Tuna bone calcium absorption promoting transport peptide and calcium chelate thereof

Abstract

The invention discloses a tuna bone calcium absorption promoting transport peptide and a calcium chelate thereof. The amino acid sequence of the tuna bone calcium absorption promoting transport peptide is GFSGLDGAK. In-vivo calcium regulation key factors can be activated, absorption and transport of human small intestine epithelial cells to calcium ions are promoted, and the bioavailability of calcium is improved; through chelation modification and other methods, an efficient calcium delivery system is constructed, the digestion stability of a sample is improved, calcium ions and dietary factors are inhibited from forming insoluble complexes, and the content of soluble calcium in a small intestine cavity is guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of biosynthesis, and in particular to a tuna bone calcium absorption promoting transport peptide and a calcium chelate thereof. Background Art

[0002] Calcium is one of the important components of the human body. It is widely distributed in various tissues and organs of the human body. Its content is second only to carbon, hydrogen, oxygen and nitrogen. It is the inorganic element with the highest content in the human body. It is commonly found in bones and teeth and is one of the most important components for maintaining the hardness of bones and teeth. Calcium ions are also a vital element for maintaining life.

[0003] Calcium deficiency is one of the more common nutrient deficiencies. The Chinese Residents' Dietary Guidelines point out that daily diets need to control salt intake and increase calcium intake, and daily calcium intake should be controlled at 800 mg-1000 mg.

[0004] There are two main reasons for calcium deficiency, one of which is the low per capita calcium intake. The Sixth China Residents' Nutrition and Health Status Survey in 2021 found that the average calcium intake of Chinese residents was about 573 mg per day. This is an increase from 388.8 mg in the previous two years. This figure is based on the results of a nationwide sampling survey, including residents in urban and rural areas. However, this intake is still far lower than the daily intake recommended by the Chinese Nutrition Society for Chinese residents. Among them, the main groups with insufficient calcium intake are middle-aged and elderly people and children.

[0005] Another major cause of calcium deficiency is the low calcium absorption rate. The best stage for calcium absorption is in the early stages of growth and development. Survey reports show that during the period of bone growth and development in infants, young children and adolescents, the calcium absorption rate reaches the highest, reaching about 70%. With growth and development, and age, the absorption rate decreases. When in middle age, growth and development are formed, the body's metabolism decreases, and the calcium absorption rate is only about 30%. Previous studies have shown that other substances such as vitamin D, protein, and amino acids can promote calcium ion absorption to a certain extent, but some dietary factors such as oxalic acid (OA) and phytic acid (PA) will form irreversible precipitation with calcium ions, resulting in a decrease in calcium ion absorption rate. my country's dietary habits include a large amount of plant-based foods, some of which contain higher amounts of oxalic acid (OA) and phytic acid (PA), but the intake of eggs and milk is relatively low. Therefore, the low calcium intake and the influence of dietary factors brought about by dietary habits are the reasons for the calcium deficiency problem in my country.

[0006] Inorganic calcium, organic acid calcium, amino acid chelate calcium and peptide chelate calcium are the four stages of the development of calcium supplements. The first generation of inorganic calcium salts mainly include calcium carbonate, calcium phosphate, calcium chloride, etc. This type of inorganic calcium mainly exists in the form of calcium ions. This inorganic calcium has a more obvious disadvantage, that is, it is easy to form precipitation during gastrointestinal digestion, which is not conducive to the body's absorption and utilization of calcium. It is also easy to cause some side effects, and its use has been gradually reduced. The second generation is organic acid calcium, including calcium lactate, calcium citrate and calcium gluconate. Compared with the first generation of inorganic calcium salts, it is more soluble, but it still has problems such as poor stability and low calcium supplementation efficiency. The third generation of amino acid chelated calcium solves the problem of low bioavailability of ionized calcium to a certain extent and overcomes some of the constraints that affect calcium ion absorption. However, in the gastrointestinal environment, when the calcium ion concentration is too high, it may be dose-related toxic and affect the health of the body. The absorption process of the third-generation calcium supplement amino acid chelate calcium does not rely on calcium binding proteins like the first and second generation calcium supplements. Instead, it surrounds calcium ions through amino acids. When they are absorbed into the blood, they will not dissociate immediately. Therefore, they are considered to be a good alternative to ionic calcium supplements. However, the cost of implementing the chelation reaction between amino acids and calcium is extremely high, and may be accompanied by unnecessary color reactions and lipid oxidation reactions. Therefore, the fourth-generation peptide chelate calcium has become an important research topic. Peptides as receptors bind to calcium with the advantages of low energy consumption, fast transportation, and low carrier saturation, and can form soluble and stable chelates with calcium ions. At present, peptide calcium chelates have received widespread attention due to their excellent stability, good absorption performance and bioavailability. They are one of the more ideal calcium supplements at present. The development of calcium preparations with good bioavailability and calcium-carrying capacity is of far-reaching significance. Summary of the invention

[0007] The purpose of the present invention is to provide a tuna bone calcium absorption transport peptide and a calcium chelate thereof, which can activate the key calcium regulation factors in the body, promote the absorption and transport of calcium ions by human small intestinal epithelial cells, and improve the bioavailability of calcium; through chelation modification and other methods, a calcium efficient delivery system is constructed to improve the digestion stability of the sample, inhibit the formation of insoluble complexes between calcium ions and dietary factors, and ensure the soluble calcium content in the small intestinal cavity.

[0008] The technical solution adopted by the present invention to solve its technical problem is: A tuna bone calcium absorption-promoting transport peptide, whose amino acid sequence is: GFSGLDGAK.

[0009] A tuna bone calcium absorption transport peptide-calcium chelate is formed by chelating the tuna bone calcium absorption transport peptide and calcium chloride in a mass ratio of 3:1. The peptide calcium chelate is formed to construct a calcium efficient delivery system to achieve effective delivery and efficient utilization of calcium; while promoting calcium absorption and transport, it improves digestion stability, inhibits calcium ions from forming insoluble complexes with dietary factors, and ensures the soluble calcium content in the small intestine.

[0010] The preparation method is as follows: tuna bone calcium absorption transport peptide is prepared into a peptide aqueous solution, which is then mixed with calcium chloride, shaken in a constant temperature water bath at 70°C and pH=6 for 2 hours, precipitated with anhydrous ethanol, allowed to stand overnight at 4°C, and collected by centrifugation. The precipitate is vacuum freeze-dried to obtain tuna bone calcium absorption transport peptide-calcium chelate.

[0011] The concentration of the peptide aqueous solution was 3-5 mg / mL.

[0012] A phosphorylated transport peptide-calcium chelate, which is formed by phosphorylating the tuna bone calcium absorption promoting transport peptide to form a phosphorylated transport peptide, and then chelating the phosphorylated transport peptide with calcium chloride at a mass ratio of 3:1. While the target peptide segment plays a role in promoting calcium absorption and transport, a calcium efficient delivery system is constructed through the phosphorylation-chelation combined technology, which, on the one hand, improves the calcium carrying capacity of the target peptide segment; on the other hand, improves digestion stability, effectively prevents calcium ions from forming insoluble complexes with dietary inhibitors, realizes targeted delivery of calcium ions, and ensures the soluble calcium content in the small intestine.

[0013] The preparation method is: A 3 mg / mL tuna bone calcium absorption transport peptide aqueous solution was prepared, and then sodium tripolyphosphate was added to a final concentration of 8 mg / mL, the solution pH was adjusted to 9, and the solution was bathed in a 40°C water bath for 2 hours, the pH was adjusted to 7, and after dialysis at 4°C for 24 hours, the phosphorylated transport peptide was obtained by vacuum freeze drying; The phosphorylated transport peptide was prepared into a phosphorylated peptide aqueous solution, which was then mixed with calcium chloride and shaken in a constant temperature water bath at 70°C and pH = 6 for 2 h. The solution was precipitated with anhydrous ethanol and allowed to stand overnight at 4°C. The precipitate was collected by centrifugation and freeze-dried in vacuum to obtain the phosphorylated transport peptide-calcium chelate.

[0014] The concentration of the phosphorylated peptide aqueous solution is 3-5 mg / mL.

[0015] The beneficial effects of the present invention are: it can activate key calcium regulation factors in the body, promote the absorption and transport of calcium ions by human small intestinal epithelial cells, and improve calcium bioavailability; through chelation modification and other methods, a calcium efficient delivery system is constructed to improve the digestion stability of the sample, inhibit the formation of insoluble complexes between calcium ions and dietary factors, and ensure the soluble calcium content in the small intestinal cavity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of HPLC test of tuna bone calcium absorption transport peptide; Figure 2 This is the LC-MS / MS analysis result of tuna bone calcium absorption transport peptide; Figure 3 This is the docking active site diagram of tuna bone calcium absorption transport peptide and CaSR; Figure 4 It is a calcium absorption promoting peptide calcium chelate that resists the effects of phytic acid (PA) and oxalic acid (OA) on calcium transport; Figure 5 This is the calcium fluorescence imaging of different forms of tuna bone calcium absorption transport peptide in Caco-2 cells at different concentrations; Figure 6 The figure shows the calcium transport of different forms of tuna bone calcium absorption transport peptide in the Caco-2 monolayer differentiation model. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below through specific embodiments.

[0018] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0019] Embodiment 1: Obtaining tuna bone calcium absorption transport peptide: The high-activity polypeptide raw material is prepared by using a mixed polypeptide sample with good calcium binding ability and calcium transport promoting ability as the raw material.

[0020] Specific preparation conditions: Tuna bone powder was hydrolyzed by composite protease (1.5% of bone powder mass, w / w) (consisting of endo-enzyme, exo-enzyme and flavor enzyme, 5:2:3) at pH 7.0 and 50 °C for 6 h at a material-liquid ratio of 1:10 (w / v, g / mL). After inactivation of the enzyme, the supernatant was concentrated by centrifugation and vacuum freeze-dried to obtain tuna bone collagen peptide. Tuna bone collagen peptide was dissolved in water to form a 5 mg / mL peptide aqueous solution, CaCl2 was added, and the mass ratio of peptide: calcium chloride reached = 3:1, pH = 6 was adjusted, and constant temperature water bath was shaken at 70 °C for 2 h. After the reaction, anhydrous ethanol was added at a volume ratio of 1 (reaction product): 9 (anhydrous ethanol), and low-temperature alcohol precipitation was carried out at 4 °C overnight. The precipitate was centrifuged and freeze-dried to obtain tuna bone collagen peptide calcium chelate.

[0021] Construction of peptide database: Ultra-high performance liquid chromatography-quadrupole-high resolution tandem mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS) was used to identify the peptide sequences in highly active fish bone peptide calcium chelate, and a tuna bone collagen peptide database was established.

[0022] The LC-MS / MS analysis parameters were set as follows: a C18 column (Acclaim PepMap 150 μm id ×150 mm) was used in combination with a UV detector; 0.1% formic acid solution was used as mobile phase A, and 0.1% formic acid, 80% ACN was used as mobile phase B; the flow rate was set at 600 nL / min, and the gradient elution was performed as follows: 0-2 min, 4% B; 2-45 min, 8% B; 45-55 min, 28% B; 55-56 min, 40% B; 56-66 min, 95% B.

[0023] High-throughput targeted screening: Calcium-sensing receptor (CaSR) was used as the target, and high-affinity peptides were screened using molecular docking technology: the interaction between the peptide sequence obtained and CaSR was measured; in order to verify the interaction between the peptide sequence and CaSR, the Autodock Vina software, one of the fastest and most widely used open source programs, was used for molecular docking simulation. A total of 225 peptide sequences were identified from the peptide sequences, and the mass of all peptides was less than 1000 Da. Three peptides with calcium absorption-promoting ability, QPGGGW (SEQID No. 1), GFSGLDGAK (SEQ ID No. 2), and GPEGAP (SEQID No. 3), were further screened out, which may have good physical and chemical properties.

[0024] The calcium absorption promoting peptides interact with CaSR mainly through hydrogen bonds and ionic bonds. The docking sites and forces of multiple peptides are different. In addition to ionic bonds and hydrogen bonds, the possible binding site of GFSGLDGAK also includes -CH- ( Figure 3 ), GPEGPA is connected to Glu251 through hydrogen bonds. Unlike other sequences, QPGGGW is connected only by hydrogen bonds. GFSGLDGAK has the best affinity with CaSR, which is -8.8 kcal / mol. The amino acid sequence of GFSGLDGAK is Gly-Phe-Ser-Gly-Leu-Asp-Gly-Ala-Lys, and the molecular weight is 850.8 Da. Figure 1 This is the HPLC test result of tuna bone calcium absorption transport peptide. Figure 2 These are the results of LC-MS analysis of tuna bone calcium absorption transporter peptide.

[0025] Calcium-sensing receptor (CaSR) is one of the important components of the calcium homeostasis system and requires the synergistic activation of calcium ions and small molecule peptides. CaSR is widely expressed in multiple organs, such as the thyroid, kidneys, and gastrointestinal system. At the same time, CaSR is also considered to be a new target for improving bone health and treating calcium metabolism diseases. Activated CaSR can sense the blood calcium level and intestinal calcium level of people with low calcium, and promote intestinal absorption of calcium ions by regulating the calcium transport mechanism and increasing the absorption of ions in the intestinal solution. In addition, activated CaSR can reduce the secretion of calcitonin and the loss of urinary calcium, which can maintain the appropriate calcium concentration in the human body to a certain extent. Therefore, based on CaSR, active peptides that promote calcium absorption and transport are screened and used as new calcium carriers, which can not only prevent calcium ions from forming insoluble complexes with dietary factors such as phytic acid and oxalic acid, but also co-activate CaSR with calcium ions to promote the absorption of calcium ions in the intestine, thereby greatly improving the bioavailability of calcium and maintaining the calcium homeostasis system.

[0026] Synthesis of target peptides: The target peptides were synthesized by Nanjing GenScript Biotechnology Co., Ltd., purified by HPLC and freeze-dried, and identified by mass spectrometry; finally, three sequences QPGGGW (SEQ ID No. 1), GFSGLDGAK (SEQ ID No. 2), and GPEGAP (SEQ ID No. 3) were synthesized.

[0027] Example 2: Tuna bone calcium absorption transport peptide-calcium chelate QPGGGW, GFSGLDGAK and GPEGAP were prepared into 5 mg / mL peptide aqueous solutions, respectively. Then the peptide and calcium chloride were mixed in a mass ratio of 3:1, and shaken in a constant temperature water bath at 70°C and pH=6 for 2 h. Anhydrous ethanol was added in a volume ratio of 1 (reaction product): 9 (anhydrous ethanol), and the mixture was precipitated at 4°C overnight. The precipitate was collected by centrifugation and vacuum freeze-dried to prepare peptide calcium chelates QPGGGW-Ca, GFSGLDGAK-Ca and GPEGAP-Ca, respectively.

[0028] The calcium ion chelation activity of the peptide was determined by atomic absorption spectrophotometry. The results showed that the calcium ion chelation amounts of QPGGGW, GFSGLDGAK and GPEGAP reached 50.54, 64.37 and 52.81 mg / g, respectively. It is speculated that GFSGLDGAK has a higher calcium binding ability and more calcium chelation sites. The peptide calcium chelate formed after chelating calcium ions has a higher calcium content and is suitable for further experiments.

[0029] Calcium retention assay Dissolve the peptide calcium chelate in a 1:1 ratio with simulated gastric fluid electrolyte. Add porcine pepsin to a 2000U / mL enzyme activity. Add the pre-prepared gastric lipase solution to a 60U / mL gastric lipase activity. Adjust the pH to 3 and react for 90 min. After the reaction is complete, incubate in a water bath at 100°C for 15 min to inactivate the enzyme in the solution.

[0030] The content of calcium ions in the reaction solution increased before and after the digestion reaction by atomic absorption spectrophotometer; the calcium retention rate of QPGGGW-Ca and GPEGAP-Ca decreased during the digestion process, and more calcium ions were dissociated. By comparison, it was found that GFSGLDGAK-Ca was relatively stable, with a calcium retention rate of more than 60%.

[0031] Table 1 Calcium retention rate of peptide calcium chelates .

[0032] However, in previous studies, it was found that after the action of pepsin, when entering the intestinal stage, the peptide calcium chelate may undergo secondary chelation in the intestine. Therefore, from the analysis of the retention rate data, it can be concluded that GFSGLDGAK-Ca has better in vitro digestion stability, higher calcium ion content, and the ability to promote calcium absorption.

[0033] GFSGLDGAK-Ca's ability to resist dietary factors in calcium transport: Caco-2 cell monolayer differentiation model was constructed (when the growth density of Caco-2 cells reached about 90% in the field of view under the microscope, they were digested with 0.25% trypsin. The cells were counted on a hemocytometer and the number of cells was calculated as 1×10 5 / well, seeded in a 12-well Transwell plate. Pipette 0.5 mL of cell suspension into the upper chamber (AP side), and pipette 1.5 mL of DMEM culture medium into the bottom chamber (BL side), and culture in an incubator. Change the medium every other day for the first week. After the 7th day, change the medium every day and culture for 21 days) to study its ability to resist dietary factors in calcium transport. Discard the culture medium and wash both sides with preheated HBSS. After adding oxalic acid (OA) and phytic acid (PA) in advance and incubate for 30 minutes, add only calcium chloride (Ca 2+ concentration of 50 µg / well)) as blank control, GFSGLDGAK-Ca (Ca 2+ After incubation for 2 h, 200 μL of the solution was taken from the upper chamber and the bottom chamber respectively. The calcium ion content was determined by atomic absorption spectrophotometer.

[0034] Effects of dietary factors coexisting in the diet on the transport of calcium ions from peptide calcium chelates Figure 4As shown. Compared with the blank and dietary factors, the calcium transport of CaCl2 was greatly affected by dietary factors, but when GFSGLDGAK-Ca co-existed with oxalic acid (OA) or phytic acid (PA), the transport amount was reduced, but not significantly. It can be inferred that compared with CaCl2, GFSGLDGAK-Ca has a good ability to resist adverse dietary factors.

[0035] Example 3: Phosphorylated transport peptide-calcium chelate Construction of an efficient calcium delivery system. While the target peptide plays a role in promoting calcium absorption and transport, a calcium efficient delivery system is constructed through phosphorylation-chelation combined technology, which, on the one hand, improves the calcium-carrying capacity of the target peptide; on the other hand, improves digestion stability, effectively prevents calcium ions from forming insoluble complexes with dietary inhibitors, achieves targeted delivery of calcium ions, and ensures the soluble calcium content in the small intestine.

[0036] The preparation method is: A 3 mg / mL tuna bone calcium absorption transport peptide (GFSGLDGAK) aqueous solution was prepared, and then sodium tripolyphosphate was added to a final concentration of 8 mg / mL, the solution pH was adjusted to 9, and the solution was bathed in a 40°C water bath for 2 h, the pH was adjusted to 7, and after dialysis at 4°C for 24 h, the phosphorylated transport peptide was obtained by vacuum freeze drying; The phosphorylated transport peptide was prepared into a 5 mg / mL phosphorylated peptide aqueous solution, and then the phosphorylated peptide was mixed with calcium chloride in a mass ratio of 3:1, and shaken in a constant temperature water bath at 70°C and pH=6 for 2 h. Anhydrous ethanol was added in a volume ratio of 1 (reaction product): 9 (anhydrous ethanol), and the solution was precipitated overnight at 4°C. The precipitate was collected by centrifugation and vacuum freeze-dried to obtain the phosphorylated transport peptide-calcium chelate. The calcium loading capacity reached 79.82 mg / g. After in vitro gastrointestinal simulated digestion, the calcium retention rate in the system reached 82.64%.

[0037] Effect of promoting calcium absorption and transport. Caco-2 small intestinal epithelial cells were used to detect the calcium absorption and transport of the target peptide (GFSGLDGAK), peptide calcium chelate (GFSGLDGAK-Ca), and phosphorylated peptide calcium chelate.

[0038] Fluorescence microscopy was used to observe the changes in calcium ion influx in Caco-2 cells by different samples, and their calcium transport capacity was quickly evaluated. Figure 5 As shown in the figure, under the same calcium content, compared with CaCl2, GFSGLDGAK and its modifications can significantly increase the calcium absorption in Caco-2 cells, and the phosphorylated peptide calcium chelate has the most significant effect.

[0039] The Caco-2 cell monolayer differentiation model with a resistance value of 450Ω after 21 days of differentiation was used as the experimental model to examine the calcium absorption and transport capacity of different samples. Figure 6 As shown, under the same calcium content, compared with CaCl2, GFSGLDGAK and its modifications can significantly increase the amount of calcium transport, with a CaCl2 transport rate of 47.69%, a target peptide transport rate of 62.09%, and a peptide calcium chelate transport rate of 77.44%. The phosphorylated peptide calcium chelate has the highest effect in promoting calcium transport, with a transport rate of 81.44%.

[0040] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A tuna bone calcium absorption transport peptide, characterized in that: Its amino acid sequence is: GFSGLDGAK.

2. A tuna bone calcium absorption transport peptide-calcium chelate, characterized in that: The calcium absorption promoting transport peptide of tuna bone according to claim 1 is chelated with calcium chloride in a mass ratio of 3:

1.

3. The tuna bone calcium absorption transport peptide-calcium chelate according to claim 2, characterized in that: The preparation method is as follows: tuna bone calcium absorption transport peptide is prepared into a peptide aqueous solution, which is then mixed with calcium chloride, shaken in a constant temperature water bath at 70°C and pH=6 for 2 hours, precipitated with anhydrous ethanol, allowed to stand overnight at 4°C, and collected by centrifugation. The precipitate is vacuum freeze-dried to obtain tuna bone calcium absorption transport peptide-calcium chelate.

4. A phosphorylated transport peptide-calcium chelate according to claim 3, characterized in that: The concentration of the peptide aqueous solution was 3-5 mg / mL.

5. A phosphorylated transport peptide-calcium chelate, characterized in that The tuna bone calcium absorption promoting transport peptide according to claim 1 is phosphorylated to form a phosphorylated transport peptide, and then the phosphorylated transport peptide is chelated with calcium chloride in a mass ratio of 3:

1.

6. A phosphorylated transport peptide-calcium chelate according to claim 5, characterized in that: The preparation method is: A 3 mg / mL tuna bone calcium absorption transport peptide aqueous solution was prepared, and then sodium tripolyphosphate was added to a final concentration of 8 mg / mL, the solution pH was adjusted to 9, and the solution was bathed in a 40°C water bath for 2 hours, the pH was adjusted to 7, and after dialysis at 4°C for 24 hours, the phosphorylated transport peptide was obtained by vacuum freeze drying; The phosphorylated transport peptide was prepared into a phosphorylated peptide aqueous solution, which was then mixed with calcium chloride and shaken in a constant temperature water bath at 70°C and pH = 6 for 2 h. The solution was precipitated with anhydrous ethanol and allowed to stand overnight at 4°C. The precipitate was collected by centrifugation and freeze-dried in vacuum to obtain the phosphorylated transport peptide-calcium chelate.

7. A phosphorylated transport peptide-calcium chelate according to claim 6, characterized in that: The concentration of the phosphorylated peptide aqueous solution is 3-5 mg / mL.

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