Nanoliposome embedding gadusolt peptide calcium chelate and application thereof

Abstract

The application provides a nano-liposome embedding cod bone peptide calcium chelate, so as to enhance the digestion stability of the cod bone peptide calcium chelate, facilitate Ca 2+ Targeted release in the small intestine, thereby improving its bioavailability. The cod bone calcium chelate active peptide has an amino acid sequence of SSGPGGERGPp. The nano-liposome of the application can guarantee the structural integrity in the stomach to protect the cod bone peptide calcium chelate from being destroyed, and can release the peptide calcium chelate in the intestinal digestion process, thereby improving the digestion stability of the cod bone peptide calcium chelate and realizing the targeted delivery.

Description

Technical Field

[0001] The invention belongs to the technical field of biological functional products, and particularly relates to a nanoliposome encapsulating cod bone peptide calcium chelate and application thereof. Background Art

[0002] The digestive stability of peptide calcium chelates is affected by many factors, such as pH, oxalic acid, phytic acid, anions, and various digestive enzymes. In the acidic environment of the stomach, the binding site of the peptide calcium chelate is protonated, resulting in the dissociation of the chelate. The alkaline pH of the intestine is conducive to the formation of peptide calcium chelates, but most free Ca 2+ Phytic acid, oxalic acid, tannins, polyphenols and long-chain fatty acids in plant foods can all compete to bind to Ca 2+ A precipitate forms, resulting in a decrease in its solubility.

[0003] In addition, the peptides used to prepare peptide calcium chelates are easily digested into oligopeptides and amino acids by pepsin, trypsin, aminopeptidase, and α-chymotrypsin. Peptides containing hydrophobic amino acids are particularly sensitive to trypsin and peptidase, which is a major factor limiting the application of calcium chelating peptides in related fields. Currently, chemical modification, microencapsulation, and nanoliposome encapsulation are mostly used to improve the stability of peptide calcium chelates in the gastrointestinal tract.

[0004] Liposomes are lipid vesicles composed of a phospholipid bilayer that can encapsulate active ingredients within their aqueous core medium. The liposomes themselves are also soluble in aqueous dispersion media. In recent years, nanoliposome (NL) technology has been widely used in the production of functional foods, nutritional supplements, cosmetics, and pharmaceuticals due to its excellent encapsulation efficiency, good stability, and targeted release. To improve the digestive stability of calcium-chelating peptides and peptide-calcium chelates, an increasing number of researchers are introducing nanoliposome technology to protect peptide-calcium chelates. Summary of the Invention

[0005] The purpose of the present invention is to provide a nanoliposome encapsulating cod bone peptide calcium chelate (PBPs-Ca), thereby enhancing the digestion stability of cod bone peptide calcium chelate, and facilitating the digestion of Ca 2+ Targeted release in the small intestine, thereby improving its bioavailability.

[0006] The present invention first provides a calcium chelating active peptide polypeptide, the amino acid sequence of which is SSGPGGERGPp, wherein p is hydroxyproline, and the calcium chelating activity of the peptide segment is 3.29±1.02 μg / mg.

[0007] The polypeptide SSGPGGERGPp provided by the present invention can be used to prepare peptide calcium chelates.

[0008] The peptide calcium chelate provided by the present invention has a preparation method as follows: Ca 2+ The concentration is 0.4-0.6 mM, the peptide concentration is 1.4-1.6 g / L, and the pH is 6.8-7.2. Chelation is carried out at room temperature for 28-32 minutes.

[0009] The peptide calcium chelate provided by the present invention can be used to prepare calcium supplement products. In an embodiment of the present invention, the product is a nanoliposome;

[0010] In another aspect, the present invention provides a nanoliposome comprising a core material, a wall material and an emulsifier, wherein the core material comprises the above-mentioned PBPs-Ca, the wall material comprises egg yolk phosphatidylcholine (EP), and the emulsifier comprises Tween 80, glyceryl monostearate, cholesterol and casein.

[0011] The nanoliposomes provided by the present invention are prepared by dissolving and mixing the wall material in anhydrous ethanol, dissolving the core material in ultrapure water, mixing the core material and the wall material in a certain proportion, shearing for 3-7 minutes, ultrasonicating for 8-10 minutes until the solution is evenly mixed, and then performing high-pressure homogenization to obtain nanoliposomes with a particle size of less than 250 nm that encapsulate the cod bone peptide calcium chelate.

[0012] The mass ratio of the core material to the wall material is 1:4-1:6, preferably 1:5.

[0013] The concentration of the core material is 14-16 mg / mL, preferably 15 mg / mL.

[0014] The emulsifiers are Tween 80, glyceryl monostearate, cholesterol and casein, preferably Tween 80.

[0015] The ultrasonic time is 8-10 min, preferably 9 min.

[0016] The present invention uses EP as the wall material to prepare a NLs capable of loading PBPs-Ca and optimizes its preparation process. It was found that the core-wall ratio was 1:5, the PBPs-Ca was 15 mg / mL, and Tween-80 was used as an emulsifier for 9 minutes of ultrasound. The prepared NLs had a high embedding rate of up to 74.55%, an average particle size of 235.03 nm, a PDI of 0.29, and a particle size potential of -31.90 mV. The NLs have a small particle size and good stability. They can maintain structural integrity in the stomach to protect the cod bone peptide calcium chelate from destruction. During intestinal digestion, the peptide calcium chelate can be released, thereby improving the digestive stability of the cod bone peptide calcium chelate and achieving its targeted delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Figure 2 is the secondary mass spectrum of the peptide SSGPGGERGPp, where a is the extracted ion chromatogram; b is the primary mass spectrum; c is the secondary mass spectrum; and d is the structure diagram.

[0018] Figure 2 This is the core-to-wall ratio optimization diagram, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the embedding efficiency.

[0019] Figure 3 This is the core material concentration optimization diagram, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the embedding rate.

[0020] Figure 4 This is the ultrasound time optimization diagram, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the encapsulation rate.

[0021] Figure 5 This is the optimization diagram of emulsifier types, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the encapsulation efficiency.

[0022] Figure 6 is the infrared spectrum of NLs.

[0023] Figure 7 is the X-ray diffraction pattern of NLs.

[0024] Figure 8 Figure 2 shows the microstructure of NLs during simulated digestion.

[0025] Figure 9 Figure 3 is a graph showing the changes in NLs during simulated gastric digestion, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the chelation rate.

[0026] Figure 10 Figure 3 is a graph showing the changes in NLs during simulated intestinal digestion, where a is the particle size distribution; b is the average particle size and PDI; c is the Zeta potential; and d is the chelation rate.

[0027] Figure 11 The graph shows the changes in the release rate of NLs during simulated gastrointestinal digestion.

[0028] Figure 12 The graph shows the changes in the embedding rate of NLs during storage. DETAILED DESCRIPTION

[0029] The present invention uses nanoliposome technology to embed the peptide calcium chelate to improve the digestion stability of the peptide calcium chelate, achieve protection of the peptide calcium chelate, and enhance Ca 2+ Targeted release and bioavailability in the small intestine.

[0030] In the following examples, the calcium chelating activity was determined as follows:

[0031] 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 continue incubating at 37°C for 30 min. Centrifuge at 8000 rpm / min for 20 min to remove calcium phosphate precipitate. After filtering the supernatant through a 0.22 μm filter, take 2 mL and add 10 mL of nitric acid for digestion. Calcium content is determined according to GB 5009.92-2016 (atomic absorption spectrometry). Set up a blank control group without adding polypeptide. The calcium chelation activity is calculated as follows:

[0032]

[0033] 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.

[0034] In the following examples, the embedding efficiency was determined as follows:

[0035] Unentrapped Ca 2+ Determination of NLs: Take an appropriate amount of NLs powder and dissolve it in ultrapure water to a final concentration of 30 mg / mL. Vortex and dissolve it into a uniform solution to wash away the unembedded cod bone peptide calcium chelate on the surface. Centrifuge at 8000 rap / min for 10 minutes and then use a 100 kDa ultrafiltration centrifuge tube for ultrafiltration. 2+ As the solution passes through the ultrafiltration membrane and is separated from the NLs solution, 1 mL of the liquid at the bottom of the ultrafiltration centrifuge tube is taken to determine the calcium content.

[0036] Total Ca 2+ Determination of calcium ion content: Add an appropriate amount of NLs powder to 5 mL of petroleum ether. Ultrasonicate for 15 minutes to disrupt the NLs structure, exposing the cod bone peptide calcium chelate inside. Evaporate the petroleum ether in a water bath. Once the petroleum ether has completely evaporated, add a sufficient amount of ultrapure water to dissolve the NLs at a concentration of 10 mg / mL. Measure the calcium ion content in 1 mL of the mixture. Calculate the embedding efficiency using the following formula:

[0037] E=(X–Y / 3) / X×100%

[0038] E is the embedding efficiency, in %; X is the Ca content in 10 mg NLs sample. 2+ The mass of NLs is expressed in μg; Y is the unentrapped Ca in 30 mg NLs sample. 2+ The mass of the substance in μg.

[0039] In the following examples, the particle size, zeta potential value and polydispersity index (PDI) of NLs were determined as follows:

[0040] After the prepared NLs emulsion was diluted 10 times, 1 mL was taken out with a pipette and added to the sample pool, and its particle size, Zeta potential value and PDI were measured using a Malvern laser particle size analyzer.

[0041] In the following examples, the determination method of Fourier transform infrared spectroscopy is as follows:

[0042] 1-2 mg of NLs powder was mixed with an appropriate amount of potassium bromide (KBr) dried to constant weight in an agate mortar, ground and mixed, and then pressed into tablets. Infrared spectra were measured using a Nicolet-iS10 Fourier transform infrared spectrometer with a scanning range of 4000-500 cm -1 , scanning resolution is 2cm -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.

[0043] In the following examples, the X-ray diffraction analysis method is as follows:

[0044] A certain amount of EP, PBPs-Ca and NLs were placed in a mold and scanned from 4 to 60° (2θ) at an accelerating voltage of 40 kV and an accelerating current of 40 mA at a speed of 6° / min.

[0045] In the following examples, the simulated digestion analysis method of NLs is as follows:

[0046] NLs were added to 80 mL of simulated gastric fluid (1.25×), adjusted to pH 3.0 with concentrated hydrochloric acid, and then the solution volume was adjusted to 100 mL with ultrapure water. The simulated gastric digestion was carried out in a shaking water bath at 37°C. At 0, 30, 60, 90, and 120 min of digestion, 10 mL of samples were taken, respectively, and the enzyme was inactivated in a boiling water bath for 5 min and labeled as G0, G30, G60, G90, and G120.

[0047] After the simulated gastric digestion was completed, 30 mL of simulated gastric fluid was taken and added with 24 mL of intestinal digestion fluid (1.25×), and the pH was adjusted to 7.0 with concentrated NaOH. Ultrapure water was added to adjust the final volume of the solution to 60 mL. The simulated intestinal digestion was carried out in a shaking water bath at 37°C. At 0, 30, 60, 90, and 120 min of digestion, 10 mL of samples were taken, and the enzyme was inactivated in a boiling water bath for 5 min. The samples were labeled as I0, I30, I60, I90, and I120.

[0048] In the following embodiments, the analysis method of transmission electron microscopy is as follows:

[0049] The NLs digestion solution was diluted to an appropriate multiple and then dropped onto a copper grid. After counterstaining with phosphotungstic acid, the sample was dried and the changes in the NLs microstructure were observed under a projection electron microscope, and photographed at different magnifications.

[0050] In the following examples, the analysis method of calcium chelation rate is as follows:

[0051] Take 1 mL of digestion solution and add 8 times the volume of anhydrous ethanol. After standing for 3 hours, the chelate is precipitated. Centrifuge at 8000 r / min for 15 minutes. Take the supernatant and concentrate it to about 1 mL. Continue to add 8 times the volume of anhydrous ethanol and repeat the above operation to completely remove the chelate precipitation. Measure the free Ca in the supernatant. 2+ content.

[0052] The chelation rate is calculated as follows:

[0053] C = (W1-W2) / W1×100%.

[0054] Where C is the chelation rate, in %, and W1 is the total Ca in 1 mL of digestive fluid. 2+ Content (μg), W2 is the free Ca in 1mL supernatant 2+ Content (μg).

[0055] In the following examples, the analysis method of calcium release rate is as follows:

[0056] L=(E2-E1) / E2×100%

[0057] Where L is the calcium release rate, in %; E2 is the embedding rate of NLs without gastrointestinal digestion; E1 is the embedding rate of NLs samples at different times of gastrointestinal simulated digestion.

[0058] In the following examples, the method for simulating the in vitro digestion of NLs is as follows:

[0059] Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared. NL was mixed with pre-prepared SGF (1.25×), adjusted to pH 3.0 with concentrated hydrochloric acid, and the solution volume was adjusted to 50 mL with ultrapure water. Simulated gastric digestion was performed in a shaking water bath at 37°C. 10 mL samples were taken at 0, 30, 60, 90, and 120 min of digestion and inactivated in a boiling water bath for 5 min. These samples were labeled G0, G30, G60, G90, and G120. After the simulated gastric digestion was completed, the simulated gastric fluid was mixed with 1.25× SIF (SIF), adjusted to pH 7.0 with concentrated NaOH, and the solution volume was adjusted to 50 mL with ultrapure water. Simulated intestinal digestion was performed in a shaking water bath at 37°C. 10 mL samples were taken at 30, 60, 90, and 120 min of digestion and inactivated in a boiling water bath for 5 min. These samples were labeled I30, I60, I90, and I120.

[0060] In the following examples, the method for storage stability of NLs is as follows:

[0061] NLs were stored at -18℃, 4℃ and 25℃ for 0, 7, 14, 21 and 28 days, respectively, and the changes in their embedding efficiency under different storage conditions and storage time were investigated.

[0062] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0063] Example 1: Preparation of calcium chelating active peptides and peptide calcium chelates

[0064] The fish bones were softened at high temperature and then crushed, and distilled water was added to fully extract at 80°C to obtain cod bone crude protein, which was then hydrolyzed for 4 hours by adding papain with a substrate protein content of 1%. After the enzyme was inactivated, cod bone calcium chelate peptide was obtained.

[0065] 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.

[0066] like Figure 1 As shown, mass spectrometry identified the sequence of the PBPs as SSGPGGERGPp, with a molecular weight of 1016.46 Da. This peptide contains 11 amino acids, of which p is hydroxyproline, a characteristic amino acid of collagen. The calcium chelation activity of SSGPGGERGPp was measured, and its calcium binding capacity was 3.29±1.02 μg / mg, significantly higher than that of the previously reported cod bone gelatin peptide KGDPGLSPGK (2.53±0.12 μg / mg).

[0067] Based on this peptide segment, peptide calcium chelate was prepared by adjusting the CaCl2 concentration to 0.5 mM and the polypeptide concentration to 1.5 g / L. The mixture was chelated at room temperature for 0.5 h under pH 7.0 and precipitated with 8 times the volume of ethanol to obtain peptide calcium chelate.

[0068] Example 2: Preparation of Nanoliposomes Encapsulating Cod Bone Peptide Calcium Chelate

[0069] First, a polypeptide with an amino acid sequence of SSGPGGERGPp is synthesized, and the corresponding peptide calcium chelate is prepared and dissolved in ultrapure water. EP is dissolved in anhydrous ethanol and an emulsifier is added and stirred evenly. The lecithin ethanol solution is added dropwise to the cod bone peptide calcium chelate aqueous solution according to a certain core-to-wall ratio, and then sheared and ultrasonically treated. The mixed solution is then magnetically stirred. After the liquid is evenly mixed, it is subjected to high-pressure homogenization treatment to finally obtain nanoliposome particles loaded with cod bone peptide calcium chelate.

[0070] Effect of core-to-wall ratio on particle size, Zeta potential, PDI and embedding efficiency Figure 2 As shown, as the core-to-wall ratio increases, the particle size of the NLs decreases, the PDI decreases, and the absolute value of the potential increases, indicating that the stability of the NLs also gradually improves. The embedding efficiency increases with the core-to-wall ratio and then stabilizes. When the core-to-wall ratio is 1:5, the embedding efficiency reaches 61.25%, at which point the NLs have a particle size of 148.20 nm, a PDI of 0.25, and a zeta potential of -35.07 mV. Therefore, NLs prepared with a core-to-wall ratio of 1:5 have good embedding efficiency and high system stability.

[0071] Effects of core material concentration on particle size, PDI, Zeta potential and embedding efficiency of NLs Figure 3 As shown. With the increase of cod bone peptide calcium chelate concentration, the particle size of NLs gradually increased. When the concentration reached 25 mg / mL, the particle size of NLs reached its maximum value. When the concentration was 30 mg / mL, the particle size of NLs decreased significantly (p<0.05), and the change trend of PDI was consistent with the particle size. The encapsulation efficiency showed a trend of first increasing and then stabilizing with the increase of cod bone peptide calcium chelate concentration. At 20 mg / mL, the encapsulation efficiency was 75.37%. Since the encapsulation efficiency of cod bone peptide calcium chelate was 72.95% at 15 mg / mL, which was not significantly different from 20 mg / mL, the average particle size and PDI of NLs prepared at this concentration were smaller. The absolute value of Zeta potential was the largest, indicating that the NLs system was more uniform and stable at this time. Therefore, 15 mg / mL was selected as the addition concentration of cod bone peptide calcium chelate in the subsequent preparation process.

[0072] Effects of ultrasound time on particle size, PDI, Zeta potential and entrapment efficiency of NLs Figure 4 When the ultrasonic time was 9 min, the NLs encapsulation rate was the maximum at 74.55%, the PDI was the minimum at 0.29, the particle size was 235.03 nm, and the potential was -31.90 mV.

[0073] The effects of emulsifier on particle size, potential and embedding efficiency of NLs are shown in the following table. Figure 5The NLs with Tween 80 and cholesterol had the highest entrapment rates of 72.73% and 75.71%, respectively. The particle size of the Tween 80 group was smaller at 224.53 nm. Therefore, Tween 80 was selected as the emulsifier for the preparation of cod bone peptide calcium chelate nanoliposomes.

[0074] Example 3: Physicochemical properties of nanoliposomes encapsulating cod bone peptide calcium chelate

[0075] The embedding efficiency of NLs prepared in Example 2 was 74.55%, the average particle size was 235.03 nm, the PDI was 0.29, and the particle potential was -31.90 mV. The Fourier transform infrared spectra (FTIR) of cod bone peptide calcium chelate, EP and NLs are shown in FIG. Figure 6 As shown in the FTIR spectrum, NLs are more similar to EP and quite different from cod bone peptide calcium chelate, which indicates that cod bone peptide calcium chelate can be embedded in EP to form nanoliposomes. In the spectrum of cod bone peptide calcium chelate, 3363.85 cm -1 The peak at 1651.27 cm is the amide A band, which is mainly formed by NH stretching; -1 The absorption peak at 1548.53 cm represents the amide I band, which is mainly related to C=O stretching vibration; -1 The absorption peak at 1243.17 cm is amide II band, which is mainly caused by NH bending vibration; -1 The absorption peak is amide III band, which is mainly caused by CN bending vibration. -1 and 2951.67cm -1 The absorption peaks of -CH2 symmetric and asymmetric stretching vibrations are mainly related to the symmetric and asymmetric stretching vibrations of -CH2, which shift to 2925.79 cm-1 and 2926.79 cm-2 in NLs, respectively. -1 and 2855.05cm -1 , indicating that there is an ionic interaction between EP and cod bone peptide calcium chelate. 1738.56cm -1 The absorption peak of EP is mainly formed by the stretching vibration of the hydrocarbon chain connecting to the phosphate group. This peak almost disappears in NLs, which indicates that the C=O in EP forms a hydrogen bond interaction with the cod bone peptide calcium chelate. EP is at 1069.80 cm -1 and 1235.64cm -1 The absorption peak at PO 2- The symmetric stretching and asymmetric stretching of NLs are respectively shifted to 1072.08 cm -1 and 1240.25cm -1 .

[0076] The X-ray diffraction patterns of PBPs-Ca, EP and NLs are shown in Figure 7. The characteristic peak of cod bone peptide calcium chelate appears at 15-30°, showing as a broad peak. The diffraction peaks of EP and NLs are similar and appear at 15-25°, showing as a sharp peak. This result shows that the cod bone peptide calcium chelate is effectively encapsulated in NLs, and the presence of the encapsulated cod bone peptide calcium chelate does not cause changes in the characteristic structure of EP. If the cod bone peptide calcium chelate exists outside the NLs, crystals will be produced due to its low water solubility and may affect the diffraction peaks of NLs. Obviously, the diffraction patterns of NLs and EP have not changed, which further indicates that the cod bone peptide calcium chelate has been successfully encapsulated into the NLs liposomes.

[0077] Example 4: Digestive stability of cod bone peptide calcium chelate

[0078] TEM can observe the shape, size distribution, uniformity and integrity of NLs. TEM images of NLs during digestion are shown in Figure 2. Figure 8 As shown. In the G0 stage, NLs are complete spherical nanoparticles. This result is consistent with the above results. NLs present uniform and stable single nanoliposome particles. However, as the digestion time prolongs, it can be clearly seen in the G120 stage that the surface structure of NLs is destroyed, but the spherical conformation is still maintained. In the I120 stage, the spherical structure of NLs has been completely destroyed. The results of this study show that although the surface structure of NLs is destroyed in the gastric environment, the basic conformation does not change, so it still has a certain protective effect on cod bone peptide calcium chelate, and the cod bone peptide calcium chelate embedded inside is completely destroyed in the intestine to release and achieve the effect of targeted delivery.

[0079] like Figure 9 As shown in the figure, the average particle size of NLs showed a trend of first being constant and then increasing during simulated digestion in the stomach from 0min to 120min. This indicates that the NLs particles are relatively stable in the stomach and can still play an important role in protecting the cod bone peptide calcium chelate after being destroyed by gastric acid and pepsin. The Zeta potential did not change much during the 120min of gastric digestion, which is similar to the particle size results. This indicates that NLs are highly stable in the stomach, not prone to leakage, and have an excellent protective effect on PBPs-C. The calcium chelation rate results show that the chelation rate of the unencapsulated cod bone peptide calcium chelate is easily affected by the acidic environment of the stomach, resulting in Ca 2+ When the cod bone peptide calcium chelate is embedded in EP to form NLs, its Ca 2+ The chelation rate is relatively stable and is not easily affected by the gastric digestive environment. This indicates that NLs particles have a good protective effect on cod bone peptide calcium chelate and can improve the digestive stability of cod bone peptide calcium chelate in the stomach.

[0080] The changes of particle size, PDI, Zeta potential and chelation rate of NLs during simulated intestinal digestion are shown in Figure 2. Figure 10 As shown. The particle size of NLs showed a downward trend during the simulated intestinal digestion process. The average particle size of the NLs sample decreased from 233.27 nm immediately after entering the simulated intestinal digestion to 182.83 nm after 120 minutes of intestinal digestion. This result indicates that NLs were completely digested in the intestine and cod bone peptide calcium chelate was released. Its Zeta potential value remained at around -20 mV during intestinal digestion. This indicates that NLs were digested into a relatively uniform liquid in the intestine, facilitating the absorption of nutrients. The calcium chelation rate results showed that the chelation rate of cod bone peptide calcium chelate did not change much in the intestine, indicating that NLs particles have a good protective effect on cod bone peptide calcium chelate, can improve the digestion stability of cod bone peptide calcium chelate in the stomach, and maintain a high calcium chelation rate when released in the intestine without being destroyed by gastric acid.

[0081] The calcium release rate of NLs during gastrointestinal digestion is as follows Figure 11 As shown, NLs maintained good stability during the simulated gastric digestion stage. The release rate of cod bone peptide calcium chelate showed a significant upward trend during intestinal digestion. At 30 minutes, the release rate of cod bone peptide calcium chelate reached 52.28%, continued to increase to 83.45% at 90 minutes, and reached a maximum of 88.14% at 120 minutes. This result is similar to the trend of particle size change, indicating that NLs maintain good stability in the stomach, play a protective role for cod bone peptide calcium chelate, and can gradually release cod bone peptide calcium chelate in the intestine.

[0082] Example 5: Storage stability of cod bone peptide calcium chelate

[0083] The changes of embedding efficiency of NLs stored at 25, 4 and -18℃ for 0, 7, 14, 21 and 28 days are shown in Figure 2. Figure 12 The embedding efficiency of NLs decreased from 76.55±3.12% to 45.96±4.97% after 28 days of storage at 25°C, from 75.34±2.98% to 57.60±0.76% after 28 days of storage at 4°C, and from 78.03±2.48% to 65.51±0.50% after 28 days of storage at -20°C. With prolonged storage, the embedding efficiency of NLs showed a significant downward trend at all storage temperatures. Compared with storage day 0, the embedding efficiency decreased by 39.96%, 23.55%, and 16.05% after 28 days of storage at 25°C, 4°C, and -18°C, respectively. During the first 7 days of storage, different storage temperatures had little effect on the embedding efficiency. However, with prolonged storage, the embedding efficiency of NLs stored at 25°C and 4°C decreased significantly.

Claims

1. A cod bone calcium chelate peptide, characterized in that: The amino acid sequence of the cod bone calcium chelating peptide is SSGPGGERGPp, wherein p is hydroxyproline.

2. Use of the cod bone calcium chelate 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 polypeptide according to claim 1.

4. Use of the peptide calcium chelate according to claim 3 in the preparation of calcium supplement products.

5. The use according to claim 4, characterized in that The calcium supplement product is nano liposome.

6. A nanoliposome, characterized in that: The core material of the nanoliposome is the peptide calcium chelate according to claim 3.

7. The nanoliposome according to claim 6, wherein The nano liposome further comprises a wall material and an emulsifier, wherein the wall material comprises EP and the emulsifier comprises Tween 80, glyceryl monostearate, cholesterol and casein.

8. The nanoliposome according to claim 6, wherein The nanoliposomes are prepared by dissolving and mixing the wall material in anhydrous ethanol, dissolving the core material in ultrapure water, mixing the core material and the wall material evenly, shearing for 3-7 minutes, ultrasonicating for 8-10 minutes until the solution is evenly mixed, and then performing high-pressure homogenization to obtain nanoliposomes encapsulating cod bone peptide calcium chelate with a particle size of less than 250nm and a potential of -30mV.

9. The nanoliposome according to claim 8, wherein The mass ratio of the core material to the wall material in the nanoliposome is 1:4-1:6, and the emulsifier is Tween 80.

10. The nanoliposome according to claim 8, wherein The concentration of the core material in the nanoliposome is 14-16 mg / mL.

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