High-calcium chelating activity collagen peptide prepared based on chicken bones, peptide calcium chelate and application

Abstract

The invention discloses a high-calcium chelating activity collagen peptide prepared based on chicken bones, a peptide calcium chelate and application, an ultrasonic-assisted enzymolysis technology is utilized to destroy a collagen cross-linked structure through an ultrasonic cavitation effect, and three-stage enzymolysis time sequence regulation is combined, so that the hydrolysis rate of the collagen peptide reaches 33.18 + / -0.84%. The collagen peptide provided by the invention has relatively high calcium chelating activity, and the peptide calcium chelate is stable in structure in an intestinal neutral environment and under a weakly alkaline condition and is beneficial to intestinal absorption of calcium.

Description

(I) TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive peptides, and specifically relates to a high-calcium chelating active collagen peptide and a peptide calcium chelate based on chicken bone preparation and application. (II) BACKGROUND

[0002] Collagen peptide is a small molecular fragment obtained by hydrolysis of collagen, which is rich in characteristic amino acids such as glycine and proline, and has good water solubility and absorption. The functional groups such as carboxyl and amino on the molecular chain can chelate calcium ions as ideal ligands, forming a peptide calcium chelate, thereby improving the bioavailability of calcium. Calcium is a key mineral element for maintaining human bone and neuromuscular function. Traditional calcium supplements such as calcium carbonate and calcium phosphate have poor solubility and low bioavailability, and their absorption is easily interfered by gastric acid and dietary factors.

[0003] The peptide calcium chelate can effectively protect calcium ions from the influence of gastric acid and anti-nutritional factors in the diet, and is synergistically absorbed through the peptide transport channel of the small intestine, thereby significantly improving the bioavailability of calcium. In the prior art, the raw materials for collagen peptide calcium chelate are mainly concentrated in mammalian bones (such as cow bones, rabbit bones, etc.) and aquatic processing by-products (fish bones, fish skins, etc.). However, there are still many bottlenecks in the related preparation process:

[0004] CN115677849A (Preparation method of collagen peptide calcium chelate of livestock and poultry bones) uses collagen peptide solution of livestock and poultry bones to prepare peptide calcium chelate, which has better stability and can better promote the proliferation and mineralization of osteoblasts, and the temperature has no significant effect on the calcium retention rate. However, the use of organic solvent degreasing and single alkaline protease enzymolysis process to prepare peptide calcium chelate has the problems of destroying the natural structure of collagen and limited hydrolysis degree, and the use of calcium chloride as calcium source and the determination of chelation conditions by simple single factor experiment result in that the product bioactivity and calcium binding capacity are not optimal.

[0005] CN116751828A (Preparation method of livestock and poultry bone polypeptide chelated calcium powder) discloses a method for preparing livestock and poultry bone polypeptide chelated calcium by using steam explosion and electrolytic water technology, which aims to realize green preparation through physical explosion and alternative acid source. However, the high temperature and high pressure pretreatment easily leads to denaturation of the product, the single enzymolysis system has low hydrolysis degree (the highest is 24.1%), and the strong acid calcium extraction environment and the chelation process which is not fully optimized restrict the quality and safety of the product.

[0006] CN119241647A (A nano-liposome embedding cod bone peptide calcium chelate and its application) provides a scheme for embedding cod bone specific peptide calcium chelate in nano-liposome. Although this method aims to achieve intestinal targeted release through a delivery system, it relies on a specific synthetic peptide segment with low calcium chelation activity (3.29 μg / mg) and requires complex nano-liposome embedding, which is cumbersome and costly, making it difficult to apply to large-scale high-value conversion of livestock and poultry bone by-products.

[0007] In summary, the prior art has different degrees of defects in raw material pretreatment, enzymatic efficiency, calcium source selection, chelation process optimization, and cost control, etc. At the same time, a large amount of chicken claw bone by-products generated in poultry processing are rich in type I collagen and bioavailable calcium, which are ideal raw materials for preparing collagen peptide calcium chelates. However, due to the low extraction efficiency and low proportion of active components of traditional technology, an effective resource utilization path has not been established. Chicken bones are the largest by-product generated during chicken processing, with an annual output of 1500-2000 million tons (Xie Shanci. Research progress of chicken bone processing and utilization [J]. Food Science and Technology, 2024). According to the data of Popular Science China in 2023, more than 20 billion chicken claws are consumed in China every year, and their high-value conversion channel needs to be opened up. Therefore, developing a chicken bone collagen peptide calcium chelate preparation method that can avoid raw material denaturation, achieve deep and efficient enzymolysis, select safe and high-quality calcium sources, and have precise and controllable process and significant cost advantage, is of great industrial significance and market value for breaking through the bottleneck of existing technology, improving the bioavailability of calcium preparations, and realizing the green value-added conversion of poultry waste. (III) SUMMARY

[0008] The purpose of the present application is to provide a high calcium chelation activity collagen peptide and peptide calcium chelate based on chicken bone preparation and application, which is prepared by ultrasonic-assisted enzymolysis technology, and has high hydrolysis degree and high calcium chelation activity. The chicken bone collagen peptide calcium chelate prepared based on the preferred calcium source has good stability, which solves the problems of low absorption rate of traditional calcium agents and waste of chicken bone resources.

[0009] The technical scheme adopted by the present application is:

[0010] In a first aspect, the present application provides a high-calcium chelating active collagen peptide prepared based on chicken bone, which is prepared by the following method: (1) raw material pretreatment: chicken paw bones are crushed, and impurity proteins, fat and minerals are removed to obtain pretreated bone powder; (2) ultrasonic-assisted primary enzymolysis: the pretreated bone powder is mixed with an acetic acid aqueous solution, and is subjected to ultrasonic treatment at 100-480 W for 10-30 min to expose the enzymolysis sites by ultrasonic cavitation effect; then pepsin (specifically hydrolyzing aromatic amino acids) is added, and primary enzymolysis is carried out at 35-39°C (preferably 37°C) and pH 1.5-2.0 (preferably 1.7) for 2-6 h (preferably 6 h) under continuous stirring to obtain a primary enzymolysis solution; (3) secondary enzymolysis: the primary enzymolysis solution is adjusted to pH 8-10 (preferably 8.5), and then alkaline protease is added, and the alkaline protease is stirred at 35-55°C (preferably 45°C) for 2-6 h (preferably 5 h) to cut the hydrophobic peptide chains to release small molecular fragments to obtain a secondary enzymolysis solution; (4) tertiary enzymolysis: the secondary enzymolysis solution is adjusted to pH 5.5-7.5 (preferably 6.0), and then complex protease is added, and the complex protease is stirred at 35-55°C (preferably 50°C) for 1-5 h (preferably 2 h) to cut the peptide bonds to enrich 0.5-1.5 kDa active peptides to obtain a tertiary enzymolysis solution; (5) purification: the tertiary enzymolysis solution is filtered, and the filtrate is subjected to 0.22 μm microfiltration and 500 Da dialysis bag pure water dialysis for 48 h in sequence, and the cut-off liquid is freeze-dried to obtain the collagen peptide, which is referred to as CBCP for short.

[0011] Further, the raw material pretreatment in step (1) is carried out by the following steps: ① the chicken paw is removed of skin, meat and tendon tissues, washed and dried at 80°C for 10 h, crushed and passed through a 40-mesh sieve to obtain bone powder; ② impurity protein removal: the bone powder is soaked in 0.1 mol / L NaOH aqueous solution at a material-to-liquid ratio of 5 mL / g for 6 h at room temperature (25-30°C), and the solution is changed every 2 h, and then washed with water until neutral, drained and dried (50°C air drying for 1 h) to obtain bone powder after impurity protein removal; ③ fat removal: the bone powder after impurity protein removal is soaked in 5% NaOH aqueous solution at a material-to-liquid ratio of 5 mL / g for 24 h at room temperature, and the solution is changed twice in the middle, and then washed with water until neutral, drained and dried (50°C air drying for 1 h) to obtain bone powder after fat removal; ④ mineral removal: the bone powder after fat removal is soaked in 0.25 mol / L EDTA (ethylenediaminetetraacetic acid disodium salt) aqueous solution (pH 7.5) at a material-to-liquid ratio of 5 mL / g for 24 h at room temperature, and the solution is changed twice in the middle, and then washed with water until the solution is clear, drained and dried (50°C air drying for 1 h) to obtain pretreated bone powder. C Further, the raw material pretreatment in step (1) is carried out by the following steps: ① the chicken paw is removed of skin, meat and tendon tissues, washed and dried at 80°C for 10 h, crushed and passed through a 40-mesh sieve to obtain bone powder; ② impurity protein removal: the bone powder is soaked in 0.1 mol / L NaOH aqueous solution at a material-to-liquid ratio of 5 mL / g for 6 h at room temperature (25-30°C), and the solution is changed every 2 h, and then washed with water until neutral, drained and dried (50°C air drying for 1 h) to obtain bone powder after impurity protein removal; ③ fat removal: the bone powder after impurity protein removal is soaked in 5% NaOH aqueous solution at a material-to-liquid ratio of 5 mL / g for 24 h at room temperature, and the solution is changed twice in the middle, and then washed with water until neutral, drained and dried (50°C air drying for 1 h) to obtain bone powder after fat removal; ④ mineral removal: the bone powder after fat removal is soaked in 0.25 mol / L EDTA (ethylenediaminetetraacetic acid disodium salt) aqueous solution (pH 7.5) at a material-to-liquid ratio of 5 mL / g for 24 h at room temperature, and the solution is changed twice in the middle, and then washed with water until the solution is clear, drained and dried (50°C air drying for 1 h) to obtain pretreated bone powder.

[0012] Further, the concentration of the acetic acid aqueous solution in step (2) is 0.1-1M (preferably 0.5M), and the volume is 1-10mL / g (preferably 5mL / g) based on the mass of the pretreated bone powder; the ultrasonic conditions are 320W for 20min; and the amount of pepsin added is 130-170U / g (preferably 150U / g) based on the mass of the pretreated bone powder. The pepsin (porcine origin) (Araldine, product number: P110927, 3000U / g) is pepsinogen secreted by the chief cells of the gastric mucosa.

[0013] Further, in step (3), the first enzyme solution is adjusted to pH 8.5, and the amount of alkaline protease added is 3000-7000U / g (preferably 5000U / g) based on the mass of the pretreated bone powder, and stirring is performed at 45°C for 5h. The alkaline protease (Yelo, product number: S10154, 200U / mg) is a protein hydrolase obtained by deep fermentation, extraction and purification of Bacillus licheniformis 2709 obtained by mutagenesis of bacterial protoplasts.

[0014] Further, in step (4), the second enzyme solution is adjusted to pH 6, and the amount of complex protease added is 3000-7000U / g (preferably 5000U / g) based on the mass of the pretreated bone powder, and stirring is performed at 50°C for 2h. The complex protease (Yelo, product number: S10155, 120U / mg) is a special complex enzyme preparation for animal protein hydrolysis, mainly composed of endo-protease, exo-protease and flavor enzyme.

[0015] Further, in step (5), the freeze-drying is pre-frozen at -80°C for 12h, and vacuum freeze-drying is performed at -50°C, 10Pa for 48h.

[0016] In a second aspect, the present application also provides a peptide calcium chelate prepared from the collagen peptide, wherein the peptide calcium chelate is prepared by dissolving the collagen peptide in pure water (brand: Wahaha), adding a calcium source, and reacting at pH 5-7 and 25-60°C for 20-60min, precipitating with anhydrous ethanol (preferably 6 times the volume of the reaction solution), centrifuging at 4000r / min for 20min at 4°C, and freeze-drying the precipitate (preferably at -50°C, 10Pa for 48h) to obtain the peptide calcium chelate (referred to as CBCP-Ca).

[0017] Further, the calcium source includes calcium citrate, calcium chloride, calcium lactate or calcium gluconate, preferably calcium citrate; the mass ratio of the collagen peptide to the calcium source is 1:1-3:1, preferably 1:1. The mass concentration of the collagen peptide in the pure water is 5g / L.

[0018] Further, the reaction is preferably performed at pH 5.5 and 40°C for 30min.

[0019] In a third aspect, the present application provides a use of the peptide calcium chelate in the preparation of a functional food.

[0020] Further, the functional food includes calcium nutritional supplements, high-calcium foods, and special medical use formula foods.

[0021] In a fourth aspect, the present application provides a functional food prepared from the peptide calcium chelate, which includes high-efficiency calcium nutritional supplements, high-calcium foods, special medical use formula foods, and other related products.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] (1) The collagen peptide preparation process based on chicken bones provided by the present application has an innovative breakthrough. The ultrasonic-assisted enzymatic hydrolysis technology is used to destroy the collagen cross-linking structure through ultrasonic cavitation effect, and the three-stage enzymatic hydrolysis time sequence control is combined to realize a collagen peptide hydrolysis rate of 33.18 ± 0.84%, which is increased by about 10.28% compared with the non-ultrasonic group.

[0024] (2) The multi-stage enzymatic hydrolysis process of CBCP provided by the present application gradually improves the hydrolysis efficiency through the synergistic effect of different enzymes. Single enzyme (pepsin) enzymatic hydrolysis realizes a collagen peptide hydrolysis rate of 18.40 ± 1.72%; two-stage enzymatic hydrolysis (pepsin + alkaline protease) time sequence control realizes a collagen peptide hydrolysis rate of 28.38 ± 1.31%, which is increased by about 54.24% compared with the single enzyme group; three-stage enzymatic hydrolysis (pepsin + alkaline protease + complex protease) time sequence control realizes a collagen peptide hydrolysis rate of 33.18 ± 0.84%, which is increased by about 16.91% compared with the two-stage enzymatic hydrolysis group. The degree of hydrolysis (33.18%) of the present application is increased by about 37.68% compared with CN116751828A (24.1%).

[0025] (3) The molecular weight distribution of CBCP provided by the present application is as follows: 0.5-1 kDa peptide segment accounts for 15%; 1-1.5 kDa peptide segment accounts for 62.5%; and 1.5-2 kDa peptide segment accounts for 22.5%. The multi-stage enzymatic hydrolysis process can decompose the original larger molecular weight collagen protein into smaller active peptide segments. Low molecular peptides usually have better biological activity and absorption efficiency.

[0026] (4) The CBCP core structure provided by the present application has the highest glycine content (16.36%), followed by glutamic acid (14.24%) and aspartic acid (9.84%). This high proportion of glycine is a typical feature of collagen protein, which plays a key role in the collagen triple helix structure. The collagen characteristic amino acids are rich in proline (12.66%), which further supports its collagen protein characteristics. These amino acids are of great significance to stabilize the collagen molecular structure and promote skin and bone health.

[0027] (5) The CBCP provided by the application has high calcium chelation activity, and the calcium binding activity is 112.5 ± 3.2 μg / mg, which reflects the chelation of the chicken bone collagen protein peptide, and the calcium binding activity is increased by about 34 times compared with CN119241647A (3.29 µg / mg).

[0028] (6) The CBCP-Ca provided by the application selects calcium citrate with higher binding force as the calcium source of the chelate, and the calcium binding force reaches 123.45 ± 3.57 µg / mg, which is 3.11 times higher than that of the traditional CaCO (6) The CBCP-Ca provided by the application selects calcium citrate with higher binding force as the calcium source of the chelate, and the calcium binding force reaches 123.45 ± 3.57 µg / mg, which is 3.11 times higher than that of the traditional CaCO

[0029] (7) In the stability determination of the CBCP-Ca provided by the application, the stability at 40℃ is optimal: the retention rate is >95% in the first 10 minutes, and is still about 83% after 50 minutes, proving that a low-temperature environment can effectively delay the dissociation of calcium ions. The retention rate is maintained at 65%-75% in the pH 6-9 interval, especially at pH 7-8, reaching a peak, indicating that the structure of the chelate is stable in the neutral and weak alkaline environment of the intestinal tract, which is beneficial to the intestinal absorption of calcium. In the in vitro simulated digestion stability analysis, the simulated stomach group is the main reason for the decrease in chelation, which is related to the low stability of the chelate in acidic conditions. Under the combined action of acidic conditions and pepsin, the chelate will decrease obviously within 15 min, and then tend to be stable. (Four) Description of Drawings

[0030] Figure 1 , Effect of ultrasonic power (a) and time (b) on the degree of hydrolysis of the first-stage enzyme hydrolysate in Example 1 Step 2.

[0031] Figure 2 , Effect of enzyme dosage (a), temperature (b), pH (c), and time (d) on the degree of hydrolysis of the first-stage enzyme hydrolysate in Example 1 Step 2.

[0032] Figure 3 , Effect of enzyme dosage (a), temperature (b), pH (c), and time (d) on the degree of hydrolysis of the first-stage enzyme hydrolysate in Example 1 Step 2.

[0033] Figure 4 , Effect of different extraction methods on the degree of hydrolysis of chicken bone collagen protein in Comparative Example 1.

[0034] Figure 5 , Effect of different enzymes on the degree of hydrolysis of chicken bone collagen protein in Comparative Example 2.

[0035] Figure 6 , Effect of enzyme type and enzyme addition order on the degree of hydrolysis of chicken bone collagen protein in Comparative Example 3 (a) and Comparative Example 4 (b).

[0036] Figure 7 Effect of calcium source (a) and peptide to calcium mass ratio (b) on the calcium binding capacity of peptides.

[0037] Figure 8 Calcium retention rate of chicken bone collagen peptide calcium chelate under different temperatures (a), pH (b) and in vitro simulation (c) conditions.

[0038] Figure 9 Physicochemical properties of chicken bone collagen peptide and peptide calcium chelate; a represents ultraviolet-visible full spectrum scanning, b represents Fourier infrared spectrum; c represents the differential scanning calorimetry of CBCP; d represents the differential scanning calorimetry of CBCP-Ca.

[0039] Figure 10 Scanning electron microscope images of chicken bone collagen peptide and peptide calcium chelate; a represents the elemental distribution point scanning of CBCP, b represents the elemental distribution point scanning of CBCP-Ca, c represents the morphology of CBCP, and e represents the morphology of CBCP-Ca. (V) SPECIFIC EMBODIMENT

[0040] The application will be further described in conjunction with specific embodiments, but the protection scope of the application is not limited to this:

[0041] In the following examples, the determination methods of protein, collagen, moisture, fat, ash, calcium and phosphorus are as follows:

[0042] Protein content was determined by Kjeldahl nitrogen determination method (GB5009.5-2016 first method) (full-automatic Kjeldahl nitrogen determination instrument and matching digestion instrument, brand Haineng, K9860+SH220F); collagen content was determined by hydroxyproline (HYP) content detection kit (spectrophotometric method, brand Solaybao, item number jgslb-BC0250-50T / 48S); water content was determined by water rapid determination instrument (brand Aind, model MS-70); crude fat was determined by Soxhlet extraction method (GB5009.6-2016 first method) fat determination instrument (brand Haineng, model SOX406); ash content was determined by incineration method (reference GB5009.4-2016 first method); calcium ion concentration was determined by ethylenediaminetetraacetic acid (EDTA) complexometric titration method (reference GB5009.92-2016 second method); and phosphorus content was determined by molybdenum blue spectrophotometric method (reference GB5009.87-2016 first method).

[0043] In the following examples, the determination method of hydrolysis degree is as follows:

[0044] The growth rate of soluble nitrogen was determined by TCA (trichloroacetic acid) precipitation method to indicate the degree of hydrolysis. 10 mL of enzyme solution was added with 10 mL of 20% trichloroacetic acid aqueous solution, and then fully oscillated. After centrifugation at 10000 r / min for 15 min, the supernatant was taken and the content of soluble nitrogen was determined by Kjeldahl method. The calculation formula of the degree of hydrolysis is as follows:

[0045]

[0046] Wherein N0 is the total nitrogen content of the original sample (mg); N1 is the soluble nitrogen content of the sample added with 20% TCA before enzyme hydrolysis (mg); N2 is the soluble nitrogen content of the supernatant added with 20% TCA after enzyme hydrolysis (mg).

[0047] In the following examples, the determination method of the extraction rate of collagen peptide is as follows:

[0048] According to the weight of the dried chicken bone powder and the freeze-dried collagen peptide powder, the calculation formula of the extraction rate is as follows:

[0049]

[0050] Wherein m1 is the mass of the dried chicken bone powder (g); m2 is the mass of the freeze-dried collagen peptide powder (g).

[0051] In the following examples, the determination method of calcium chelating activity is as follows:

[0052] 3 mg of CBCP was dissolved in 2 mL of 5 mmol / L CaCl2 aqueous solution, and incubated at 37 °C for 20 min. Then 4 mL of sodium phosphate buffer (20 mmol / L, pH 7.5) was added, and the mixture was further incubated at 37 °C for 30 min. After centrifugation at 8000 r / min for 20 min to remove the calcium phosphate precipitate, the calcium content in the supernatant was determined. The sample without CBCP was used as a control group. The calculation formula of calcium chelating activity is as follows:

[0053]

[0054] Wherein, Ca s is the calcium content in the supernatant of each sample group, μg; Ca c is the calcium content in the supernatant of the control group, μg; m s is the mass of the sample, mg.

[0055] In the following examples, the determination method of calcium binding capacity is as follows:

[0056] The calcium content in CBCP-Ca and the calcium content in CBCP were determined by EDTA titration method according to GB 5009.92-2016. The calculation formula of calcium binding capacity is as follows: ​

[0057]

[0058] In the following examples, the method for measuring the calcium retention rate is as follows:

[0059] The calcium content is measured according to GB 5009.92-2016 EDTA titration method. The calcium retention rate calculation formula is as follows:

[0060]

[0061] Wherein, m1 is the total calcium content before reaction; m2 is the calcium content of the supernatant after reaction of the solution.

[0062] The brand of pure water used in the embodiments of the application is Wahaha.

[0063] Example 1: Preparation of chicken bone collagen peptide (CBCP) with high hydrolysis degree and high calcium chelation activity.

[0064] 1. Raw material pretreatment

[0065] The white feather chicken boneless claw by-product was removed of skin, meat and tendon and the like, washed, dried at 80°C for 10h, crushed through a 40 mesh sieve, and bone powder was obtained (containing water 5.32%, ash 40.16%, fat 18.22%, protein 30.92%, calcium 12.59% and phosphorus 7.48%);

[0066] Impure protein: 50.29g of the bone powder was soaked in 0.1 mol / L NaOH aqueous solution at room temperature (25-30°C, the same below) for 6h according to the amount of 5mL / g, the solution was changed every 2h, washed with water until neutral, and then dried (50°C air drying for 1h) to obtain the bone powder after removing impure protein; the protein mass content of the chicken bone powder was determined to be 81.46%, which was increased by 2.63 times.

[0067] Defatting: the bone powder after removing impure protein was soaked in 5% N C aqueous solution at room temperature for 24h according to the amount of 5mL / g, the solution was changed twice, washed with water until neutral, and then dried (50°C air drying for 1h) to obtain the defatted bone powder; the crude fat mass content was determined to be 4.75%, and the defatting rate was 73.93%.

[0068] Decalcification: the defatted bone powder was soaked in 0.25 mol / L EDTA aqueous solution (pH 7.5) at room temperature for 24h according to the amount of 5mL / g, the solution was changed twice, washed with water until clear, and then dried (50°C air drying for 1h) to obtain 10.15g of pretreated bone powder; the calcium content and phosphorus mass content were determined to be 12.59% and 7.48%, the decalcification rate was 83.80%, and the dephosphorization rate was 83.29%.

[0069] 2. Ultrasonic-assisted three-stage enzymatic hydrolysis

[0070] (1) First-stage enzymatic hydrolysis

[0071] Optimization of ultrasonic power: After pretreatment, the bone powder was added to 0.5 mol / L acetic acid solution at a solid-liquid ratio of 5 mL / g. Ultrasonic washing machine was used to expose the enzymatic cleavage site through ultrasonic cavitation effect at different powers (0, 100, 320, 400, and 480 W) for 20 min. Then, pepsin (porcine origin) (Aldrin, Catalog No.: P110927, 3000 U / g) was added at a dosage of 150 U / g based on the mass of the pretreated bone powder. The first-stage enzymatic hydrolysis was carried out at 37°C and pH 1.7 with continuous stirring for 6 h. Pepsin specifically hydrolyzes aromatic amino acids, and the first-stage enzymatic hydrolysate was obtained. The degree of hydrolysis was detected, and the results are shown in Table Figure 1 a.

[0072] Optimization of ultrasonic time: The ultrasonic power was fixed at 320 W, and the ultrasonic time was changed to 0, 10, 15, 20, 25, and 30 min. The other operations were the same, and the degree of hydrolysis is shown in Table Figure 1 b. The degree of hydrolysis under 320 W ultrasonic for 20 min was increased by 10.28% compared to that without ultrasonic assistance, indicating that ultrasonic assistance significantly improved the efficiency of enzymatic hydrolysis.

[0073] Under the optimized conditions (ultrasonic power 320 W, time 20 min), the first-stage enzymatic hydrolysis was carried out with three repeats, and the degree of hydrolysis was 18.40 ± 1.72%.

[0074] (2) Second-stage enzymatic hydrolysis

[0075] Optimization of enzyme addition amount: The first-stage enzymatic hydrolysate under the optimal conditions of step (1) was adjusted to pH 8.5, and alkaline protease (source leaf, Catalog No.: S10154, 200 U / mg) was added at dosages of 3000, 4000, 5000, 6000, and 7000 U / g based on the mass of the pretreated bone powder. The second-stage enzymatic hydrolysis was carried out at 45°C for 5 h. Alkaline protease cut the hydrophobic peptide chain to release small molecular fragments, and the second-stage enzymatic hydrolysate was obtained. The degree of hydrolysis was detected, and the results are shown in Table Figure 2 a.

[0076] Optimization of enzymatic hydrolysis temperature: In the enzyme addition amount optimization step above, the enzyme addition amount was fixed at 5000 U / g, and the temperature was changed to 35, 40, 45, 50, and 55°C. The other operations were the same, and the results are shown in Table Figure 2 b.

[0077] Optimization of enzymatic hydrolysis pH: In the enzyme addition amount optimization step above, the enzyme addition amount was fixed at 5000 U / g, and the pH was changed to 8, 8.5, 9, 9.5, and 10. The other operations were the same, and the results are shown in TableFigure 2 c.

[0078] Enzymatic hydrolysis time optimization: The enzyme dosage in the above enzyme addition optimization step was fixed at 5000 U / g, and the stirring time was changed to 2, 3, 4, 5, and 6 hours. Other operations remained the same. The results are shown in […]. Figure 2 d.

[0079] Under optimized conditions (enzyme addition of 5000 U / g, pH 8.5, temperature 45℃, time 5h), a secondary enzymatic hydrolysis was performed with 3 replicates. The degree of hydrolysis was 28.38±1.31%, which was 54.24% higher than that of the primary enzymatic hydrolysate.

[0080] (3) Three-stage enzymatic hydrolysis

[0081] Enzyme addition optimization: In step (2), the pH of the secondary enzymatic hydrolysate after enzymatic hydrolysis was adjusted to 6.0, and then a compound protease (Yuan Ye, product number: S10155, 120U / mg) was added. The addition amounts were 3000, 4000, 5000, 6000, and 7000 U / g, respectively, based on the mass of the pretreated bone meal. The tertiary enzymatic hydrolysate was carried out by stirring at 50℃ for 2 hours. The compound protease broadly cleaved peptide bonds and enriched 0.5-1.5kDa active peptides to obtain the tertiary enzymatic hydrolysate. The degree of hydrolysis of the tertiary enzymatic hydrolysate was detected, and the results are shown in the figure. Figure 3 a.

[0082] Enzymatic hydrolysis temperature optimization: The enzyme dosage in the above enzyme addition optimization step was fixed at 5000 U / g, and the temperature was changed to 35, 40, 45, 50, and 55℃. All other operations remained the same. Results are shown below. Figure 3 b.

[0083] Enzymatic hydrolysis pH optimization: The enzyme dosage was fixed at 5000 U / g in the above enzyme addition optimization steps, and the pH was changed to 5.5, 6, 6.5, 7, and 7.5. All other operations remained the same. Results are shown below. Figure 3 c.

[0084] Enzymatic hydrolysis time optimization: The enzyme dosage in the above enzyme addition optimization step was fixed at 5000 U / g, and the stirring time was changed to 1, 2, 3, 4, and 5 hours. Other operations remained the same. The results are shown in [the original text]. Figure 3 d.

[0085] Under optimized conditions (enzyme addition of 5000 U / g, pH 6.0, temperature 50℃, time 2h), a three-stage enzymatic hydrolysis was performed with three replicates. The final degree of hydrolysis was 33.18±0.84%, which was 16.91% higher than that of the two-stage enzymatic hydrolysate.

[0086] 3. Purification and freeze-drying

[0087] The tertiary enzymatic hydrolysate prepared under the optimal conditions of step 2 was centrifuged, and the filtrate was subjected to 0.22 μm microfiltration and 500 Da dialysis bag dialysis in pure water for 48 h. The above-mentioned retentate was pre-frozen at -80°C for 12 h, and vacuum freeze-dried (-50°C, 10 Pa) to obtain CBCP freeze-dried powder. The above-mentioned operation was repeated three times, and the mass extraction rate was 5.32 ± 1.51%, the protein content was 84.85 ± 0.85%, and the collagen content was 58.02 ± 2.33%.

[0088] 4. Determination of chelating activity

[0089] The calcium chelating activity of the CBCP freeze-dried powder sample was detected, and the experimental results showed that the calcium chelating activity of the sample was 112.5 μg / mg, indicating that each milligram of collagen peptide could chelate 112.5 micrograms of calcium ions. The calcium content of the supernatant of the control group (without sample) was 168.75 μg, while the calcium content of the supernatant of the experimental group (with sample) was as high as 506.25 μg. This difference (+337.5 μg) proves that the collagen peptide effectively inhibited the formation of calcium precipitation, and more calcium ions remained in the supernatant in the form of free ions, reflecting the chelation effect, and having significant calcium chelating activity.

[0090] Comparative Example 1: Comparison of bone collagen extracted by different methods

[0091] The difference between the enzymatic hydrolysate prepared under the optimal conditions of step 2 of Example 1 and this embodiment is that acid hydrolysis and alkali hydrolysis are used to extract chicken bone collagen.

[0092] Control group: The pretreated bone powder prepared by the method of Example 1 was dissolved in pure water solution at a material-liquid ratio of 10 mL / g, and continuously stirred at room temperature for 48 h, and then filtered. The filtrate was the collagen of the control group.

[0093] Acid hydrolysis: The pretreated bone powder prepared by the method of Example 1 was mixed with 0.5 mol / L acetic acid aqueous solution at a material-liquid ratio of 10 mL / g, and continuously stirred at room temperature for 48 h, and then filtered. The obtained filtrate was the acid-soluble collagen.

[0094] Alkali hydrolysis: The pretreated bone powder prepared by the method of Example 1 was mixed with 0.12 mol / L NaOH aqueous solution at a material-liquid ratio of 10 mL / g, and continuously stirred at room temperature for 48 h, and then filtered. The filtrate was the alkali-soluble collagen.

[0095] The degrees of hydrolysis were detected, and the results are shown in Table 1. Figure 4 The degrees of hydrolysis of chicken bone collagen by acid hydrolysis and alkali hydrolysis were 5.05% and 11.19%, respectively. Compared with enzyme hydrolysis, the degrees of hydrolysis were increased by 3.72 times and 1.68 times, respectively, indicating that the use of enzyme hydrolysis significantly improved the enzyme hydrolysis efficiency.

[0096] Comparative Example 2: Effect of enzyme type on degree of hydrolysis

[0097] This example uses a first-stage enzymatic hydrolysis method, and four different enzymes are used, namely pepsin, alkalin protease, papain and composite protease.

[0098] The pretreated bone powder is added to purified water at a solid-liquid ratio of 5 mL / g, and then the following four enzymes are added (the amount of enzyme added is based on the pretreated bone powder), and the first-stage enzymatic hydrolysis is continuously stirred for 5 h under different conditions, and the degree of hydrolysis of the enzymatic hydrolysate is detected.

[0099] Pepsin: added at a concentration of 5000 U / g, pH 1.7, enzymatic hydrolysis at 37°C for 5 h;

[0100] Alkalin protease: added at a concentration of 5000 U / g, pH 8.0, enzymatic hydrolysis at 45°C for 5 h;

[0101] Papain (source leaf, product number S10011, 800 u / mg): added at a concentration of 5000 U / g, pH 7.0, enzymatic hydrolysis at 65°C for 5 h;

[0102] Composite protease (same as in Example 1): added at a concentration of 5000 U / g, pH 5.5, enzymatic hydrolysis at 50°C for 5 h.

[0103] As shown in Table 1, the degree of hydrolysis of composite protease is 14.96%, the degree of hydrolysis of alkalin protease is 10.94%, the degree of hydrolysis of pepsin is 9.81%, and the degree of hydrolysis of papain is 8.26%, indicating that the use of different enzymes has a significant difference in enzymatic hydrolysis efficiency. Figure 5 Comparative Example 3: Effect of enzyme type and addition order on degree of hydrolysis in second-stage enzymatic hydrolysis method

[0104] The difference between this example and Example 1 is that this example uses a second-stage enzymatic hydrolysis method and the addition order of the enzymes is different.

[0105] The pretreated bone powder is added to purified water at a solid-liquid ratio of 5 mL / g, and then the following four enzymes are added (the amount of enzyme added is based on the pretreated bone powder), and the first-stage enzymatic hydrolysis is continuously stirred for 5 h under different conditions, and the degree of hydrolysis of the enzymatic hydrolysate is detected.

[0106] Group A: first pepsin hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 1.7, temperature 37°C, then alkalin protease hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 8.0, temperature 45°C;

[0107] Group B: first alkalin protease hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 8.0, temperature 45°C, then pepsin hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 1.7, temperature 37°C;

[0108] Group C: first papain hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 7.0, temperature 65°C, then pepsin hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 1.7, temperature 37°C; Group D: first pepsin hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 1.7, temperature 37°C, then papain hydrolysis for 5 h, added at a concentration of 5000 U / g, pH 7.0, temperature 65°C.Group B: First, pepsin was used for hydrolysis for 5 h, with a concentration of 5000 U / g, pH 1.7, and temperature 37℃. Then, complex protease was used for hydrolysis for 5 h, with a concentration of 5000 U / g, pH 5.5, and temperature 50℃.

[0109] Group C: First, alkaline protease hydrolysis was performed for 5 h, with a concentration of 5000 U / g, pH 8.0, and temperature 45℃. Then, pepsin hydrolysis was performed for 5 h, with a concentration of 5000 U / g, pH 1.7, and temperature 37℃.

[0110] Group D: First, alkaline protease hydrolysis was performed for 5 h, with a concentration of 5000 U / g, pH 8.0, and temperature 45℃. Then, complex protease hydrolysis was performed for 5 h, with a concentration of 5000 U / g, pH 5.5, and temperature 50℃.

[0111] Group E: First, hydrolyze with compound protease for 5 h, add 5000 U / g, pH 5.5, temperature 50℃, then hydrolyze with pepsin for 5 h, add 5000 U / g, pH 1.7, temperature 37℃;

[0112] Group F: First, hydrolyze with compound protease for 5 h, add 5000 U / g, pH 5.5, temperature 50℃, then hydrolyze with alkaline protease for 5 h, add 5000 U / g, pH 8.0, temperature 45℃.

[0113] like Figure 6 As shown in a, AP is an alkaline protease, and CP is a complex protease (same as in Example 1). The degree of hydrolysis in the AF group was 22.64%, 18.95%, 19.23%, 17.98%, 20.70%, and 17.32%, respectively, indicating that the type of enzyme and the order of enzyme addition have significant differences in hydrolysis efficiency during secondary enzymatic hydrolysis.

[0114] Comparative Example 4: Effect of enzyme type and addition order on the degree of hydrolysis in a three-stage enzymatic hydrolysis method

[0115] In Example 1, after pretreatment, bone meal was added to purified water at a ratio of 5 mL / g. Then, enzymes were added according to the following groups (the amount added was based on the pretreated bone meal). Three-stage enzymatic hydrolysis was carried out under different conditions with continuous stirring, and the degree of hydrolysis of the enzymatic hydrolysate was detected.

[0116] Group A: First, pepsin was used for hydrolysis for 5 h, with a concentration of 5000 U / g, pH 1.7, and temperature 37℃. Then, alkaline protease was used for hydrolysis for 5 h, with a concentration of 5000 U / g, pH 8.0, and temperature 45℃. Finally, compound protease (same as in Example 1) was used for hydrolysis for 5 h, with a concentration of 5000 U / g, pH 5.5, and temperature 50℃.

[0117] Group B: First, pepsin hydrolyzes for 5 h, with a concentration of 5000 U / g, pH 1.7, and temperature 37℃; then, compound protease hydrolyzes for 5 h, with a concentration of 5000 U / g, pH 5.5, and temperature 50℃; finally, alkaline protease hydrolyzes for 5 h, with a concentration of 5000 U / g, pH 8.0, and temperature 45℃.

[0118] Group C: First, alkaline protease hydrolysis was performed for 5 hours at a concentration of 5000 U / g, pH 8.0, and temperature 45℃. Then, pepsin hydrolysis was performed for 5 hours at a concentration of 5000 U / g, pH 1.7, and temperature 37℃. Finally, complex protease hydrolysis was performed for 5 hours at a concentration of 5000 U / g, pH 5.5, and temperature 50℃.

[0119] Group d: First, alkaline protease hydrolysis for 5 h, with a concentration of 5000 U / g, pH 8.0, and temperature 45℃; then, complex protease hydrolysis for 5 h, with a concentration of 5000 U / g, pH 5.5, and temperature 50℃; finally, pepsin hydrolysis for 5 h, with a concentration of 5000 U / g, pH 1.7, and temperature 37℃.

[0120] Group e: First, hydrolyze with compound protease for 5 h, add 5000 U / g, pH 5.5, temperature 50℃, then hydrolyze with pepsin for 5 h, add 5000 U / g, pH 1.7, temperature 37℃, and finally hydrolyze with alkaline protease for 5 h, add 5000 U / g, pH 8.0, temperature 45℃.

[0121] Group f: First, hydrolyze with compound protease for 5 h, add 5000 U / g, pH 5.5, temperature 50℃, then hydrolyze with alkaline protease for 5 h, add 5000 U / g, pH 8.0, temperature 45℃, and finally hydrolyze with pepsin for 5 h, add 5000 U / g, pH 1.7, temperature 37℃.

[0122] like Figure 6 As shown in b, P represents pepsin. The degrees of hydrolysis in groups af were 29.51%, 26.21%, 28.28%, 27.68%, 27.92%, and 24.65%, respectively, indicating that the type of enzyme and the order of enzyme addition significantly affected the hydrolysis efficiency during the three-stage enzymatic hydrolysis.

[0123] Example 2: Preparation of CBCP lyophilized powder and molecular weight distribution of peptides

[0124] (1) Preparation of CBCP lyophilized powder

[0125] 100 g of the pretreated bone powder prepared according to the method of Example 1 was added to 0.5 mol / L acetic acid aqueous solution at a ratio of 5 mL / g, and an ultrasonic cleaner was used to perform ultrasonic treatment at 320 W for 20 min. Then, pepsin was added (the amount of pepsin added was 150 U / g based on the mass of the pretreated bone powder), and first-stage enzymatic hydrolysis was performed under the conditions of 37 °C and pH 1.7 for 6 h with continuous stirring. The pH was adjusted to 8.5, and then alkaline protease was added (the amount of alkaline protease added was 5000 U / g based on the mass of the pretreated bone powder), and second-stage enzymatic hydrolysis was performed under the conditions of 45 °C for 5 h with stirring. The pH was adjusted to 6.0, and then complex protease was added (the amount of complex protease added was 5000 U / g based on the mass of the pretreated bone powder), and third-stage enzymatic hydrolysis was performed under the conditions of 50 °C for 2 h with stirring. The enzymatic hydrolysate was filtered, and the filtrate was subjected to 0.22 μm microfiltration and dialysis in a 500 Da dialysis bag for 48 h. The retained liquid was pre-frozen at -80 °C for 12 h, and then vacuum freeze-dried (-50 °C, 10 Pa) to obtain 5.32 g of CBCP freeze-dried powder.

[0126] (2) Molecular weight distribution of the peptide segments of CBCP

[0127] A Thermo UltiMate 3000 RSLCnano nanoliter liquid chromatograph coupled with a Q Exactive HF mass spectrometer was used. The peptide segment sample was injected through an automatic injector, combined with a C18 trapping column (75 µm 2 cm, 3 µm particle size, 100 Å pore size, Thermo), and then separated in an analysis column (75 µm 25 cm, 1.9 µm particle size, 100 Å pore size). An analysis gradient was established using mobile phase A (0.1% formic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formic acid / 3% DMSO / 97% acetonitrile (ACN)), with a gradient program of 7% B phase initially, linearly increased to 12% B over 5 min, 20 min to 15% B, 43 min to 25% B, 57 min to 47% B, 57.5 min to 90% B and maintained for 7.5 min. The analysis flow rate was set to 300 nL / min. Mass spectrometry was performed in DDA mode for data acquisition. The raw mass spectrometry data were analyzed using MaxQuant (2.2.0.0) software.

[0128] A total of 40 peptide segments were identified in the CBCP sample, of which 0.5-1 kDa peptide segments accounted for 15%, 1-1.5 kDa peptide segments accounted for 62.5%, and 1.5-2 kDa peptide segments accounted for 22.5%.

[0129] Example 3: Preparation of high calcium-binding chicken bone collagen peptide calcium chelate (CBCP-Ca)

[0130] 1 g of CBCP lyophilized powder prepared according to the method in Example 2 was dissolved in purified water to a concentration of 5 g / L to obtain a collagen peptide solution. Then, calcium citrate was added to the collagen peptide solution to make a peptide-to-calcium mass ratio of 1.16:1. The pH of the solution was adjusted to 5.5 with 0.1 M NaOH aqueous solution, and the mixture was stirred at 36°C for 32 min. After the chelation reaction was completed, 6 volumes of anhydrous ethanol were added to remove free calcium and precipitate the peptide-calcium chelate. The mixture was centrifuged at 4000 rpm for 20 min at 4°C, the precipitate was collected, and freeze-dried under vacuum at -50°C for 48 h to obtain 0.574 g of the peptide-calcium chelate product, CBCP-Ca, with a calcium binding capacity of 116.75 µg / mg.

[0131] Comparative Example 5: Effect of calcium source on peptide calcium chelates

[0132] The peptide-calcium chelate was prepared using the method of Example 3. The difference from Example 3 is that the calcium source used in this example is calcium lactate, calcium gluconate and calcium chloride, the peptide-calcium mass ratio is 1:1, the calcium chelation pH is 7, the calcium chelation temperature is 50℃ and the calcium chelation time is 40 min.

[0133] like Figure 7 As shown in Figure a, the calcium binding capacities of calcium chloride, calcium lactate, calcium citrate, and calcium gluconate were 39.68 µg / mg, 29.93 µg / mg, 123.35 µg / mg, and 52.19 µg / mg, respectively. The calcium citrate group showed a 3.11-fold increase in calcium binding capacity compared to CaCl2 as a calcium source, indicating that different calcium sources have significant differences in the calcium binding capacity of peptides.

[0134] Comparative Example 6: Effect of peptide-calcium feed ratio on peptide-calcium chelates

[0135] The peptide-calcium chelate was prepared using the method of Example 3. The difference from Example 3 is that the peptide-calcium mass ratio was set to 1:3, 1:2, 1:1, 2:1 and 3:1 in this example.

[0136] like Figure 7 As shown in b, the calcium binding forces of peptide-calcium mass ratios of 1:3, 1:2, 1:1, 2:1, and 3:1 were 106.63 µg / mg, 102.41 µg / mg, 123.85 µg / mg, 96.01 µg / mg, and 76.15 µg / mg, respectively, indicating that a peptide-calcium mass ratio of 1:1 significantly improves the peptide-calcium binding force.

[0137] Comparative Example 7: Effect of pH on peptide calcium chelates

[0138] The peptide calcium chelate was prepared by the method of Example 3, except that the chelation conditions were set as follows: peptide calcium mass ratio 1:1, calcium chelation temperature 50℃, calcium chelation time 40 min, and pH 5.0, 5.5, 6.0, 6.5 and 7.0, respectively. The calcium binding capacity was measured to be 125.14 µg / mg, 126.63 µg / mg, 123.57 µg / mg, 120.46 µg / mg and 121.9 µg / mg, respectively. This indicates that the calcium binding capacity is significantly improved at a chelation pH of 5.5.

[0139] Comparative Example 8: Effect of temperature on peptide calcium chelate

[0140] The peptide calcium chelate was prepared by the method of Example 3, except that the chelation conditions were set as follows: peptide calcium mass ratio 1:1, calcium chelation pH 5.5, calcium chelation time 40 min, and temperature 25, 30, 40, 50 and 60℃, respectively. The calcium binding capacity was measured to be 125.06 µg / mg, 129.58 µg / mg, 132.97 µg / mg, 130.63 µg / mg and 121.29 µg / mg, respectively. This indicates that the calcium binding capacity is significantly improved at a chelation temperature of 40℃.

[0141] Comparative Example 9: Effect of time on peptide calcium chelate

[0142] The peptide calcium chelate was prepared by the method of Example 3, except that the chelation conditions were set as follows: peptide calcium mass ratio 1:1, calcium chelation pH 5.5, calcium chelation temperature 40℃, and time 20, 30, 40, 50 and 60 min, respectively. The calcium binding capacity was measured to be 129.06 µg / mg, 132.32 µg / mg, 126.6 µg / mg, 126.19 µg / mg and 123.97 µg / mg, respectively. This indicates that the calcium binding capacity is significantly improved at a chelation time of 30 min.

[0143] Example 4: Stability test of CBCP-Ca

[0144] 1. Preparation of CBCP-Ca

[0145] 0.5 g of the CBCP lyophilized powder prepared by the method in Example 2 was dissolved in purified water to a concentration of 5 g / L to obtain a collagen peptide solution. Then, calcium citrate was added to the collagen peptide solution to make the peptide-calcium mass ratio 1:1. The pH of the solution was adjusted to 5.5 with 0.1 M NaOH aqueous solution, and the mixture was stirred at 40 °C for 30 min. After the chelation reaction was completed, 6 volumes of anhydrous ethanol were added to remove free calcium and precipitate the peptide-calcium chelate. The mixture was centrifuged at 4000 rpm for 20 min at 4 °C, the precipitate was collected, and freeze-dried under vacuum at -50 °C for 48 h to obtain 0.291 g of peptide-calcium chelate, denoted as CBCP-Ca.

[0146] 2. Thermal stability analysis

[0147] Dissolve the CBCP-Ca prepared in step 1 in pure water at a concentration of 5 mg / mL, and react in a water bath at 40, 50, 60, 70 and 80°C for 40 min. Take 1 mL of sample every 10 min, cool it to room temperature with water, precipitate it in 6 times anhydrous ethanol, centrifuge at 5000 rpm for 3 min, take the supernatant, and measure the calcium retention rate.

[0148] like Figure 8 As shown in Figure a, the calcium retention rate of the chelate decreases with increasing temperature. The degradation is most severe at 80℃: the retention rate drops sharply from 98% to 75% within 50 minutes. The best stability is observed at 40℃: the retention rate is >95% for the first 10 minutes and remains at 83% after 50 minutes, demonstrating that a low-temperature environment effectively delays the dissociation of calcium ions.

[0149] 3. Acid-base stability analysis

[0150] Dissolve the CBCP-Ca prepared in step 1 in purified water to a concentration of 5 mg / mL, adjust the pH to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, incubate at 37°C for 2 hours, precipitate in 6 times anhydrous ethanol, centrifuge at 5000 rpm for 3 minutes, and take the supernatant to measure the calcium retention rate.

[0151] like Figure 8 As shown in b, a strongly acidic environment disrupts the chelate structure, with calcium retention below 60% at pH 2-3. This indicates that the acidic environment of the stomach (pH≈2.5) leads to the dissociation of the peptide-calcium chelate, possibly related to the competition for calcium binding sites by collagen peptide protonation. The retention rate remains at 65%-75% in the pH 6-9 range, peaking particularly at pH 7-8, indicating that the chelate is structurally stable under neutral and weakly alkaline conditions in the intestine, facilitating intestinal calcium absorption.

[0152] 4. Stability analysis in in vitro simulation experiments

[0153] Gastric digestion group: CBCP-Ca prepared in step 1 was dissolved in purified water at a concentration of 5 mg / mL. The pH was adjusted to 2. The mixture was prepared according to a mass ratio of pepsin (3000 U / mg) to substrate CBCP-Ca of 1:50. The mixture was then placed in a 37°C water bath. 2 mL of the sample solution was taken at 0, 15, 30, 45, and 60 min, respectively, and precipitated in 6 times anhydrous ethanol. After centrifugation at 5000 rpm for 3 min, the supernatant was collected, and the calcium retention rate was measured.

[0154] Intestinal digestion: CBCP-Ca prepared in step 1 was dissolved in purified water at a concentration of 5 mg / mL. The pH was adjusted to 7.8. The mixture was prepared according to a mass ratio of trypsin (250 U / mg) to substrate CBCP-Ca of 1:25. The mixture was then incubated in a 37°C water bath. 2 mL of the sample solution was taken at 0, 15, 30, 45, and 60 min, and precipitated in 6 times anhydrous ethanol. After centrifugation at 5000 rpm for 3 min, the supernatant was collected to determine the calcium retention rate.

[0155] Gastric + Intestinal digestion: In the gastric simulation reaction, 2 mL of sample solution was taken at 0, 15, 30, 45, and 60 min, respectively. 1 M sodium bicarbonate (approximately 15–20% of the gastric digestive fluid volume) was added to terminate the reaction. The pH was adjusted to 7.8. Trypsin was added at a mass ratio of 1:25 between trypsin and substrate CBCP-Ca. The mixture was incubated in a 37°C water bath. 2 mL of sample solution was taken at 0, 15, 30, 45, and 60 min, respectively. The sample solution was precipitated in 6 times anhydrous ethanol. After centrifugation at 5000 rpm for 3 min, the supernatant was collected to determine the calcium retention rate.

[0156] like Figure 8 As shown in c, the gastric simulated group was the main cause of the decreased chelating force, which is related to Figure 8 The lower stability of the chelate in acidic conditions is related to the fact that, under the combined action of acidic conditions and pepsin, the chelate showed a significant decrease within 15 minutes, and then tended to stabilize.

[0157] Example 5: Physicochemical properties of CBCP and CBCP-Ca

[0158] 1. Determination of amino acid composition:

[0159] Weigh 100 mg each of the CBCP lyophilized powder prepared by the method in Example 2 and the CBCP-Ca sample prepared by the method in Example 4. Carefully add 10 mL of a solution of concentrated hydrochloric acid and purified water in a 1:1 volume ratio (approximately 6 mol / L). After blowing with nitrogen for 15 min, seal the tube. Place the sealed sample in an oven at 110 °C for 24 h for hydrolysis, then remove and cool before opening the tube. Dilute the sample to 25 mL with purified water. Accurately pipette 1 mL of the diluted sample and place it in a nitrogen evaporator at 55 °C until dry. Accurately add 1.0 mL of pH 2.2, 0.2 mol / L sodium citrate buffer solution to a vortex mixer and mix well. Filter through a 0.22 µm aqueous filter and run on an amino acid analyzer (Hitachi LA8080).

[0160] Table 1 shows that there are differences in the proportion of certain characteristic amino acids among the total amino acids in CBCP and CBCP-Ca. The content of glycine decreased by 21.6%, indicating a reduction in collagen-specific amino acids, suggesting that its side chain (-H) participates in calcium coordination. Proline decreased by 48.0%, indicating that the rigid ring structure was disrupted, facilitating calcium ion insertion into the collagen peptide space. Aspartic acid increased by 30.3%, indicating that the key chelating site -COOH provides coordinating oxygen atoms. Glutamic acid increased by 5.7%, indicating that the carboxyl group forms a stable coordination bond with calcium ions. Alanine appeared from none, indicating enhanced hydrophobicity and improved lipid solubility of the chelate.

[0161] Table 1 shows the proportion of amino acids in CBCP and CBCP-Ca.

[0162]

[0163] 2. Ultraviolet-Visible Full Spectrum Scan

[0164] At room temperature, the CBCP lyophilized powder prepared by the method in Example 2 and the CBCP-Ca sample prepared by the method in Example 4 were dissolved in purified water to prepare solutions of 2 mg / mL. Undissolved substances were removed by centrifugation at 10000 r / min. The supernatant was then scanned on a UV spectrophotometer (Thermo Fisher Scientific, GENESYS 150) with a scanning range of 190-400 nm and a scanning rate of 1 nm / s. Baseline calibration was performed using purified water before measurement.

[0165] like Figure 9 As shown in Figure a, there are differences in the UV-Vis full-spectrum scans of CBCP and CBCP-Ca. Chelation causes spectral changes. Before chelation, CBCP has a strong absorption peak near 237 nm, while the peptide-calcium chelate is slightly shifted to the short-wavelength direction at 234 nm, and the peak absorbance is significantly reduced, indicating that the chelation process changes the electronic structure of collagen peptides.

[0166] 3. Fourier transform infrared spectroscopy

[0167] Fourier transform infrared spectrometer (Thermo Fisher Nicolet iS20) was used, with a resolution of 4 cm⁻¹. -1 The number of scans was 32, and the test wavenumber range was 400-4000 cm⁻¹. -1 The CBCP prepared by the method in Example 2 and the CBCP-Ca prepared by the method in Example 4 were tested under the specified conditions.

[0168] like Figure 9 As shown in b, the Fourier transform infrared spectra of CBCP and CBCP-Ca differ. The overall enhancement of characteristic peaks after chelation (especially amide I / II) supports the formation of coordination bonds between calcium ions and the carboxyl and amino groups of collagen peptides, leading to changes in peptide bond vibration modes. The difference in the amide III region further confirms that the peptide chain conformation undergoes adaptive adjustment, which may be related to the stability of the chelate.

[0169] 4. Differential scanning calorimetry

[0170] Differential scanning calorimetry (TA Q2000, USA) was used to test the CBCP prepared by the method of Example 2 and the CBCP-Ca prepared by the method of Example 4 under the condition of equilibration heating at a rate of 10℃ / min from 40℃ to 200℃, with an empty aluminum crucible as a blank reference.

[0171] like Figure 9 As shown in figures c and d, the differential scanning calorimetry (DSC) patterns of CBCP and CBCP-Ca differ, with the endothermic peak shifting towards higher temperatures after chelation: this indicates that the introduction of calcium enhances the thermal stability of collagen peptides. The increase in peak area (endothermic enthalpy (ΔH)) suggests that more energy is required to break down the structure, further supporting the improved thermal stability.

[0172] 5. Scanning electron microscope image

[0173] Using a scanning electron microscope (ZEISS Sigma 360, Germany), the samples were magnified 200 times under an accelerating voltage of 3.00 kV. Spot scanning was performed on CBCP prepared by the method in Example 2 and CBCP-Ca prepared by the method in Example 4, with a total of 8 elements selected: N, O, Na, P, S, Cl, Ca, and C.

[0174] like Figure 10 As shown, there are differences between the scanning electron microscope images of CBCP and CBCP-Ca. Figure 10 In samples a and b, calcium was successfully introduced and uniformly bound into CBCP-Ca, and a characteristic peak of calcium (Ca) was detected, which was lower in CBCP. This indicates that calcium ions have successfully bound to collagen peptides to form chelates. Figure 10The morphology of the collagen peptide and the chelate changed obviously. The collagen peptide showed porous, fibrous and sponge-like structure, while the chelate showed more compact structure and granular aggregates. It indicated that the introduction of calcium changed the aggregation state of the collagen peptide, and possibly formed cross-linked or complex structure.

Claims

1. A high-calcium chelated active collagen peptide prepared from chicken bones, characterized in that, The collagen peptides were prepared as follows: (1) Raw material pretreatment: Chicken claw bone powder was pretreated by removing impurities, defatting and demineralizing to obtain pretreated bone powder; (2) Ultrasonic-assisted primary enzymatic hydrolysis: The pretreated bone powder was mixed with acetic acid aqueous solution and ultrasonically treated for 10-30 min at 100-480 W to expose the enzyme cleavage sites through ultrasonic cavitation effect; then pepsin was added and the mixture was continuously stirred for 2-6 h at 35-39℃ and pH 1.5-2.0 to obtain primary enzymatic hydrolysate; (3) Secondary enzymatic hydrolysis: The pH of the primary enzymatic hydrolysate was adjusted to 8-10, and then alkaline protease was added and stirred for 2-6 h at 35-55℃ to obtain secondary enzymatic hydrolysate; (4) Tertiary enzymatic hydrolysis: The pH of the secondary enzymatic hydrolysate was adjusted to 5.5-7.5, and then compound protease was added and stirred for 1-5 h at 35-55℃ to obtain tertiary enzymatic hydrolysate; (5) Purification: The tertiary enzymatic hydrolysate was filtered, and the filtrate was subjected to 0.22 μm microfiltration, dialysis with pure water using a 500 Da dialysis bag for 48 hours, and freeze-drying of the retentate to obtain the collagen peptides.

2. The collagen peptide as described in claim 1, characterized in that, Step (1) Raw material pretreatment is carried out as follows: ① Remove the skin, meat and tendons from the chicken feet, wash them, dry them at 80℃ for 10 hours, and pulverize them through a 40-mesh sieve to obtain bone meal; ② Remove impurities and protein: Soak the bone meal in 0.1 mol / L NaOH aqueous solution at room temperature for 6 hours at a material-to-liquid ratio of 5 mL / g, changing the solution every 2 hours, washing with water until neutral, draining and drying to obtain bone meal after removing impurities and protein; ③ Defatting: Defatt the bone meal after removing impurities and protein at a material-to-liquid ratio of 5 mL / g with 5% NaOH aqueous solution. C ④ Demineralization: The defatted bone meal was soaked in 0.25 mol / L EDTA aqueous solution at room temperature for 24 hours, with the solution changed twice in between. After washing with water until the solution was neutral, the bone meal was drained and dried to obtain the defatted bone meal.

3. The collagen peptide as described in claim 1, characterized in that, Step (2) The concentration of acetic acid aqueous solution is 0.1-1M, and the volume is 1-10mL / g based on the mass of the pretreated bone meal; the amount of pepsin added is 130-170U / g based on the mass of the pretreated bone meal.

4. The collagen peptide as described in claim 1, characterized in that, Step (3) The amount of alkaline protease added is 3000-7000 U / g based on the weight of the pretreated bone meal.

5. The collagen peptide as described in claim 1, characterized in that, Step (4) The amount of compound protease added is 3000-7000 U / g based on the weight of the pretreated bone meal.

6. A peptide-calcium chelate prepared from the collagen peptide of claim 1, characterized in that, The peptide-calcium chelate is obtained by dissolving the collagen peptide in purified water, adding a calcium source, reacting at pH 5-7 and 25-60 ℃ for 20-60 min, precipitating with anhydrous ethanol, centrifuging, and freeze-drying the precipitate.

7. The peptide-calcium chelate according to claim 6, characterized in that, The calcium source includes calcium citrate, calcium chloride, calcium lactate, or calcium gluconate; the mass ratio of the collagen peptide to the calcium source is 1:1 to 3:

1.

8. The use of the peptide-calcium chelate of claim 6 in the preparation of functional foods.

9. The application as described in claim 8, characterized in that, The functional foods include calcium supplements, high-calcium foods, and foods for special medical purposes.

10. A functional food prepared from the peptide-calcium chelate of claim 6.

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