Casein-sourced heptapeptide for promoting calcium mineral absorption and application of casein-sourced heptapeptide

Abstract

The invention belongs to the technical field of biology, and particularly relates to casein-sourced heptapeptide for promoting calcium mineral absorption and application of the casein-sourced heptapeptide. The CPP containing the high-activity peptide fragment is obtained through an optimization process, and the influence of the CPP on mineral absorption and conversion is evaluated by utilizing a mineral deficiency rat model; then heptapeptide with calcium absorption promoting activity is screened from the CPP in combination with peptidomics and molecular docking technologies, functional verification is carried out, it is found that when the GPFPIIV is 2 mu M, the Ca < 2 + > transport amount is remarkably increased by 21.21%, meanwhile, zebra fish experiments show that intervention of the GPFPIIV effectively reverses accumulated optical density value reduction caused by dexamethasone, and compared with a module making set, the method has the advantages that the effect is good, and the effect is good. And the skull accumulated optical density value of a GPFPIIV (20 mu M) processing group is obviously increased. It is shown that GPFPIIV can effectively promote Ca < 2 + > transfer and absorption, improve the osteoporosis symptom of zebra fish and increase deposition of calcium in bones.

Description

TECHNICAL FIELD

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

[0002] Calcium is the most abundant mineral element in the human body, accounting for 1.5-2.2% of the body weight of an adult. About 1.2 kg of calcium exists in the body of an adult, of which more than 99% exists in the bones and teeth, and the remaining calcium exists in the form of free calcium and participates in blood circulation. Calcium is essential for the normal physiological functions of an organism, and is involved in multiple physiological processes such as nerve signal transmission, muscle tissue contraction, cell differentiation and apoptosis, enzyme activity activation and immune response. However, the problem of mineral deficiency is still serious worldwide, and the insufficient intake or absorption of key minerals such as calcium, iron and zinc has become an important factor affecting human health. Therefore, it is of great theoretical and practical significance to explore functional ingredients that can promote the absorption of minerals. 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. Studies have isolated three active peptides GPAGPHGPVG, FDHIVY and YQEPVIAPKL from tilapia bone collagen hydrolysate, and the calcium chelating activities of the three peptides are 18.80±0.49 mg / g, 35.73±0.74 mg / g and 28.4±0.94 mg / g, respectively.

[0003] Casein phosphopeptide (CPP) is a hydrolyzate derived from casein, which has the ability to bind to divalent minerals due to the enrichment of phosphoserine residues, and is considered as a potential mineral absorption promoter. Studies have shown that CPP can bind to mineral ions to form soluble complexes, thereby improving the solubility of these minerals in the intestinal tract and promoting their absorption in the small intestinal mucosa. In addition, CPP may also indirectly affect the absorption of minerals by regulating the expression of intestinal-related genes.

[0004] However, the research on small peptides in CPP that affect mineral absorption is insufficient, and new casein-derived non-phosphoric peptides with mineral absorption-promoting efficacy need to be further developed. SUMMARY

[0005] In order to solve the above technical problems, the application obtains a casein phosphopeptide rich in high-activity peptide segments by optimizing the process, and takes six mineral elements of calcium, magnesium, copper, iron, zinc and manganese as the research objects. First, the mineral deficiency rat model is used to evaluate the absorption and utilization of the six minerals by CPP. Second, the bioactive peptides with calcium absorption activity are screened from CPP by combining peptidomics and molecular docking technology and the function is verified. The present study aims to reveal the effect of CPP and small molecule peptides on mineral absorption and utilization at the cellular and animal levels and their application potential, and to provide a theoretical basis for the functional development of CPP.

[0006] The application provides a heptapeptide, the heptapeptide is GPFPIIV, and the amino acid sequence of the heptapeptide is Gly-Pro-Phe-Pro-Ile-Ile-Val.

[0007] Further, the heptapeptide GPFPIIV can be prepared by a chemical solid-phase synthesis method. Further, the heptapeptide GPFPIIV can also be obtained by separation from casein enzymolysis products. Further, the preparation method of the casein enzymolysis products comprises the following steps. S1. casein and water are put into a reaction kettle in a mass ratio of 1:5-10 and mixed uniformly. Further, the pH of the material solution is adjusted to 6.5-7.0 by using a NaOH solution, and the solution is stirred and dissolved for 30-90 min. S2. After the dissolution is completed, 0.1-2.0% (w / w) of the complex protease of the mass of the casein is added to the solution, and the enzymolysis is performed at 45-50 DEG C for 4-8 h to obtain an enzymolysis solution. Further, the complex protease comprises a protease, a papain, a bromelain, a carboxypeptidase and an aminopeptidase. 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. Preferably, the protease is derived from Bacillus subtilis. Preferably, the carboxypeptidase is derived from Aspergillus niger. Preferably, the aminopeptidase is derived from Aspergillus oryzae. S3. The supernatant is separated from the enzymolysis solution. S4. The supernatant is filtered through a membrane with a molecular weight cutoff of 5000 Da, and the filtrate is obtained. S5. The filtrate is concentrated to a sugar content of 35-40 DEG, and a casein phosphopeptide concentrate is obtained. Further, the filtrate is concentrated to a sugar content of 35-40° by vacuum low-temperature thin film evaporator. Further, the concentrated solution is sterilized at high temperature and spray dried to obtain casein phosphopeptide; the casein phosphopeptide comprises a heptapeptide GPFPIIV.

[0008] The second technical solution provided by the present application is a composition comprising the heptapeptide GPFPIIV, wherein the composition takes GPFPIIV as the only active ingredient, and can further comprise other components having the activity of promoting absorption of calcium mineral elements and / or excipients.

[0009] The third technical solution provided by the present application is the application of the heptapeptide GPFPIIV in the first technical solution or the composition in the second technical solution, in particular, the application in promoting absorption of calcium mineral elements, and more particularly, the application in preparing food, health products or drugs for promoting absorption of calcium mineral elements. Further, the promotion of absorption of calcium mineral elements can be any of the following aspects: increasing Ca 2+ transportation, improving osteoporosis symptoms, increasing bone density or increasing deposition of calcium in bones. Further, the heptapeptide GPFPIIV can be used alone in the preparation of the food, health products or drugs; or can be used in combination with other components having the activity of promoting absorption of calcium mineral elements or supplements containing calcium mineral elements. Preferably, the heptapeptide GPFPIIV and the supplement containing calcium mineral elements are used in combination, and the absorption effect of calcium mineral elements is better than that of using casein phosphopeptide alone or supplementing calcium mineral elements alone. Further, the heptapeptide GPFPIIV is prepared into beverages, oral solutions, capsules, microcapsule powders, tablets, granules or emulsion tablets. Preferably, the heptapeptide GPFPIIV is prepared into microcapsules to improve its stability in gastrointestinal digestion, bioavailability and shelf life, so that it can be better applied in the food industry and health care field.

[0010] Advantages: (1) The present application obtains a casein phosphopeptide rich in high-activity peptide segments by optimizing the process, and takes six mineral elements of calcium, magnesium, copper, iron, zinc and manganese as the research objects, and verifies the influence of the casein phosphopeptide prepared by the present application on mineral absorption through a mineral deficiency rat model. Specifically as follows: In the mineral deficiency rat model, compared with the MD group, the content of six kinds of minerals in the femur of each intervention group was increased, among which the Ca, Mg, Cu, Fe, Zn and Mn contents in the femur of the CPP group were increased by 7.12%, 3.56%, 20.76%, 15.84%, 9.94% and 67.12% respectively, which indicated that casein phosphopeptide could effectively promote the absorption of minerals by rats and promote the deposition of minerals on the skeleton; the Ca, Mg, Cu, Fe, Zn and Mn contents in the femur of the MS group were increased by 0.42%, 3.79%, 13.20%, 18.51%, 4.31% and 24.80% respectively, which indicated that the supplement of minerals could effectively alleviate the influence of low-mineral diet on the bone mineral deficiency of rats; the Ca, Mg, Cu, Fe, Zn and Mn contents in the femur of the CM group were increased by 10.56%, 14.92%, 19.48%, 13.68%, 19.28% and 21.06% respectively. By comparing the absorption of Ca, Mg, Cu, Fe, Zn and Mn in the CPP group, the MS group and the CM group, it was found that the casein phosphopeptide in the CM group could further promote the absorption of minerals, and the overall effect was better than that of using CPP or supplementing mineral elements alone.

[0011] (2) Through the function verification of the peptide segment GPFPIIV screened from the casein phosphopeptide prepared by the application, it is found that: i. GPFPIIV significantly increases Ca 2+ transportation at 2 μM and 10 μM. 2+ The Ca

[0012] ii. The influence of the synthetic peptide GPFPIIV on the expression of calcium transport related genes is that: compared with the control group, GPFPIIV significantly up-regulates the gene expression of TRPV6. This indicates that they mainly promote the transport of calcium ions through the TRPV6 calcium ion channel.

[0013] iii. Zebrafish experiments show that: the intervention of the synthetic peptide GPFPIIV effectively reverses the decrease of the cumulative optical density value caused by dexamethasone. Specifically, compared with the modeling group, the cumulative optical density value of the skull of the GPFPIIV (20 μM) treatment group is significantly increased. These results indicate that the synthetic peptide GPFPIIV can effectively improve the symptoms of dexamethasone-induced osteoporosis in zebrafish and increase the deposition of calcium in the skeleton. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a total ion chromatogram of casein phosphopeptide.

[0015] Figure 2 To analyze the cytotoxicity of synthetic peptide GV-7.

[0016] Figure 3 To analyze the effect of synthetic peptide GV-7 on Ca 2+ transport.

[0017] Figure 4 To analyze the effect of synthetic peptide GV-7 on the expression of calcium transport-related genes.

[0018] Figure 5 To analyze the effect of synthetic peptide GV-7 on bone mineralization in osteoporotic zebrafish.

[0019] Note: (1) Figures 2-3 Among them, different letters represent significant differences between groups (p < 0.05).

[0020] (2) Figures 4-5 Among them, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.00001 compared with the blank group, # represents p < 0.05, ## represents p < 0.01, ### represents p < 0.001, ### represents p < 0.00001 compared with the model group, and ns represents no statistical difference. DETAILED DESCRIPTION

[0021] The present application will be described below through specific embodiments. The technical means not specifically explained in the present application are methods well known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present application, and the essence and scope of the present application are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0022] The present application will be further explained and described below through specific embodiments.

[0023] Example 1 Preparation and property determination of casein phosphopeptide (CPP) 1. A preparation method of casein phosphopeptide S1. According to the ratio of casein: water = 1:10 (W / W), casein (manufacturer: Hengtianr Dairy) and water were put into a reaction kettle and mixed uniformly, the pH of the material liquid was adjusted to 6.5-7.0 with NaOH solution, and stirred and dissolved for 30 min; S2. After the dissolution is completed, 2.0% of the compound protease (the compound protease comprises: protease + papain + bromelain + carboxypeptidase + aminopeptidase = 5:5:1:0.1:0.1 (mass ratio)) is added to the solution according to the casein mass ratio, and enzymolysis is performed at 45°C for 6h to prepare an enzymolysis liquid; The sources of the protease, papain, bromelain, carboxypeptidase, and aminopeptidase comply with the relevant provisions of the National Food Safety Standard Standard for Use of Food Additives (GB 2760-2024).

[0024] S3. The enzymolysis liquid is separated using a disc separator to obtain supernatant; S4. The supernatant is filtered through a membrane with a molecular weight cutoff of 5000 Da to obtain a filtrate; S5. The filtrate is evaporated and concentrated to a sugar content of 40° by a vacuum low-temperature thin film evaporator to prepare a casein phosphopeptide concentrate; the concentrate is sterilized at high temperature and spray dried to prepare casein phosphopeptide.

[0025] The degree of hydrolysis of the casein phosphopeptide is 25.58%.

[0026] 2. Determination of casein phosphopeptide molecular weight distribution The molecular weight distribution of the casein phosphopeptide was determined by high performance liquid chromatography. The molecular weight distribution showed a diversified characteristic. As shown in Table 1, the CPP molecular weight distribution range was relatively wide, among which the part with a molecular weight of 180-500 Da accounted for the highest proportion of 47.14%, indicating that the number of peptide segments in this interval was relatively rich. In addition, the proportion of <180 Da was 6.87%, 500-1000 Da was 22.13%, 1000-2000 Da was 18.61%, 2000-3000 Da was 4.48%, and >3000 Da was only 0.77%.

[0027] Table 1 CPP molecular weight distribution

[0028] 3. Amino acid composition of casein phosphopeptide 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 them, glutamic acid and lysine can be used as binding sites for mineral ions. In addition, the content of acidic amino acids (aspartic acid and glutamic acid) is positively correlated with the binding ability of mineral ions. The proportion of acidic amino acids in CPP is 28.29%, indicating that CPP may have good binding ability with minerals, affecting the transport process of minerals.

[0029] Table 2 CPP amino acid composition

[0030] 4. Mass spectrometry analysis The casein phosphopeptide prepared in step 1 was used as sample for sample pretreatment, and an appropriate amount of sample was dissolved with 50 mM ammonium bicarbonate solution. Dithiothreitol solution was removed and added to the sample solution to make the final concentration of dithiothreitol 10 mM, and reduced at 56 ℃ for 1 h. Iodoacetamide solution was removed and added to the sample solution to make the final concentration of iodoacetamide 20 mM, and reacted in the dark for 40 min, followed by neutralizing the unreacted iodoacetamide with dithiothreitol. Desalting column was used for desalting, and vacuum dried at 45 ℃.

[0031] The mass spectrum raw file was obtained after the pretreated sample was analyzed by LC / MS-MS, and the polypeptide sequence was analyzed by the method of PEAKS De novo, and the search parameters were as follows: fixed modification (Fixed modifications): Carbamidomethyl (C); variable modification (Variable modifications): Oxidation (M), Acetyl (Peptide N-term), Phospho (S, T, Y); enzyme (Enzyme): Non specific; primary mass spectrum deviation (Peptide Mass Tolerance): 20 ppm; secondary mass spectrum deviation (Fragment Mass Tolerance): 0.02 Da.

[0032] The total ion chromatogram is shown in Figure 1 .

[0033] Among them, the top 20 peptides in abundance are shown in Table 3.

[0034] Table 3

[0035] 5. Molecular docking Calcium is absorbed into the blood from the intestinal lumen through two pathways: paracellular transport and transcellular transport. Paracellular transport usually occurs in the ileum and jejunum, and calcium ions are passively absorbed along the concentration gradient through tight junctions. Transcellular transport is an active saturation process against the concentration gradient, which is the main way of calcium absorption in the initial segment of the intestine (duodenum and jejunum), and mainly consists of three steps: first, Ca 2+ is transported into the intestinal epithelial cells through the brush border membrane epithelial Ca 2+ channel. Ca 2+The channels mainly include transient receptor potential vanilloid member 6 (TRPV6) and voltage gated Ca channel v1.3 (Cav1.3). Subsequently, Ca 2+ is transported from the apical side to the basolateral side of the cell by binding to a calcium-binding protein (mainly Calbindin-D9K) with high calcium affinity. Finally, Ca 2+ is pumped out of the cell into the blood by PMCA1b / Ca 2+ pump (Plasma membrane calcium-transporting ATPase1b, PMCA1b) and Na + / Ca 2+ exchanger (Sodium-calcium exchanger 1, NCX1).

[0036] Therefore, the peptide segments with a relative abundance greater than 10 9 were selected, and the specific action mode of the candidate peptide segments with calcium ions and calcium ion channel transport proteins TRPV6 and NCX1 was explored by molecular docking technology. The peptide segments with calcium absorption activity were screened and synthesized.

[0037] When the peptide segment and calcium ions are docked, the calcium ions are ligands and the peptide segment is the receptor. The numerical value is adjusted to cover the entire peptide segment, and a pocket is established for molecular docking.

[0038] When the peptide segment and TRPV6 and NCX1 proteins are docked, the peptide segment is the ligand and the protein is the receptor. The numerical value is adjusted to cover the active site of the protein, and a pocket is established for molecular docking.

[0039] The results are shown in Tables 4, 5 and 6. According to the binding energy (≤-5.0) as the standard, it is determined that 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 used as potential active peptides for promoting mineral absorption.

[0040] Table 4 Molecular docking results with calcium ions

[0041] Table 5 Molecular docking results of peptide segments and TRPV6 protein

[0042] Table 6 Peptide segment and NCX1 protein molecule docking results

[0043] Example 2 Effect of casein phosphopeptide on rat femur 1. Rat experiment 50 three-week-old male Wister rats were raised in the animal experiment center of Zhejiang Chinese Medical University. After one week of adaptive feeding, the formal experiment was carried out. According to the body weight, the rats were randomly divided into 5 groups, 10 rats in each group. The normal group was fed with normal feed, and the rest of the treatment groups were fed with low mineral feed. The mineral composition in the feed is shown in Table 7 (the composition in the normal feed and the low mineral feed is the same except for the mineral composition), and the specific grouping and dosing are shown in Table 8. Among them, the dose of mineral supplement was calculated according to the recommended human intake of 2 tablets per person per day and then gavaged (market purchase: Nuotai Lei multiple vitamins and mineral tablets). According to the relevant provisions of “Health Food Raw Material Directory Nutrient Supplement (2023 Edition)”, the dose of CPP was selected as 5:1 for Ca:CPP. CPP and mineral supplements were ground into powder and suspended in 0.5% carboxymethylcellulose sodium for gavage, with a gavage dose of 1 mL / 100 g. The normal group and the model group were gavaged with the same dose of carboxymethylcellulose sodium every day, and the other groups were gavaged with the corresponding test substances according to the sequence number 3, 4 and 5 in Table 8, respectively, for 6 weeks of continuous intervention. After the end of the 6th week experiment, the rats were anesthetized with pentobarbital sodium, and the right femur was dissected after removing the soft tissue. Then the right femur was quickly frozen in liquid nitrogen and placed in a -80 ℃ refrigerator for analysis.

[0044] The research protocol has been reviewed by the Animal Ethics Committee of Zhejiang Chinese Medical University, and all animal experiments strictly follow the animal welfare and ethical standards, with the animal ethics number IACUC-20231225-07.

[0045] Table 7 Mineral content in feed

[0046] Table 8 Animal experiment group setting

[0047] 2. Experimental results (1) Determination of femur mineral content The femur was dried in a 105 ℃ oven, ground in a mortar, and digested by wet method. After digestion, deionized water was added to constant volume, membrane was passed, and the corresponding dilution multiple was determined by machine for the content of calcium, magnesium, copper, iron, zinc and manganese.

[0048] The results are shown in Table 9. Compared with the NCD group, the contents of Ca, Mg, Cu, Fe, Zn and Mn in the femur of the MD group were reduced, indicating that feeding with low mineral feed could cause disorder of mineral distribution in the femur of rats. Compared with the MD group, the contents of the six minerals in the femur of each intervention group were increased, among which the contents of Ca, Mg, Cu, Fe, Zn and Mn in the femur of the CPP group were increased by 7.12%, 3.56%, 20.76%, 15.84%, 9.94% and 67.12% respectively, indicating that casein phosphopeptide could effectively promote the absorption of minerals by rats and promote the deposition of minerals on the skeleton; the contents of Ca, Mg, Cu, Fe, Zn and Mn in the femur of the MS group were increased by 0.42%, 3.79%, 13.20%, 18.51%, 4.31% and 24.80% respectively, indicating that supplementing minerals could effectively alleviate the effect of low-mineral diet on the lack of minerals in the skeleton of rats; the contents of Ca, Mg, Cu, Fe, Zn and Mn in the femur of the CM group were increased by 10.56%, 14.92%, 19.48%, 13.68%, 19.28% and 21.06% respectively. Comparing the absorption of Ca, Mg, Cu, Fe, Zn and Mn in the CPP group, the MS group and the CM group, it was found that casein phosphopeptide in the CM group could further promote the absorption of minerals, and the overall effect was better than that of using CPP alone or supplementing mineral elements alone.

[0049] Table 9 Effect of CPP on the content of femur minerals of rats

[0050] Example 3 Verification of the function of the peptide segment The peptide segment GPFPIIV screened in Example 1 was artificially synthesized, and then the Caco-2 cell model was used to verify the calcium absorption activity and mechanism of action of the synthesized peptide segment. Finally, the zebrafish model of osteoporosis was used to verify the effect of the synthesized peptide segment on calcium absorption in vivo.

[0051] 1. Cell toxicity test of synthetic peptide Cell toxicity test method: CCK-8 staining method was used to detect cell viability, and the specific steps were as follows: 100 μL of Caco-2 cells were seeded in a 96-well plate at a concentration of 1.0×10 5cells / mL were seeded in 96-well plates and incubated for 24 h to allow cells to adhere completely. Then the culture medium was discarded and the cells were washed twice with PBS, and 100 μL of a solution containing different concentrations of the peptide (the synthetic peptide was administered at concentrations of 0, 1, 5, 20, 50, and 100 μM) was added, with 6 replicates in each 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, and incubated for 60 min. The absorbance was measured at 450 nm using a microplate reader. The cell survival rate was calculated according to the following formula: Cell survival rate (%) = (OD t - OD0) / (OD s - OD0) where OD s is the absorbance of the control group (the group administered with the synthetic peptide at a concentration of 0), OD t is the absorbance of the sample group, and OD0is the absorbance of the blank group (the group without cells and without sample).

[0052] The results are shown in Table 1. Figure 2 As can be seen, within the concentration range of 0-100 μM, the synthetic peptide GPFPIIV had no significant effect on the viability of Caco-2 cells.

[0053] 2. Effect of synthetic peptide on calcium transport Establishment of Caco-2 cell monolayer model: Caco-2 cells were seeded at a density of 1.0 × 10 5 cells / cm 2 in the apical chambers (AP) of 12-well Transwell plate nests, 500 μL per well, and 1.5 mL of complete medium was added to the basolateral side (BL), and the plates were incubated in an incubator, with medium changed every other day, until day 21.

[0054] Caco-2 cell monolayer transport: TEER values higher than 400 Ω•cm 2transport experiments. To better simulate the acidic microenvironment of the intestinal lumen surface, the pH of the AP side HBSS was adjusted to 6.0 using 25 mM MES and Tris phosphate solution, and the pH of the BL side HBSS was adjusted to 7.4 using 25 mM HEPES and Tris phosphate solution. The Caco-2 cells were gently washed with pre-warmed HBSS for 3 times and incubated for 30 min. Then 0.5 mL of sample (HBSS solution, pH 6.0, the dosing concentration of synthetic peptide on the AP side was 0, 1, 2, 10 μΜ, and a certain concentration of CaCl2 solution was added to each well so that the final concentration of Ca2+ was 1.5 mM) was added to the AP side, and 1.5 mL of HBSS (pH 7.4) was added to the BL side, and the transport experiment was carried out in the incubator for 120 min. 2+ The final concentration in the culture solution was 200 μg / mL), 1.5 mL of HBSS (pH 7.4) was added to the BL side, and the transport experiment was carried out in the incubator for 120 min.

[0055] GV-7 at concentrations of 1, 2 and 10 μΜ was co-transported with Ca2+ at a concentration of 1.5 mM for 120 min, and the results are shown in 2+ Figure 3 GV-7 significantly increased the transport of Ca2+ at concentrations of 2 μΜ and 10 μΜ (p<0.05), with a significant increase of 22.21% at 2 μΜ and a significant increase of 22.73% at 10 μΜ. 2+

[0056] In summary, the effective concentration of GV-7 is 2 μΜ, and subsequent experiments can be carried out.

[0057] 3. Effect of synthetic peptide on the expression of calcium transport related genes (1) RNA extraction After the transport experiment was completed, the Caco-2 cells were washed with pre-cooled PBS for 2 times, 1 mL of Trizol was added to each well, the cells were lysed on ice for 5 min, and then centrifuged at 12,000 rpm for 5 min. The supernatant was mixed with 200 μL of chloroform, and after standing for 5 min, it was centrifuged at 12,000 rpm for 15 min. The upper liquid was mixed with 500 μL of isopropanol, and after standing for 30 min, it was centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and the RNA precipitate at the bottom of the tube was washed with 75% ethanol for 2 times. Finally, 30 μL of DEPC water was added for dissolution, and the concentration and purity of RNA were determined by Nano Drop.

[0058] (2) RNA reverse transcription ​​The RNA reverse transcription was performed according to the reverse transcription reagent instruction, and the specific process was as follows: 1 μL RNA solution, 4 μL 4×gDNA wiperMix and 16 μL ddH2O were mixed, and incubated at 42 ℃ for 2 min. Then, 4 μL 5×Hiscript III qRT SuperMix was added, incubated at 37 ℃ for 15 min, and incubated at 85 ℃ for 5 s, to obtain the cDNA stock solution.

[0059] (3) Real-time fluorescent quantitative PCR The real-time fluorescent quantitative PCR reaction system was shown in Table 10, and the target gene and primer sequence were shown in Table 11. The reaction parameters were as follows: pre-denaturation at 95 ℃ for 30 s, one cycle; cycle reaction at 95 ℃ for 10 s and at 60 ℃ for 30 s, 40 cycles; melting curve at 95 ℃ for 15 s, at 60 ℃ for 60 s and at 95 ℃ for 15 s, one cycle. The relative expression amount of gene was determined by 2-ΔΔCT method.

[0060] Table 10 Real-time fluorescent quantitative PCR reaction system

[0061] Table 11 Primer sequence table

[0062] The results were shown in Table 12. Figure 4 Compared with the control group, GV-7 significantly up-regulated the gene expression of TRPV6 (p<0.05); at the same time, it had no significant effect on the gene expression of Cav1.3, Calbindin-D9k and PMCA1b (p>0.05). This indicated that GV-7 mainly promoted the transport of calcium ions through the TRPV6 calcium ion channel, and did not affect the normal function of the L-type calcium ion channel.

[0063] Example 4 Effect of synthesized peptide on bone mineralization of zebrafish Calcium is the most abundant mineral element in the human body, accounting for 1.5% to 2.2% of the body weight of an adult. There are about 1.2 kg of calcium in the body of an adult, and more than 99% of the calcium exists in the bones and teeth. Calcium metabolism disorder can cause bone diseases. Dexamethasone belongs to glucocorticoid drugs, which can reduce intestinal calcium absorption, increase urinary calcium excretion, indirectly induce secondary hyperparathyroidism, and further aggravate bone loss.

[0064] Zebrafish, a commonly used model organism today, has shown its unique value in the study of bone diseases. Its structural and physiological characteristics provide a flexible and efficient animal model for the study of bone diseases. In terms of bone development, zebrafish has many similarities with mammals, including endochondral ossification and intramembranous ossification.

[0065] Alizarin red staining is a method of fixation and staining, which produces coloration by reacting with calcium nodules in the bone, and is used to detect the bone morphology and bone density of fish.

[0066] This example will use zebrafish as experimental organisms, and dexamethasone to construct an osteoporosis model. Alizarin red staining is used to verify the promoting effect of the peptide segment GPFPIIV (hereinafter referred to as GV-7) screened in Example 1 on calcium absorption.

[0067] Zebrafish were raised in the Zebrafish Platform of the Medical College of Zhejiang University, and the feeding conditions were as follows: light / dark cycle 14:10 h, water temperature 28℃.

[0068] Male and female zebrafish were paired to spawn, and the obtained fertilized eggs were placed in a 28.5℃ constant temperature incubator for culture. At 3 days post fertilization (dpf), the zebrafish embryos were randomly transferred to 6-well plates, with 15 embryos per well. The following solutions were added to each well: Control group: water containing 0.2% (m / v) dimethyl sulfoxide (DMSO); Model group: water containing 0.2% DMSO + dexamethasone (dexamethasone was dissolved in water containing 0.2% DMSO to a final concentration of 20 μM); Synthetic peptide GV-7 group: water containing 0.2% DMSO + dexamethasone (dexamethasone was dissolved in water containing 0.2% DMSO to a final concentration of 20 μM) + synthetic peptide GV-7 (final concentration 0, 0.5, 1, 2, 10, 20 μM); All groups were replaced with the solution every 24 h until the zebrafish larvae developed to 8 dpf. Subsequently, the zebrafish larvae were anesthetized to death with anesthetics, and the following treatments were performed in turn: 4% paraformaldehyde fixation for 24 h, 50% ethanol rinsing for 10 min, 3% H2O2+0.5% KOH solution for 30 min, 25% glycerol+0.1% KOH for 3 times, 0.01% alizarin red staining solution for 2 h, 50% glycerol+0.1% KOH for 3 times, and finally stored in 100% glycerol. The skull images of zebrafish were collected using a stereomicroscope, and the cumulative optical density of the skull was quantitatively analyzed using Image J software.

[0069] The results are shown in Table 1. Figure 5 Compared with the control group, the cumulative optical density value of the zebrafish skull in the model group was significantly reduced (P<0.05). p<0.05), indicating that dexamethasone successfully induced osteoporosis in zebrafish, leading to severe calcium loss in the skull. The intervention of synthetic peptide GV-7 effectively reversed the decrease in cumulative optical density value caused by dexamethasone. Specifically, compared with the modeling group, the cumulative optical density value of the skull in the GV-7 (20 μM) treatment group was significantly increased (P < 0.05). p <0.05). These results show that synthetic peptide GV-7 can effectively improve the symptoms of dexamethasone-induced osteoporosis in zebrafish and increase calcium deposition in the skeleton.

[0070] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various forms and details of changes, modifications, replacements and variations to these embodiments without departing from the spirit and principles of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A heptapeptide, characterized in that, The heptapeptide is GPFPIIV, which has the amino acid sequence Gly-Pro-Phe-Pro-Ile-Ile-Val.

2. A composition containing the heptapeptide GPFPIIV.

3. The heptapeptide as described in claim 1, characterized in that, The heptapeptide GPFPIIV is prepared by chemical solid-phase synthesis or isolated from casein hydrolysis products.

4. The heptapeptide as described in claim 3, characterized in that, The method for preparing the casein hydrolysis product 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 them 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; 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 heptapeptide GPFPIIV.

5. The heptapeptide 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 heptapeptide 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.

7. The heptapeptide as described in claim 4, characterized in that, The protease is derived from Bacillus subtilis, the carboxypeptidase is derived from Aspergillus niger, and the aminopeptidase is derived from Aspergillus oryzae.

8. The use of the heptapeptide GPFPIIV of claim 1 or the composition of claim 2 in promoting the absorption of calcium mineral elements.

9. The application as described in claim 8, characterized in that, It is used in the preparation of foods, health products, or medicines that promote the absorption of calcium minerals.

10. The application as described in claim 8, 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.

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