Nano-liposome embedded with cod bone peptide calcium chelate and application of nano-liposome

Abstract

The invention provides the nano-liposome for embedding the cod bone peptide calcium chelate, so that the digestion stability of the cod bone peptide calcium chelate is enhanced, targeted release of Ca < 2 + > in small intestines is facilitated, and the bioavailability of the cod bone peptide calcium chelate is further improved. The amino acid sequence of the cod bone calcium chelating active peptide is SSGPGGERGPp. The nano-liposome disclosed by the invention can ensure the structural integrity in the stomach to protect the cod bone peptide calcium chelate from being damaged, and can release the peptide calcium chelate in the intestinal digestion process, so that the digestion stability of the cod bone peptide calcium chelate is improved, and the targeted delivery of the cod bone peptide calcium chelate is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biological functional products, and in particular relates to a nano liposome 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 of the free Ca 2+ Phytic acid, oxalic acid, tannins, polyphenols and long-chain fatty acids in plant foods can all compete for 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, especially peptides containing hydrophobic amino acids are sensitive to trypsin and peptidase, which is an important factor limiting the application of calcium chelating peptides in related fields. At present, chemical modification, microcapsule 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 phospholipid bilayers, which can encapsulate active ingredients in the aqueous core medium inside them, and the liposomes themselves can be dissolved in aqueous dispersion media. In recent years, nanoliposome (NLs) technology has been widely used in the production of functional foods, nutritional supplements, cosmetics and pharmaceutical products due to its excellent encapsulation efficiency, good stability and targeted release. In order to improve the digestion stability of calcium chelating peptides and peptide calcium chelates, more and more researchers have introduced nanoliposome technology to achieve the protection of 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 the 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 invention can be used to prepare peptide calcium chelates.

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

[0009] The peptide calcium chelate provided by the present invention can be used to prepare calcium supplement products. In the 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 nano liposome provided by the present invention has a preparation method comprising the following steps: dissolving the wall material in anhydrous ethanol and mixing them uniformly, dissolving the core material in ultrapure water, mixing the core material and the wall material uniformly in a certain ratio, shearing for 3-7 minutes, ultrasonicating for 8-10 minutes until the solution is uniformly mixed, and then subjecting the solution to high pressure homogenization treatment to obtain nano liposomes with a particle size of less than 250 nm and encapsulating 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 a wall material to prepare a NLs capable of loading PBPs-Ca and optimizes its preparation process. It is found that the core-wall ratio is 1:5, PBPs-Ca is 15 mg / mL, and Tween-80 is used as an emulsifier for 9 minutes of ultrasound. The prepared NLs have 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. In the stomach, the structural integrity can be ensured to protect the cod bone peptide calcium chelate from being destroyed. The peptide calcium chelate can be released during intestinal digestion, thereby improving the digestion stability of the cod bone peptide calcium chelate and achieving its targeted delivery. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 It 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 rate.

[0019] Figure 3 It 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; 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 embedding rate.

[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] Fig. 9 The graph shows the changes of 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] Fig.10 The graph shows the changes of 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] Fig.11 The graph shows the changes in the release rate of NLs during simulated gastrointestinal digestion.

[0028] Fig.12 This is a graph showing 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 method for determining calcium chelating activity is as follows:

[0031] 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 solution 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 adding polypeptide was set up. The calculation formula of calcium chelation activity is 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 embodiments, the embedding rate was determined as follows:

[0035] Unentrapped Ca 2+ Determination of: Take an appropriate amount of NLs powder and dissolve it in ultrapure water to a final concentration of 30 mg / mL, vortex and dissolve 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 centrifugation. 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: Take an appropriate amount of NLs powder and add it to 5mL petroleum ether. Ultrasonic treatment for 15 minutes destroys the NLs structure to expose the internal cod bone peptide calcium chelate. Volatilize the petroleum ether in a water bath. After the petroleum ether is completely evaporated, add a certain amount of ultrapure water to dissolve the NLs concentration to 10mg / mL. Take 1mL of the mixed solution to determine the calcium ion content. The embedding rate is calculated according to the following formula:

[0037] E=(X–Y / 3) / X×100%

[0038] E is the embedding rate, in %; X is the Ca content in 10 mg NLs sample 2+ The unit is 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 with a pipette and added into the sample pool, and its particle size, Zeta potential value and PDI were measured by Malvern laser particle size analyzer.

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

[0042] 1-2 mg of NLs powder and an appropriate amount of potassium bromide (KBr) dried to constant weight were placed 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 embodiments, 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 4-60° (2θ) at a speed of 6° / min under the conditions of an accelerating voltage of 40 kV and an accelerating current of 40 mA.

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

[0046] Take NLs and add 80 mL of simulated gastric fluid (1.25×), adjust the pH to 3.0 with concentrated hydrochloric acid, and then add ultrapure water to adjust the solution volume to 100 mL. Perform simulated gastric digestion in a 37°C water bath shaker. Take 10 mL of samples at 0, 30, 60, 90, and 120 min of digestion, inactivate the enzyme in a boiling water bath for 5 min, and mark them 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×), adjusted to pH 7.0 with concentrated NaOH, and then ultrapure water was added to adjust the final volume of the solution to 60 mL. The simulated intestinal digestion was carried out in a 37°C water bath shaker. 10 mL of samples were taken at 0, 30, 60, 90, and 120 min of digestion, respectively, and the enzymes were inactivated in a boiling water bath for 5 min, and marked 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 photographs were taken 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 precipitates. Centrifuge at 8000r / 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 precipitate. Determine 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 1 mL supernatant 2+ Content (μg).

[0055] In the following embodiments, 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] Prepare simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Mix NL with the prepared SGF (1.25×), adjust the pH to 3.0 with concentrated hydrochloric acid, and then add ultrapure water to adjust the solution volume to 50 mL. Perform simulated gastric digestion in a 37°C water bath shaker. Take 10 mL samples at 0, 30, 60, 90, and 120 min of digestion, and inactivate the enzyme in a boiling water bath for 5 min, and mark them as G0, G30, G60, G90, and G120. After the simulated gastric digestion is completed, take the simulated gastric fluid and mix it with 1.25×SIF SIF, adjust the pH to 7.0 with concentrated NaOH, and then add ultrapure water to adjust the final volume of the solution to 50 mL. Perform simulated intestinal digestion in a 37°C water bath shaker. Take 10 mL samples at 30, 60, 90, and 120 min of digestion, and inactivate the enzyme in a boiling water bath for 5 min, and mark them as 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 the embedding rate under different storage conditions and storage time were investigated.

[0062] The present invention is described in detail below in conjunction with specific embodiments and 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, and papain with a substrate protein content of 1% was added to hydrolyze for 4 hours, and cod bone calcium chelate peptide was obtained after the enzyme was inactivated.

[0065] The amino acid sequence of calcium chelating peptide was identified by UHPLC-Q-Orbitrap. UHPLC parameters: mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile, flow rate: 0.15mL / min, injection volume: 10μL. Column temperature: 40℃, gradient elution program: 0-20min, 2%-25% B; 20-30min, 25-45% B; 30-35min, 45%-85% B; 35-40min, 85% B; 40-42min, 85-2% B; 42-52min, 2% B. Scanning range: 100-1200m / z, electrospray mode: electrospray positive ion, electrospray voltage 3600v.

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

[0067] Based on the peptide segment, peptide calcium chelate was further prepared, the CaCl2 concentration was adjusted to 0.5 mM, the polypeptide concentration was adjusted to 1.5 g / L, chelation was carried out at room temperature for 0.5 h under pH 7.0 conditions, and alcohol precipitation was carried out with 8 times the volume of ethanol to prepare the 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 a 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 dropped into the cod bone peptide calcium chelate aqueous solution according to a certain core-to-wall ratio, and then ultrasonic treatment is performed after shearing. 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 in the figure, with the increase of the core-to-wall ratio, the particle size of NLs shows a trend of gradually decreasing, the PDI gradually decreases, and the absolute value of the potential gradually increases, indicating that the stability of NLs is also gradually improved. The embedding rate shows a trend of first increasing and then stabilizing with the increase of the core-to-wall ratio. When the core-to-wall ratio is 1:5, the embedding rate reaches 61.25%, at which time the particle size of NLs is 148.20nm, the PDI is 0.25, and the Zeta potential is -35.07mV. Therefore, the NLs prepared with a core-to-wall ratio of 1:5 have a good embedding effect and high stability of the system.

[0071] Effect of core material concentration on particle size, PDI, Zeta potential and embedding efficiency of NLs Figure 3 As shown. With the increase of the concentration of cod bone peptide calcium chelate, the particle size of NLs gradually increased. When the concentration reached 25 mg / mL, the particle size of NLs reached the 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 embedding rate showed a trend of first increasing and then stabilizing with the increase of the concentration of cod bone peptide calcium chelate. The embedding rate was 75.37% at 20 mg / mL. Since the embedding rate of cod bone peptide calcium chelate was 72.95% at 15 mg / mL, there was no significant difference with 20 mg / mL, but 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] Effect of ultrasound time on particle size, PDI, Zeta potential and embedding efficiency of NLs Figure 4 When the ultrasonic time was 9 min, the NLs embedding rate was 74.55% at the maximum, the PDI was 0.29 at the minimum, 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 Figure 5As shown. The highest embedding rates of NLs with Tween 80 and cholesterol were 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 preparing 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. From the FTIR shape, NLs are more similar to EP and are 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 1651.27cm 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 1243.17cm is the amide II band, which is mainly caused by NH bending vibration; -1 The absorption peak is the 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 in NLs. -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 connected to the phosphate group in EP. 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 at 1069.80 cm -1 and 1235.64cm -1 The absorption peak at PO 2- The symmetric stretching and asymmetric stretching of -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 a broad peak. The diffraction peaks of EP and NLs are similar and appear at 15-25°, showing 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 increases, 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, achieving the effect of targeted delivery.

[0079] like Fig. 9 As shown in the figure, the average particle size of NLs in the simulated digestion in the stomach from 0min to 120min showed a trend of being constant at first and then increasing, which shows that NLs particles are relatively stable in the stomach and can still play an important role in protecting cod bone peptide calcium chelate after being destroyed by gastric acid and pepsin. The Zeta potential did not change much within 120min of gastric digestion, which is similar to the particle size results. This shows that NLs are highly stable in the stomach, not prone to leakage, and have excellent protective effects 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] Changes in particle size, PDI, Zeta potential and chelation rate of NLs during simulated intestinal digestion Fig.10 As shown. The particle size of NLs showed a downward trend during the simulated intestinal digestion. The average particle size of the NLs sample just after entering the simulated intestinal digestion decreased from 233.27nm to 182.83nm after 120 minutes of intestinal digestion. This result shows that NLs were completely digested in the intestine and the cod bone peptide calcium chelate was released. During the intestinal digestion process, its Zeta potential value remained at around -20mV. This shows that NLs are digested into a relatively uniform liquid in the intestine, which is convenient for the absorption of nutrients. The calcium chelation rate results show that the chelation rate of cod bone peptide calcium chelate does not change much in the intestine, which shows that NLs particles have a good protective effect on cod bone peptide calcium chelate, which can improve the digestion stability of cod bone peptide calcium chelate in the stomach, and it is not destroyed by gastric acid and still maintains a high calcium chelation rate when released in the intestine.

[0081] The calcium release rate of NLs during gastrointestinal digestion is as follows Fig.11 As shown in the figure, 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%, and continued to increase to 83.45% at 90 minutes, reaching a maximum of 88.14% at 120 minutes. The results of this study are similar to the trend of particle size changes, indicating that NLs maintain good stability in the stomach, play a protective role on 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. Fig.12 As shown in the figure, the embedding rate of NLs decreased from 76.55±3.12% to 45.96±4.97% after 28 days of storage at 25℃, from 75.34±2.98% to 57.60±0.76% after 28 days of storage at 4℃, and from 78.03±2.48% to 65.51±0.50% after 28 days of storage at -20℃. With the extension of storage time, the embedding rate of NLs under various storage temperature conditions showed a significant downward trend. Compared with the storage day 0, the embedding rate of NLs stored at 25℃, 4℃ and -18℃ for 28 days decreased by 39.96%, 23.55% and 16.05%, respectively. In the first 7 days of storage, different storage temperatures had little effect on the embedding rate. With the extension of storage time, the embedding rate of NLs stored at 25℃ and 4℃ 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 as claimed in claim 3.

7. The nanoliposome according to claim 6, characterized in that The nano liposome also contains wall material and emulsifier, wherein the wall material contains EP, and the emulsifier contains Tween 80, glyceryl monostearate, cholesterol and casein.

8. The nanoliposome according to claim 6, characterized in that The preparation method of the nano liposome is as follows: the wall material is dissolved and mixed evenly with anhydrous ethanol, the core material is dissolved in ultrapure water, the core material and the wall material are mixed evenly, shearing for 3-7 minutes, ultrasonicating for 8-10 minutes until the solution is evenly mixed, and then high-pressure homogenization treatment is performed to obtain nano liposomes with a particle size of less than 250nm and a potential of -30mV that encapsulate the cod bone peptide calcium chelate.

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

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