Calcium absorption promoting milk-derived pentapeptide and use thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a milk-derived pentapeptide for promoting absorption of calcium mineral and application thereof. The application obtains a CPP containing a high-activity peptide segment by optimizing a process, and verifies the influence of the CPP on mineral absorption by using a zebrafish model. Afterwards, a pentapeptide GPFPI with the activity of promoting calcium absorption is screened from the CPP by combining peptidomics and molecular docking technology and function verification is performed, and it is found that the pentapeptide can effectively promote Ca 2+ transport in a dose-dependent manner, and the Ca 2+ transport is increased by 23.23%, and the zebrafish experiment shows that the intervention of GPFPI effectively reverses the decrease of the cumulative optical density value caused by dexamethasone, and compared with the modeling group, the cumulative optical density value of the skull of the GPFPI (2-20 μM) treatment group is significantly increased. 2+ Therefore, the synthetic peptide GPFPI can effectively promote Ca 2+ transport and absorption, improve the osteoporosis symptoms of zebrafish, and increase the deposition of calcium in the bone.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a milk-derived pentapeptide for promoting absorption of calcium minerals and application thereof. BACKGROUND

[0002] Divalent minerals are trace nutrients essential for maintaining normal physiological functions of the human body, and play an important role in bone health, immune regulation, nerve conduction, etc. However, the problem of mineral deficiency is still serious worldwide, especially the insufficient intake or absorption of key minerals such as calcium, iron and zinc, which has become an important factor affecting human health. Therefore, it is of great theoretical and practical significance to explore functional ingredients that can promote mineral absorption. In recent years, bioactive peptides have shown broad application prospects in the field of promoting mineral absorption due to their unique physiological functions and good biocompatibility. Some studies have isolated and identified 14 calcium-chelating peptides from Antarctic krill hydrolysate, among which VERG shows the strongest calcium-chelating ability, and the calcium-chelating complex of this peptide has been confirmed to significantly improve calcium transport in an in vitro cell model.

[0003] Casein phosphopeptides (CPP) are a hydrolysate derived from casein, and studies have shown that they have the ability to bind to divalent minerals due to the high content of phosphoserine residues, can bind to mineral ions to form soluble complexes, and improve the solubility of these minerals in the intestinal tract, thereby promoting their absorption in the small intestinal mucosa. However, there is still insufficient research on the non-phosphopeptides with a high proportion of CPP products for mineral absorption and utilization, and further research and development of new active peptides with the function of promoting mineral absorption are needed. SUMMARY

[0004] To solve the above technical problems, the present application obtains a casein phosphopeptide containing a high-activity peptide segment by optimizing the process. First, the influence of the casein phosphopeptide prepared by the present application on mineral absorption is verified using a zebrafish model. Second, bioactive peptides with calcium absorption activity are screened from CPP by combining peptidomics and molecular docking technology and their functions are verified. This study aims to reveal the effects of CPP and small molecule peptides in mineral absorption and utilization and their application potential at the cellular and animal levels, and to provide a theoretical basis for the functional development of CPP.

[0005] One of the technical solutions provided by the present application is the application of a pentapeptide, particularly in promoting the absorption of calcium mineral elements, and more particularly in the preparation of health food or medicine for promoting the absorption of calcium mineral elements.

[0006] The pentapeptide is GPFPI, i.e., the amino acid sequence is Gly-Pro-Phe-Pro-Ile; the pentapeptide is separated from casein phosphopeptides produced by casein enzymolysis.

[0007] Further, the pentapeptide GPFPI can be used alone in the preparation of the health food or drug; or can be used in combination with other components having calcium mineral absorption promoting activity or calcium mineral element-containing supplements;

[0008] Preferably, the pentapeptide GPFPI and the calcium mineral element-containing supplement are used in combination, and the absorption effect of the calcium mineral element is better than that of casein phosphopeptide alone or calcium mineral element supplementation alone;

[0009] Further, the pentapeptide GPFPI is prepared into a beverage, an oral solution, a capsule, a microcapsule powder, a tablet, a granule, or an emulsion tablet, etc.

[0010] Preferably, the pentapeptide GPFPI is prepared into a microcapsule to improve its gastrointestinal digestion stability, bioavailability, and shelf life, so that it can be better applied in the food industry and health care field;

[0011] Further, the pentapeptide GPFPI can be prepared by a chemical solid-phase synthesis method;

[0012] Further, the pentapeptide GPFPI can also be obtained by separation from casein proteolysis products;

[0013] Still further, the preparation method of the casein proteolysis products comprises the following steps:

[0014] S1. The casein and water are mixed in a reaction kettle according to a mass ratio of casein: water = 1:5-10;

[0015] Further, the pH of the material solution is adjusted to 6.5-7.0 with a NaOH solution, and stirring and dissolving are performed for 30-90 min;

[0016] S2. After the dissolving is completed, a compound protease with a mass fraction of 0.1-2.0% (w / w) of the casein is added to the solution, and the enzyme hydrolysis is performed at 45-50°C for 4-8 h to obtain an enzyme hydrolysate;

[0017] Further, the compound protease comprises a protease, a papain, a bromelain, a carboxypeptidase, and an aminopeptidase;

[0018] Still further, the protease, the papain, the bromelain, the carboxypeptidase, and the aminopeptidase are compounded in a mass ratio of 5-10:5-10:0.1-1:0.1-1:0.1-1;

[0019] Preferably, the protease is derived from Bacillus subtilis;

[0020] Preferably, the carboxypeptidase is derived from Aspergillus niger;

[0021] Preferably, the aminopeptidase is derived from Aspergillus oryzae;

[0022] S3. Separate the supernatant from the enzymatic hydrolysate;

[0023] S4. Pass the supernatant through a membrane with a molecular weight cutoff of 5000 Da, and collect the filtrate;

[0024] S5. Concentrate the filtrate until the sugar content of the liquid reaches 35-40° to obtain a casein phosphopeptide concentrate;

[0025] Furthermore, the filtrate is evaporated and concentrated using a vacuum low-temperature thin-film evaporator until the sugar content of the liquid reaches 35-40°.

[0026] Furthermore, the concentrate is sterilized at high temperature and spray-dried to obtain casein phosphopeptides; the casein phosphopeptides contain the pentapeptide GPFPI.

[0027] Beneficial effects:

[0028] (1) This application obtains a casein phosphopeptide containing highly active peptide fragments through process optimization. The effect of the casein phosphopeptide prepared in this invention on mineral absorption was verified by zebrafish bone density experiments. Details are as follows:

[0029] In zebrafish experiments, skull bone mineral density analysis revealed that casein phosphopeptides (CPS) of 1–10 μg / ml significantly increased bone mineral density in osteoporosis models. Specifically, 2 μg / ml CPS significantly increased bone mineral density in zebrafish (p < 0.00001). Compared to the normal control group, 2 μg / ml CPS increased bone mineral density by 20.6%, and compared to the osteoporosis model group, 2 μg / ml CPS increased bone mineral density by 56.1%.

[0030] (2) Functional verification of the GPFPI peptide screened from the casein phosphopeptide prepared in this invention revealed that:

[0031] i. GPFPI for Ca 2+ Transport showed a dose-dependent trend, and at 10 μM Ca 2+ The transport volume increased significantly by 23.23%. Therefore, the effective concentration of GPFPI is 10 μM.

[0032] ii. The effect of the synthetic peptide GPFPI on the expression of calcium transport-related genes was as follows: compared with the control group, GPFPI significantly upregulated the gene expression of TRPV6 and NCX1. This indicates that GPFPI mainly promotes calcium ion transport through the TRPV6 calcium ion channel and does not affect the normal function of L-type calcium ion channels.

[0033] iii. Zebrafish experiments showed that intervention with the synthetic peptide GPFPI effectively reversed the dexamethasone-induced decrease in cumulative optical density. Specifically, compared with the model group, the cumulative optical density of the skull in the GPFPI (2–20 μM) treatment group was significantly increased. These results indicate that the synthetic peptide GPFPI can effectively improve dexamethasone-induced osteoporosis symptoms in zebrafish and increase calcium deposition in bones. Attached Figure Description

[0034] Figure 1 This is a total ion diagram of casein phosphopeptides.

[0035] Figure 2 The effect of casein phosphopeptide on bone density in zebrafish, an osteoporosis model.

[0036] Figure 3 Cytotoxicity analysis of the synthetic peptide GI-5.

[0037] Figure 4 To synthesize peptide GI-5 for Ca 2+ The impact of transshipment.

[0038] Figure 5 The effect of GI-5 on the expression of calcium transport-related genes.

[0039] Figure 6 The effect of synthetic peptide GI-5 on bone mineralization in osteoporotic zebrafish.

[0040] Note:

[0041] (1) Figure 2 In the comparison with the control group, # indicates p < 0.05; compared with the model group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.00001, and no marker indicates no statistical difference.

[0042] (2) Figures 3-4 In the study, there were significant differences between the groups represented by different letters (p<0.05).

[0043] (3) Figures 5-6 In the comparison with the control group, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.00001; and # indicates p<0.00001. Compared with the model group, # indicates p<0.05; ## indicates p<0.01; ### indicates p<0.001; ### indicates p<0.00001; and ns indicates no statistical difference. Detailed Implementation

[0044] The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0045] The present invention will be further explained and described below through specific embodiments.

[0046] Example 1: Preparation and property determination of casein phosphopeptide (CPP)

[0047] 1. A method for preparing casein phosphopeptides

[0048] S1. Add casein (manufacturer: Fonterra Dairy) and water into the reactor according to the ratio of casein:water = 1:10 (W / W), mix evenly, adjust the pH of the solution to 6.5-7.0 with NaOH solution, and stir to dissolve for 30 minutes;

[0049] S2. After dissolution, add 2.0% of complex protease (complex protease contains: protease + papain + bromelain + carboxypeptidase + aminopeptidase = 5:5:1:0.1:0.1 (mass ratio)) to the solution according to the casein mass ratio, and enzymatically hydrolyze for 6 hours at 45℃ to obtain the enzymatic hydrolysate;

[0050] The sources of the aforementioned proteases, papain, bromelain, carboxypeptidase, and aminopeptidase comply with the relevant provisions of the National Food Safety Standard for the Use of Food Additives (GB 2760-2024).

[0051] S3. The supernatant of the enzymatic hydrolysate is obtained by separating it using a disc separator;

[0052] S4. Pass the supernatant through a membrane with a molecular weight cutoff of 5000 Da, and collect the filtrate;

[0053] S5. The filtrate is evaporated and concentrated in a vacuum low-temperature thin-film evaporator until the sugar content of the liquid reaches 40° to obtain casein phosphopeptide concentrate; the concentrate is sterilized at high temperature and spray-dried to obtain casein phosphopeptide.

[0054] The degree of hydrolysis of casein phosphopeptide was determined to be 25.58%.

[0055] 2. Determination of molecular weight distribution of casein phosphopeptides

[0056] The molecular weight distribution of casein phosphopeptides (CPPs) was determined using high-performance liquid chromatography (HPLC). The molecular weight distribution exhibited diverse characteristics. As shown in Table 1, the CPP molecular weight distribution range was relatively wide, with the highest proportion (47.14%) occurring in the 180–500 Da range, indicating a relatively abundant number of peptides within this range. Furthermore, peptides with molecular weights <180 Da accounted for 6.87%, 500–1000 Da for 22.13%, 1000–2000 Da for 18.61%, 2000–3000 Da for 4.48%, and >3000 Da for only 0.77%.

[0057] Table 1. Molecular weight distribution of CPP

[0058]

[0059] 3. Amino acid composition of casein phosphopeptides

[0060] As shown in Table 2, CPP is rich in glutamic acid (24.26%), leucine (12.54%), lysine (11.62%), alanine (7.17%), and valine (5.91%). Among these, glutamic acid and lysine can serve as binding sites for mineral ions. Furthermore, the content of acidic amino acids (aspartic acid and glutamic acid) is positively correlated with the binding capacity of mineral ions. The proportion of acidic amino acids in CPP is 28.29%, indicating that CPP may have a good binding capacity with minerals, influencing the mineral transport process.

[0061] Table 2. Amino acid composition of CPP

[0062]

[0063] 4. Mass spectrometry analysis

[0064] The casein phosphopeptide prepared in step 1 was used as the sample for pretreatment. An appropriate amount of sample was dissolved in 50 mM ammonium bicarbonate. Dithiothreitol solution was transferred to the sample solution to bring the final concentration of dithiothreitol to 10 mM, and reduction was carried out at 56 ℃ for 1 h. Iodoacetamide solution was transferred to the sample solution to bring the final concentration of iodoacetamide to 20 mM, and the reaction was carried out in the dark for 40 min. Subsequently, unreacted iodoacetamide was neutralized with dithiothreitol. The sample was desalted using a desalting column and dried under vacuum at 45 ℃.

[0065] After processing, the samples were analyzed by LC / MS-MS to obtain raw mass spectrometry files. Peptide sequence resolution was performed using the PEAKS De novo method, with the following search parameters: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term), Phospho (S,T,Y); Enzyme: Non-specific; Peptide Mass Tolerance: 20 ppm; Fragment Mass Tolerance: 0.02 Da.

[0066] Total ion chromatogram as follows Figure 1 As shown.

[0067] The top 20 most abundant peptides are shown in Table 3.

[0068] Table 3

[0069]

[0070] 5. Molecular docking

[0071] Calcium is absorbed from the intestinal lumen into the bloodstream through two pathways: bypass transport and transcellular transport. Bypass transport typically occurs in the ileum and jejunum, where calcium ions are passively absorbed along their concentration gradient via tight junctions. Transcellular transport is an active saturation process against the concentration gradient and is the primary mode of calcium absorption in the initial intestinal segments (duodenum and jejunum). It mainly involves three steps: First, Ca... 2+ via brush border membrane via epithelial Ca 2+ The passage enters the intestinal epithelial cells. Ca 2+ The channels primarily include transient receptor potential vanilloid member 6 (TRPV6) and voltage-dependent L-type calcium channels (Cav1.3). Subsequently, Ca... 2+ It is transported from the apical side to the basal side of the cell by binding to calcium-binding proteins with high calcium affinity (mainly Calbindin-D9K). Finally, Ca 2+ via PMCA1b / Ca 2+ The pump (Plasma membrane calcium-transporting ATPase1b, PMCA1b) and Na + / Ca 2+ The exchanger (Sodium-calcium exchanger 1, NCX1) is expelled from the cell into the bloodstream.

[0072] Therefore, we selected those with relative abundance greater than 10. 9 The specific interaction mechanisms between candidate peptides and calcium ions, as well as calcium ion channel transport proteins TRPV6 and NCX1, were explored using molecular docking technology. Peptides with calcium absorption-promoting activity were screened and synthesized.

[0073] When a peptide docks with a calcium ion, the calcium ion acts as the ligand and the peptide acts as the acceptor. The values ​​are adjusted to cover the entire peptide, and a pocket is created for molecular docking.

[0074] When docking peptides with TRPV6 and NCX1 proteins, the peptides act as ligands and the proteins as acceptors. The values ​​are adjusted to cover the active sites of the proteins, and a pocket is created for molecular docking.

[0075] The results are shown in Tables 4, 5, and 6. Based on the binding energy (≤-5.0), GPFPI (GI-5, SEQ ID NO.1), FYPEL (FL-5, SEQ ID NO.2), YPVEPF (YF-6, SEQ ID NO.3), GPFPIIV (GV-7, SEQ ID NO.4), and VAPFPEV (VV-7, SEQ ID NO.5) can be considered as potential bioactive peptides that promote mineral absorption.

[0076] Table 4. Molecular docking results with calcium ions

[0077]

[0078] Table 5. Results of docking of peptides with TRPV6 protein molecules

[0079]

[0080] Table 6. Results of docking of peptides with NCX1 protein molecules

[0081]

[0082] Example 2: Effect of casein phosphopeptide on zebrafish bone density

[0083] Calcium is the most abundant mineral element in the human body, accounting for 1.5% to 2.2% of an adult's body weight. An adult body contains approximately 1.2 kg of calcium, with over 99% of it found in bones and teeth. Disorders of calcium metabolism can lead to bone diseases. Dexamethasone is a glucocorticoid that reduces intestinal calcium absorption and increases urinary calcium excretion, indirectly inducing secondary hyperparathyroidism and further exacerbating bone loss.

[0084] Zebrafish, a commonly used model organism, demonstrates unique value in skeletal disease research. Their structural and physiological characteristics provide a flexible and efficient animal model for studying skeletal diseases. In terms of skeletal development, zebrafish share many similarities with mammals, including both endochondral and intramembranous ossification—two modes of bone formation.

[0085] Alizarin red staining is a fixation staining method that produces a colorimetric phenomenon by reacting with calcium nodules in bone, and is used to detect bone morphology and bone density in fish.

[0086] In this embodiment, zebrafish will be used as the experimental organism, and an osteoporosis model will be constructed using dexamethasone. Alizarin red staining will be used to verify the promoting effect of casein phosphopeptide on calcium absorption.

[0087] The specific experiment is as follows:

[0088] The zebrafish were housed at the Zebrafish Platform of Zhejiang University School of Medicine under the following conditions: a light / dark cycle of 14:10h and a water temperature of 28℃.

[0089] Male and female zebrafish were paired and spawned. The resulting fertilized eggs were cultured in a 28.5°C incubator. Three days post-fertilization (dpf), zebrafish embryos were randomly transferred to 6-well plates, 15 embryos per well. These were designated as a control group, a model group, and a casein phosphopeptide group (sample: casein phosphopeptide prepared in Example 1), with 6 wells in each group. The following solutions were added to each group:

[0090] Control group: Water containing 0.2% (m / v) dimethyl sulfoxide (DMSO);

[0091] Model group: water containing 0.2% dimethyl sulfoxide + dexamethasone (dexamethasone dissolved in water containing 0.2% DMSO to a final concentration of 20 μM);

[0092] Casein phosphopeptide group: water containing 0.2% dimethyl sulfoxide + dexamethasone (dexamethasone dissolved in water containing 0.2% DMSO to a final concentration of 20 μM) + casein phosphopeptide (final concentrations of casein phosphopeptide in water: 0, 1, 2, 5, 10, 20, 50, 100 μg / mL).

[0093] All groups underwent solution changes every 24 hours until zebrafish larvae reached 8 days per fish (dpf). Subsequently, the zebrafish larvae were euthanized by anesthesia and subjected to the following treatments: fixation with 4% paraformaldehyde for 24 hours, rinsing with 50% ethanol for 10 minutes, eluting with 3% H₂O₂ + 0.5% KOH solution for 30 minutes, rinsing three times with 25% glycerol + 0.1% KOH, staining with 0.01% alizarin red solution in the dark for 2 hours, rinsing three times with 50% glycerol + 0.1% KOH, and finally storing in 100% glycerol. Images of the zebrafish skulls were acquired using a stereomicroscope, and bone mineral density (expressed as relative fluorescence intensity) was quantitatively analyzed using ImageJ software.

[0094] The results are as follows Figure 2 As shown, dexamethasone intake significantly reduced the relative fluorescence intensity of bones (p<0.05), indicating the successful establishment of the zebrafish osteoporosis model. Casein phosphopeptide (1-10 μg / ml) significantly improved bone mineral density in the osteoporosis model, with 2 μg / ml significantly increasing bone mineral density in zebrafish (p<0.00001). Compared to the normal control group, 2 μg / ml casein phosphopeptide increased bone mineral density by 20.6%, and compared to the osteoporosis model group, 2 μg / ml casein phosphopeptide increased bone mineral density by 56.1%.

[0095] Example 3: Peptide Function Verification

[0096] The peptide GPFPI (hereinafter referred to as GI-5) screened in Example 1 was artificially synthesized. Subsequently, the calcium absorption-promoting activity and mechanism of action of the synthesized peptide were verified using the Caco-2 cell model. Finally, the effect of the synthesized peptide on calcium absorption and utilization in vivo was verified using an osteoporosis-like zebrafish model.

[0097] 1. Assay for the cytotoxicity of synthetic peptides

[0098] Cytotoxicity assay: Cell viability was detected using the CCK-8 staining method. The specific steps are as follows: 100 μL of Caco-2 cells were inoculated at 1.0 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of [number] cells / mL in 96-well plates and incubated for 24 h to allow complete cell adhesion. The culture medium was then discarded, and the cells were washed twice with PBS. 100 μL of a solution containing different concentrations of peptides (synthetic peptide concentrations were set at 0, 1, 5, 20, 50, and 100 μM) was added to each well, with six replicates per group, and incubated for 24 h. After incubation, the culture medium was discarded, the cells were washed with PBS, and CCK-8 reagent was added, followed by incubation for 60 min. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula:

[0099] Cell viability (%) = (OD) t - OD0) / (OD s - OD0)

[0100] Among them, OD s The absorbance and OD of the control group (the group where the synthetic peptide was administered at a concentration of 0) are shown. t OD0 represents the absorbance of the sample loading group, while OD0 represents the absorbance of the blank group (the group containing neither cells nor samples).

[0101] The results are as follows Figure 3 As shown, within the concentration range of 0–100 μM, the synthetic peptide GPFPI had no significant effect on the viability of Caco-2 cells.

[0102] 2. The effect of synthetic peptides on calcium transport

[0103] Establishment of a Caco-2 cell monolayer model: Caco-2 cells were layered at a density of 1.0 × 10⁻⁶. 5 cells / cm 2 The culture medium was seeded at a density of 500 μL into the apical chambers (AP) of 12-well Transwell plates, and 1.5 mL of complete culture medium was added to the basolateral side (BL). The plates were then incubated in an incubator, with the medium changed every other day until day 21.

[0104] Caco-2 cell monolayer transport: Cells with a TEER value higher than 400 Ω•cm were selected. 2Transport experiments were conducted using wells. To better simulate the acidic microenvironment of the intestinal lumen, the pH of the HBSS on the AP side was adjusted to 6.0 using 25 mM MES and Tris phosphate solution, and the pH of the HBSS on the BL side was adjusted to 7.4 using 25 mM MES and Tris phosphate solution. Caco-2 cells were gently washed three times with pre-warmed HBSS and incubated for 30 min. Subsequently, 0.5 mL of sample (HBSS dissolved, pH 6.0) was added to the AP side, and the synthetic peptide administration concentrations on the AP side were 0, 1, 2, and 10 μM. Simultaneously, a certain concentration of CaCl2 solution was added to each well to ensure that CaCl2 was properly dissolved. 2+ The final concentration in the culture medium was 200 μg / mL. 1.5 mL of HBSS (pH 7.4) was added to the BL side, and the mixture was placed in an incubator for a transport experiment for 120 min. After the experiment, the solution sample from the BL side was collected, and the mineral ion concentration was determined using inductively coupled plasma mass spectrometry.

[0105] 1, 2, and 10 μM GI-5 were respectively reacted with Ca 2+ The two were transported together for 120 minutes, and the results were as follows: Figure 4 As shown. GI-5 affects Ca 2+ Transport showed a dose-dependent trend, and at 10 μM Ca 2+ The transport capacity was significantly increased (p<0.05), reaching 2.44±0.20 μg / well, representing a 23.23% increase compared to the control group. Based on these results, the effective concentration of GI-5 is 10 μM, which is suitable for further experiments.

[0106] 3. Effects of synthetic peptides on the expression of calcium transport-related genes

[0107] (1) RNA extraction

[0108] After the transport experiment, Caco-2 cells were harvested, washed twice with pre-chilled PBS, and 1 mL of Trizol was added to each well. Cells were lysed on ice for 5 min, collected, and centrifuged at 12000 rpm for 5 min. The supernatant was collected, mixed with 200 μL of chloroform, allowed to stand for 5 min, and then centrifuged at 12000 rpm for 15 min. The upper layer was collected, mixed with 500 μL of isopropanol, allowed to stand for 30 min, and then centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and the RNA precipitate at the bottom of the ion exchange tube was obtained. The precipitate was washed twice with 75% ethanol. Finally, 30 μL of LDPPC water was added to dissolve the precipitate, and the concentration and purity of the RNA were determined using Nano Drop.

[0109] (2) RNA reverse transcription

[0110] RNA reverse transcription was performed according to the reverse transcription reagent instructions. The specific procedure was as follows: 1 μL of RNA solution, 4 μL of 4×gDNAwiperMix, and 16 μL of ddH2O were mixed and incubated at 42 °C for 2 min. Subsequently, 4 μL of 5×HiscriptⅢqRTSuperMix was added, and the mixture was incubated at 37 °C for 15 min, followed by incubation at 85 °C for 5 s to obtain the cDNA stock solution.

[0111] (3) Real-time quantitative PCR

[0112] The real-time quantitative PCR reaction system is shown in Table 7, and the target gene and primer sequences are shown in Table 8. The reaction parameters were: pre-denaturation at 95℃ for 30 s, one cycle; cycling at 95℃ for 10 s, 60℃ for 30 s, 40 cycles; melting curve at 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s, one cycle. Relative gene expression levels were determined using the 2-ΔΔCT method.

[0113] Table 7 Real-time Quantitative PCR Reaction System

[0114]

[0115] Table 8 Primer sequence list

[0116]

[0117] The results are as follows Figure 5 As shown in the figure, compared with the control group, GI-5 significantly upregulated TRPV6 gene expression (p<0.05). GI-5 significantly upregulated NCX1 gene expression (p<0.05). GI-5 had no significant effect on the gene expression of Cav1.3, Calbindin-D9k, and PMCA1b (p>0.05). This indicates that GI-5 mainly promotes calcium ion transport through the TRPV6 calcium ion channel and does not affect the normal function of L-type calcium ion channels.

[0118] Example 6: Effect of Synthetic Peptides on Zebrafish Bone Mineralization

[0119] The zebrafish were housed at the Zebrafish Platform of Zhejiang University School of Medicine under the following conditions: a light / dark cycle of 14:10h and a water temperature of 28℃.

[0120] Male and female zebrafish were paired and spawned. The resulting fertilized eggs were cultured in a 28.5℃ incubator. Three days post-fertilization (dpf), zebrafish embryos were randomly transferred to 6-well plates, 15 embryos per well. These were designated as a control group, a model group, and a synthetic peptide group (GI-5), with 6 wells per group. The following solutions were added to each group:

[0121] Control group: Water containing 0.2% (m / v) dimethyl sulfoxide (DMSO);

[0122] Model group: water containing 0.2% dimethyl sulfoxide + dexamethasone (dexamethasone dissolved in water containing 0.2% DMSO to a final concentration of 20 μM);

[0123] Synthetic peptide GI-5 group: water containing 0.2% dimethyl sulfoxide + dexamethasone (dexamethasone dissolved in water containing 0.2% DMSO to a final concentration of 20 μM) + synthetic peptide GI-5 (final concentrations of 0, 0.5, 1, 2, 10, and 20 μM).

[0124] All groups had their solutions changed every 24 hours until the zebrafish juveniles reached 8 days per fish (dpf). Subsequently, the zebrafish juveniles were anesthetized and euthanized, and then subjected to the following treatments: fixation with 4% paraformaldehyde for 24 hours, rinsing with 50% ethanol for 10 minutes, eluting with 3% H₂O₂ + 0.5% KOH solution for 30 minutes, rinsing three times with 25% glycerol + 0.1% KOH, staining with 0.01% alizarin red solution in the dark for 2 hours, rinsing three times with 50% glycerol + 0.1% KOH, and finally storing in 100% glycerol. Images of the zebrafish skulls were acquired using a stereomicroscope, and the cumulative optical density of the skulls was quantitatively analyzed using ImageJ software.

[0125] The results are as follows Figure 6 As shown. Compared with the control group, the cumulative optical density value of the zebrafish skull in the model group was significantly reduced ( p <0.05 indicates that dexamethasone successfully induced osteoporosis in zebrafish, leading to severe calcium loss from the skull. Intervention with GI-5 effectively reversed the dexamethasone-induced decrease in cumulative optical density. Specifically, compared to the model group, the cumulative optical density of the skull in the GI-5 (2–20 μM) treatment group was significantly increased (…). p <0.05). These results indicate that the synthetic peptide GI-5 can effectively improve dexamethasone-induced osteoporosis symptoms in zebrafish and increase calcium deposition in bones.

[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. The application of the pentapeptide GPFPI, characterized in that, This refers to the application of pentapeptide GPFPI in the preparation of health foods that increase bone density, or the application of pentapeptide GPFPI in the preparation of drugs that promote the absorption of calcium mineral elements; the amino acid sequence of the pentapeptide GPFPI is: Gly-Pro-Phe-Pro-Ile.

2. The application as described in claim 1, characterized in that, The promotion of calcium and mineral element absorption manifests itself in any of the following ways: increasing Ca... 2+ It can transport and improve osteoporosis symptoms, increase bone density, or increase calcium deposition in bones.

3. The application as described in claim 1, characterized in that, The pentapeptide GPFPI was prepared by chemical solid-phase synthesis.

4. The application as described in claim 1, characterized in that, The pentapeptide GPFPI was isolated from casein hydrolysate; The method for preparing the casein hydrolysate includes the following steps: S1. Add casein and water to the reaction vessel according to the mass ratio of casein:water = 1:5-10 and mix evenly; S2. After dissolution, add 0.1-2.0% of casein complex protease to the solution and hydrolyze at 45-50℃ for 4-8 hours to obtain the hydrolysate. The complex protease comprises protease, papain, bromelain, carboxypeptidase, and aminopeptidase; The protease is derived from Bacillus subtilis, the carboxypeptidase is derived from Aspergillus niger, and the aminopeptidase is derived from Aspergillus oryzae. S3. Separate the supernatant from the enzymatic hydrolysate; S4. Pass the supernatant through a membrane with a molecular weight cutoff of 5000 Da, and collect the filtrate; S5. The filtrate is concentrated to a sugar content of 35-40° to obtain a casein phosphopeptide concentrate; the concentrate is sterilized at high temperature and spray-dried to obtain casein phosphopeptides; the casein phosphopeptides contain pentapeptide GPFPI.

5. The application as described in claim 4, characterized in that, In S1, adjust the pH of the solution to 6.5-7.0 with NaOH solution and stir to dissolve for 30-90 minutes.

6. The application as described in claim 4, characterized in that, In S2, the protease, papain, bromelain, carboxypeptidase and aminopeptidase are compounded in a mass ratio of 5-10:5-10:0.1-1:0.1-1:0.1-1.

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