0079]

[0080] Compared with Comparative Examples 1 and 2, the composition obtained after fermentation of the raw materials significantly released the activities of strain protease, amylase, cellulase, and β-glucosidase. By promoting the catalytic activity of these enzymes, the release of small molecule compounds such as polyphenols and flavonoids was further promoted.

[0081] 2. Determination of short-chain fatty acids in fermentation broth

[0082] The content of short-chain fatty acids (acetic acid, propionic acid, and n-butyric acid) in the supernatant before and after fermentation was analyzed by gas chromatography-liquid chromatography with external standard method. A DB-5MS column (30m × 0.25mm × 0.25μm) was used. The column temperature was set to 95℃ and held for 5 min, then increased to 210℃ at a rate of 25℃ / min and held for 3 min. The injection port temperature was set to 250℃. 1 μL of sample was injected into the instrument at a split ratio of 1:10. N2 was used as the carrier gas at a flow rate of 1 mL / min, and the detector temperature was set to 250℃.

[0083] Table 5. Short-chain fatty acid content in different groups

[0084]

[0085] After fermentation, the contents of acetic acid, propionic acid, and butyric acid in the fermentation broth increased significantly, especially propionic acid, which increased by 87.5%, and butyric acid, which doubled in content. Compared with control groups 1 and 2, the composition after fermentation showed a significant increase in propionic acid and butyric acid. Literature reports that acetic acid inhibits the NF-κB pathway through the GPR43 receptor, reducing the damage of inflammatory factors to the mucosal barrier; butyric acid can accelerate intestinal epithelial renewal, inhibit cell apoptosis, and improve villous morphology, thereby enhancing nutrient absorption. Therefore, the composition obtained after raw material fermentation can improve the intestinal mucosal barrier and enhance nutrient absorption by increasing the content of propionic and butyric acids.

[0086] The absorption rates of the raw materials themselves and the composition obtained after fermentation were determined according to the method in Example 1, and the permeability index of sodium fluorescein was used for evaluation.

[0087] Table 6. Effect of supernatant before and after fermentation on the permeability of sodium fluorescein.

[0088]

[0089]

[0090] It is evident that the supernatant after fermentation can increase the intestinal absorption rate of sodium fluorescein by improving its permeability. The main reasons are likely: *Lactobacillus plantarum* PPL19 converts macromolecules such as bovine spleen peptides into smaller amino acids through its secretory enzyme system, significantly increasing leucine and lysine content; secondly, it releases oligosaccharides or free phenolic acids by cleaving plant glycosidic bonds, reducing molecular weight and improving bioavailability; finally, the fermentation broth contains high concentrations of acetic acid, propionic acid, and butyric acid. Butyric acid, as an energy substrate for the intestinal mucosa, can regulate tight junction proteins, enhance intestinal mucosal permeability, promote transmembrane diffusion of nutrients, and achieve a breakthrough in absorption rate.

[0091] Under normal conditions, intestinal mucosal permeability reflects the ability of substances to be transported across the mucosa, mainly relying on passive diffusion or carrier-mediated active transport. The higher the permeability, the faster the nutrients are absorbed into the bloodstream. Under pathological conditions, damage to the intestinal barrier leads to a pathological increase in intestinal mucosal permeability, especially when tight junctions are disrupted, resulting in abnormally high permeability and leaky gut. Secondly, mucosal damage can impair the function of carrier proteins, reducing the efficiency of active transport of key nutrients (such as glucose and amino acids).

[0092] To further explore whether the fermentation composition can improve the symptoms of spleen-deficient mice by improving intestinal mucosal permeability, an efficacy study was conducted in Example 4.

[0093] Example 4

[0094] Animal test efficacy evaluation

[0095] Animal Experiment Design: Fifty BALB / c young mice (3-4 weeks old) were acclimatized for one week, then divided into groups of 10. Except for the normal control group, the other groups were modeled as spleen-deficient mice using a purgative method, receiving 4 g / kg / day of senna leaves via gavage. When mice exhibited symptoms of spleen and stomach weakness, such as loose stools, huddling, arched backs, reduced appetite, lethargy, and anal discharge, their growth was inhibited. In contrast, the normal control group showed normal activity, glossy and dense fur, alertness, and granular feces, indicating successful modeling. Group Design:

[0096] Normal group: No modeling treatment was performed; normal saline was administered by gavage; no dietary restrictions were imposed for 14 consecutive days.

[0097] Model group: Senna leaf composition group administered by gavage at 4 g / kg daily; senna leaf composition group administered by gavage at 4 g / kg daily for modeling, and simultaneously administered by gavage at 0.2 mL / kg pre-fermentation composition group for treatment (Example 2).

[0098] Fermented composition group: Senna model was established by daily gavage administration of 4 g / kg, and the fermented composition group was treated simultaneously by daily gavage administration of 0.2 mL / kg (Example 2).

[0099] Comparative Example 3

[0100] Sijunzi Decoction: Herbal composition: Codonopsis pilosula 12g, Poria cocos 12g, stir-fried Atractylodes macrocephala 12g, prepared Glycyrrhiza uralensis 6g. Soak in 8 times the amount of water for 1 hour, simmer over low heat for 1 hour, then continue simmering in 6 times the amount of water for 1 hour. Combine the decoctions and administer 0.2mL / kg of the decoction by gavage daily.

[0101] After two weeks of continuous gavage, blood was collected through the orbital cavity. After the blood was allowed to stand for 1 hour, it was centrifuged at 4°C and 3000 r / min for 10 minutes to obtain serum.

[0102] Evaluation indicators:

[0103] ① Mouse weight and length

[0104] ② Changes in gastric emptying rate and small intestinal propulsion rate in mice

[0105] ③ Intestinal mucosal permeability: Permeability was detected by assessing the concentration of fluorescein isothiocyanate-dextran (FITC-Dextran) in plasma.

[0106] ④ Measurement of femur length and weight in mice

[0107] After the final gavage, the mice were euthanized by dislocation, dissected, and the bilateral femurs were separated. The left femur was sealed and stored at -20°C, while the right femur was fixed in paraformaldehyde for later analysis. The femur length was measured using calipers. Bone samples were taken, and attached muscle tissue was removed. The samples were then soaked in anhydrous ether for 12 hours to defatted the bone, placed in a 105°C drying oven for 72 hours, and allowed to equilibrate at room temperature for 24 hours before weighing the mice.

[0108] ⑤ Bone mineral density measurement in mice

[0109] After anesthetizing the experimental mice, a disposable sheet was placed on the mice and they were placed on a bone density testing bed. Bone density was detected and analyzed using X-ray bone density detection. The whole-body animal scanning mode was selected to scan each experimental mouse individually. Bone density analysis was performed on the head, spine and whole body of the mice, and the experimental data were recorded.

[0110] ⑥ Detection of mouse serum markers

[0111] On day 14 of the experiment, blood was collected intravenously, and the serum levels of insulin-like growth factor (IGF-1), osteocalcin (OCN), gastrin (GAS), motilin (MTL), vasoactive intestinal peptide (VIP), and substance P (SP) in mice were measured using an ELISA kit.

[0112] ⑦ Expression of G protein-coupled receptor 109A (GPR109A) mRNA.

[0113] Table 7 Changes in mouse body weight and body length

[0114] During the experiment, changes in the weight and height of young mice were monitored weekly. The model group mice showed an increase in weight of only 52.80% and a increase in length of only 11.75%; while the fermented composition group showed an increase in weight of 79.15% and a increase in length of 17.07%. This indicates that the fermented composition group mice were able to recover to normal levels, and compared with the model group, the fermented composition group significantly increased their weight and length, which was superior to the control group 3.

[0115] Table 8. Changes in gastric emptying rate and small intestinal propulsion rate in mice.

[0116]

[0117] Compared with the normal group, the gastric motility of mice in the model group was significantly reduced. Compared with the model group, the gastric emptying rate and small intestinal propulsion rate of the composition were significantly improved before and after fermentation, and the fermented composition group was more effective than the control group 3 (Sijunzi Decoction), indicating that the composition can significantly improve the gastric emptying rate and small intestinal propulsion rate of spleen-deficient mice.

[0118] Based on the apparent properties, the composition obtained after fermentation of the raw materials can increase the weight and length of young mice with spleen deficiency, and improve the gastric emptying rate and intestinal propulsion rate of the mice. In order to further study whether the fermented composition has the function of improving the apparent properties of mice with spleen deficiency and promoting growth, further research will be conducted.

[0119] One of the most common characteristics of spleen and stomach weakness is gastrointestinal dysfunction. Gastrointestinal hormones play a role in regulating gastrointestinal motility. Motilin (MTL) and substance P (SP) are excitatory gastrointestinal hormones that can accelerate gastric emptying, shorten small intestinal transit time, and promote gastrointestinal function. Vasoactive intestinal peptide (VIP) is an inhibitory gastrointestinal hormone that primarily inhibits gastrointestinal motility and reduces gastrointestinal activity. Therefore, measuring changes in these indicators is important.

[0120] Table 9. Changes in serum markers in different groups

[0121]

[0122]

[0123] The combination group obtained after fermentation of the raw materials in this study can restore the mice to the normal young mouse state, and promote gastrointestinal motility by increasing the levels of MTL and SP and decreasing the level of VIP, thereby improving the function of the spleen and stomach.

[0124] Spleen deficiency is an important cause of increased intestinal mucosal permeability (leaky gut). Under pathological conditions, damage to the intestinal barrier leads to a pathological increase in intestinal mucosal permeability. In particular, when tight junctions are damaged, permeability increases abnormally, resulting in intestinal leakage. Therefore, it is necessary to detect intestinal mucosal permeability indicators and explore whether fermentation compositions can improve spleen deficiency based on these indicators.

[0125] Table 10. Changes in intestinal mucosal permeability in different groups of spleen-deficient mice

[0126]

[0127] Compared with the normal group, the serum FITC-Dextran fluorescence concentration in the model group increased, indicating that the model group caused a significant increase in intestinal mucosal permeability in mice. After intervention, the combined composition group and the three control groups could effectively reduce the intestinal mucosal permeability of damaged mice. The FITC-Dextran fluorescence concentration in the fermented composition group was not significantly different from that in the normal group, indicating that the fermented composition could restore the intestinal mucosa of spleen-deficient mice to a normal state.

[0128] To further explore the molecular mechanism by which the fermentation composition affects the intestinal mucosal permeability of mice, the expression levels of Occludin, Claudin-2, ZO-1 genes and genes related to the ERK / MLCK signaling pathway were measured.

[0129] Occludin, Claudin-2, and ZO-1 are core structural genes that constitute tight junctions in intestinal mucosal epithelial cells. Occludin is the transmembrane "building block" that maintains the integrity of tight junctions, Claudin-2 is responsible for forming selective channels that allow water and small molecules to pass through, and ZO-1 acts as an intracellular "bridge" to anchor the former two to the cytoskeleton. The expression levels of these three genes (e.g., elevated Occludin and ZO-1 enhance the barrier, and elevated Claudin-2 increases permeability) directly determine the function and permeability of the intestinal mucosal barrier.

[0130] The ERK / MLCK signaling pathway is a key pathway for regulating the above-mentioned tight junctions: the ERK pathway indirectly alters barrier integrity by affecting the expression and modification of tight junction proteins, while the MLCK pathway creates gaps by activating cytoskeleton contraction and pulling tight junctions. Changes in the activity of both (such as when they are inhibited) will ultimately affect intestinal mucosal permeability by regulating the function of structural genes.

[0131] like Figure 1 As shown, compared with the blank group, the expression levels of Claudin-2 and ERK / MLCK signaling pathway-related genes in the model group were increased. After 2 weeks of intervention, compared with the model group, the combined group, the fermentation combination, and comparative example 3 all significantly reduced the level of Claudin-2 and significantly increased the expression levels of Occludin and ZO-1 genes. Figure 2 As shown, compared with the model group, the expression of p-ERK1 / 2, ERK1 / 2, p-MLC2, and MLCK genes in the intestinal tissue of mice in each intervention group was significantly reduced and returned to normal levels.

[0132] In summary, the composition obtained after raw material fermentation can promote gastrointestinal motility and improve epigenetic indicators such as gastric emptying and intestinal propulsion in mice by increasing serum MTL and SP levels and decreasing VIP levels. Secondly, it can improve the expression of tight junction-related genes by regulating the ERK / MLCK signaling pathway, ultimately improving intestinal mucosal permeability in spleen-deficient mice and restoring them to normal.

[0133] Furthermore, based on the above results, it was found that the composition obtained after fermentation of the raw materials could promote the growth of height and weight in mice. Therefore, bone density, bone length, and other indicators of mice were measured. Femur length and bone weight are generally indicators of skeletal development level; the higher the femur length and the greater the bone weight, the better the skeletal development. After modeling treatment, the fermented mice were able to recover to normal levels and had a higher femur length and greater bone weight than the normal group.

[0134] Table 11 Changes in femoral length, bone weight, and bone mineral density in mice from different groups

[0135]

[0136] Compared with the model group, the femur length of mice in the fermented composition group increased by 24.46%, while that in control group was only 10.49%; bone weight increased by 20.38%, while that in control group was only 12.07%, indicating that the fermented composition can significantly promote bone growth and development. Mice in the fermented composition recovered to normal bone mineral density levels, and compared with the model group, bone mineral density increased significantly by 36.16%, while the bone mineral density of mice in the unfermented composition increased by only 13.40%. This shows that fermentation can improve the absorption of effective substances in the raw materials and promote a significant increase in bone mineral density.

[0137] Table 12 Serum levels of IGF-1 and OCN in different groups

[0138]

[0139] IGF-1 is a key hormone required for growth and a regulator of bone growth. The serum IGF-1 levels in mice treated with the fermented composition returned to normal levels and were more potent than those with the unfermented composition. This may be because the short-chain fatty acids (especially butyric acid) produced during fermentation can induce a significant increase in IGF-1, thereby promoting bone growth and remodeling.

[0140] OCN is a marker of bone formation and bone turnover. The OCN content in the serum of mice treated with the fermented composition was restored to normal levels and was higher than that in the Sijunzi decoction group, indicating that the fermented composition can promote osteoblast activity and bone turnover rate after gavage, thereby promoting bone growth.

[0141] To further investigate the mechanism by which the composition affects growth, G protein-coupled receptors (GPCRs) were detected. GPCRs are cell surface molecules involved in signal transduction and cell differentiation, and have become key participants in child development, growth, and maturation. To assess whether the fermentation composition could activate the expression of GPCR 109A (GPR109A), its RNA was isolated from vertebral bones, tibias, and mouse-derived bone marrow cells. Real-time quantitative PCR was used to determine the expression level of GPR109A mRNA in tissues and cells.

[0142] Table 13 Effects of different groups on GPR109A mRNA expression in mice

[0143]

[0144]

[0145] Compared with the normal group, the model group showed a significant decrease in GPR109A mRNA expression. The combined composition and Comparative Example 3 showed no significant difference in GPR109A mRNA expression. However, the fermented combination significantly increased GPR109A mRNA expression. This indicates that the fermentation combination largely preserved the effective components of the GPR109A receptor agonist. Furthermore, the expression of GPR109A markers showed a significant positive correlation with IGF-1 and OCN.

[0146] This patent provides a composition obtained by fermenting a raw material similar to a GPR109A receptor agonist, which can significantly increase the content of bone growth regulator factor (IGF-1) and osteocalcin (OCN) in young mice, and promote their growth and development.

[0147] In summary, the composition obtained after fermentation of raw materials can improve spleen deficiency in young mice and promote their growth and development.

Claims

1. A composition for improving spleen deficiency and promoting growth in children, characterized in that: It is obtained by fermentation with Lactobacillus plantarum PPL19, consisting of 5-20 parts by weight of bovine spleen peptide, 5-20 parts of hawthorn powder, 5-15 parts of yam and 1-10 parts of radish seed powder.

2. The composition as described in claim 1, which has the functions of improving spleen deficiency and promoting children's growth, is characterized in that: The molecular weight of the bovine spleen peptide is 3000-8000 Da.

3. The method for preparing the composition with the functions of improving spleen deficiency and promoting children's growth as described in claim 1, characterized in that: Bovine spleen peptides, hawthorn powder, yam and radish seed powder are mixed, water is added, and the mixture is sterilized to obtain fermentation raw materials; Inoculate the fermentation raw material with a bacterial culture of *Lactobacillus plantarum* PPL19, centrifuge after fermentation, and collect the supernatant.

4. The method for preparing the composition with the functions of improving spleen deficiency and promoting children's growth as described in claim 3, characterized in that: The ratio of the fermentation raw material to water is 1g:(5-10mL); The sterilization temperature is 115℃ and the sterilization time is 20 minutes.

5. The method for preparing the composition with the functions of improving spleen deficiency and promoting children's growth as described in claim 3, characterized in that: The bacterial solution was *Lactobacillus plantarum* PPL19, with a viable count of 10-1. 7 CFU / mL; The fermentation temperature is 32-37℃, the fermentation time is 12-24h, and the rotation speed is 50-100r / min; The centrifuge speed is 3000-5000 r / min; Centrifugation time is 5 to 10 minutes.

6. The application of the composition for improving spleen deficiency and promoting children's growth as described in claim 1, characterized in that: It can be used to prepare food or medicine that improves the intestinal mucosal permeability of mice with spleen deficiency.

7. The application of the composition for improving spleen deficiency and promoting children's growth as described in claim 1, characterized in that: It can be applied to the preparation of food or drugs that improve intestinal mucosal permeability in spleen-deficient mice by regulating the expression of intestinal tight junction genes mediated by the ERK / MLCK signaling pathway.

8. The application of the composition for improving spleen deficiency and promoting children's growth as described in claim 1, characterized in that: It can be used to prepare foods or medicines that increase the levels of IGF-1 and OCN and promote bone growth.

9. The application of the composition for improving spleen deficiency and promoting children's growth as described in claim 1, characterized in that: It can be used to prepare food or medicine that promotes growth and development by efficiently retaining the active ingredients of GPR109A receptor agonists.

Images