A novel cod bone calcium absorption promoting peptide and its preparation method and application

Abstract

The present invention discloses a novel cod bone calcium absorption promoting peptide and a preparation method and application thereof, belonging to the technical field of biological polypeptides. The amino acid sequence of the fish bone calcium absorption promoting peptide disclosed in the present invention is shown in SEQ ID No. 1, and the specific sequence is GRGNEGPQ. The fish bone calcium absorption promoting peptide provided by the present invention has very high calcium absorption promoting activity and calcium chelating activity. The peptide segment can be used for the development of calcium supplement preparations to improve the bioavailability of calcium, and can be used as a functional food ingredient in the preparation of special dietary foods, health foods, and calcium nutrition fortified foods or medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of biological polypeptides, and in particular relates to a novel cod bone calcium absorption promoting peptide and a preparation method and application thereof. Background Art

[0002] Calcium is the most abundant trace element in the human body. It is involved in many physiological functions such as cell metabolism, bone growth, and blood coagulation. Therefore, it is essential to the health of the body. Insufficient calcium intake can cause osteoporosis, rickets, and osteomalacia, which affect human health.

[0003] Studies have shown that peptides can compete with anti-nutritional factors such as oxalic acid, phytic acid and tannins in the gastrointestinal tract to bind Ca 2+ , so that Ca 2+ It can remain dissolved in the small intestine and then be absorbed and utilized by the small intestinal epithelial cells. 2+ After binding, it can promote Caco-2 cells to Ca 2+ The absorption of calcium is different, and the effect of peptides with different molecular weights on promoting calcium absorption is different. Peptides with 10-30 kDa have the best effect on promoting calcium absorption. Sea cucumber egg peptide can maintain Ca in the presence of oxalic acid. 2+ It has high solubility and can promote Ca in both Caco-2 cell model and HT-29 cell model. 2+ Duck egg white calcium chelate peptide can reverse the effects of common calcium absorption interference factors in the diet, such as oxalic acid, phosphate and zinc ions, and maintain a good calcium absorption promoting effect.

[0004] Chinese patent CN118452475A discloses a preparation method and application of a calcium absorption promoting postbiotic, wherein a postbiotic containing a calcium absorption promoting peptide is prepared by inoculating Lactobacillus helveticus CCFM1263 into an αs1-casein solution and fermenting the fermented liquid. The postbiotic can increase the calcium absorption rate of rats and thereby improve the blood calcium and bone calcium content of calcium-deficient rats.

[0005] Chinese patent CN118388602A discloses an egg yolk peptide that promotes calcium absorption, and its preparation method and application. Five egg yolk peptides were prepared by enzymatic hydrolysis of fertilized eggs. The results of in vitro simulated digestion showed that the calcium retention rate of the egg yolk peptide calcium chelate was significantly higher than that of CaCl2, indicating that the egg yolk peptide calcium chelate is more conducive to promoting calcium absorption and utilization.

[0006] Chinese patent CN113999283A discloses a shrimp-derived heptapeptide with calcium absorption promoting activity and its application. Through the rat everted intestinal sac model, it was found that compared with CaCl2, the shrimp-derived heptapeptide calcium complex (QEELIS-Ca) increased the calcium transport in the duodenum by 6.44 times and the calcium transport in the ileum by 4.74 times, playing a good role in promoting calcium absorption.

[0007] Chinese patent CN117843718A discloses an egg-derived bifunctional bioactive peptide, its preparation method and application. A bifunctional peptide KILDDTDNQ with antioxidant and calcium absorption promoting functions was prepared by hydrolyzing egg yolk with trypsin and alkaline protease. This peptide can reduce the oxidative stress of MC osteoblasts, thereby promoting calcium absorption and forming mineralized nodules. Summary of the invention

[0008] The purpose of the present invention is to provide a novel cod bone calcium absorption promoting peptide and a preparation method thereof. The polypeptide provided by the present invention is separated and extracted from cod bones. By identifying its amino acid sequence, it is determined to be a novel polypeptide sequence with significant calcium absorption promoting activity.

[0009] Furthermore, the novel cod bone calcium absorption promoting peptide provided by the present invention consists of eight amino acids, the sequence of which is shown in SEQ ID No. 1, and the specific sequence is GRGNEGPQ (F21-3).

[0010] The fish bone calcium absorption promoting peptide provided by the present invention has good calcium absorption promoting activity. In the Caco-2 cell model, the calcium transport amount reached a maximum value of 42.30±2.88 μg / well at 120 min of transport, which was significantly higher than CaCl2. The peptide segment can be used for the development of calcium supplement preparations to improve the bioavailability of calcium.

[0011] The fish bone calcium absorption promoting peptide provided by the present invention also has a very high calcium chelating activity, and its calcium chelating activity reaches 4.36±0.57 μg / mg, and can be used to prepare peptide calcium chelates.

[0012] The invention also provides application of the fish bone calcium absorption promoting peptide and the peptide calcium chelate prepared therefrom in food.

[0013] Furthermore, the food may be special dietary food, health food, calcium-fortified food, and other foods related to calcium supplementation.

[0014] The present invention also provides a method for preparing a novel cod bone calcium absorption promoting peptide. The peptide segment can be separated and obtained through enzymatic hydrolysis, hydroxyapatite affinity chromatography, Sephadex G25 gel filtration chromatography, C18 reverse phase high performance liquid chromatography and the like, or can be prepared through artificial chemical synthesis, such as chemical solid phase synthesis.

[0015] Beneficial effects of the present invention:

[0016] The polypeptide provided by the present invention has very high calcium absorption promoting activity and calcium chelating activity. The peptide segment can be used for the development of calcium supplement preparations to improve the bioavailability of calcium, and can be used as a functional food ingredient in the preparation of special dietary foods, health foods and calcium-fortified foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 is the mass spectrum of the peptide GRGNEGPQ, where a is the extracted ion current diagram; b is the primary mass spectrum diagram; c is the secondary mass spectrum diagram; and d is the structure diagram.

[0018] Figure 2 This is the physicochemical property analysis of the peptide GRGNEGPQ, where a is the UV-visible spectrum, b is the Fourier transform infrared spectrum, c is the circular dichroism spectrum and secondary structure composition diagram, and d is the Raman spectrum.

[0019] Figure 3 This is the growth morphology of the Caco-2 cell monolayer membrane structure.

[0020] Figure 4 The changes of transmembrane resistance of Caco-2 cells.

[0021] Figure 5 This is the effect of peptide GRGNEGPQ on calcium transport in Caco-2 cells.

[0022] Figure 6 The effect of peptide GRGNEGPQ on calcium bioavailability in Caco-2 cells. DETAILED DESCRIPTION

[0023] The present invention extracts and identifies a novel polypeptide, cod bone calcium absorption promoting peptide, through hydroxyapatite affinity chromatography, Sephadex G25 gel filtration chromatography and C18 reverse phase high performance liquid chromatography combined with Q-Orbitrap, the amino acid sequence of which is GRGNEGPQ. The peptide segment has very high calcium chelating activity, and its calcium chelating activity reaches 4.36±0.57 μg / mg.

[0024] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0025] In the following examples, the method for peptide sequence identification is as follows:

[0026] The amino acid sequence of the peptide was identified by UHPLC-Q-Orbitrap, and the chromatographic column used was Agilent Advance Bio Peptide Map C18 column. UHPLC parameters: mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile, flow rate: 0.15 mL / min, injection volume: 10 μL. Column temperature: 40 °C, gradient elution program: 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. Scanning range: 100-1200 m / z, electrospray mode: electrospray positive ion, electrospray voltage 3600 v.

[0027] In the following examples, the method for determining calcium chelating activity is as follows:

[0028] 3 mg of sample was dissolved in 2 mL of 5 mmol / L CaCl2 and incubated at 37 °C for 20 min. 4 mL of 20 mmol / L sodium phosphate buffer (pH 7.5) was added to the reaction system and incubated at 37 °C for 30 min. The mixture was centrifuged at 8000 rpm / min for 20 min to remove calcium phosphate precipitate. After the supernatant was filtered through a 0.22 μm filter, 2 mL was taken and digested with 10 mL of nitric acid. The calcium content was determined according to GB 5009.92-2016 (atomic absorption method). A blank control group without polypeptide was set up. The calculation formula for calcium chelation activity is as follows:

[0029]

[0030] 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.

[0031] In the following embodiments, the UV-visible spectrum is measured as follows:

[0032] The sample was dissolved in deionized water to a concentration of 1 mg / mL, and the UV-visible spectrum of the sample was measured using a UV-visible spectrophotometer in the wavelength range of 190-350 nm, and deionized water was used for blank correction.

[0033] In the following embodiments, the determination method of Fourier transform infrared spectroscopy is as follows:

[0034] After the sample was repeatedly freeze-dried to remove moisture, 1-2 mg was taken and placed in an agate mortar with an appropriate amount of potassium bromide (KBr) dried to constant weight, ground and mixed, and then pressed into tablets. The infrared spectrum was measured using a Nicolet-iS10 Fourier transform infrared spectrometer with a scanning range of 4000-500 cm -1 , the scanning resolution is 2 cm -1 The number of scans was 64. KBr background was subtracted from all samples. To reduce the influence of moisture in the air, all operations were performed under an infrared lamp.

[0035] In the following embodiments, the circular dichroism spectrum is measured as follows:

[0036] The samples were dissolved in deionized water to 0.1 mg / mL. The circular dichroism spectra of the peptide components and peptide calcium chelates were collected using Jasco-1500. The scanning range was set to 180-260 nm, the scanning speed was 100 nm / min, and the resolution was 0.5 nm. Deionized water was used as the background and each sample was calibrated.

[0037] In the following embodiments, the Raman spectrum is measured as follows:

[0038] Spread the sample powder on a glass slide and place it on the sample measuring platform. Focus the sample and record the excitation wavelength of 532 nm at 200 cm -1 Up to 2000 cm -1 Raman spectra in the range.

[0039] In the following embodiments, the assay method for promoting calcium absorption activity is as follows:

[0040] (1) Cultivation, passaging and cryopreservation of Caco-2 cells

[0041] Caco-2 cells were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37 °C, 5% CO2 and 90% relative humidity. When the cell density reached about 80%, the cells were passaged, the old culture medium was removed, the cells were rinsed with HBSS buffer, 1 mL of trypsin was added for 5 min, the trypsin was aspirated, 2 mL of complete medium was added, the cells were blown with a pipette to form a cell suspension, the cell suspension was transferred to a centrifuge tube, and centrifuged at 1200 r / min at room temperature for 5 min. The supernatant was discarded and 1 mL of complete medium was added to blow the cells, and the cell suspension was evenly divided into 2 parts, and 6 mL of complete medium was added to each T25 culture flask.

[0042] When freezing Caco-2 cells, first remove the old culture medium, add HBSS buffer solution to rinse the cells, digest the cells with trypsin for 5 min, then remove the trypsin, add 2 mL of DMEM high-glucose complete medium, blow the cells with a pipette to form a cell suspension, centrifuge and collect the cells, add 1 mL of freezing solution (containing 72% DMEM high-glucose medium + 20% FBS + 8% dimethyl sulfoxide (DMSO)) to the cells, blow evenly, transfer to a cryopreservation tube, and mark it. After the cryopreservation tube containing the cells is gradually cooled, it is placed in a -80 °C ultra-low temperature refrigerator overnight, and finally placed in a liquid nitrogen tank for long-term storage.

[0043] (2) Establishment of intestinal epithelial cell model

[0044] Caco-2 cells were digested and diluted to 1×10 5 cells / mL, take 0.5 mL and inoculate it in the upper chamber (AP side) of a 12-well Transwell plate to make the cells evenly spread on the polycarbonate membrane, add 1.5 mL of complete culture medium to the lower chamber (BL side) of the Transwell plate, and place the inoculated culture plate in a cell culture incubator for routine culture. Replace the culture medium every two days in the first week, and replace the culture medium every day after one week, and continue to culture for 21 days.

[0045] (3) Validation of the intestinal epithelial cell model

[0046] The growth status of Caco-2 cells in Transwell plates at 1, 3, 5, 7, 14 and 21 days after inoculation was observed under an inverted microscope and photographed.

[0047] The cells in the Transwell plate were cultured to measure the TEER values ​​using a cell resistance meter on the 1st, 3rd, 5th, 8th, 12th, 16th, 19th, and 21st days, and the wells without inoculated cells were set as blank controls. The cell TEER calculation formula is as follows:

[0048] TEER (Ω·cm 2 ) = (T - T0) × S

[0049] Wherein, T is the resistance value of the Transwell chamber inoculated with Caco-2 cells, T0 is the resistance value of the blank control group, and S is the area of ​​the polycarbonate membrane of the Transwell chamber (1.12 cm in this experiment). 2 ).

[0050] (4) Determination of calcium absorption promoting activity

[0051] The resistance of Caco-2 cells cultured in Transwell plates for 21 days was measured, and cells with a TEER value > 300 Ω×cm were selected. 2The above Caco-2 cell monolayer model was used for calcium transport experiments. The upper and lower chambers were gently rinsed three times with 37 °C preheated HBSS solution (without calcium and magnesium ions), 0.5 mL of sample was added to the AP side, and 1.5 mL of HBSS solution (without calcium and magnesium ions) was added to the BL side. The Transwell plate was placed in the incubator for continued culture. At 30, 60, and 90 min of transport, 1.0 mL of BL test culture medium was taken, and 1.0 mL of HBSS solution (without calcium and magnesium ions) was added to continue culture for 120 min, the experiment was terminated, and the culture medium on the AP and BL sides was collected for the determination of calcium ion content.

[0052] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0053] Example 1 Preparation of a novel cod bone peptide promoting calcium absorption

[0054] Fish bone hydrolysates (PBPs) were prepared according to the optimized enzymatic hydrolysis method in the previously disclosed patent CN116806917A, a fish bone gelatin calcium chelate peptide and its preparation method. PBPs were separated step by step by hydroxyapatite affinity chromatography, Sephadex G25 gel filtration chromatography and C18 reversed-phase high-performance liquid chromatography, and finally the peptide sequence of PBPs was identified by high-resolution mass spectrometry.

[0055] like Figure 1 As shown, the polypeptide sequence of PBPs determined by mass spectrometry was GRGNEGPQ (F21-3).

[0056] Example 2 Synthesis of GRGNEGPQ and Verification of Chelating Activity

[0057] The identified peptide GRGNEGPQ was produced by solid phase peptide synthesis, and the peptide was desalted and had a purity of >95%. The calcium binding capacity of GRGNEGPQ prepared by solid phase synthesis was verified, and its calcium chelation capacity was 4.36±0.57 mg / g.

[0058] Example 3 Physicochemical properties of GRGNEGPQ and GRGNEGPQ-Ca

[0059] The calcium ion concentration was adjusted to 0.5 mmol / L, the GRGNEGPQ (F21-3) concentration was adjusted to 1.5 g / L, and the pH was adjusted to 7.0. The chelation was carried out at room temperature for 0.5 h to prepare GRGNEGPQ-Ca (F21-3-Ca). The physical and chemical properties of F21-3 and F21-3-Ca were studied. Figure 2 As shown in a, the λmax of F21-3 is 192 nm, which is similar to that of Ca 2+After chelation, the λmax of the chelates all showed a red shift, and the λmax of F21-3-Ca shifted to 193 nm, and the absorbance of the chelates increased. The red shift may be due to the conjugation effect between the lone pair electrons on the -NH2 and -OH groups and the π electrons on the carbonyl double bond, resulting in a decrease in the transition energy of n→π*. FTIR spectra such as Figure 2 As shown in b, F21-3 is at 3350.26 cm -1 The peak at is the characteristic peak of the amide A band, which is caused by the stretching vibration of NH. However, this peak shifts to 3323.55 cm in F21-3-Ca. -1 , indicating that Ca 2+ The peptide structure changes and thus affects the stretching vibration of the NH bond. -1 The absorption peak at is the characteristic peak of amide I band, which is generated by C=O stretching vibration. This absorption peak shifts to 1661.27 cm in F21-3-Ca. -1 , and the intensity is significantly weakened, which indicates that the carboxyl group is involved in the Ca 2+ Chelated. F21-3 at 1434.29 cm -1 The peak at -COO - The displacement in F21-3-Ca is 1409.14 cm due to stretching vibration. -1 , Description - COO - The non-bonding free electron pairs of Ca 2+ The oxygen atoms of the carboxyl and carbonyl side chain hydroxyl groups participate in the reaction of F21-3 with Ca 2+ Chelation. Figure 2 The random coil in C is the main secondary structure of F21-3 and F21-3-Ca. 2+ After chelation, the random coil content of F21-3 increased from 41.4% to 42.6%, the α-helix content increased from 8.4% to 12.0%, the β-sheet content decreased from 25.9% to 23.8%, and the β-turn content decreased from 24.3% to 21.6%. Figure 2 The d in the figure is the Raman spectra of F21-3 and F21-3-Ca. It can be seen from the figure that after calcium chelation, 1663.93 cm -1 The intensity of the characteristic peak of the amide I band of 2+ Chelated. 919.10 cm -1 The characteristic peak caused by CC bond stretching at 724.25 cm-1 basically disappeared, indicating that the change in secondary structure caused by calcium chelation affected the CC bond stretching vibration. -1 The characteristic peaks of amide IV / V at 594.75 cm -1The characteristic absorption peak of amide VI at the position disappears, indicating that O=CN, NH, and C=O may be Ca 2+ binding site.

[0060] Example 4 Calcium absorption promoting activity of GRGNEGPQ

[0061] like Figure 3 The morphological changes during the establishment of the Caco-2 monolayer cell model. On the first day of model establishment, since the cells had just been inoculated into the Transwell chamber, the cells were observed to be round and sparse, and the large intercellular gaps were clearly observed. On the third day of model establishment, the cells had attached to the polycarbonate membrane of the Transwell chamber. The cells grew in sheets but were not fully grown and the intercellular gaps were still clearly observed. On the fifth day of modeling, the cells gradually merged without obvious boundaries. Culture continued until 21 days. At this time, the Caco-2 cells showed a typical intestinal cell-like morphology, with a minimal cell surface and a complete brush edge with tall and regular microvilli. The integrity of the Caco-2 monolayer was evaluated by TEER values. The results are as follows Figure 4 As shown in Figure 2, the TEER value reached 459.20 Ω·cm on the 8th day of model establishment. 2 After that, it was maintained at 500 Ω·cm 2 Around 523.04 Ω·cm on the 21st day 2 Studies have shown that when the TEER value of the monolayer is >300 Ω·cm 2 When , it indicates that the cells have formed a complete monolayer membrane and can be used for transport experiments.

[0062] The results of F21-3 promoting calcium absorption are as follows Figure 5 As shown in the figure, with the extension of transport time, the calcium transport amount of F21-3 under the conditions of 1 and 3 mM calcium concentration increased significantly. When the transport time was 120 min, the calcium transport amount reached the maximum value of 37.45±2.09 and 42.30±2.88 μg / well, respectively, which was significantly higher than that of the 1 and 3 mM CaCl2 control groups (p<0.05). 2+ Bioavailability Figure 6 As shown, compared with 1 mM CaCl2, 1 mg / mL F21-3 2+ The bioavailability of F21-3 increased by 25.52% compared with 3 mM CaCl2. 2+ The bioavailability was increased by 28.91%.

[0063] Example 5 Transport and absorption characteristics of GRGNEGPQ

[0064] In order to explore the transport of peptides in the Caco-2 monolayer cell model, the peptide composition on the AP side and the BL side after 120 min of transport was identified by UPLC-Q-Orbitrap, and the transmembrane transport of peptides while promoting calcium absorption was analyzed. As shown in Table 1, three peptides GRGNEGPQ, RGNEGPQ, and GNEGPQ were identified on the AP side of F21-3, and four peptides GNE, RGN, NE, and GRG were identified on the BL side. The peptide was partially degraded on the AP side. This is because Caco-2 cells can express multiple enzymes such as aminopeptidase N, aminopeptidase A, carboxypeptidase P, and dipeptidyl peptidase IV in the brush border after 21 days of differentiation. Therefore, the peptide will be further degraded into smaller fragments or free amino acids on the AP side. Therefore, F21-3 is mainly transported to the BL side through fragments smaller than the tripeptide.

[0065] Table 1 shows the transmembrane transport of the peptide GRGNEGPQ

[0066]

[0067] In summary, F21-3 has good calcium absorption promoting activity and can be used as a functional food ingredient in the preparation of special dietary foods, health foods and calcium-fortified foods.

[0068] The above embodiments show and describe the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for illustrating the principles of the present invention, and are not intended to limit the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the protection claimed.

Claims

1. An application of a fish bone calcium absorption promoting peptide in the preparation of health food, characterized in that: The amino acid sequence of fish bone calcium absorption promoting peptide is shown in SEQ ID No. 1, and the specific sequence is GRGNEGPQ.

2. Use of a peptide calcium chelate in the preparation of health food, characterized in that: The calcium chelating activity of the peptide calcium chelate is 4.36±0.57 μg / mg, and the peptide calcium chelate contains the fish bone calcium absorption promoting peptide according to claim 1.

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