High-activity calcium chelating peptide derived from cod bone and preparation method of high-activity calcium chelating peptide

Abstract

The invention discloses a cod bone-derived high-activity calcium chelating peptide and a preparation method thereof, and belongs to the technical field of functional biological product preparation. The calcium chelating peptide provided by the invention is derived from a cod bone collagen enzymatic hydrolysate and is obtained through affinity chromatography separation of hydroxyapatite, the calcium chelating activity is 4.9-5.2 mu g / mg, and the calcium chelating peptide contains aspartic acid and glutamic acid enriched peptide fragments, through deep processing of cod bones, high-valued utilization of cod bone resources is realized, the high-activity calcium chelating peptide is prepared, and the calcium chelating peptide has a wide application prospect. And a high-quality raw material is provided for the development of a novel calcium supplement.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional biological product preparation, and particularly relates to a calcium chelating peptide derived from cod bones and a preparation method thereof. Background Art

[0002] Calcium is an essential mineral for the human body, playing a key role in physiological processes such as bone development, nerve conduction, and muscle contraction. Calcium deficiency can lead to osteoporosis, rickets, and osteomalacia. However, insufficient calcium intake is common among Chinese residents. The Chinese Dietary Reference Intake (DRI) recommends a daily calcium intake of 800 mg for adults, but the average daily calcium intake per standard person in China is only 356.3 mg / day.

[0003] Traditional calcium supplements such as calcium carbonate and calcium gluconate have drawbacks such as low absorption rate and severe gastrointestinal irritation, making it difficult to meet the body's calcium nutritional needs. Studies have found that calcium chelate peptides can form complexes with calcium, preventing calcium precipitation in the gastrointestinal tract, thereby improving calcium absorption and bioavailability. They are considered ideal calcium supplements and can be used as ingredients in the development of functional foods. Currently, the preparation of calcium chelate peptides has problems such as low efficiency, high cost, and low product activity. Therefore, an efficient and low-cost preparation process for calcium chelate peptides is urgently needed.

[0004] Cod bones, as the main by-product of cod processing, account for about 15% of the total weight of cod and are rich in collagen and minerals. However, most of them are currently discarded or used in the production of low-value-added products, resulting in waste of resources. Utilizing cod bone resources to obtain high-quality products has become an urgent problem to be solved.

[0005] Chinese patent CN116806917A discloses a fishbone gelatin calcium chelate peptide and its preparation method. The fishbone gelatin calcium chelate peptide is prepared by enzymatic hydrolysis of cod bone gelatin with papain, affinity chromatography of the gelatin peptide using 60 nm needle-shaped hydroxyapatite, and elution with 120 mM phosphate buffer. The fishbone gelatin calcium chelate peptide provided by the invention has a calcium chelating activity of 4.36±0.72 μg / mg and the sequence is SSGPFG, HyP-GTK, SSGPLG, L-HyP GPQ, A-HyP-GFL, HyP-GPK, GPAGP-HyP, S-HyP-GS-HyP-GPDGKL, TSGPQ or MTGPQ, wherein HyP is hydroxyproline and binds to calcium ions to form a calcium peptide chelate. The activity of the calcium chelate peptide obtained by enzymatic hydrolysis of the prior art needs to be further improved, and it is not possible to carry out targeted enrichment and efficient separation of amino acids. Summary of the Invention

[0006] To address the problems of the prior art, the present invention aims to provide a method for preparing and isolating highly active calcium chelating peptides from cod bones, thereby realizing high-value utilization of cod bone resources and preparing calcium chelating peptides with a calcium chelating activity of 4.9-5.2 μg / mg, providing high-quality raw materials for the development of new calcium supplements.

[0007] The polypeptide provided by the present invention is prepared by the following method:

[0008] (1) Extraction of cod bone gelatin

[0009] The cod bones with the fish meat removed are softened at high temperature, and then the softened fish bones are crushed with water and extracted, and then centrifuged to obtain fish bone gelatin solution;

[0010] The fish meat is removed by enzymatically hydrolyzing the cod bones with 0.4% to 0.6% of 2500 USP u / g trypsin and 0.4% to 0.6% of 200 USP u / g neutral protease at 45 to 65° C. and a material-liquid ratio of 1:1.1 to 1:1.3 for 1 to 2 hours to remove the fish meat;

[0011] The high temperature softening is carried out at a temperature of 115-125°C and a high pressure for 45-55 minutes.

[0012] The extraction after adding water and crushing is carried out at 20-30°C, a material-liquid ratio of 1:3-1:5, crushing at 20000-40000 r / min for 2-4 minutes, and then extracting at 75-85°C for 3.5-4.5 hours.

[0013] (2) Preparation of cod bone gelatin peptide

[0014] Adding protease to the cod bone gelatin solution for enzymatic hydrolysis, inactivating the enzyme after the enzymatic hydrolysis, centrifuging the enzymatic hydrolyzate, and concentrating and drying to obtain cod bone gelatin peptide;

[0015] The cod bone gelatin solution has a protein concentration of 80-120 g / L; the protease comprises 0.5%-2.5% of 2500 USP u / g trypsin and 0.5%-2.5% of 200 USP u / g neutral protease.

[0016] The enzymatic hydrolysis is carried out at 45-55°C and pH 7.2-7.8 for 2.5-3.5 h; preferably, 2.0% trypsin and 2.0% neutral protease are added to a cod bone gelatin solution with a mass concentration of 100 g / L, and the solution is hydrolyzed at 50°C and pH 7.5 for 4 h.

[0017] (3) Preparation of cod bone highly active calcium chelating peptide

[0018] The cod bone gelatin peptide solution is added to activated and balanced hydroxyapatite for binding, and the unbound cod bone gelatin peptide is repeatedly eluted with phosphate buffer to remove the unbound cod bone gelatin peptide. Then, the cod bone gelatin calcium chelate peptide specifically bound to the hydroxyapatite is gradient eluted and collected with phosphate buffer. After concentration, dialysis desalination, and freeze-drying, the cod bone highly active calcium chelate peptide is obtained.

[0019] The cod bone gelatin peptide solution, wherein the mass concentration of the polypeptide is 2.5% to 3.0%;

[0020] The activated and balanced hydroxyapatite is prepared by activating 40-200 nm needle-shaped hydroxyapatite using a phosphate buffer solution of 0.4-0.6 mol / L Na2HPO4∙12H2O and NaH2PO4∙2H2O, and balancing it using a buffer solution containing 4-6 mmol / L Na2HPO4∙12H2O, NaH2PO4∙2H2O, and 80-120 mmol / L sodium chloride;

[0021] The combination comprises statically combining the hydroxyapatite with the cod bone gelatin peptide at 35-40° C. and a rotation speed of 160-180 r / min for 2.5-3.5 hours;

[0022] The elution is performed by centrifugation at 800-1200 r / min for 8-12 min, followed by addition of a buffer solution containing 4-6 mmol / L Na2HPO4∙12H2O, NaH2PO4∙2H2O, and 80-120 mmol / L sodium chloride, and centrifugation for 2-4 times;

[0023] The gradient elution and collection are performed by centrifugation 2 to 4 times using a buffer solution containing 40 to 120 mmol / L Na2HPO4∙12H2O, NaH2PO4∙2H2O, and 80 to 120 mmol / L sodium chloride.

[0024] The present invention also provides a use of the cod bone highly active calcium chelate peptide, which is used in the preparation of peptide calcium chelates.

[0025] The present invention also provides a peptide calcium chelate, which is prepared by using the above-mentioned polypeptide as a chelating peptide.

[0026] The peptide calcium chelate has a preparation method comprising adding CaCl2 to a cod bone calcium chelate peptide solution with a mass fraction of 4% to 6% so that the mass ratio of peptide to calcium is 6:1 to 10:1, performing oscillation and combining at 45 to 55°C, pH 5.2 to 5.8, and a rotation speed of 160 to 180 r / min for 0.5 to 1.5 hours, precipitating with ethanol 6 to 10 times the volume of the system for 10 to 15 hours, centrifuging at a speed of 8,000 to 10,000 r / min for 10 to 20 minutes, washing the precipitate with ethanol 2 to 4 times, and drying to obtain the cod bone peptide calcium chelate.

[0027] Beneficial effects of the present invention

[0028] Compared with the prior art CN116806917A, the present invention optimizes the enzymatic hydrolysis conditions. Cod bone gelatin is hydrolyzed by a combination of neutral protease and trypsin, followed by hydroxyapatite affinity chromatography to prepare highly active calcium chelating peptides, increasing their activity to 4.9-5.2 μg / mg. The invention also achieves targeted enrichment and efficient separation of acidic amino acids in the calcium chelating active peptide segment, thereby improving the performance of the product and providing broader possibilities for the application of calcium chelating peptides in the fields of food, health products, etc.

[0029] The present invention realizes the high-value utilization of cod bone resources by providing a method for preparing and isolating highly active calcium chelating peptides from cod bones. Calcium chelating peptides with a calcium chelating activity of 4.9-5.2 μg / mg are prepared, providing high-quality raw materials for the development of new calcium supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of cod bone gelatin peptide are shown in Figure 1, where A represents the calcium chelating activity of cod bone gelatin peptide; and B represents the morphology of cod bone gelatin peptide.

[0031] Figure 2 The figure is a result diagram of hydroxyapatite affinity chromatography, wherein A is the hydroxyapatite affinity chromatography process; B is the calcium chelating ability of each elution component.

[0032] Figure 3 This is the result of the physical and chemical property analysis of cod bone highly active calcium chelating peptide, where A is the molecular weight distribution and B is the amino acid composition.

[0033] Figure 4 The peptide mass fingerprint spectra of cod bone gelatin peptide and high-activity calcium chelating peptide, where A is the number of peptides retrieved in the peptide, B is the Venn diagram of the retrieved peptides, C is the amino acid frequency in the peptide sequence, and D is the terminal amino acid frequency in the peptide sequence. DETAILED DESCRIPTION

[0034] The present invention separates a highly active cod bone-derived calcium chelating peptide by hydroxyapatite affinity chromatography, and the calcium chelating activity thereof reaches 4.9-5.2 μg / mg.

[0035] In the following examples, the calcium chelating activity was determined as follows:

[0036] 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 another 30 min. The mixture was centrifuged at 8000 r / 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 GB5009.92-2016 (atomic absorption method). A blank control group without peptide was set up.

[0037]

[0038] Among them, C s is the calcium content in the supernatant of each sample group, μg; C b is the calcium content in the supernatant of the control group, μg; M is the sample mass, mg.

[0039] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0040] Example 1: Preparation of cod bone gelatin peptides (PBPs)

[0041] (1) Extraction of cod bone gelatin: Distilled water was added to the cod bones at a solid-liquid ratio of 1:1.2, and 0.5% by mass of trypsin and neutral protease were added. The cod bones were enzymatically hydrolyzed at 55 °C for 1.5 h. After washing, the clean cod bones were obtained. The cod bones were then softened by high-pressure treatment at 121 °C for 50 min. Distilled water was added at a solid-liquid ratio of 1:4, and the solution was stirred and extracted at 80 °C for 4 h. The gelatin solution was obtained after centrifugation and filtration to remove the bone residue. The gelatin solution was concentrated and dried to obtain cod bone gelatin.

[0042] (2) Enzymatic hydrolysis of cod bone gelatin: The concentration of cod bone gelatin was adjusted to 100 g / L, and the pH was adjusted to 7.5. Trypsin and neutral protease were added, and the enzyme addition levels were set at 0.5%, 1%, 1.5%, 2% and 2.5% of the substrate mass. After enzymatic hydrolysis at 50 °C for 3 h, the enzyme was boiled for 15 min to inactivate the enzyme. The enzymatic hydrolyzate was centrifuged to remove the precipitate, concentrated and freeze-dried to obtain cod bone gelatin peptides (PBPs).

[0043] like Figure 1As shown in Figure A, when the amount of trypsin added was 2.0%, the calcium chelation activity of the enzymatic hydrolysate was 1.12 ± 0.18 μg / mg, which was not significantly different from that when the amount of trypsin added was 2.5% (p > 0.05). When the amount of neutral protease added was 2.0%, the calcium chelation activity of the product was the highest, which was 1.28 ± 0.14 μg / mg. Figure 1 As shown in B, the prepared cod bone gelatin peptide is a uniform light yellow powder.

[0044] Example 2: Separation of cod bone highly active calcium chelating peptides (HACPs) by hydroxyapatite affinity chromatography

[0045] (1) Solution preparation:

[0046] Buffer A (pH 6.7): contains 5 mmol / L NaH2PO4·2H2O, 5 mmol / L Na2HPO4·12H2O, and 100 mmol / L NaCl;

[0047] Buffer B1: contains 40 mmol / L NaH2PO4·2H2O, 40 mmol / L Na2HPO4·12H2O, and 100 mmol / L NaCl;

[0048] Buffer B2: contains 80 mmol / L NaH2PO4·2H2O, 80 mmol / L Na2HPO4·12H2O, and 100 mmol / L NaCl;

[0049] Buffer B1: contains 120 mmol / L NaH2PO4·2H2O, 120 mmol / L Na2HPO4·12H2O, and 100 mmol / L NaCl;

[0050] Buffer C (pH 6.8): contains 0.5 mol / L NaH2PO4·2H2O and 0.5 mol / L Na2HPO4·12H2O.

[0051] (2) Hydroxyapatite affinity chromatography:

[0052] 100 g of 60 nm needle-shaped hydroxyapatite was activated and eluted by repeated centrifugation with 60 mL of Buffer C. The hydroxyapatite was then eluted by centrifugation with 60 mL of Buffer A. 1 g of cod bone gelatin peptide was dissolved in 35 mL of Buffer A, filtered through a 0.22 μm filter, added to the hydroxyapatite, and mixed thoroughly. Binding was allowed to proceed at 37°C and 170 rpm for 3 h. The sample was then centrifuged at 1000 rpm for 10 min to remove unbound components. 60 mL of Buffer A was added and the solution was centrifuged repeatedly to remove weakly bound components. 60 mL of Buffer B1 was added and the solution was centrifuged three times. The eluate containing the calcium-chelating peptide was collected. Buffer B2 and Buffer B3 were then centrifuged three times each, and the eluate was collected. The eluates were concentrated, dialyzed for desalination, and freeze-dried to obtain the highly active cod bone calcium-chelating peptide sample.

[0053] like Figure 2 As shown, the calcium chelating activities of the fractions eluted with 40 mmol / L, 80 mmol / L, and 120 mmol / L phosphate were 1.24 ± 0.23 μg / mg, 3.29 ± 0.37 μg / mg, and 4.99 ± 0.33 μg / mg, respectively.

[0054] Example 3: Structural Characterization of Cod Bone Highly Active Calcium Chelating Peptide

[0055] like Figure 3 As shown in A, the molecular weight of the cod bone high-activity calcium chelating peptide is relatively low, with more than 90% of the peptide being below 2000 Da, 31.97% of which is below 500 Da, 36.38% of which is within the range of 500-1000 Da, and 22.58% of which is within the range of 1000-2000 Da. Figure 3 As shown in B, the amino acid with the highest content in cod bone high-activity calcium chelating peptide is glycine, which accounts for about 1 / 3 of all amino acids, and contains more proline and hydroxyproline. This is a typical (Gly-XY) n The protein is determined by a repetitive sequence, where X and Y are typically proline and hydroxyproline, respectively. This sequence allows collagen to form a stable triple helical structure. Aspartic acid and glutamic acid, present in concentrations of 68.64 mol / kmol and 83.65 mol / kmol, respectively, are widely recognized calcium ion binding sites.

[0056] Example 4: Sequence Identification of Cod Bone Gelatin Peptide and Highly Active Calcium Chelating Peptide

[0057] like Figure 4As shown in Figures A and 4B, in order to characterize the primary structure of the highly active calcium chelating peptide derived from cod bone, 1493 and 1087 non-repeating peptides (cross-correlation score XCorr ≥ 1.2) were identified from cod bone gelatin peptide and highly active calcium chelating peptide, respectively, through mass spectrometry acquisition and database search. Of these, 164 peptides were present in both. As shown in Table 1, the sequences –Gly–Pro– and –Gly–Pro–Pro– frequently appeared in both cod bone gelatin peptide and highly active calcium chelating peptide, which is consistent with the (Gly-XY) sequence of collagen. n The repeat sequences matched.

[0058] Table 1 shows the partial peptide sequences (XCorr≥4) present in both cod bone gelatin peptide and cod bone highly active calcium chelating peptide

[0059] Serial number Sequence PSM XCorr 1 FTGMQGPPGQAGSSGDQGPAGSAGPAGPR 7 10.28 2 TGMQGPPGQAGSSGDQGPAGSAGPAGPR 3 9.12 3 MQGPPGQAGSSGDQGPAGSAGPAGPR 6 8.27 4 SGEMGPAGPPGPPGPPGPPGAPGGGFDMGF 4 7.14 5 GPSGSSGSAGKDGMSGLPGPSGPSGPR 5 6.67 6 QGPPGQAGSSGDQGPAGSAGPAGPR 1 6.27 7 GSSGDQGPAGSAGPAGPR 3 5.51 8 GPAGPHGPPGKDGR 13 5.38 9 AGSSGDQGPAGSAGPAGPR 4 5.38 10 FTGPAGEPGEPGASGPMGSR 5 5.07 11 GESGPGGPTGPSGM 2 4.89 12 LRGDSGPAGPPGEQGMLGPAG 14 4.78 13 SGDSGSAGPAGPSGPR 7 4.75 14 GAPGSPGAPGPVGPAGK 3 4.68 15 AGSSGPGGERGPPGPMGPSG 4 4.66 16 GAPGPSGAPGPVGPAGK 4 4.60 17 SGERGPAGATGPAGPR 3 4.57 18 [[ID=3D]]GGNRGESGPGGPTGPSGM 2 4.57 19 FTGPAGEPGEPGASGPM 3 4.52 20 LGPAGPAGPAGQ 14 4.47 21 FSGLDGAKGDSGPAGPK 4 4.37 22 GGNRGESGPGGPTGPSGMAG 3 4.36 23 GSPGSAGSSGPQG 2 4.33 24 GDQGPAGSAGPAGPR 4 4.30 25 SAGPQGPAGPAGPK 7 4.23 26 AGPQGPAGPAGPK 10 4.21 27 PGAIGAPGPAGGA 2 4.19 28 FSGLDGAKGDAGAPGPK 2 4.14 29 ASGPQGPAGAPGPK 4 4.11 30 GAPGSPGAPGPVGPA 2 4.08 31 TGPAGEPGEPGASGPM 1 4.07 32 QGPAGPAGPAGPK 4 4.06 33 FTGPPGESGEAG 2 4.06 34 GAPGPSGAPGPVGPA 2 4.06 35 PQGNKGSVGSPGHQGPP 8 4.00

[0060] like Figure 4 As shown in Figures C and 4D, the frequency of amino acids in the peptide sequences of cod bone gelatin peptide and high-activity calcium chelating peptide was statistically analyzed. Glycine accounts for approximately one-third of the total amino acids, and proline accounts for slightly more than one-sixth, which is consistent with the typical characteristics of collagen peptides. The frequencies of aspartic acid and glutamic acid in the high-activity calcium chelating peptide are 37.11% and 83.68% higher than those in gelatin peptide, respectively. The frequencies of peptide terminal aspartic acid (N-terminus: 2.39%, C-terminus: 1.56%) and glutamic acid (N-terminus: 3.86%, C-terminus: 2.94%) are also higher than those in gelatin peptide (aspartic acid: N-terminus: 1.00%, C-terminus: 1.14%; glutamic acid: N-terminus: 1.14%, C-terminus: 0.74%). This indicates that the peptides enriched in the high-activity calcium chelating peptide after hydroxyapatite affinity chromatography contain more acidic amino acids that are beneficial for calcium binding.

[0061] The invention uses cod bone as raw material to separate and prepare high calcium chelating active cod bone peptide, which is of great significance for the development of calcium supplement preparations and provides a new idea for the high-value utilization of cod bone.

[0062] 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 highly active calcium chelating peptide derived from cod bones, characterized by: Derived from the enzymatic hydrolysis product of cod bone collagen, it was obtained by hydroxyapatite affinity chromatography. It has a calcium chelating activity of 4.9-5.2 μg / mg and contains peptides enriched in aspartic acid and glutamic acid. The fraction with a molecular weight below 2000 Da exceeds 90%, of which the fraction below 500 Da accounts for 31.97%, the fraction within the range of 500-1000 Da accounts for 36.38%, and the fraction within the range of 1000-2000 Da accounts for 22.58%.

2. A method for preparing a highly active calcium chelating peptide derived from cod bones, characterized in that: The following steps are involved: (1) Cod bone gelatin extraction: The cod bones were enzymatically hydrolyzed with trypsin and neutral protease to remove the fish meat, and then enzymatically hydrolyzed with 0.4%~0.6% trypsin and neutral protease for 1~2 h. Then, the bones were softened at high temperature and high pressure at 115~125 °C for 45~55 min. Water was then added at a material-liquid ratio of 1:3~1:5, and the bones were crushed at 20000~40000 r / min for 2~4 min. The bones were extracted at 75~85 °C for 3.5~4.5 h, and the gelatin solution was obtained after centrifugation. (2) Preparation of cod bone gelatin peptide: The gelatin solution concentration was adjusted to 80-120 g / L, 0.5%-2.5% trypsin and neutral protease were added, and enzymatic hydrolysis was performed at 45-55 °C and pH 7.2-7.8 for 2.5-3.5 h. The gelatin peptide was obtained after enzyme inactivation, centrifugation, concentration and drying. (3) Separation of highly active calcium-chelating peptides: Gelatin peptides were prepared into a solution with a mass concentration of 2.5% to 3.0%, and statically bound to 60 nm needle-shaped hydroxyapatite after activation equilibrium at 35 to 40 °C and 160 to 180 r / min for 2.5 to 3.5 h. Unbound peptides were first eluted with a buffer containing 4 to 6 mmol / L phosphate and 80 to 120 mmol / L sodium chloride, and then gradient eluted with a buffer containing 40 to 120 mmol / L phosphate and 80 to 120 mmol / L sodium chloride. The highly active components were collected and the calcium-chelating peptides were obtained after concentration, desalination by dialysis, and lyophilization.

3. The preparation method according to claim 2, wherein: The preferred enzymatic hydrolysis conditions in step (2) are: gelatin solution concentration of 100 g / L, addition of 2.0% trypsin and 2.0% neutral protease, and enzymatic hydrolysis at 50 °C and pH 7.5 for 4 h.

4. The preparation method according to claim 2, wherein: During the gradient elution in step (3), the phosphate concentration is 40 mmol / L, 80 mmol / L, and 120 mmol / L, respectively, and elution is performed 2 to 4 times each time. The centrifugal speed is 800 to 1200 r / min, and the elution time is 8 to 12 min.

5. Use of the highly active calcium chelating peptide according to claim 1 in the preparation of calcium supplement preparations.

6. A peptide calcium chelate prepared using the above-mentioned highly active calcium chelating peptide.

7. The method for preparing the peptide calcium chelate according to claim 6, characterized in that: The specific steps are as follows: adding CaCl2 to a cod bone calcium chelate peptide solution with a mass fraction of 4% to 6% so that the mass ratio of peptide to calcium is 6:1 to 10:1; oscillating and combining for 0.5 to 1.5 hours at 45 to 55°C, pH 5.2 to 5.8, and a rotation speed of 160 to 180 r / min; precipitating with ethanol 6 to 10 times the volume of the system for 10 to 15 hours; centrifuging at a speed of 8000 to 10000 r / min for 10 to 20 minutes; washing the precipitate with ethanol 2 to 4 times; and drying to obtain the cod bone peptide calcium chelate.

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