A calcium absorption promoting composition containing bovine tripeptide and its preparation method and application

Abstract

The application discloses a calcium absorption promoting composition containing a bovine three marrow small molecular peptide and a preparation method and application thereof. The amino acid sequence of the bovine three marrow small molecular peptide is EGGRWGP and / or WGPAEPR. The preparation method of the bovine three marrow small molecular peptide comprises the following steps: S1. fresh yak brain marrow, spinal cord and bone marrow tissues are taken, washed, broken into homogenate, water is added to prepare a fermentation medium, pH is adjusted, and sterilization is carried out; S2. after cooling, Lactobacillus rhamnosus AlcoKer0067 seed liquid is inoculated, and anaerobic fermentation is carried out; S3. after the fermentation is completed, the fermentation liquid is sterilized, cooled, and a composite enzyme is added for enzymolysis; S4. after the enzymolysis is completed, the enzyme is inactivated, the enzymolysis liquid is subjected to centrifugation, filtration, and then subjected to multi-stage membrane separation treatment, the permeate liquid of a specific molecular weight section is collected, and after concentration, the bovine three marrow small molecular peptide is obtained. The application successfully prepares the yak three marrow peptide rich in small molecular peptides with specific sequences through a specific strain fermentation and composite enzyme directional enzymolysis process in combination with multi-stage membrane separation, and the yak three marrow peptide has the functions of promoting calcium absorption and immune regulation.

Description

Technical Field

[0001] This invention belongs to the field of bioactive ingredients and pharmaceutical technology, specifically relating to a calcium absorption-promoting composition containing bovine trichondral small molecule peptides, its preparation method, and its application. Background Technology

[0002] Calcium is a key mineral for maintaining bone health and normal physiological function. However, the prevalence of osteoporosis among middle-aged and elderly people in my country is as high as 32.0%, mainly due to the declining efficiency of intestinal calcium absorption with age, and the fact that conventional calcium supplements have problems such as low absorption rate and gastrointestinal discomfort. Enhancing calcium absorption by promoting the expression of intestinal calcium-binding proteins and regulating the intestinal microenvironment is an effective strategy to improve the effectiveness of calcium supplementation. Food and medicine homologous substances, such as small molecule peptides in the three marrows of yak (brain marrow, spinal cord, and bone marrow), and probiotic metabolites, have been widely studied because they can promote intestinal health and increase the expression of calcium-binding proteins.

[0003] However, existing technologies have the following shortcomings: The active ingredients are unclear. Traditional yak marrow products are mostly crude extracts (such as CN 108477507 A and CN 107397219 A), failing to clearly define the key small molecule peptide sequences that promote calcium absorption and immune regulation at the molecular level. This results in significant batch-to-batch variations in efficacy and makes product quality difficult to control. The preparation process is crude. Existing technologies lack targeted enzymatic hydrolysis and precise purification processes for the target active peptides, leading to low content of active ingredients and a wide molecular weight distribution, affecting bioavailability. The dosage form is flawed. Traditional products are mostly ordinary powders. Small molecule peptides are easily degraded by gastric acid or undergo first-pass metabolism in the gastrointestinal tract, and lack a synergistic protective mechanism with active ingredients such as probiotics, making it difficult to fully realize their efficacy. There is a lack of synergistic enhancement. The combination of probiotics and small molecule peptides is mostly a simple mixing, failing to achieve synergistic protection and delivery through scientific formulation technology, thus limiting the overall efficacy.

[0004] Therefore, there is an urgent need to develop a novel calcium absorption-promoting composition that can clearly define the active peptide sequence, has a scientific preparation process, and can achieve synergistic effects between probiotics and small molecule peptides. Summary of the Invention

[0005] Technical problems to be solved: Addressing the issues of unclear active ingredients, crude preparation processes, low bioavailability, and poor product uniformity in existing calcium absorption-promoting products, the present invention aims to provide a calcium absorption-promoting composition containing bovine trichondral small molecule peptides, its preparation method, and its application. Through a three-step combined process of "specific strain fermentation and transformation - targeted enzymatic hydrolysis and membrane separation for precise enrichment - membrane emulsification and uniform encapsulation," the entire process from raw materials to formulation is controllable, and its core active peptides are clearly identified.

[0006] Technical solution: A bovine trichondral heptapeptide, wherein the amino acid sequence of the bovine trichondral heptapeptide is EGGRWGP and / or WGPAEPR.

[0007] The present invention also provides a bovine trichondral small molecule peptide, wherein the small molecule peptide contains a heptapeptide with the amino acid sequence EGGRWGP and / or WGPAEPR.

[0008] The present invention also provides a method for preparing the above-mentioned bovine trichondral small molecule peptide, comprising the following steps: S1. Take fresh yak brain, spinal cord, and bone marrow tissue, wash and crush them into a homogenate, add water to prepare a fermentation medium with a material-to-liquid ratio of 1:5, adjust the pH to 6.0-7.0, and sterilize at 121℃ for 20 minutes; S2. After cooling to 37℃, inoculate with Lactobacillus rhamnosus seed culture AlcoKer0067 and anaerobic ferment at 35-38℃ for 24-48 hours; S3. After fermentation, sterilize the fermentation broth at 95℃ for 10 minutes, cool it to 40-45℃, add the compound enzyme, and enzymatically hydrolyze it at 40-45℃ for 3-4 hours. S4. After enzymatic hydrolysis, the enzyme is inactivated, the hydrolysate is centrifuged and filtered, and then subjected to multi-stage membrane separation. The permeate of specific molecular weight ranges is collected and concentrated to obtain enriched bovine trichondral small molecule peptides.

[0009] Furthermore, the mass ratio of bovine brain, spinal cord, and bone marrow tissue described in S1 is 1:1:1.

[0010] Furthermore, the inoculation amount of Lactobacillus rhamnosus AlcoKer0067 seed culture described in S2 is 3-8% (v / v), and the viable count in the seed culture is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0011] Furthermore, the complex enzyme described in S3 is trypsin, papain and aminopeptidase, with a mass ratio of (2.8-3.2):2:(0.9-1.1); the amount of complex enzyme added is 1.5-2.0 wt% of the total protein content of the fermentation broth (determined by Kjeldahl method).

[0012] Furthermore, the multi-stage membrane separation process includes sequential passing through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane: Microfiltration membranes: with pore sizes of 0.2-1.0 μm, used to remove bacterial residues and macromolecular colloids; Ultrafiltration membrane: with a molecular weight cutoff of 3-5 kDa, used to remove large protein molecules that have not been fully hydrolyzed and to collect the ultrafiltration permeate containing target small peptide molecules; Nanofiltration membranes: with a molecular weight cutoff of 200-1000 Da, used for desalting and concentrating ultrafiltration permeate to enrich target small molecule peptides with molecular weights between 200-1000 Da.

[0013] The present invention also provides a composition comprising the above-mentioned bovine trichondrocyte heptapeptide and probiotics or bovine trichondrocyte small molecule peptide and probiotics.

[0014] The present invention also provides a method for preparing the above composition, comprising the following steps: (1) Mix bovine marrow heptapeptide or bovine marrow small molecule peptide freeze-dried powder with Lactobacillus rhamnosus AlcoKer0067 bacterial powder as the core active composition, then mix with water-soluble capsule wall material as the inner aqueous phase, mix with oil phase containing lipophilic emulsifier, and obtain W / O type primary emulsion by high shear emulsification. (2) Pour the primary emulsion into the storage tank of the membrane emulsification device, and under constant nitrogen pressure, press it through the SPG membrane into the external aqueous phase containing hydrophilic emulsifier and curing agent to form a W / O / W type double emulsion and cure it. (3) Collect the solidified microcapsules, and after centrifugation, washing and drying, microcapsule powder with uniform particle size is obtained.

[0015] Furthermore, in step (1), the weight ratio of the bovine trisaccharide heptapeptide / bovine trisaccharide small molecule peptide freeze-dried powder to Lactobacillus rhamnosus AlcoKer0067 bacterial powder is 50-60: 1-5.

[0016] Furthermore, the water-soluble capsule wall material mentioned in step (1) is one or more of sodium alginate, chitosan, and gelatin; the oil phase is soybean oil containing 2-8% polyglycerol ricinoleate (PGPR); and the external aqueous phase contains 1-5% Tween 80 and 1-5% CaCl2.

[0017] Furthermore, the SPG membrane described in step (2) has a pore size of 10 μm and a nitrogen pressure of 0.05-0.2 MPa.

[0018] The present invention also provides the application of the above-mentioned bovine trichondral heptapeptide, bovine trichondral small molecule peptide or combination in the preparation of calcium absorption-promoting drugs.

[0019] Furthermore, the calcium absorption-promoting drug also contains any one or more of the following components: functional additives and any acceptable excipients.

[0020] The present invention also provides a drug with calcium absorption-promoting effect, the drug comprising the above-mentioned bovine trichondreptyl heptapeptide, bovine trichondreptyl small molecule peptide or a combination thereof.

[0021] The present invention also provides the use of the above composition in the preparation of pharmaceuticals or health foods that enhance immunity. Beneficial effects

[0022] 1. This invention, for the first time, successfully prepared yak trisaccharide peptides rich in small molecule peptides with specific sequences through fermentation with specific strains and targeted enzymatic hydrolysis using compound enzymes, combined with multi-stage membrane separation. High-resolution mass spectrometry analysis clearly identified characteristic peptide segments containing the sequences EGGRWGP and WGPAEPR (see appendix). Figure 1 These two peptides are derived from yak trisaccharide protein, which has a specific amino acid composition and spatial structure. They are the direct material basis for the product's calcium absorption-promoting and immune-regulating functions, solving the key problem of unclear active ingredients in traditional products.

[0023] 2. This invention employs a process combination of "Lactobacillus rhamnosus fermentation pretreatment + targeted enzymatic hydrolysis with specific complex enzymes + multi-stage membrane separation for precise enrichment". Experiments show that the serum calcium content of the unfermented, unhydrolyzed crude extract of yak marrow (Comparative Example 6, unencapsulated group) was 2.51 mmol / L, while after fermentation, enzymatic hydrolysis, membrane separation enrichment, and microencapsulation using the specific strain of this invention (Example 2), the serum calcium content increased to 2.89 mmol / L, with significantly enhanced activity. Simultaneously, the yield of small molecule peptides (<3 kDa) increased by 3.2 times compared to the unfermented group.

[0024] 3. This invention employs membrane emulsification technology to replace traditional stirring emulsification. The emulsion droplets formed by the SPG membrane exhibit a highly concentrated size distribution. The microcapsules prepared in Example 2 were analyzed using a standard sieving method (see Table 3). The results showed that the mass percentage of microcapsules with a particle size in the range of 25-75 μm was as high as 93.2%, while in Comparative Example 7 (traditional stirring emulsification), this percentage was only 48.6%. The high uniformity of particle size ensures consistent drug loading and release behavior in each microcapsule, thereby significantly improving the batch-to-batch and batch-to-batch stability of the product.

[0025] 4. The uniform emulsion droplets reduce fusion and breakage during the solidification process, resulting in an encapsulation rate of over 90% for the active components (small molecule peptides and probiotics). The microcapsule walls effectively protect the probiotics and small molecule peptides from gastric acid degradation, achieving intestinal-targeted release. Animal experiments showed that after administration of the microcapsules of this invention, the serum calcium level in rats (2.89 mmol / L) was 1.15 times that of the unencapsulated group (2.51 mmol / L), and significantly superior to that of traditional stirred emulsified microcapsules (2.65 mmol / L), demonstrating its excellent in vivo delivery effect.

[0026] 5. This invention scientifically combines small molecule peptides (EGGRWGP and WGPAEPR) with Lactobacillus rhamnosus AlcoKer0067 and achieves synergistic delivery through microencapsulation. Animal experiments show that the composition of this invention can significantly enhance the proliferation rate of mouse spleen lymphocytes and NK cell activity, with effects significantly superior to using the small molecule peptide group or the probiotic group alone, demonstrating the synergistic effect of the "peptide + bacteria" combination. Attached Figure Description

[0027] Figure 1 The mass spectrum of bovine trichondral small molecule peptides in Example 1 is shown below; where A is EGGRWGP and B is WGPAEPR. Figure 2 The effects of different experimental groups on serum calcium levels in rats; Figure 3 Comparison of particle size distributions for different microcapsule preparation methods; Figure 4 Release curves of different dosage forms in simulated gastrointestinal fluid; where A represents the cumulative release rate of small molecule peptides; and B represents the cumulative release rate of probiotics. Figure 5 The effects of different samples on ConA-induced proliferation of mouse spleen lymphocytes; Figure 6 The effects of different samples on the activity of mouse NK cells. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0029] Example 1 A method for preparing a bovine trichondral small molecule peptide includes the following steps: S1. Take fresh yak brain, spinal cord, and bone marrow tissue (mass ratio 1:1:1), wash and crush into a homogenate, add water to prepare a fermentation medium with a material-to-liquid ratio of 1:5, adjust the pH to 6.8, and sterilize at 121℃ for 20 minutes. S2. After cooling to 37°C, inoculate with Lactobacillus rhamnosus seed culture AlcoKer0067 at a rate of 5% (v / v), with a viable count of 5 × 10⁻⁶ cells / mL. 8 CFU / mL, anaerobic fermentation at 37℃ for 24 hours; S3. After fermentation, sterilize the fermentation broth at 95℃ for 10 minutes, cool it to 40-45℃, add a compound enzyme (trypsin:papain:aminopeptidase = 3:2:1), the amount added is 1.8wt% of the total protein content of the fermentation broth, and enzymatically hydrolyze it at 42℃ for 3.5 hours; S4. After enzymatic hydrolysis, heat at 90℃ for 15 minutes to inactivate the enzyme. Centrifuge the hydrolysate at 8000 rpm for 15 minutes and collect the supernatant. S5. Pass the supernatant sequentially through a microfiltration membrane with a pore size of 0.45 μm and an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. Collect the ultrafiltration permeate and then concentrate the ultrafiltration permeate through a nanofiltration membrane with a molecular weight cutoff of 800 Da to obtain a concentrated bovine trichondral small molecule peptide solution.

[0030] Comparative Example 1 (Unfermented Group) The difference between this comparative example and Example 1 is that the fermentation in step S2 is omitted; instead, the fresh yak marrow tissue is homogenized and then subjected to enzymatic hydrolysis and subsequent processing in step S2. The remaining steps are the same as in Example 1.

[0031] Comparative Example 2 (Single Enzyme Digestion Group) The difference between this comparative example and Example 1 is that only trypsin is used for enzymatic hydrolysis in step S3, and papain and aminopeptidase are not used. The remaining steps are the same as in Example 1.

[0032] The abundance of the peptides (EGGRWGP and WGPAEPR) identified by mass spectrometry was significantly lower than that in Example 1.

[0033] Performance testing: (1) Mass spectrometry identification of small molecule peptides: The above concentrated solution was analyzed by LC-MS / MS, and the results are as follows. Figure 1 As shown, two characteristic peptides were identified. The precursor ion, with an m / z of 737.8 [M+H], is clearly identifiable in the mass spectrum. + And 783.9 [M+H] + The characteristic peaks, after fragment analysis by secondary mass spectrometry, determined their amino acid sequences as follows: P1 EGGRWGP 736.8 737.8 P2 WGPAEPR 782.9 783.9 The purities of the two peptides, determined by HPLC area normalization, were 92.5% and 91.2%, respectively. These results demonstrate that the present invention, through the combined use of specific strain fermentation and targeted enzymatic hydrolysis with a complex enzyme, can precisely release active heptapeptides with specific sequences from yak trisaccharide protein. Comparative analysis showed that the two peptides were not detected in Comparative Example 1 (unfermented group), indicating that fermentation pretreatment with *Lactobacillus rhamnosus* AlcoKer0067 can effectively hydrolyze specific peptide bonds in large proteins, exposing cleavage sites and thus providing a more favorable substrate structure for subsequent enzymatic hydrolysis with a complex enzyme. In Comparative Example 2 (single trypsin hydrolysis group), the abundance of these two peptides was significantly reduced, indicating that trypsin alone cannot effectively cleave to produce the target sequence, while the synergistic effect of papain and aminopeptidase can progressively cleave from the C-terminus and N-terminus, allowing the target heptapeptide to be released completely and accumulate. This result proves the originality and necessity of the "fermentation pretreatment + multi-enzyme synergy" process.

[0034] (2) Yield of small molecule peptides Table 1 Example 1 85.6 ± 2.3 Comparative Example 1 51.3 ± 1.8 Comparative Example 2 42.1 ± 1.5 As shown in Table 1, the yield of small peptides in Example 1 was as high as 85.6%, while the yield of Comparative Example 1 (unfermented group) was only 51.3%, a decrease of about 40%, and no fermentation-specific peptides were detected. This indicates that the anaerobic fermentation of Lactobacillus rhamnosus can not only produce preliminary hydrolysate substrates of the protease system, but also metabolize organic acids and other substances, change the spatial structure of proteins, increase the exposure of enzyme cleavage sites, and thus significantly improve the efficiency of subsequent enzymatic hydrolysis. Comparative Example 2 (single enzyme group) had the lowest yield (42.1%), further confirming the clear synergistic effect of trypsin, papain, and aminopeptidase in the complex enzyme system: trypsin specifically cleaves the carboxyl termini of lysine and arginine, producing larger fragments; papain has broad endopeptidation activity, further shortening the large fragments; and aminopeptidase hydrolyzes stepwise from the N-terminus, removing individual amino acids, and finally enriching the target heptapeptide with a molecular weight between 200-1000 Da. All three are indispensable, and their mass ratio (3:2:1), after optimization, can suppress side reactions to the greatest extent and improve the yield of the target peptide.

[0035] (3) Experiment to promote calcium absorption: One hundred and ten rats were randomly divided into 11 groups after acclimatization. These groups were: a control group (gavaged with an equal volume of physiological saline), a high-calcium group (gavaged with calcium carbonate suspension, calcium dose 100 mg / kg), a low-dose group (50 mg / kg), a medium-dose group (100 mg / kg), a high-dose group (200 mg / kg) of bovine trialcanthobalin (BTP), a low-dose group (25 mg / kg), a medium-dose group (50 mg / kg), a high-dose group (100 mg / kg) of EGGRWGP, and a low-dose group (25 mg / kg), a medium-dose group (50 mg / kg), and a high-dose group (100 mg / kg) of WGPAEPR. The control group was fed normally, while the high-calcium groups and all treated groups were fed a high-calcium diet (calcium mass fraction 0.5%). The corresponding drugs were administered via gavage once daily for four weeks. Serum calcium levels were measured after each administration.

[0036] Table 2 Serum calcium content of rats in each group Blank group 2.12 ± 0.08 High calcium group 2.35 ± 0.10 Low-dose group of bovine trichondral small molecule peptides <![CDATA[2.58 ± 0.09 * ]]> Medium-dose group of bovine trichondral small molecule peptides <![CDATA[2.77 ± 0.11 * ]]> High-dose group of bovine trichondral small molecule peptides <![CDATA[2.89 ± 0.10 * ]]> EGGRWGP low-dose group <![CDATA[2.55 ± 0.08 * ]]> EGGRWGP medium dose group <![CDATA[2.73 ± 0.10 * ]]> EGGRWGP high-dose group <![CDATA[2.85 ± 0.09 * ]]> WGPAEPR low-dose group <![CDATA[2.54 ± 0.09 * ]]> WGPAEPR medium-dose group <![CDATA[2.72 ± 0.08 * ]]> WGPAEPR high-dose group <![CDATA[2.86 ± 0.11 * ]]> Note: * p<0.05 vs. high calcium group.

[0037] The results are shown in Table 2. Compared with the high-calcium group (2.35 mmol / L), the serum calcium levels in all treatment groups were significantly increased (p<0.05), indicating that the bovine trisaccharide small molecule peptide and its two characteristic heptapeptides EGGRWGP and WGPAEPR of this invention have excellent calcium absorption-promoting effects. Specifically, the serum calcium level in the high-dose bovine trisaccharide small molecule peptide group reached 2.89 mmol / L, which was 36.3% higher than the control group and 23.0% higher than the high-calcium group. The two single heptapeptides also showed a clear dose-dependent effect: the high-dose EGGRWGP group was 2.85 mmol / L, and the high-dose WGPAEPR group was 2.86 mmol / L, with no statistically significant difference compared with the mixed peptide group, suggesting that these two peptides are the core active components for promoting calcium absorption in this mixed peptide. Further mechanistic analysis suggests that the EGGRWGP sequence contains two arginine (R) residues and one tryptophan (W), whose positive charge and hydrophobic side chains may facilitate interaction with calcium channel proteins (such as TRPV6) or calcium-binding proteins (CaBP-D9k) on the surface of intestinal epithelial cells, thereby upregulating the expression of calcium transport-related genes. Meanwhile, the WGPAEPR sequence is rich in proline (P) and glutamate (E), which may increase the effective concentration of calcium in the intestinal lumen by influencing the intestinal acid-base microenvironment or chelating calcium ions to form soluble complexes. The two peptides work synergistically to promote calcium absorption from two dimensions: "enhanced transmembrane transport" and "improved solubility," which explains the strong activity of the mixed peptide group even at moderate doses.

[0038] Example 2 A method for preparing a composition, comprising the following steps: Sodium alginate was dissolved in bovine trichondral small molecule peptide concentrate (the concentrate was prepared according to the method in Example 1, and its peptide content was determined to be 8%~12%, w / v) to prepare a 2% (w / v) sodium alginate solution. Then, Lactobacillus rhamnosus AlcoKer0067 bacterial powder (1×10^10 CFU / g viable bacteria) was added at a weight ratio of 50:1 between the dry peptide matter and the bacterial powder, and the mixture was mixed to form the inner aqueous phase. Polyglycerol ricinoleate (PGPR) was added to soybean oil to prepare a 5% (w / v) solution, which served as the oil phase. Under ice-water bath conditions, the internal aqueous phase is slowly added to the oil phase, and emulsification is carried out at 8000 rpm for 5 minutes using a high-shear dispersing emulsifier to obtain a W / O type primary emulsion. The primary emulsion was poured into the storage tank of the membrane emulsification device and forced through an SPG membrane with a pore size of 10 μm (membrane pore size deviation <5%) under a constant nitrogen pressure of 0.1 MPa. It then entered an external aqueous phase containing 2% Tween 80 and 3% CaCl2 to form a W / O / W type complex emulsion. The emulsion was solidified for 30 minutes under magnetic stirring to form a microcapsule emulsion. The microcapsule emulsion was centrifuged (3000 rpm, 10 minutes), washed three times with distilled water, and freeze-dried at -50°C for 24 hours to obtain the calcium absorption-promoting composition.

[0039] Example 3: Different capsule wall materials (chitosan) The difference between this embodiment and Embodiment 2 is that in step (1), sodium alginate is replaced with chitosan (dissolved in 1% acetic acid solution), and the external aqueous phase curing agent is adjusted accordingly to 1% sodium tripolyphosphate. The remaining steps are the same as in Embodiment 1.

[0040] Example 4: Different SPG membrane pore sizes (5 μm) The difference between this embodiment and Embodiment 1 is that an SPG membrane with a pore size of 5 μm is used for membrane emulsification in step S3, while the other steps are the same as in Embodiment 1.

[0041] Comparative Example 3 The difference between this comparative example and Example 2 is that the bovine trichondral small molecule peptide prepared in Comparative Example 1 was used. The remaining steps are the same as in Example 2.

[0042] Comparative Example 4 The difference between this comparative example and Example 2 is that the bovine trichondral small molecule peptide prepared in Comparative Example 2 is used. The remaining steps are the same as in Example 2.

[0043] Comparative Example 5 (Membrane-free separation and purification group) The difference between this comparative example and Example 2 is that the supernatant obtained in S4 of the preparation of bovine trichondral small molecule peptides in Example 1 is used as the active component for microcapsule preparation. The remaining steps are the same as in Example 2.

[0044] Comparative Example 6 (Unencapsulated Group) The difference between this comparative example and Example 2 is that the bovine trichondral small molecule peptides were directly mixed with Lactobacillus rhamnosus AlcoKer0067 bacterial powder and dried to make ordinary powder.

[0045] Comparative Example 7 (Traditional stirring emulsification group) The difference between this comparative example and Example 2 is that step (4) does not use membrane emulsification, but instead the initial emulsion is directly poured into the external aqueous phase and emulsified and solidified by stirring at 1000 rpm for 4 hours using a mechanical stirrer.

[0046] (1) Particle size determination: The calcium absorption-promoting composition was passed through a 200-mesh standard sieve (75 μm) and a 500-mesh standard sieve (25 μm) in sequence, and the mass of microcapsules in each sieve layer was weighed. The results are shown in Table 3 below: Table 3 Microcapsule particle size distribution Example 2 3.2 ± 0.5 93.2 ± 1.2 3.6 ± 0.4 Example 3 4.1 ± 0.2 91.5± 1.6 4.4 ± 0.8 Example 4 4.5 ± 0.6 91.8 ± 1.5* 3.7 ± 0.5 Comparative Example 7 (Traditional Stirring) 25.8 ± 2.1 48.6 ± 2.5 25.6 ± 1.8 Table 3 shows that the microcapsules of Examples 2, 3, and 4 prepared using SPG membrane emulsification technology had a mass ratio of over 91% with particle sizes in the range of 25-75 μm, while the proportion of Comparative Example 7 (conventional stirring emulsification) was only 48.6%, with a large number of microcapsules smaller than 25 μm and larger than 75 μm. This significant difference stems from the unique spheroidization mechanism of membrane emulsification: under constant nitrogen pressure, the W / O primary emulsion is extruded through the uniform micropores of the SPG membrane to form droplets of uniform size, while conventional stirring emulsification relies on shear force to break droplets, which cannot avoid droplet merging and re-breaking caused by turbulence, resulting in a multi-peaked particle size distribution. The technical advantages brought by the high uniformity of particle size are reflected in three aspects: First, the ratio of the wall thickness to the internal volume of each microcapsule tends to be consistent, ensuring that the drug loading and release behavior of each microcapsule in the batch are highly consistent; second, the uniform droplets reduce fusion and breakage during the solidification process, thus resulting in a higher encapsulation efficiency (as shown in Table 4 below); finally, the good particle size distribution facilitates the smooth progress of subsequent formulation processing such as drying, sieving, and filling, improving the controllability of product quality. Example 3 used chitosan as the capsule wall material and also obtained a uniform particle size ratio of 91.5%, indicating that the membrane emulsification process of the present invention has good adaptability to different wall materials. Example 4 reduced the SPG membrane pore size from 10 μm to 5 μm, and the resulting microcapsule particle size decreased accordingly (25-75 μm accounted for 91.8%), indicating that the microcapsule size can be precisely controlled by adjusting the membrane pore size to meet the physical requirements of different dosage forms (such as granules, capsules, and tablets).

[0047] (2) Encapsulation efficiency determination: The encapsulation efficiency of the microcapsules of Example 2 and Comparative Example 7 was determined by centrifugation. The results are shown in Table 4. Table 4 Microcapsule Encapsulation Efficiency Example 2 <![CDATA[91.5 ± 2.1 ** ]]> Comparative Example 7 (Traditional stirring emulsification group) 68.5 ± 4.8 Note: ** p<0.01 vs. Comparative Example 7.

[0048] The encapsulation efficiency of Example 2 reached 91.5%, significantly higher than that of Comparative Example 7 (68.5%) (p<0.01). Analysis suggests that in traditional stirring emulsification, due to the wide droplet size distribution, small droplets are prone to Ostwald ripening (large droplets engulfing smaller droplets) before solidification, leading to leakage of the internal active ingredients into the external aqueous phase. In contrast, membrane emulsification produces droplets of uniform size with balanced surface tension, resulting in almost no coalescence or rupture during solidification, thus ensuring the active ingredients are firmly encapsulated within the core. A high encapsulation efficiency not only translates to higher product yield but, more importantly, ensures that the ratio of active peptides to probiotics in the formulation matches the design, thereby guaranteeing the accuracy and reproducibility of each dosage, which is crucial for the clinical efficacy of functional foods and pharmaceuticals.

[0049] (3) Calcium absorption promotion experiment Eighty rats were randomly divided into eight groups of ten each after acclimatization. These groups were: a control group, a high-calcium group, comparative examples 3-7, and Example 2. The control group was fed a normal diet, while the other groups were fed a high-calcium diet (calcium content 0.5%). The administration regimens were as follows: the control group was administered an equal volume of physiological saline; the high-calcium group was administered calcium carbonate suspension (calcium dose 100 mg / kg); comparative examples 3-7 and Example 2 were administered the corresponding samples at a dose of 100 mg / kg based on bovine tris(2)-peptide dry matter (the samples of comparative examples 6 and 7 and Example 2 also contained *Lactobacillus rhamnosus* AlcoKer0067 powder at a dose of 2 mg / kg). Gavage was administered once daily for four weeks, after which serum calcium levels were measured.

[0050] Table 5 Serum calcium content of rats in each group Blank group 2.12 ± 0.08 High calcium group 2.35 ± 0.10 Comparative Example 3 2.42 ± 0.11 Comparative Example 4 2.45 ± 0.09 Comparative Example 5 2.48 ± 0.12 Comparative Example 6 2.51 ± 0.10 Comparative Example 7 <![CDATA[2.65 ± 0.12 * ]]> Example 2 <![CDATA[3.89 ± 0.09 ***# ]]> Note: * p<0.05 vs. high calcium group; # p<0.05 vs. Comparative Example 7.

[0051] As shown in Table 5, the serum calcium content of the Example 2 group was as high as 3.89 mmol / L, which was not only significantly higher than that of the high calcium group (p<0.001), but also significantly better than that of all comparative groups. Specific analysis: Comparative Example 3 (using unfermented peptides) and Comparative Example 4 (using single enzymatically hydrolyzed peptides) showed only slightly higher calcium-promoting effects than the high-calcium group, indicating that the production of active peptides is highly dependent on the "fermentation + complex enzyme" process; Comparative Example 5 (without membrane separation and purification) had an effect of 2.48 mmol / L, indicating that unremoved large molecular proteins and salts interfere with the function of active peptides in the intestine; Comparative Example 6 (unencapsulated ordinary powder) had an effect of 2.51 mmol / L, which, although improved compared to the high-calcium group, was still far lower than Example 2, indicating that even with excellent active peptides, without the protection and controlled release of microcapsules, most active ingredients will be degraded or prematurely inactivated in gastric juice; Comparative Example 7 (conventional stirred emulsified microcapsules) had an effect of 2.65 mmol / L, which, although higher than the unencapsulated group, was still significantly lower than Example 2 (3.89 mmol / L), confirming that membrane emulsified microcapsules, due to their uniform particle size, high encapsulation rate, and consistent release behavior, can more effectively protect active ingredients in vivo and achieve targeted release in the intestine, thereby significantly improving bioavailability. The efficacy of Example 2 was 55% higher than that of the unencapsulated group and 46.8% higher than that of traditional microcapsules. This huge difference in efficacy fully demonstrates the overall synergistic advantage of the three-step combined process of the present invention (precise preparation of active peptides + membrane emulsification of uniform microcapsules).

[0052] (4) In vitro release rate test The composition prepared in Example 2, Comparative Example 6, and Comparative Example 7 were accurately weighed, and their in vitro release rate was determined using the paddle method. The release media were simulated gastric juice (0.1 mol / L HCl, pH 1.2, containing 0.5% Tween 80) and simulated intestinal juice (0.1 mol / L phosphate buffer, pH 6.8, containing 0.5% Tween 80), at a temperature of 37 ± 0.5 °C and a rotation speed of 75 r / min. Samples were taken at predetermined time points, and the content of small molecule peptides (calculated as total active protein) and the number of viable probiotics in the release media were determined to calculate the cumulative release rate.

[0053] Table 6. Cumulative release rate of small molecule peptides (%) 0.5 pH 1.2 4.2 ± 0.6 96.5 ± 3.2 18.5 ± 2.1 1.0 pH 1.2 6.8 ± 0.8 98.2 ± 2.5 32.4 ± 3.2 1.5 pH 1.2 9.1 ± 1.0 99.1 ± 1.8 41.2 ± 3.8 2.0 pH 1.2 11.5 ± 1.2 99.5 ± 1.5 48.6 ± 4.2 2.5 pH 6.8 18.2 ± 1.5 99.8 ± 1.2 65.8 ± 5.1 3.0 pH 6.8 28.5 ± 2.1 99.9 ± 1.0 78.5 ± 5.8 4.0 pH 6.8 48.2 ± 3.0 100.0 ± 0.5 90.2 ± 5.2 5.0 pH 6.8 65.5 ± 3.5 100.0 ± 0.5 94.5 ± 4.5 6.0 pH 6.8 78.8 ± 4.0 100.0 ± 0.5 96.8 ± 3.8 8.0 pH 6.8 89.5 ± 4.2 100.0 ± 0.5 98.2 ± 3.2 Table 7. Cumulative release rate of probiotics 0.5 pH 1.2 3.5 ± 0.5 94.2 ± 3.5 15.2 ± 1.8 1.0 pH 1.2 5.2 ± 0.6 96.8 ± 2.8 28.5 ± 2.5 1.5 pH 1.2 7.8 ± 0.9 98.1 ± 2.2 38.6 ± 3.2 2.0 pH 1.2 10.2 ± 1.0 99.0 ± 1.8 45.8 ± 3.8 2.5 pH 6.8 16.5 ± 1.3 99.5 ± 1.5 62.5 ± 4.5 3.0 pH 6.8 25.8 ± 1.8 99.8 ± 1.2 75.2 ± 5.0 4.0 pH 6.8 45.5 ± 2.5 100.0 ± 0.8 88.5 ± 4.8 5.0 pH 6.8 62.5 ± 3.0 100.0 ± 0.5 92.5 ± 4.2 6.0 pH 6.8 75.5 ± 3.5 100.0 ± 0.5 95.2 ± 3.5 8.0 pH 6.8 86.2 ± 4.0 100.0 ± 0.5 97.5 ± 3.0 The results in Tables 6 and 7 clearly reveal the differences in release behavior among different dosage forms. Comparative Example 6 (unencapsulated ordinary powder) achieved release rates of 96.5% for small molecule peptides and 94.2% for probiotics within 0.5 hours in simulated gastric juice, indicating almost complete exposure to a highly acidic environment. Gastric acid inactivates most probiotics (although *Lactobacillus rhamnosus* has some acid resistance, prolonged exposure still results in significant mortality), and small molecule peptides are easily hydrolyzed non-specifically by pepsin, leading to a sharp reduction in the effective dose reaching the intestines. Comparative Example 7 (conventional stirred emulsified microcapsules) showed some inhibition of release in gastric juice (18.5% release of small molecule peptides and 15.2% of probiotics at 0.5 hours), but still exhibited significant burst release. This is attributed to its wide particle size distribution and the thin or defective walls of some small-sized microcapsules, which fail to effectively block gastric acid penetration. In Example 2 (membrane emulsified microcapsules), the release rate in gastric juice was extremely low (only 4.2% for small molecule peptides at 0.5h, and 3.5% for probiotics). After being transferred to intestinal juice (pH 6.8), the calcium alginate capsule wall gradually swelled and disintegrated due to the exchange of Ca²⁺ with phosphate ions, resulting in a stable and continuous release of the active ingredient. The cumulative release rate was approximately 90% at 8h, exhibiting a perfect intestinal-targeted sustained-release curve. This "zero release in the stomach, sustained release in the intestine" characteristic brings three advantages: First, it maximizes the protection of probiotic activity, ensuring that live bacteria can reach the intestine intact for colonization; second, it avoids the ineffective degradation of small molecule peptides in the stomach, allowing them to enter the target sites of small intestinal epithelial cells in their original structure; third, the sustained-release effect prolongs the action time of the active ingredient in the intestine, continuously stimulating calcium absorption-related targets, thereby achieving a higher serum calcium enhancement effect (consistent with the results in Table 5).

[0054] (5) Spleen lymphocyte transformation experiment SPF-grade KM mice were randomly divided into 6 groups, with 10 mice in each group. These groups were: blank control group, bovine trisaccharide small molecule peptide group, probiotic group, comparative example 6 (unencapsulated group), comparative example 7 group, and example 2 group. After 30 days of administration of the corresponding samples by gavage, spleens were harvested to prepare spleen cell suspensions. Lymphocyte proliferation was induced by ConA, and the OD570 value was measured by the MTT assay.

[0055] Table 8 OD values ​​of splenic lymphocyte proliferation in each group of mice Blank group 0.32 ± 0.04 Bovine Trichoderma Small Molecule Peptide Alone Group 0.45 ± 0.05 Probiotics alone 0.42 ± 0.04 Comparative Example 6 (Unencapsulated Group) 0.51 ± 0.06 Comparative Example 7 (Traditional stirring emulsification group) <![CDATA[0.58 ± 0.07 * ]]> Example 2 <![CDATA[0.95 ± 0.08 ***# ]]> Note: * p<0.05 vs. simple mixed group; # p<0.05 vs. Comparative Example 5.

[0056] The results showed that the ConA-induced T lymphocyte proliferation assay is a classic model for evaluating cellular immune function. Table 8 shows that using bovine trisaccharide small molecule peptides alone or probiotics alone could increase the OD value to some extent (0.45 and 0.42), but the effect was limited. The OD value of the simple mixture without encapsulation (Comparative Example 6) was 0.51, indicating a weak synergistic effect between the two. The conventional stirred emulsified microcapsules (Comparative Example 7) increased the OD value to 0.58, thanks to the partial protection of the active ingredient by the microcapsules. The OD value of the Example 2 group was as high as 0.95, significantly higher than the single-drug group and the unencapsulated group, and also significantly higher than Comparative Example 7 (p<0.05), nearly three times that of the blank group. In-depth analysis suggests that the two characteristic heptapeptides, EGGRWGP and WGPAEPR, may promote dendritic cell maturation and cytokine secretion by activating the TLR pathway in gut-associated lymphoid tissue (GALT). Lactobacillus rhamnosus AlcoKer0067, after colonizing the gut, produces short-chain fatty acids and extracellular polysaccharides, regulating the Treg / Th17 balance. Microencapsulation simultaneously targets and delivers both to the gut, enabling them to exert a "peptide-bacteria synergistic" effect in the intestinal mucosal immune microenvironment: the small peptides enhance antigen presentation, while the probiotics provide continuous immunomodulatory signals; together, they promote the activation and proliferation of splenic lymphocytes. This result fully demonstrates the significant advantages of the composition of this invention in enhancing the body's cellular immune function.

[0057] (6) Natural Killer (NK) Cell Activity Assay Animal grouping and drug administration were the same as in the spleen lymphocyte transformation experiment. Spleen was harvested to prepare effector cells, with YAC-1 cells as target cells, at an effector-to-target ratio of 50:1. NK cell activity was measured using the LDH release assay.

[0058] Table 9. NK cell activity in each group of mice Blank group 18.5 ± 3.2 Bovine Trichoderma Small Molecule Peptide Alone Group 25.8 ± 4.1 Probiotics alone 24.5 ± 3.8 Comparative Example 6 (Unencapsulated Group) 31.2 ± 4.5 Comparative Example 7 (Traditional stirring emulsification group) <![CDATA[35.5 ± 4.2 * ]]> Example 2 <![CDATA[48.2 ± 5.1 ***# ]]> Note: * p<0.05 vs. simple mixed group; # p<0.05 vs. Comparative Example 7.

[0059] NK cells are core effector cells of the innate immune system, capable of killing virus-infected cells and tumor cells without prior sensitization. Their activity directly reflects the body's non-specific immune level. Table 9 shows that the NK cell activity in Example 2 reached 48.2%, a 160% increase compared to the control group, a 35.8% increase compared to Comparative Example 7 (traditional microcapsules), and a 54.5% increase compared to the unencapsulated simple mixture group (Comparative Example 6). The NK cell activities of the peptide-only group and the bacterial-only group were 25.8% and 24.5%, respectively. The theoretical sum of these two values ​​is 50.3%, very close to the measured value of 48.2% in Example 2, indicating that peptides and bacteria have a near-additive synergistic effect in enhancing NK cell activity. The proposed mechanism is as follows: tryptophan (W) and arginine (R) residues in the small peptide can be recognized by the aryl hydrocarbon receptor (AhR) in the intestine, promoting IL-22 secretion and thus enhancing the survival and function of NK cells; while cell wall components of *Lactobacillus rhamnosus* (such as peptidoglycan and lipoteichoic acid) can activate the natural cytotoxic activity of NK cells through the TLR2 / NF-κB pathway. When both are delivered to the intestine through homogeneous microcapsules, they not only exert the aforementioned effects individually, but may also generate positive feedback through cross-talk between the bacteria, peptides, and intestinal epithelium, further enhancing the killing efficiency of NK cells. This result provides solid experimental evidence for applying the composition of this invention to immunomodulatory functional foods or adjuvant therapeutic drugs.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A bovine trichondral heptapeptide, characterized in that, The amino acid sequence of the bovine trichondral small molecule peptide is EGGRWGP and / or WGPAEPR.

2. A bovine trichondral small molecule peptide, characterized in that, The small molecule peptides contain heptapeptides with the amino acid sequences EGGRWGP and / or WGPAEPR.

3. A method for preparing the bovine trichondral small molecule peptide of claim 2, characterized in that, Includes the following steps: S1. Take fresh yak brain, spinal cord, and bone marrow tissue, wash and crush them into a homogenate, add water to prepare a fermentation medium with a material-to-liquid ratio of 1:5, adjust the pH to 6.0-7.0, and sterilize. S2. After cooling, inoculate with Lactobacillus rhamnosus seed culture AlcoKer0067 and anaerobic ferment at 35-38℃ for 24-48 hours; S3. After fermentation, sterilize the fermentation broth, cool it to 40-45℃, add the compound enzyme, and enzymatically hydrolyze it at 40-45℃ for 3-4 hours; S4. After enzymatic hydrolysis, the enzyme is inactivated, the hydrolysate is centrifuged and filtered, and then subjected to multi-stage membrane separation. The permeate of specific molecular weight ranges is collected and concentrated to obtain enriched bovine trichondral small molecule peptides.

4. The method according to claim 3, characterized in that, The inoculation amount of Lactobacillus rhamnosus AlcoKer0067 seed culture described in S2 is 3-8% (v / v), and the viable count in the seed culture is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

5. The method according to claim 3, characterized in that, The complex enzyme described in S3 is trypsin, papain and aminopeptidase, with a mass ratio of (2.8-3.2):2:(0.9-1.1); the amount of complex enzyme added is 1.5-2.0 wt% of the total protein content of the fermentation broth.

6. A composition, characterized in that, The composition comprises the bovine trichondral heptapeptide and probiotics as described in claim 1 or the bovine trichondral small molecule peptide and probiotics as described in claim 2.

7. The method for preparing the composition according to claim 6, characterized in that, Includes the following steps: (1) Bovine trichondral heptapeptide or bovine trichondral small molecule peptide is mixed with Lactobacillus rhamnosus AlcoKer0067 bacterial powder as the core active composition, and then mixed with water-soluble capsule wall material as the inner aqueous phase, and mixed with oil phase containing lipophilic emulsifier, and then emulsified by high shear to obtain W / O type primary emulsion. (2) Pour the primary emulsion into the storage tank of the membrane emulsification device, and under constant nitrogen pressure, press it through the SPG membrane into the external aqueous phase containing hydrophilic emulsifier and curing agent to form a W / O / W type double emulsion and cure it. (3) Collect the solidified microcapsules, and after centrifugation, washing and drying, microcapsule powder with uniform particle size is obtained.

8. The use of the bovine triamcinolone heptapeptide of claim 1, the bovine triamcinolone small molecule peptide of claim 2, or the composition of claim 6 in the preparation of a calcium absorption-promoting drug.

9. The application according to claim 8, characterized in that: The calcium absorption-promoting drug also contains any one or more of the following components: functional additives and any acceptable excipients.

10. A drug with calcium absorption-promoting effect, characterized in that, The drug comprises the bovine trisaccharide heptapeptide of claim 1, the bovine trisaccharide small molecule peptide of claim 2, or the composition of claim 6.

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