Co-production preparation method of fish scale gelatin, hydroxyapatite and calcium peptide chelate

Abstract

The invention discloses a co-production preparation method of fish scale gelatin, hydroxyapatite and calcium peptide chelate, which comprises the following steps: sequentially soaking fish scales of tilapia in a NaCl solution and a NaOH solution for pretreatment, then drying, putting the dried fish scales into water, extracting the fish scale gelatin by using a hot water method, and carrying out freeze-drying to obtain the fish scale gelatin. Fish scale residues are fished out after extraction is completed, and fish scale glue is obtained; fish scale residues obtained after fish scale glue is extracted serve as raw materials, and the fish scale residues are sequentially dried and carbonized to obtain a fish scale hydroxyapatite product. Taking the obtained fish scale gelatin as a raw material, and sequentially performing enzymolysis reaction, chelation reaction, precipitation and freeze-drying on the fish scale gelatin to obtain the calcium peptide chelate. The fish scale gelatin prepared by the invention is verified to accord with the specific characteristics of gelatin and has relatively high gel strength; fTIR (Fourier transform infrared spectroscopy) and XRD (X-Ray Diffraction) prove that the prepared hydroxyapatite meets the structural characteristics of the hydroxyapatite and has relatively high purity; structural activity analysis shows that the prepared peptide calcium chelate is successfully prepared.

Description

Technical Field

[0001] The invention relates to a high-efficiency co-production preparation method of tilapia-derived fish scale glue, hydroxyapatite and calcium peptide chelate, and belongs to the technical field of high-value utilization of aquatic product processing waste. Background Art

[0002] During the fish processing process, many by-products are produced, such as fish skin, fish bones, fish scales, etc., which are usually directly discarded or processed into low-economic-value products, causing environmental pollution and economic losses. Therefore, the high-value utilization of fish by-products has become an urgent problem to be solved. my country has abundant tilapia farming production, and tilapia scales are a wide source of by-products, with low prices. In addition, compared with raw materials such as fish bones, the pre-treatment steps of fish scales are simple and time-consuming. At present, there are relatively few studies on the comprehensive extraction of fish scale glue and hydroxyapatite from fish scales, and most of the research results on biologically derived hydroxyapatite come from foreign scholars, while domestic research is relatively rare. In addition, fish scale glue has a wide range of applications due to its unique functional properties. Studies have shown that collagen peptides prepared from it have calcium chelating activity. In this paper, fish scale glue is hydrolyzed by protease to obtain fish scale collagen peptides, and fish scale glue is further developed and utilized.

[0003] Currently, targeted preparation of calcium-chelating peptides is mostly achieved through hydroxyapatite column chromatography. However, this type of hydroxyapatite is expensive, with an average market price ranging from 50 to 200 yuan per gram. Furthermore, the column chromatography volume is small, resulting in low peptide yields and making it unsuitable for large-scale preparation. Hydroxyapatite prepared from fish scale residue is low-cost and simple to operate, providing a promising approach for the preparation of bio-hydroxyapatite.

[0004] Gelatin is a denatured protein obtained by thermal hydrolysis of collagen. It possesses emulsifying, gelling, foaming, and film-forming properties. Global demand for gelatin is increasing due to its growing application across various industries. Currently, gelatin is primarily obtained from mammalian byproducts, with common types including pigskin gelatin, cowhide gelatin, and bovine bone gelatin. However, due to the risk of zoonotic diseases and religious beliefs, mammalian gelatin has certain limitations in its practical application. Compared to mammalian gelatin, fish gelatin offers advantages such as abundant sources, no zoonotic risks, and no special dietary requirements. Hydroxyapatite, with a composition similar to natural bone and teeth, is a key component in bone scaffold development. Bio-hydroxyapatite extracted from fish scales offers a simpler process and lower cost than chemically synthesized hydroxyapatite. Research has shown that hydroxyapatite can be used to isolate and purify active peptides and increase their calcium-binding capacity, demonstrating that fish scale hydroxyapatite possesses affinity filler properties. Calcium is a vital element in the human body, and calcium deficiency can lead to a variety of diseases. Therefore, it is important to ensure adequate calcium intake in daily life. Traditional calcium supplements have low bioavailability and are ineffective. In recent years, studies have found that many bioactive peptides derived from fish scales have a certain calcium-binding capacity, which can be used as raw materials to develop calcium supplements and enhance their bioavailability.

[0005] Currently, the methods for preparing fish scale glue, hydroxyapatite and calcium peptide chelate are independent and lack the combined use of raw materials. The characteristics of the prepared products are unclear and the purity is not ideal. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the deficiencies in the prior art and to provide a tilapia-derived fish scale glue, hydroxyapatite and calcium peptide chelate and an efficient co-production method thereof. The fish scale glue is prepared using tilapia scales as raw materials, and then the remaining fish scale residue is used as raw material to prepare hydroxyapatite, and then the prepared fish scale glue is used as raw material to prepare calcium peptide chelate. The raw materials are fully utilized, and waste from aquatic product processing is utilized in a high-value manner.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a highly efficient co-production method for tilapia fish scale glue, hydroxyapatite, and calcium peptide chelate. The method first uses a hot water method to obtain fish scale glue, then uses the fish scale residue remaining after extracting the fish scale glue to produce hydroxyapatite by high-temperature oxidation, then enzymatically hydrolyzes the fish scale glue to produce fish scale calcium chelate peptide, utilizes the affinity filler properties of hydroxyapatite to separate and purify the fish scale calcium chelate peptide, prepares affinity peptide, and finally prepares peptide calcium chelate through chelation. The method mainly comprises the following steps: (1) Tilapia scale pretreatment: Tilapia scales were sequentially immersed in NaCl solution and NaOH solution, with the NaCl solution being used for deproteinization and the NaOH solution being used for degreasing, decolorization, and deodorization, and finally the scales were cleaned with distilled water until neutrality and dried for later use; (2) Extraction of fish scale glue: The dried fish scales in step (1) are placed in water, and fish scale glue is extracted using a hot water method. After the extraction is completed, the fish scale residue is removed to obtain fish scale glue, a portion of which is used as a raw material for preparing calcium peptide chelate, and a portion is collected as a fish scale glue product; (3) Preparation of fish scale hydroxyapatite: The fish scale residue in step (2) is used as a raw material, and the fish scale residue is dried and oxidized at high temperature to obtain a fish scale hydroxyapatite product; (4) Preparation of calcium peptide chelate: The fish scale glue in step (2) is used as a raw material, and the fish scale glue is subjected to an enzymatic hydrolysis reaction, a chelation reaction, and precipitation in sequence to obtain a calcium peptide chelate.

[0008] In the above technical solution, in step (1), the concentration of the NaCl solution is 1% (w / v), the soaking time is 18-24 hours, the pH is 7, the solution is stirred during soaking at a stirring rate of 200 r / min, the material-liquid ratio is 1:20 (w / v), and the NaCl solution is replaced every 6 hours.

[0009] In the above technical solution, in step (1), the concentration of the NaOH solution is 0.1 M, the soaking time is 24-36 h, the pH is 12-14, the solution is stirred during soaking at a stirring rate of 200 r / min, the material-liquid ratio is 1:20 (w / v), and the NaOH solution is replaced every 6 h.

[0010] In the above technical solution, in step (2), when using the hot water method to extract fish scale glue, the gelatin extraction rate and gel strength are used as indicators to optimize the influence of three factors: solid-liquid ratio, extraction temperature, and extraction time. The optimal extraction process is determined as follows: the solid-liquid ratio range is 1:9 - 1:13 (w / v), the extraction temperature range is 50 - 90 ° C, and the extraction time range is 1 - 5 h.

[0011] In the above technical solution, in step (3), the method for preparing fish scale hydroxyapatite (FSHA) specifically includes the following steps: The fish scale residue remaining after the fish scale glue is extracted in step (2) is washed several times and then placed in an oven for drying. After drying, it is transferred to a crucible for high-temperature carbonization. The fish scale residue is carbonized until smokeless, and then subjected to high-temperature oxidation. After the high-temperature oxidation, high-purity fish scale hydroxyapatite (FSHA) is obtained.

[0012] In the above technical solution, in step (3), the drying is carried out at a temperature of 30-40°C and for a time of 18-24 h; the carbonization is carried out by placing the crucible on a heating plate at a heating temperature of 100°C, and carbonization is carried out until the fish scale residue is smokeless; the preparation conditions of hydroxyapatite are optimized by Fourier transform infrared spectroscopy and X-ray diffraction, and the high-temperature oxidation is carried out in a muffle furnace, the high-temperature oxidation time is 1-5 h, preferably 2 h, and the high-temperature oxidation temperature is 600-1000°C, preferably 800°C.

[0013] In the above technical solution, in step (4), the method for preparing the calcium peptide chelate specifically comprises the following steps: (A) Preparation of fish scale calcium chelating peptide (FSP) The fish scale glue obtained in step (2) is added with water to prepare a 3% protein solution, and protease is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated, the enzymatic hydrolysis solution is centrifuged and the supernatant is collected. The supernatant is concentrated and dried to obtain fish scale calcium chelating peptide (FSP); (B) Preparation of calcium peptide chelate: The fish scale calcium chelate peptide (FSP) obtained in step (A) is thoroughly mixed with water to prepare a fish scale calcium chelate peptide solution, and calcium chloride dihydrate is added and mixed to obtain a solution system for chelation reaction. The chelation reaction is carried out in a water bath. After the reaction is completed, anhydrous ethanol is added, and the mixture is allowed to stand, centrifuged, precipitated, and freeze-dried to obtain a fish scale peptide calcium chelate.

[0014] In the above technical solution, in step (A), the protease is any one of pepsin, trypsin, alkaline protease, composite protease, flavor protease, papain, and animal protease, and the protease is preferably alkaline protease; the protease is added in an amount of 3% of the mass of the solution system; the enzymatic hydrolysis refers to hydrolysis at a pH of 9 and a temperature of 55°C for 4 hours; the enzyme inactivation refers to boiling water bath for 10 minutes immediately after the enzymatic hydrolysis is completed; and the centrifugation refers to centrifugation at 8000 r / min for 10 minutes.

[0015] In the above technical solution, in step (A), after obtaining the fish scale calcium chelating peptide, the fish scale calcium chelating peptide is separated and purified using the homemade fish scale hydroxyapatite in step (3) to obtain affinity peptide (FHAC); the separation and purification refers to using the homemade hydroxyapatite in step (3) to statically adsorb the fish scale calcium chelating peptide by static affinity chromatography; the temperature during static adsorption is 30°C and the adsorption time is 60 min; the fish scale calcium chelating peptide is configured into a peptide solution and then adsorbed, the peptide concentration of the peptide solution is 40 mg / mL, and the material-liquid ratio (m:V) of hydroxyapatite (FSHA) and peptide solution (FSP) is 2.5:1; after the static adsorption is completed, the low calcium binding amount component is eluted three times using 5 mM and 60 mM phosphate solutions respectively (speed of 6000-8000 r / min), and then desorbed using 120 mM phosphate buffer solution for 30 min, and finally centrifuged (speed of 6000-8000 The eluate was collected and concentrated and lyophilized to obtain the affinity peptide (FHAC).

[0016] In the above technical solution, in step (B), the fish scale calcium chelate peptide solution is a solution prepared by thoroughly mixing the fish scale calcium chelate peptide obtained by concentration and drying with water; the solution system for the chelation reaction is a mixed solution system of the fish scale calcium chelate peptide solution and calcium chloride dihydrate, and the mass ratio of the fish scale calcium chelate peptide to calcium ions in the solution system is 9:1 (w / w).

[0017] In the above technical solution, in step (B), the chelation reaction is carried out in a water bath at a pH of 7.5-8.0 and a temperature of 25-30°C for 25-35 minutes.

[0018] In the above technical solution, in step (B), the ichthyopeptide calcium chelate is prepared by adding eight times the volume of anhydrous ethanol after the chelation reaction is completed, mixing, standing for 8-12 hours, and then centrifuging to collect the precipitate. The powdered solid obtained by freeze-drying the precipitate is the ichthyopeptide calcium chelate.

[0019] The present invention also provides fish scale glue, hydroxyapatite and calcium peptide chelate prepared by the above-mentioned joint production method.

[0020] Compared with the prior art, the present invention has the following characteristics: The prepared fish scale glue has been verified to meet the unique characteristics of gelatin and has high gel strength; the prepared hydroxyapatite has been verified by FTIR and XRD to meet the structural characteristics of hydroxyapatite and has high purity; the prepared peptide calcium chelate has been successfully prepared through structure-activity analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description: Figure 1 The optimized diagram for the extraction conditions of fish scale glue in screening Example 1 is shown below: Figure 1 a is the effect of different material-liquid ratios on the extraction of fish scale glue; Figure 1 b is the effect of different extraction temperatures on the extraction of fish scale glue; Figure 1 c is the effect of different extraction times on fish scale glue extraction; Figure 2 The UV spectrum of the fish scale glue prepared by the optimal process in screening Example 1; Figure 3 The infrared spectrum of the fish scale glue prepared by the optimal process in screening Example 1; Figure 4 The XRD patterns of hydroxyapatite prepared under different conditions in screening Example 2 are as follows: Figure 4 a is the XRD pattern of hydroxyapatite prepared at different high temperature oxidation temperatures; Figure 4 b is the XRD pattern of hydroxyapatite prepared at different high temperature oxidation times; Figure 5 The FTIR images of hydroxyapatite prepared under different conditions in screening Example 2 are as follows; wherein: Figure 5 a is the FTIR image of hydroxyapatite prepared at different high temperature oxidation temperatures; Figure 5 b is the FTIR image of hydroxyapatite prepared at different high temperature oxidation times; Figure 6 This is the optimization diagram for the single-factor experiment of screening different peptide calcium chelation in Example 3, wherein: Figure 6 a is the effect of different chelation times on the calcium content of fish scale calcium peptide chelate; Figure 6 b is the effect of different chelation temperatures on the calcium content of fish scale calcium peptide chelate; Figure 6 c is the effect of different pH on the calcium content of fish scale calcium peptide chelate; Figure 6 d is the effect of different peptide-calcium ratios on the calcium binding content of fish scale calcium peptide chelate; Figure 7 This is a 3D diagram of the three-factor, three-level response surface experiment for peptide calcium chelation in screening Example 3, wherein: Figure 7 a is a 3D graph of the peptide calcium chelation response surface experiment at different peptide-calcium ratios and pH; Figure 7 b is a 3D graph of the peptide calcium chelation response surface experiment at different chelation times and pH values; Figure 7 c is a 3D graph of the peptide calcium chelation response surface experiment at different peptide-calcium ratios and chelation times; Figure 7 d is the plane diagram of the peptide calcium chelation response surface experiment at different peptide-calcium ratios and pH; Figure 7 e is the plane diagram of the peptide calcium chelation response surface experiment at different chelation times and pH values; Figure 7 f is the plane diagram of the peptide calcium chelation response surface experiment under different peptide-calcium ratios and chelation times; Figure 8 This is a thermodynamic analysis diagram of the affinity peptide and Ca2+ in screening Example 3. DETAILED DESCRIPTION

[0022] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description: The experimental raw materials of the present invention are fish scales, which are by-products of Hainan tilapia processing. First, the tilapia scales are pretreated with NaCl and NaOH solutions, and the pretreated fish scales are placed in an oven and dried at low temperature. With gelatin yield and gel strength as indicators, the effects of three factors, namely, solid-liquid ratio, extraction temperature, and extraction time, on the extraction of fish scale glue are optimized. The fish scale residue from which the fish scale glue is extracted is carbonized until smokeless, and then oxidized at high temperature to prepare hydroxyapatite. Using fish scale glue as raw material, affinity peptides and calcium peptide chelates are prepared through separation and purification of homemade fish scale hydroxyapatite. Thus, the comprehensive utilization of Hainan tilapia processing by-products can be achieved, and its economic value can be improved.

[0023] The present invention will be described below in conjunction with specific embodiments and screening examples: Screening Example 1: Optimization of conditions for preparing fish scale glue This screening example screens and optimizes the conditions of the solid-liquid ratio, extraction temperature, and extraction time when extracting fish scale glue, and explores the effects of different solid-liquid ratios, extraction temperatures, and extraction times on the extraction of fish scale glue, comprising the following steps: (1) Rinse fresh tilapia scales several times with running water to remove blood, fins and other impurities. Soak the cleaned scales in a 1% mass concentration NaCl solution for 20 h to remove impurities, changing the NaCl solution three times during this period. Rinse the scales several times with pure water and soak them in a 0.1M NaOH solution for 24 h to remove fat and decolorize, changing the NaOH solution every 6 h. After 24 h, remove the scales and wash them several times until the washing solution is neutral. Dry the pretreated scales in a 30°C oven to obtain dry scales.

[0024] (2) The dried fish scales in step (1) were placed in water, and the fish scale glue was extracted using the hot water method. A single-factor experiment was conducted on the extraction of fish scale glue: ① The extraction temperature was controlled at 70 °C and the extraction time was 3 h. The effects of different solid-liquid ratios of 1:9, 1:10, 1:11, 1:12, and 1:13 on the extraction of fish scale glue were investigated; ② The solid-liquid ratio was controlled at 1:11 and the extraction time was 3 h. The effects of different temperatures of 50, 60, 70, 80, and 90 °C on the extraction of fish scale glue were investigated; ③ The solid-liquid ratio was controlled at 1:11 and the extraction temperature was 70 °C. The effects of different extraction times of 1, 2, 3, 4, and 5 h on the extraction of fish scale glue were investigated.

[0025] like Figure 1 As shown in the data, the solid-liquid ratio had no obvious effect on the gelatin yield and gel strength, and there was no significant difference between the data of each group (P < 0.5). The selected solid-liquid ratio was 1:11; the extraction temperature had a significant effect on the gelatin yield and gel strength. With the increase of extraction temperature, the gelatin yield increased, and the gel strength first increased and then decreased, reaching the highest at 70℃. The selected extraction temperature was 70℃; the extraction time also had a great effect on the extraction of fish scale glue. With the increase of extraction time, the gel strength first increased and then decreased, and the gelatin yield increased accordingly. The selected extraction time was 3 h.

[0026] It can be concluded that the optimal extraction conditions for fish scale glue are a solid-liquid ratio of 1:11, an extraction temperature of 70℃, and an extraction time of 3h. The fish scale glue (FSG) was obtained by the optimal process preparation according to the optimal submission conditions obtained by screening.

[0027] Commercial pig skin gelatin (CPSG) and commercial cold water fish skin gelatin (CFSG) were used for comparison, and the structure of fish scale gelatin (FSG) prepared by the optimal process was characterized: the UV spectrum is shown in the figure below: Figure 2 As shown in the figure, CPSG, FSG and CFSG have strong absorption peaks at 193, 198 and 196 nm respectively, which are characteristic absorption peaks of peptide bonds, indicating that the extracted FSG has gelatin characteristics and has no significant difference from commercially available gelatin; the infrared spectrum is shown in the figure Figure 3 As shown, the extracted FSG has the characteristic infrared absorption peak of gelatin.

[0028] Screening Example 2: Optimization of conditions for preparing fish scale hydroxyapatite The fish scale residue remaining after preparing the fish scale glue by the optimal process in Screening Example 1 was dried (drying temperature was 30° C. and time was 24 h), then placed in a crucible, and subjected to high-temperature carbonization on a hot plate until smokeless (heating temperature was 100° C. and heating time was 0.5 h), and then placed in a muffle furnace for high-temperature oxidation. The conditions for high-temperature oxidation were screened and optimized as follows: ① The high-temperature oxidation time was controlled to be 2 h, and the high-temperature oxidation temperature was 600, 700, 800, 900, and 1000°C; ② The temperature was controlled to be 800°C, and the high-temperature oxidation time was 1, 2, 3, 4, and 5 h. The purity of the products obtained under different high-temperature oxidation conditions was verified by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD): The XRD patterns of hydroxyapatite prepared under different conditions are shown in Figure 4. Figure 4 It can be seen that the prepared hydroxyapatite meets the XRD peaks and angles of standard HA. The FTIR images of hydroxyapatite prepared under different conditions are as follows: Figure 5 As shown by Figure 5It can be seen that the prepared hydroxyapatite contains P043-characteristic absorption peak.

[0029] The results show that the optimal preparation process of fish scale hydroxyapatite is oxidation at 700℃ for 3 h. Under this condition, hydroxyapatite with higher purity can be prepared.

[0030] Screening Example 3: Optimization of conditions for preparing fish scale calcium peptide chelate (A) The fish scale glue prepared under the optimal process conditions in Screening Example 1 was added with water to prepare a 3% protein solution, the solution temperature was adjusted to 55°C, the pH was adjusted to 9, 3% alkaline protease was added for enzymatic hydrolysis for 4 h, and the enzyme was immediately inactivated in a boiling water bath for 10 min after the enzymatic hydrolysis was completed. The enzymatic hydrolyzate was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The supernatant was concentrated and lyophilized to obtain fish scale calcium chelating peptide (FSP).

[0031] The fish scale calcium chelating peptide (FSP) in this example can also be used to prepare affinity peptide (FHAC): the fish scale calcium chelating peptide is added with water to prepare a 40 mg / mL peptide solution, and the peptide solution is statically adsorbed using the hydroxyapatite obtained after preparation under the optimal process in Example 2. At 30°C, when the ratio of hydroxyapatite to peptide (m / v) is 2.5:1, static adsorption is carried out for 60 minutes, and then the low calcium binding fraction is eluted by repeated centrifugation (at a speed of 8000 r / min) 3 times with 5 mM and 60 mM phosphate solutions, respectively, and then the high calcium binding fraction is eluted with 120 mM phosphate solution (at a speed of 8000 r / min). The eluate is collected and concentrated and lyophilized to obtain the affinity peptide (FHAC).

[0032] (B) The fish scale calcium chelating peptide (FSP) obtained in step (A) is thoroughly mixed with water to prepare a fish scale calcium chelating peptide solution, and calcium chloride dihydrate is added and mixed to obtain a solution system for the chelation reaction. The chelation reaction is carried out in a water bath. After the reaction is completed, eight times the volume of anhydrous ethanol is added, and the mixture is allowed to stand for 8-12 hours, and then centrifuged at 8000 r / min. After centrifugation for 10 minutes, the precipitate is collected, and the powder obtained by freeze-drying the precipitate is the fish scale peptide calcium chelate.

[0033] In this screening example, the calcium chelation conditions of fish scale peptide were optimized: ① The peptide calcium mass ratio was 9:1 (w / w), the pH was 8, and the chelation temperature was 30°C. The effects of different chelation times of 15, 25, 35, 45, and 55 min on the calcium binding content were explored; ② The peptide calcium mass ratio was 9:1 (w / w), the pH was 8, and the chelation time was 40 min. The effects of different chelation temperatures of 10, 20, 30, 40, 50, 60, and 70 min on the calcium binding content were explored; ③ The peptide calcium mass ratio was 9:1 (w / w), the chelation temperature was 30°C, and the chelation time was 40 min. The effects of different pH values ​​of 4, 5, 6, 7, 8, 9, and 10 on the calcium binding content were explored; ④ The pH was 8, the chelation temperature was 30°C, and the chelation time was 40 min. The effects of different peptide calcium mass ratios of 4.5:1, 6:1, 7.5:1, 9:1, and 10.5:1 on the calcium binding content were explored: The optimization results of the four factors are as follows Figure 6 As shown in the figure, four factors, namely chelation temperature, chelation time, peptide calcium ratio and pH, were selected to conduct single factor experiment. Figure 6 It can be seen that the optimal conditions for obtaining peptide calcium chelation are a peptide calcium mass ratio of 9:1 (w / w), pH 8, a chelation temperature of 30°C, and a chelation time of 40 min.

[0034] On the basis of the single-factor experiment, three factors, namely, chelation time, peptide-calcium ratio and pH, were selected to conduct a three-factor three-level response surface experiment. Table 1 shows the results of the response surface experiment.

[0035] Table 1 Response surface experiment results Serial number pHX1 Peptide Calcium Ratio X2 Time / minX3 Calcium binding capacity / (μg / mg)Y 1 7 7.5:1 35 3.23 ± 0.39 2 9 7.5:1 35 2.73 ± 0.28 3 7 10.5:1 35 3.57 ± 0.35 4 9 10.5:1 35 2.26 ± 0.37 5 7 9:1 25 4.05 ± 0.39 6 9 9:1 25 2.99 ± 0.16 7 7 9:1 45 3.95 ± 0.68 8 9 9:1 45 2.86 ± 0.27 9 8 7.5:1 25 3.74 ± 0.82 10 8 10.5:1 25 3.88 ± 0.31 11 8 7.5:1 45 3.79 ± 0.17 12 8 10.5:1 45 3.90 ± 0.22 13 8 9:1 35 4.27 ± 0.40 14 8 9:1 35 4.41 ± 0.43 15 8 9:1 35 4.34 ± 0.22 16 8 9:1 35 4.15 ± 0.15 17 8 9:1 35 4.21 ± 0.28 Multiple regression analysis of the response surface experiment results yielded a quadratic multivariate fit equation: Y = 4.28–0.4954X1 + 0.0122X2–0.0195X3–0.2025X1X2–0.0098X1X3–0.0073X2X3–0.8472X1X2–0.04837X2X2 + 0.0337X3X2. Table 2 shows the regression analysis results. The P value for this model was very low (P < 0.0001), and the lack-of-fit term (P = 0.3852) was not significant (>0.05), indicating that the regression model was highly significant. The coefficient of determination (R2) was 0.9869, indicating that the model fit was good. The adjusted coefficient (R2Adj) was 0.9702, indicating that the model explained 97.02% of the variation in the response values. The results showed that one linear term (X1), one interaction term (X1X2) and two quadratic terms (X12 and X22) were all significant (P < 0.05), while two linear terms (X2 and X3), two interaction terms (X1X3 and X2X3) and one quadratic term (X32) were not significant (P > 0.05).

[0036] Table 2 Analysis of variance of regression model source sum of squares degrees of freedom mean square F-number P Significance Model 6.34 9 0.7044 58.80 ＜0.0001 ** X1-pH 1.96 1 1.96 163.86 ＜0.0001 ** X2-Peptide Calcium Ratio 0.0012 1 0.0012 0.0994 0.7617 X3-Time 0.0030 1 0.0030 0.2545 0.6294 X1X2 0.1641 1 0.1641 13.70 0.0076 * X1X3 0.0004 1 0.0004 0.0318 0.8635 X2X3 0.0002 1 0.0002 0.0179 0.8974 X12 3.02 1 3.02 252.29 ＜0.0001 ** X22 0.9849 1 0.9849 82.21 ＜0.0001 ** X32 0.0048 1 0.0048 0.3986 0.5479 residual 0.0839 7 0.0120 Lack of Fit 0.0417 3 0.0139 1.32 0.3852 Pure error 0.0422 4 0.0105 sum 6.42 16 Note: * indicates significant P < 0.05; ** indicates extremely significant P < 0.001 The coupling effect of the three factors on the response surface experiment of calcium binding capacity is as follows Figure 7 As shown, the optimal chelation conditions predicted by the software were pH 7.70302, a peptide-to-calcium ratio of 9.12376:1, and a duration of 25 minutes, resulting in a calcium binding capacity of 4.40263 μg / mg. Considering practical feasibility in actual production, the predicted values ​​were adjusted to pH 8, a peptide-to-calcium ratio of 9:1, and a duration of 25 minutes. Calcium binding was measured under these conditions, and validation experiments were repeated three times. The actual calcium binding capacity was 4.38 ± 0.90 μg / mg, which was not significantly different from the predicted value (P > 0.05), confirming that these conditions are the optimal for peptide calcium chelation.

[0037] Affinity peptide and Ca 2 +Thermodynamic analysis diagram Figure 8 As shown by Figure 8 It can be seen that the titration of CaCl2 into the affinity peptide produces an exothermic binding isotherm, and the interaction produces negative ΔH and ΔG values, indicating that the affinity peptide binds to CaCl2. 2 Chelation of + occurs spontaneously at 37°C.

[0038] After the above screening examples, the optimal process conditions for preparing tilapia fish scale glue, hydroxyapatite and calcium peptide chelate of the present invention were obtained. The following examples were carried out using the optimal conditions as parameters: Example 1: A method for efficiently co-producing tilapia scale glue, hydroxyapatite and calcium peptide chelate, characterized by comprising the following steps: (1) Tilapia scale pretreatment: Fresh tilapia scales were rinsed several times with running water to remove impurities such as blood and fins. The cleaned scales were then soaked in a 1% NaCl solution for 20 hours to remove impurities, changing the NaCl solution three times. The scales were then rinsed several times with pure water and then soaked in a 0.1M NaOH solution for 24 hours to remove fat and decolorize the scales, changing the NaOH solution every six hours. After 24 hours, the scales were removed and rinsed several times until the rinse solution was neutral. The pretreated scales were then dried in a 30°C oven to obtain dried scales.

[0039] (2) Extraction of fish scale glue: The dried fish scales in step (1) are placed in water, and fish scale glue is extracted using a hot water method with a solid-liquid ratio of 1:11, an extraction temperature of 70°C, and an extraction time of 3 h. After the extraction is completed, the fish scale residue is removed to obtain fish scale glue.

[0040] (3) Preparation of fish scale hydroxyapatite: The fish scale residue remaining after preparing the fish scale glue in step (2) was dried (drying temperature was 30°C and time was 24 h), then placed in a crucible, and carbonized on a heating plate until smokeless (heating temperature was 100°C and heating time was 0.5 h), and then placed in a muffle furnace for high-temperature oxidation at a temperature of 700°C. After oxidation for 3 h, hydroxyapatite with high purity was obtained.

[0041] (4) Preparation of calcium peptide chelate: (A) The fish scale glue obtained in step (2) was added with water to prepare a 3% protein solution, the solution temperature was adjusted to 55°C, the pH was adjusted to 9, 3% alkaline protease was added for enzymatic hydrolysis for 4 h, and the enzyme was immediately inactivated in a boiling water bath for 10 min after the enzymatic hydrolysis was completed. The enzymatic hydrolyzate was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The supernatant was concentrated and lyophilized to obtain fish scale calcium chelating peptide (FSP).

[0042] (B) The fish scale calcium chelate peptide (FSP) obtained in step (A) is thoroughly mixed with water to prepare a fish scale calcium chelate peptide solution, and calcium chloride dihydrate is added and mixed to obtain a solution system for the chelation reaction. The chelation reaction is carried out in a water bath under the following reaction conditions: peptide calcium mass ratio is 9:1 (w / w), pH is 8, chelation temperature is 30°C, and chelation time is 40 min; after the reaction is completed, eight times the volume of anhydrous ethanol is added, and the mixture is allowed to stand for 8-12 h. After centrifugation at 8000 r / min for 10 min, the precipitate is collected, and the powder obtained after the precipitate is freeze-dried is the fish scale peptide calcium chelate.

[0043] In the embodiments of the present invention, tilapia scale glue, hydroxyapatite and calcium peptide chelate are obtained. The prepared fish scale glue is verified to meet the unique characteristics of gelatin and has high gel strength; the prepared hydroxyapatite is verified to meet the structural characteristics of hydroxyapatite by FTIR and XRD and has high purity; the prepared peptide calcium chelate is successfully prepared as shown by structure-activity analysis.

[0044] The above examples are only for illustrating the technical concept and technical features of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent transformation or modification made based on the essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for the efficient co-production of tilapia scale glue, hydroxyapatite and calcium peptide chelate, characterized in that: The following steps are involved: (1) Tilapia scale pretreatment: Tilapia scales were sequentially immersed in NaCl solution and NaOH solution, with the NaCl solution being used for deproteinization and the NaOH solution being used for degreasing, decolorization, and deodorization, and finally the scales were cleaned with distilled water until neutrality and dried for later use; (2) Extraction of fish scale glue: The dried fish scales in step (1) are placed in water, and fish scale glue is extracted using a hot water method. After the extraction is completed, the fish scale residue is removed to obtain fish scale glue, a portion of which is used as a raw material for preparing calcium peptide chelate, and a portion is collected as a fish scale glue product; (3) Preparation of fish scale hydroxyapatite: The fish scale residue in step (2) is used as a raw material, and the fish scale residue is dried, carbonized and oxidized at high temperature to obtain a fish scale hydroxyapatite product; (4) Preparation of calcium peptide chelate: The fish scale glue in step (2) is used as a raw material, and the fish scale glue is subjected to an enzymatic hydrolysis reaction, a chelation reaction, and precipitation in sequence to obtain a calcium peptide chelate.

2. The high-efficiency co-production preparation method according to claim 1, characterized in that: In step (1), the concentration of the NaCl solution is 1% (w / v), the soaking time is 18-24 h, the pH is 7, the solution is stirred during soaking at a stirring rate of 200 r / min, the material-liquid ratio is 1:20 (w / v), and the NaCl solution is replaced every 6 h; the concentration of the NaOH solution is 0.1 M, the soaking time is 24-36 h, the pH is 12-14, the solution is stirred during soaking at a stirring rate of 200 r / min, the material-liquid ratio is 1:20 (w / v), and the NaOH solution is replaced every 6 h.

3. The high-efficiency co-production preparation method according to claim 1, characterized in that: In step (2), when the fish scale glue is extracted using the hot water method, the process conditions are: the solid-liquid ratio range is 1:9 - 1:13 (w / v), the extraction temperature range is 50 - 90 ° C, and the extraction time range is 1 - 5 h.

4. The high-efficiency co-production preparation method according to claim 1, characterized in that: In step (3), the method for preparing fish scale hydroxyapatite (FSHA) specifically includes the following steps: The fish scale residue remaining after the fish scale glue is extracted in step (2) is washed several times and then placed in an oven for drying. After drying, it is transferred to a crucible for high-temperature carbonization. The fish scale residue is carbonized until smokeless, and then subjected to high-temperature oxidation. After the high-temperature oxidation, high-purity fish scale hydroxyapatite (FSHA) is obtained.

5. The high-efficiency co-production preparation method according to claim 4, characterized in that: In step (3), the drying is carried out at a temperature of 30-40°C and a time of 18-24 h; the carbonization is carried out by placing the crucible on a heating plate at a heating temperature of 100°C until the fish scale residue is smokeless; the high-temperature oxidation is carried out in a muffle furnace at a time of 1-5 h and a temperature of 600-1000°C.

6. The high-efficiency co-production preparation method according to claim 1, characterized in that: In step (4), the method for preparing the calcium peptide chelate specifically comprises the following steps: (A) Preparation of fish scale calcium chelating peptide (FSP) The fish scale glue obtained in step (2) is added with water to prepare a 3% protein solution, and protease is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated, the enzymatic hydrolysis solution is centrifuged and the supernatant is collected. The supernatant is concentrated and dried to obtain fish scale calcium chelating peptide (FSP); (B) Preparation of calcium peptide chelate: The fish scale calcium chelating peptide (FSP) obtained in step (A) is thoroughly mixed with water to prepare a fish scale calcium chelating peptide solution, and calcium chloride dihydrate is added and mixed to obtain a solution system for chelation reaction. The chelation reaction is carried out in a water bath. After the reaction is completed, anhydrous ethanol is added, and the mixture is allowed to stand, centrifuged, precipitated, and freeze-dried to obtain a fish scale peptide calcium chelate.

7. The high-efficiency co-production preparation method according to claim 6, characterized in that: In step (A), the protease is any one of pepsin, trypsin, alkaline protease, composite protease, flavor protease, papain, and animal protease; the protease is added in an amount of 3% of the mass of the solution system; the enzymatic hydrolysis refers to hydrolysis at a pH of 9 and a temperature of 55°C for 4 hours; the enzyme inactivation refers to boiling water bath for 10 minutes immediately after the enzymatic hydrolysis is completed; and the centrifugation refers to centrifugation at 8000 r / min for 10 minutes.

8. The high-efficiency co-production preparation method according to claim 6, characterized in that: In step (A), after obtaining the fish scale calcium chelating peptide, the fish scale calcium chelating peptide is separated and purified using the homemade fish scale hydroxyapatite in step (3) to obtain the affinity peptide (FHAC); the separation and purification refers to using the homemade hydroxyapatite in step (3) to statically adsorb the fish scale calcium chelating peptide by static affinity chromatography; the temperature during static adsorption is 30°C and the adsorption time is 60 min; the fish scale calcium chelating peptide is configured into a peptide solution and then adsorbed, the peptide concentration of the peptide solution is 40 mg / mL, and the material-liquid ratio (m:V) of hydroxyapatite (FSHA) and peptide solution (FSP) is 2.5:1; after the static adsorption is completed, the low calcium binding amount component is eluted by repeated centrifugation three times using 5 mM and 60 mM phosphate solutions respectively, and then desorbed by centrifugation for 30 min using 120 mM phosphate buffer solution, and the eluate is collected and concentrated and freeze-dried to obtain the affinity peptide (FHAC); the centrifugation speed is 8000 r / min.

9. The high-efficiency co-production preparation method according to claim 6, characterized in that: In step (B), the mass ratio of the fish scale calcium chelate peptide to calcium ions in the solution system is 9:1 (w / w); the chelation reaction is carried out at a pH of 7.5-8.0 and in a water bath at 25-30°C for 25-35 minutes. After the chelation reaction, the fish scale peptide calcium chelate is prepared by adding eight times the volume of anhydrous ethanol, mixing, and standing for 8-12 hours. The mixture is then centrifuged and the precipitate is collected. The resulting powder, which is freeze-dried, is the fish scale peptide calcium chelate.

10. Fish scale glue, hydroxyapatite and calcium peptide chelate prepared by the co-production method according to any one of claims 1 to 9.

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