Novel peptide for promoting calcium transport and absorption under calcium-dependent concentration and application thereof

Abstract

The invention discloses a novel peptide for promoting calcium transport and absorption under calcium-dependent concentration and application thereof, belongs to the field of polypeptide preparation, the novel peptide is derived from cod bone calcium chelating peptide, the amino acid sequence of the novel peptide is DYGGEY, and the invention further provides a peptide calcium chelate which is prepared from novel pure peptide chelated calcium ions. The novel pure peptide disclosed by the invention can ensure that most of peptide structures are not damaged in small intestines, and the generated short peptide can promote the transport and absorption of calcium in Caco-2 cells, improve the bioavailability of the calcium, supplement the peptide, realize the targeted delivery of the calcium and improve the absorption of the calcium in a human body. According to the preparation method provided by the invention, the calcium chelating peptide is successfully obtained, product resources can be fully utilized, the operation is simple, and a new way is provided for high-value utilization of cod bones.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide preparation, and particularly relates to a novel peptide capable of promoting calcium transport and absorption under calcium-dependent concentration conditions and application thereof. Background Art

[0002] Calcium ions are mainly absorbed in the intestine in the form of ions, but phytic acid, oxalic acid, tannins and polyphenols in plant foods can competitively bind to Ca 2+ The formation of precipitates reduces the solubility of calcium, thereby reducing the absorption of calcium in the body. However, it is still unclear what kind of peptides can promote calcium transport and improve bioavailability under conditions of calcium ion gradient concentrations.

[0003] Studies have shown that peptides with certain amino acid profiles can chelate calcium and improve its bioavailability. For example, peptides extracted from minced octopus, casein, and fission yeast protein hydrolysate can chelate calcium to form peptide-calcium chelates, thereby enhancing calcium absorption by Caco-2 cells.

[0004] In addition, studies have reported that Pacific cod bone collagen peptides and crucian carp skin collagen peptides improve calcium bioavailability. Cod bones are an ideal raw material for producing bioactive peptides due to their low cost and high protein content. Fish bones have been extensively studied, particularly their calcium-binding capacity. However, little research has been conducted on the bioavailability of monomeric cod bone peptides.

[0005] Therefore, developing a common structured backbone peptide with high bioavailability for cod bones has important research significance and economic value for the further utilization of cod bones. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel peptide that promotes calcium transport and absorption under calcium-dependent concentration, reduces calcium precipitation, thereby enhancing calcium transport and absorption in the intestine, and is beneficial to Ca 2+ Targeted release in the small intestine, thereby improving its bioavailability.

[0007] The present invention uses cod bones as raw materials, and obtains high-purity common structural skeleton peptides through cleaning, de-fleshing, enzymatic hydrolysis, purification, identification and synthetic purification. The amino acid sequence of the peptides is DYGGEY, and the calcium chelating activity is 3-5 μg / mg, which can be used to prepare peptide calcium chelates.

[0008] The pure peptide DYGGEY provided by this invention can be used to prepare a peptide calcium chelate (DYGGEY-Ca). The preparation method is as follows: a peptide concentration of 20-80 mg / mL, a peptide:CaCl2·2H2O mass ratio of 1:2-3:1, a pH of 6.0-8.0, and a reaction temperature of 37-50°C for 0.5-2 hours.

[0009] The present invention uses a Caco-2 monolayer cell model (resistance value reaches 400-600 Ω·cm 2 ), the concentration range of pure peptide was designed to be 0-2 mg / mL, and the calcium concentration was 1-5 mM.

[0010] The high-purity, universal-structured peptide DYGGEY provided by the present invention exhibits excellent calcium absorption-promoting activity. In a Caco-2 cell model, calcium transport reached a maximum of 37.05 ± 5.63 mg / well after 120 minutes of transport, significantly higher than that of CaC12.

[0011] The high-purity, universal-structured peptide DYGGEY provided by this invention significantly improves calcium bioavailability. In a Caco-2 cell model, calcium bioavailability peaked at 49.17 ± 6.43% after 120 minutes of transport, a significant 47% increase compared to the control group.

[0012] Beneficial effects of the present invention:

[0013] This invention uses DYGGEY as a peptide source to prepare a peptide calcium chelate (DYGGEY-Ca) that promotes calcium transport and absorption in Caco-2 cells. This peptide calcium chelate exhibits excellent calcium chelation capacity. The peptide calcium chelate provided by this invention is safe and non-toxic, exhibiting superior transport activity compared to traditional calcium chloride, reaching a maximum of 37.05 ± 5.63 mg / well, and significantly improving its bioavailability. Following transport, the peptide calcium chelate can be absorbed and utilized by the human body, effectively utilizing the peptide while replenishing calcium ions.

[0014] The novel peptide provided by the present invention can significantly promote the transport and absorption of calcium in cells, improve the bioavailability of calcium, fully utilize fish product resources, is simple to operate, and provides a new approach for the high-value utilization of cod bones. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the secondary mass spectrum of peptide DYGGEY, where a is the primary mass spectrum; b is the secondary mass spectrum; and c is the molecular formula diagram.

[0016] Figure 2 Structural characterization diagrams of DYGGEY and DYGGEY-Ca, where a is the circular dichroism spectrum and secondary structure diagram, and b is the particle size diagram.

[0017] Figure 3 Graph showing the effect of DYGGEY on Caco-2 cell viability in a calcium-dependent manner.

[0018] Figure 4Figure 3 is a graph showing the absorption and transport of calcium in Caco-2 cells by DYGGEY under calcium-dependent concentrations. (a) is DYGGEY at 0.5 mg / mL, and (b) is DYGGEY at 1.0 mg / mL.

[0019] Figure 5 Graph showing the effect of DYGGEY on the bioavailability of intracellular calcium in Caco-2 cells in a calcium-dependent manner. DETAILED DESCRIPTION

[0020] The present invention uses the technology of synthesizing pure peptides to chelate with calcium to ensure the ionic form of calcium ions, thereby improving the stability of calcium in Caco-2 cells and promoting Ca 2+ Transport and absorption in cells enhances their bioavailability.

[0021] In the following examples, the calcium chelating activity was determined as follows:

[0022] Dissolve 3 mg of sample in 2 mL of 5 mmol / L CaCl2 and incubate at 37°C for 20 min. Add 4 mL of 20 mmol / L sodium phosphate buffer (pH 7.5) to the reaction system and incubate at 37°C for another 30 min. Centrifuge at 8000 rpm for 20 min to remove calcium phosphate precipitate. After filtering the supernatant through a 0.22 μm filter, take 2 mL and digest with 10 mL of nitric acid. Calcium content was determined according to GB5009.92-2016 (atomic absorption spectrometry). A blank control group without peptide was set up. Calcium chelation activity was calculated as follows:

[0023] ;

[0024] Wherein, Cs is the calcium content in the supernatant of each sample group, μg; Cc is the calcium content in the supernatant of the control group, μg; Ms is the sample mass, mg.

[0025] In the following examples, the circular dichroism spectrum was determined as follows:

[0026] At room temperature, circular dichroism spectra of the peptide and peptide-calcium chelate were collected using a circular dichroism spectrometer. Using a cuvette with a 0.5 cm pathlength, DYGGEY and DYGGEY-Ca were dissolved in ultrapure water to a concentration of 0.05 mg / mL each. The acquisition range was 190-260 nm, with a scan speed of 1.0 nm / s repeated three times at a 0.5 nm acquisition interval. After scanning, the data was processed using Young's algorithm to analyze the secondary structure and calculate the proportions of α-helices, β-sheets, β-turns, and random coils in each sample.

[0027] In the following examples, the particle size of DYGGEY and DYGGEY-Ca was determined as follows:

[0028] 1 mg / mL solutions of DYGGEY and DYGGEY-Ca were prepared in deionized water, transferred to a U-shaped cell, and stabilized at room temperature for 2.0 min. All experimental operations were carried out at 25 °C, and the average particle size was studied using a particle size analyzer.

[0029] In the following examples, the method for determining the effect of DYGGEY on Caco-2 cell viability at different calcium ion concentrations is as follows:

[0030] 1×10 4 Caco-2 cells were evenly plated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. A blank control group was treated with an equal volume of culture medium, without cells; a control group was treated with an equal volume of cell culture medium and cultured under the same conditions. After 24 hours of culture, the cells were treated with 100 μL of blank DMEM medium (calcium-free) containing DYGGEY at different calcium concentrations and incubated for 24 hours. At this point, the blank and control groups were also treated with 100 μL of blank DMEM medium (calcium-free). After 24 hours of culture, 10 μL of 5.0 mg / mL MTT was added and incubated for 4 hours. After this, 150 μL of DMSO was added to each well, mixed, and shaken. The same procedure was repeated for the blank and control groups. Finally, the OD value was measured at a wavelength of 570 nm using a microplate reader. Cell viability was calculated relative to the untreated control; a cell viability below 70% was considered toxic.

[0031] ;

[0032] Wherein, OD sample group is the OD value of cells after sample treatment, OD control group is the OD value of cells without sample treatment, and OD blank group is the OD value without cells and sample.

[0033] In the following examples, the method for determining the calcium ion transport activity of DYGGEY on Caco-2 cells at different calcium ion concentrations is as follows:

[0034] Experiments were performed on Caco-2 cell membranes cultured in Transwell plates for 21 days. HBSS, preheated to 37°C, was used to gently rinse each side of the Transwell chamber two to three times. The cells were then incubated in HBSS buffer at 37°C and 5% CO₂ for 30 minutes. DYGGEY (0.5 and 1.0 mg / mL) was dissolved in HBSS containing 2.0 and 4.0 mM calcium, respectively, and preheated to the membrane. Following this, 1.5 mL of HBSS was added to the BL side, followed by 500 μL of the prepared, pre-transmembrane-passed sample to be tested on the AP side. At 30, 60, 90, and 120 minutes after the start of transwelling, 1.0 mL of solution was removed from the lower chamber for calcium ion determination, and 1.0 mL of HBSS was added to the volume until constant. The calcium content of the liquid removed from the lower chamber was measured by atomic absorption spectrometry according to GB / T 5009.92-2016. The calcium content at each time point was calculated as follows:

[0035] ;

[0036] where Bn is the calcium content on the BL side of each well transferred at different sampling times, 1.5 is a constant indicating that there is 1.5 mL of HBSS buffer on the BL side of each well, An is the calcium concentration on the BL side of each well at different sampling times, 1.0 is a constant indicating that the sample volume removed at each time point is 1.0 mL, and n is an independent variable of 1, 2, 3, or 4, indicating that the sampling time is 30 min, 60 min, 90 min, and 120 min, respectively.

[0037] In the following examples, the method for determining the bioavailability of DYGGEY on intracellular calcium in Caco-2 cells at different calcium ion concentrations is as follows:

[0038] The calcium content of the liquid taken out from the lower chamber is measured by atomic absorption spectrometry, and the bioavailability of calcium is calculated as follows:

[0039] ;

[0040] Where C t represents the total calcium ion content added to the upper chamber before transport (μg), C AP Represents the calcium ion content (μg) in the upper chamber 120 minutes after the end of transport.

[0041] Example 1: Preparation of calcium chelating active peptides and peptide calcium chelates

[0042] Cod bones were softened at high temperature and then crushed. Distilled water was added and fully extracted at 70 ℃ to obtain cod bone crude protein. 0.5 g / L trypsin and 0.5 g / L neutral protease were added for 2.0 h of enzymatic hydrolysis. After enzyme inactivation, cod bone calcium chelating peptide was obtained.

[0043] The amino acid sequence of the calcium-chelating peptide was identified using UHPLC-Q-Orbitrap. UHPLC parameters were as follows: mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile; flow rate: 0.15 mL / min; injection volume: 10 μL. Column temperature: 40°C; gradient elution: 0–20 min, 2%–25% B; 20–30 min, 25–45% B; 30–35 min, 45%–85% B; 35–40 min, 85% B; 40–42 min, 85–2% B; 42–52 min, 2% B. Scan range: 100–1200 m / z; electrospray mode: positive electrospray ionization; electrospray voltage: 3600 V.

[0044] like Figure 1 As shown, mass spectrometry identified the purified peptide sequence as DYGGEY with a molecular weight of 702.25 Da. This peptide contains six amino acids, of which D and E are acidic amino acids in collagen, which enhance calcium chelation. The calcium chelation activity of DYGGEY was measured, and its calcium binding capacity was 4.66 ± 1.37 μg / mg, significantly higher than the reported calcium chelation activity of the cod bone gelatin peptide KGDPGLSPGK (2.53 ± 0.12 μg / mg).

[0045] Based on this peptide fragment, a peptide calcium chelate was prepared. A 50 mg / mL DYGGEY solution was prepared, and CaCl2·2H2O was added at a peptide:CaCl2·2H2O mass ratio of 2:1. The pH was adjusted to 7.0-7.5. The solution was incubated at 37°C for 1.0 h, precipitated with ethanol, and lyophilized to obtain the peptide calcium chelate. The structure of the chelate was characterized by circular dichroism spectroscopy and particle size.

[0046] like Figure 2As shown, the negative peaks between 190 and 260 nm for Peptide D and Peptide D-Ca are not distinct, likely due to the short length of the peptides (6 amino acids) and the lack of a well-defined secondary structure. Nevertheless, the chromatogram of Peptide D-Ca shifts compared to Peptide D, suggesting that calcium chelation may have occurred, generating new species. The secondary structure diagram shows the changes in the proportions of α-helices and β-sheets before and after calcium chelation. The results show that the proportion of α-helices decreases after DYGGEY chelates with calcium, decreasing from 2% to 0.4%, a decrease of 1.6%. The β-sheets remain almost unchanged, while the proportion of β-turns increases from 22.2% to 22.5%, an increase of 0.3%. The proportion of random coils increases from 23% to 23.4%, an increase of 0.4%. After binding with calcium ions, the particle size of DYGGEY becomes larger, the particle size distribution is consistent, and the range distribution is relatively uniform. The particle size distribution range is relatively narrow. The change in particle size before and after indicates the generation of new substances, indicating that chelation may have occurred between DYGGEY and calcium ions.

[0047] Example 2: Effect of DYGGEY on calcium transport in Caco-2 cells in a calcium-dependent concentration-dependent manner.

[0048] Effects of DYGGEY on Caco-2 cell viability in a calcium-dependent manner Figure 3 As shown in Figure 2, when the calcium ion concentration was 2.0 mM, as the DYGGEY concentration increased from 0 to 1 mg / mL, the overall cell viability first increased and then decreased. When the peptide concentration was 0, at 2.0 mM Ca 2+ Under the condition of 400 μg / mL, the cell viability was 102.40 ± 5.24%. When the peptide concentration was 0.5 mg / mL, the cell viability changed to 101.17 ± 7.77%. The difference was not significant (p > 0.05). However, it can be seen that under this concentration condition, the cell proliferation ability was certain. When the peptide concentration increased to 1.0 mg / mL, the cell viability decreased significantly to 92.81 ± 3.88% (p < 0.05), but it was not toxic to the cells. When the calcium ion concentration was increased to 4.0 mM, compared with 2.0 mM Ca 2+ The cell viability change trends were generally consistent with those of the control group. When the DYGGEY concentration was 0.5 mg / mL, the cell viability reached 99.25 ± 4.49%. When the DYGGEY concentration increased to 1.0 mg / mL, the cell viability decreased significantly to 87.44 ± 3.20% (p < 0.05), but was still greater than the cytotoxic threshold of 70%.

[0049] like Figure 4As shown in (a), at 30 minutes, the calcium transport capacity of the 2.0 mM control group was 6.36 ± 0.14 μg / well. Addition of 0.5 mg / mL DYGGEY significantly increased this capacity (p < 0.05) to 15.93 ± 3.15 μg / well. When the DYGGEY concentration was increased to 1.0 mg / mL, the calcium transport capacity also increased to 19.43 ± 2.94 μg / well, but the difference was not significant (p > 0.05). At 60 minutes of transport, the calcium transport capacity of the control group was 13.25 ± 3.61 μg / well. Both the 0.5 mg / mL and 1.0 mg / mL DYGGEY groups significantly increased their calcium transport capacity (p < 0.05), reaching 18.18 ± 1.01 μg / well and 26.24 ± 1.31 μg / well, respectively. Calcium transport capacity gradually increased in each group with increasing transport time. At 120 min, the calcium transport amount of the 2.0 mM control group reached 26.95 ± 1.20 μg / well, while the calcium transport amount of the 0.5 mg / mL and 1.0 mg / mL peptide concentrations reached 32.35 ± 3.62 μg / well and 37.05 ± 5.63 μg / well, respectively, which were significantly different from the calcium transport amount at 30 min (p < 0.05), indicating that the addition of DYGGEY can promote calcium transport in Caco-2 cells. Figure 4 In (b), as transport time increased, the amount of calcium transported in the 4.0 mM control group increased from 10.41 ± 0.40 μg / well at 30 min to 14.32 ± 5.77 μg / well at 120 min, representing an approximate 37.56% increase. Calcium transport in the 0.5 mg / mL DYGGEY group increased from 14.30 ± 2.69 μg / well at 30 min to 30.12 ± 4.86 μg / well at 120 min, demonstrating a significant difference compared to the control group (p < 0.05).

[0050] Figure 5The effect of DYGGEY on calcium bioavailability at different concentrations was shown. The bioavailability of the 2.0 mM control was 33.45 ± 3.38%. Addition of 0.5 mg / mL and 1.0 mg / mL DYGGEY increased the bioavailability to 40.49 ± 8.80% and 49.17 ± 6.43%, respectively, both exceeding the control. The bioavailability of the 4.0 mM group (19.32 ± 0.90%) was significantly lower than that of the 2.0 mM group (33.45 ± 3.38%). However, addition of 1.0 mg / mL DYGGEY significantly increased the bioavailability to 30.25 ± 3.48% (p < 0.05). This indicates that DYGGEY can significantly increase calcium bioavailability (p < 0.05).

[0051] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention and are not intended to limit the scope of the present invention in any way. Various changes and modifications may be made to the present invention without departing from the scope of the present invention, and all such changes and modifications fall within the scope of the claims.

Claims

1. A new type of pure peptide derived from cod bone calcium chelate peptide, characterized in that: The amino acid sequence of the novel peptide is shown in SEQ ID No. 1, and the specific amino acid sequence is DYGGEY.

2. Use of the novel pure peptide according to claim 1 in the preparation of peptide calcium chelates.

3. A peptide calcium chelate, characterized in that The peptide calcium chelate is prepared by chelating calcium ions with the novel pure peptide described in claim 1.

4. The peptide calcium chelate according to claim 3, characterized in that The peptide calcium chelate is a peptide calcium chelate prepared from a new pure peptide and is a mixed state of chelate, peptide and calcium ions.

5. The peptide calcium chelate according to claim 4, characterized in that The mixed state is formed by chelation at different calcium ion concentrations.

6. The peptide calcium chelate according to claim 5, characterized in that The calcium ion concentration is 2 mM-4 mM.

7. The peptide calcium chelate according to claim 3, wherein The peptide calcium chelate can promote the transport, absorption and bioavailability of calcium in Caco-2 cells.

8. The peptide calcium chelate according to claim 7, wherein The Caco-2 cells are human colon adenocarcinoma cells.

9. The method for preparing the peptide calcium chelate according to any one of claims 3 to 8, characterized in that: The steps are: The peptide concentration is 20-80 mg / mL, the peptide:CaCl2·2H2O mass ratio is 1:2-3:1, the pH is 6.0-8.0, and the reaction is carried out at 37-50 ℃ for 0.5-2 h.

10. Use of the peptide calcium chelate according to any one of claims 3 to 8 in the preparation of a calcium supplement preparation.

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