A method for in situ preparation of calcium chelating peptides that promote intestinal calcium absorption and osteoblast development by high-pressure homogenization-assisted enzymatic hydrolysis of fish scales.

Abstract

This invention discloses a method for in-situ preparation of calcium chelate peptides that promote intestinal calcium absorption and osteoblast development from fish scales using high-pressure homogenization-assisted enzymatic hydrolysis. The method uses fish scales as raw material, which are pulverized and then pretreated with high-pressure homogenization to break down the dense cross-linked structure of the scales. Pepsin is used as the hydrolytic enzyme to achieve efficient enzymatic hydrolysis of collagen and in-situ chelation with natural calcium, resulting in a one-step preparation of collagen calcium chelate peptides. The product has a calcium retention rate of ≥84.16%, is rich in affinity amino acids, and shows a ≥62.97% increase in calcium transport compared to calcium chloride in a small intestinal epithelial cell absorption and transport model, while being safe and non-cytotoxic. This invention is a green and efficient process that eliminates the need for decalcification steps, achieving a collagen calcium chelate peptide yield of ≥83.4%. It realizes the high-value utilization of aquatic by-products and can be directly applied to various calcium supplements such as food and health products that enhance intestinal calcium absorption and promote bone development, solving problems such as low bioavailability of traditional calcium preparations and severe calcium loss in existing fish scale utilization processes.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically to a method for in situ preparation of calcium chelating peptides that promote intestinal calcium absorption and osteoblast development by high-pressure homogenization-assisted enzymatic hydrolysis of fish scales. Background Technology

[0002] Calcium is a core macromineral for maintaining human bone health, nerve conduction, muscle contraction, and other physiological functions. 99% of the body's calcium is deposited in bones and teeth, making it crucial for the growth and development of infants and adolescents, as well as the bone health of the elderly. Insufficient calcium intake leads to calcium deficiency, and the low bioavailability of traditional calcium supplements exacerbates this problem. Inorganic calcium (such as calcium carbonate and calcium chloride) has a bioavailability of only 20%-30%, and ordinary organic calcium (such as calcium lactate) is less than 40%. A large amount of calcium cannot be absorbed because it forms insoluble precipitates in the intestines, resulting in resource waste and potentially causing gastrointestinal discomfort such as constipation.

[0003] With consumers becoming increasingly health-conscious, the market demand for new calcium supplements that are "highly absorbable, naturally sourced, and safe and burden-free" is growing rapidly. In 2023, the global market size for functional calcium supplements exceeded US$20 billion, with a significant shortage of natural calcium-based raw materials with a bioavailability of ≥60%. The aquatic product processing industry generates over ten million tons of byproducts such as fish scales and bones annually. Fish scales are rich in type I collagen and hydroxyapatite (a natural calcium source), making them excellent raw materials for preparing functional calcium chelate peptides. However, currently over 90% of fish scales are directly discarded or used as low-value feed, resulting in severe resource waste and environmental pollution.

[0004] Existing processes for preparing calcium-related products from fish scales face several insurmountable technical bottlenecks: First, the collagen in fish scales forms a dense cross-linked structure with hydroxyapatite, resulting in low efficiency and limited product yield from conventional enzymatic hydrolysis processes. Second, to improve enzymatic hydrolysis, existing technologies often employ pre-decalcification treatments such as acid solution soaking and EDTA chelation. This not only involves cumbersome procedures and increases the risk of chemical reagent residues but also leads to the loss of over 80% of the natural calcium in fish scales. Furthermore, acid treatment damages the collagen structure, further reducing product yield. Third, while some processes attempt to simplify the procedures, they fail to address the insufficient activity of the product in promoting intestinal calcium absorption and bone development, making it difficult for the product to demonstrate its functional advantages when applied to calcium supplements.

[0005] In existing technologies, such as patent CN120665975A, a method for "high-pressure crushing to promote the dissolution of eggshell membrane proteins and prepare chelated calcium" is disclosed. This method uses eggshells as raw materials, requiring the separation of eggshells and eggshell membranes, followed by high-pressure homogenization and enzymatic hydrolysis twice with pepsin and trypsin, and then obtaining chelated calcium through alcohol precipitation. This process is cumbersome, requiring raw material separation and multiple enzymatic hydrolysis steps. Furthermore, the calcium transport capacity of the product in the Caco-2 cell model is only 5.21 μg, indicating that its activity needs improvement. Simultaneously, this method relies on the separation of eggshells and eggshell membranes, failing to utilize the natural complex system in the raw materials, which is fundamentally different from the natural properties of fish scales. Another existing technology (CN114920825B) uses a mixture of fish scales and fish skin as raw materials, requiring high-temperature hydrolysis with dilute sulfuric acid and chemical precipitation to remove heavy metals, posing a risk of chemical reagent residues. Moreover, the process is complex and energy-intensive. High-pressure homogenization, as a novel non-thermal processing technology, can disrupt the dense structure of materials through the shear force and cavitation effect generated by instantaneous high pressure, achieving physical modification without chemical reagents. It has already shown potential for synergistic effects in the pretreatment of protein raw materials. However, there are currently no reports on the precise coupling of high-pressure homogenization and enzymatic hydrolysis processes to achieve integrated preparation of various fish scales through "non-decalcification-efficient enzymatic hydrolysis-in-situ peptide calcium chelation." Furthermore, there is a lack of universal fish scale-derived calcium chelate peptide preparation technologies that are specifically designed for calcium supplement applications and demonstrate clear activity in promoting intestinal calcium absorption.

[0006] Therefore, developing a simple, green, efficient, and widely applicable method for preparing fish scale collagen calcium chelate peptides with clearly defined product activity is of great practical significance and industrial value for solving the waste of aquatic by-products, breaking through the technical bottlenecks of traditional calcium supplement products, and meeting the functional needs of the market. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies in preparing chelated calcium, such as complex raw material pretreatment, multiple enzymatic / chemical treatments, reliance on exogenous calcium sources, and poor environmental friendliness. It provides fish scale collagen calcium peptide chelates, their preparation method, and their application in enhancing intestinal calcium absorption and bone development. This achieves efficient utilization of endogenous collagen and calcium ions from fish scales, simplifying the process, reducing costs, and improving product safety. By optimizing the range of high-pressure homogenization and enzymatic hydrolysis process parameters, this invention expands the applicable raw material range (covering various freshwater and marine fish scales), achieving a one-step preparation without decalcification. This solves problems such as low enzymatic hydrolysis efficiency, severe calcium loss, limited raw materials, and unclear product activity in traditional processes. Simultaneously, it provides applications of this collagen calcium chelate peptide in various calcium supplements. Comparative experiments based on a unified standard confirm its technological advancement, providing core raw materials and technical support for developing highly absorbable, naturally sourced calcium supplements, thereby enhancing product market competitiveness.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention utilizes an integrated process of "high-pressure homogenization - single-pass pepsin hydrolysis - in-situ chelation," eliminating the need for raw material separation, decalcification, chemical hydrolysis, or membrane separation steps. It fully leverages the collagen and hydroxyapatite inherent in fish scales to achieve the targeted chelation of small molecule peptides with endogenous calcium ions. The specific scheme is as follows:

[0010] A method for preparing calcium chelating peptides that promote intestinal calcium absorption and osteoblast development through in-situ preparation of fish scales using high-pressure homogenization-assisted enzymatic hydrolysis includes the following steps:

[0011] (1) Raw material pretreatment: The fish scales are cleaned and impurities are removed, dried and then crushed and sieved to obtain dried fish scale powder;

[0012] (2) High pressure homogenization pretreatment: The dried fish scale powder obtained in step (1) is prepared into a suspension with water, and then circulated 8 to 12 times under a pressure of 45 to 55 MPa using a high pressure homogenizer. After freeze drying, high pressure homogenized fish scale powder is obtained.

[0013] (3) Enzymatic hydrolysis preparation: using pepsin as the hydrolytic enzyme, the high-pressure homogenized fish scale powder obtained in step (2) is placed in the enzymatic hydrolysis system, and the enzymatic hydrolysis conditions are controlled as follows: pH 1.5-2.5, temperature 35-45℃, enzyme-to-substrate ratio 0.05%-0.15%, substrate concentration 3%-7%, and the enzymatic hydrolysis is carried out for 1-3 h to obtain the enzymatic hydrolysate;

[0014] (4) Product separation: Centrifuge the enzymatic hydrolysate obtained in step (3) to obtain the supernatant, freeze-dry it to obtain collagen calcium chelate peptide hydrolysate.

[0015] Furthermore, the fish scales mentioned in step (1) are the scales of freshwater or saltwater fish (byproducts generated during processing), including tilapia, grass carp, carp, cod, and salmon. No pre-decalcification treatment is required.

[0016] Furthermore, the drying process described in step (1) involves drying in an oven at 55–65°C for 4–8 hours. The moisture content of the dried fish scale powder is ≤10%.

[0017] Furthermore, the sieving mentioned in step (1) is sieving through a 50-70 mesh sieve.

[0018] Furthermore, the concentration of the suspension mentioned in step (2) is 3% to 7% by mass.

[0019] Furthermore, in step (2), the pressure of the high-pressure homogenization process is 48–52 MPa, and the number of cycles is 9–11.

[0020] Furthermore, in step (2), the pressure of the high-pressure homogenization process is 50±0.5 MPa, and the number of cycles is 10.

[0021] Furthermore, the pepsin activity described in step (3) is 2.0 × 10⁻⁶. 5 ~3.0×10 5 U / g, and stir once every 20 to 40 minutes during enzymatic hydrolysis, with a stirring time of 1 to 3 minutes.

[0022] Furthermore, the pepsin described in step (3) has an enzyme activity of 2.5 × 10⁻⁶. 5 The enzymatic hydrolysis conditions were: pH 2, temperature 40℃, enzyme-to-substrate ratio 0.1%, substrate concentration 5%, and hydrolysis time 2 h.

[0023] Furthermore, the centrifugation conditions described in step (4) are 3500-4500 rpm for 8-12 min.

[0024] Furthermore, the centrifugation conditions described in step (4) are 4000 rpm for 10 min.

[0025] Furthermore, the freeze-drying temperature in step (4) is -55℃ to -35℃, the vacuum degree is 10 to 30 Pa, and the time is 20 to 30 h.

[0026] Furthermore, the freeze-drying temperature in step (4) is -40°C, the vacuum degree is 20 Pa, and the time is 24 h.

[0027] A fish scale collagen calcium chelate peptide hydrolysate was obtained by the above method. Based on dried fish scale powder as the starting material, the yield of the collagen calcium chelate peptide hydrolysate was ≥83.4%. The hydrolysate contained 18 amino acids, of which aspartic acid accounted for 5.63% and glutamic acid accounted for 9.86%, and it was rich in calcium-affinity amino acids. It showed no cytotoxicity to Caco-2 cells and exhibited significant activity in promoting intestinal calcium absorption.

[0028] A method for preparing fish scale collagen calcium peptide chelate includes steps (1), (2), and (3) of the above method, as well as the following steps:

[0029] Chelation reaction: The obtained enzymatic hydrolysate was adjusted to a temperature of 40-60℃ and a pH of 6.0-8.0, and chelation reaction was carried out. After alcohol precipitation, the precipitate was freeze-dried to obtain fish scale collagen calcium peptide chelate.

[0030] Furthermore, the enzymatic hydrolysate is adjusted to a temperature of 40±5℃ and a pH of 7.0±0.5.

[0031] Furthermore, the chelation reaction takes place for 1 ± 0.5 h, with stirring every fifteen minutes.

[0032] Furthermore, the alcohol precipitation temperature is 4±0.5℃.

[0033] A fish scale collagen calcium peptide chelate was obtained by the above method. Based on dried fish scale powder as the starting material, the yield of the fish scale collagen calcium peptide chelate was ≥81.41%, and the calcium content of the product was 71.44±3.01 μg / mg. In the Caco-2 cell monolayer model, it exhibited significant activity in promoting intestinal calcium absorption; it significantly promoted the proliferation, differentiation, and mineralization of MC3T3-E1 osteoblasts, with a cell proliferation rate of 127.79%–130.76% at a concentration of 10–50 μg / mL.

[0034] Furthermore, the calcium peptide chelate comprises:

[0035] (1) The peptide Asp-Leu-Gly-Asp-Asp-Asp-Gly-Phe (DLGDDDGF), which binds to 3 calcium ions, has the following binding sites:

[0036] The fourth position contains aspartic acid (Asp), which binds to one calcium ion;

[0037] The fifth position contains aspartic acid (Asp), which binds to one calcium ion;

[0038] The 6th position contains aspartic acid (Asp), which binds to one calcium ion;

[0039] (2) The peptide Glu-Asp-Gln-Glu-Glu (EDQEE) which binds to 3 calcium ions has the following binding sites:

[0040] The fourth glutamate (Glu) binds to one calcium ion;

[0041] Glutamic acid (Glu) at position 5 binds to two calcium ions.

[0042] A calcium chelating peptide is an active peptide having any of the following amino acid sequences: Asp-Leu-Gly-Asp-Asp-Asp-Gly-Phe, Glu-Asp-Gln-Glu-Glu.

[0043] The above-mentioned fish scale collagen calcium chelate peptide hydrolysate, fish scale collagen calcium peptide chelate, or calcium chelate peptide are used in the preparation of products for calcium supplementation, calcium absorption promotion, bone development promotion, and / or osteoporosis prevention.

[0044] Furthermore, the products mentioned include functional foods, health products, dietary supplements, foods for special medical purposes, or pharmaceuticals.

[0045] Furthermore, the products mentioned include foods, health supplements, or medicines that help improve bone density function.

[0046] Furthermore, the amount of fish scale collagen calcium chelate peptide hydrolysate, fish scale collagen calcium peptide chelate, or calcium chelate peptide added to the product is 1% to 20% by mass.

[0047] Furthermore, the dosage form of the product is powder, capsule, tablet, oral liquid, granule or emulsion.

[0048] The present invention has the following advantages and effects compared with the prior art:

[0049] (1) Innovative use of raw materials: For the first time, the natural composite system of fish scales "collagen-hydroxyapatite" is clearly used. No separation pretreatment is required. Endogenous calcium ions are directly chelated in situ, avoiding waste and purity problems caused by the addition of exogenous calcium sources. At the same time, the high-value utilization of fish by-products is realized. The raw material cost is only 1 / 3 to 1 / 2 of that of traditional calcium raw materials. The calcium retention rate of raw materials is ≥84.16%, which is more than 60% higher than that of traditional decalcification process.

[0050] (2) The process is extremely simple, advanced and efficient, and has great industrialization potential: Compared with the "two-time enzymatic hydrolysis + alcohol precipitation" of patent CN120665975A and the "chemical hydrolysis + impurity removal" of other fish scale related technologies, the present invention adopts "high pressure homogenization + single pepsin enzymatic hydrolysis", which shortens the process by more than 30%, eliminates the need for chemical reagents and membrane separation equipment, and reduces energy consumption and production costs; the yield of collagen calcium chelate peptides reaches 83.4% (based on 100 g of dried starting fish scale powder), which is 32.05% higher than the traditional process without homogenization (yield 63.16%) and 233.6% higher than the traditional acid decalcification process (yield 25.0%); the equipment used are all commonly used equipment in the food industry, and the process parameter range is suitable for large-scale production.

[0051] (3) Product safety and environmental protection and energy saving: There are no chemical hydrolysis, heavy metal removal and other steps, and the product has no chemical residue; there is no wastewater or waste residue discharge throughout the process, and high pressure homogenization and spray drying are low energy consumption operations, which meet the requirements of green production and have better overall performance than existing technology products.

[0052] (4) The product has a clear function and strong market competitiveness: The prepared collagen calcium chelate peptide is rich in calcium-affinity amino acids. In the Caco-2 cell model, the calcium transport capacity is increased by ≥60% compared with the calcium chloride control group, and is also significantly higher than that of eggshell membrane-derived chelate calcium (prepared by CN120665975A method). This solves the core pain point of low absorption efficiency of traditional calcium supplements and provides direct experimental evidence for the functional claims of calcium supplements.

[0053] (5) Wide range of applications and significant transformation value: The product can be added as a core functional ingredient to various calcium supplements such as powders, capsules, and tablets. The amount added is flexible (1%-20%), which can meet the needs of people of different age groups. It conforms to the market trend of natural, efficient and safe products and has broad prospects for industrialization. Attached Figure Description

[0054] Figure 1 The images show scanning electron microscope (SEM) images (6000×) of fish scale powder homogenized under high pressure and fish scale powder homogenized under high pressure in Example 1, the effect of four proteases on the yield of fish scale collagen protein hydrolysate, and the effect of single factors on the yield of fish scale collagen protein hydrolysate.

[0055] Figure 2 The graph shows the effect of high-pressure homogenization on the yield of collagen calcium chelate peptides in Example 1.

[0056] Figure 3 This is a comparison chart of the calcium content of fish scale powder and fish scale collagen calcium chelate peptide hydrolysate in Example 1.

[0057] Figure 4 The graph shows the effect of fish scale collagen calcium chelate peptide hydrolysate on the survival rate of Caco-2 cells in Example 1.

[0058] Figure 5 This is a graph showing the transepithelial resistivity of Caco-2 cells cultured for different times in Example 1.

[0059] Figure 6 Morphological images of Caco-2 cells cultured for different times in Example 1.

[0060] Figure 7 This is a comparison of calcium transport in the Caco-2 cell model between fish scale collagen calcium chelate peptide hydrolysate and calcium chloride in Example 1.

[0061] Figure 8 The graph shows the effect of a single factor on the yield of calcium peptide chelates in Example 2.

[0062] Figure 9 The graph shows the effect of alcohol precipitation temperature on the yield of calcium peptide chelates in Example 2.

[0063] Figure 10 This is a comparison chart of the calcium content of fish scale powder and fish scale collagen calcium peptide chelate in Example 2.

[0064] Figure 11 The figure shows the characterization results of the fish scale collagen calcium peptide chelate in Example 2.

[0065] Figure 12The figure shows the effect of fish scale collagen calcium peptide chelate on the proliferation of MC3T3-E1 cells and the effect on ALP activity of MC3T3-E1 cells in Example 2.

[0066] Figure 13 The figure shows the effect of fish scale collagen calcium peptide chelate on the mineralization of MC3T3-E1 cells in Example 2.

[0067] Figure 14 This is a diagram showing the effect of fish scale collagen calcium chelate peptide treatment on osteoblast proliferation in Example 3.

[0068] Figure 15 This is a graph showing the effect of fish scale collagen calcium chelating peptide on osteoblast ALP activity in Example 3.

[0069] Figure 16 This is a diagram showing the effect of fish scale collagen calcium chelating peptide on osteoblast mineralization in Example 3.

[0070] Figure 17 This is a quantitative analysis diagram of the effect of fish scale collagen calcium chelating peptide on osteoblast mineralization in Example 3.

[0071] Figure 18 The figure shows the effect of fish scale collagen calcium chelate peptide treatment on the survival rate of Caco-2 cells for 24 hours in Example 3.

[0072] Figure 19 This is a graph showing the effect of fish scale collagen calcium chelating peptide treatment on calcium transport in Caco-2 cell monolayers in Example 3.

[0073] Figure 20 This is a diagram showing the effect of fish scale collagen calcium chelating peptide on calcium retention in Caco-2 cells in Example 3.

[0074] Figure 21 This is a relative fluorescence intensity diagram of fish scale collagen calcium chelate peptide in Caco-2 cells in Example 3.

[0075] Figure 22 This is a diagram showing the molecular docking results of fish scale collagen calcium chelate peptide in Example 3.

[0076] Figure 23 This is a secondary mass spectrum of the fish scale collagen calcium chelate peptide in Example 3. Detailed Implementation

[0077] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0078] All experiments in this invention were performed in triplicate. Excel software (2024) was used for calculations. The differences between samples were analyzed using SPSS software (IBM, NY, USA) for one-way ANOVA. Data plots were created using Prism 10.

[0079] Example 1-1: Preparation of fish scale collagen calcium chelate peptide hydrolysate (tilapia scale raw material)

[0080] 1. Raw material pretreatment: Take 1 kg of tilapia processing by-product fish scales, remove impurities, rinse 3 times with tap water, dry in a 60℃ oven for 6 hours, pulverize and pass through a 60-mesh sieve to obtain about 310 g of dried fish scale powder with a moisture content of 8.5%.

[0081] 2. High-pressure homogenization pretreatment: Take 100 g of dried fish scale powder (as the starting reference material), add 1867 mL of deionized water to prepare a suspension with a mass fraction of 5%, mix evenly at a stirring rate of 200 r / min, and cycle 10 times at a pressure of 50 MPa using a KS-2000M high-pressure homogenizer. The treated suspension is then freeze-dried using an Alpha 2-4 LD plus freeze dryer to obtain 91 g of high-pressure homogenized fish scale powder.

[0082] 3. Enzymatic hydrolysis preparation: Add all 91 g of the high-pressure homogenized fish scale powder to deionized water, adjust the substrate concentration to 5%, and add 0.091 g of pepsin (enzyme-to-substrate ratio 0.1%, enzyme activity 2.5 × 10⁻⁶). 5 The system (U / g) was adjusted to pH 2.0 with hydrochloric acid and placed in a 40℃ constant temperature water bath for 2 h of enzymatic hydrolysis, with stirring for 2 min every 30 min during the process.

[0083] 4. Product separation: After enzymatic hydrolysis, the hydrolysate was poured into an L530-cence benchtop centrifuge and centrifuged at 4000 rpm for 10 min. The supernatant was collected and freeze-dried at -45℃ and 20 Pa for 6 h to obtain 83.4 g of collagen calcium chelate peptide product.

[0084] 5. Yield calculation: Based on the unified standard (weight of collagen calcium chelate peptide ÷ initial weight of dried fish scale powder × 100%), the yield = (83.4 g ÷ 100 g) × 100% = 83.4%.

[0085] The microstructure differences between untreated fish scale powder and fish scale powder homogenized under 50 MPa high pressure were compared and analyzed using SEM at 6000x magnification. The results are as follows: Figure 1As shown, the original fish scale powder without high-pressure homogenization exhibits a tightly packed, sheet-like structure with a rough and dense surface, containing only a few irregular small fragments and no obvious pores. After high-pressure homogenization at 50 MPa, the microstructure of the fish scale powder undergoes a radical change: the original dense sheet-like structure is completely destroyed, forming a loose and porous fibrous network structure, accompanied by numerous branched protrusions and a few uniformly dispersed spherical particles, resulting in a highly dispersed overall structure. High-pressure homogenization at 50 MPa can effectively destroy the dense composite structure of fish scales, achieving a loosened microstructure and exposing enzyme cleavage sites. This is a key pretreatment method to improve the efficiency of fish scale enzymatic hydrolysis and ensure the efficient subsequent peptide-calcium chelation reaction.

[0086] Examples 1-2: Preparation of fish scale collagen calcium chelate peptide enzymatic hydrolysate (grass carp scales as raw material, to verify the universality of the process)

[0087] 1. Raw material pretreatment: Take 1 kg of grass carp processing by-product fish scales, remove impurities, rinse 3 times with tap water, dry in a 60℃ oven for 6 hours, pulverize and pass through a 60-mesh sieve to obtain about 305 g of dried fish scale powder with a moisture content of 8.2%.

[0088] 2. High-pressure homogenization pretreatment: Take 100 g of dried grass carp scale powder (starting standard raw material) and treat it according to the high-pressure homogenization conditions of Example 1 (10 cycles at 50 MPa pressure) to obtain 89 g of high-pressure homogenized fish scale powder.

[0089] 3. Enzymatic hydrolysis and product separation: 89 g of high-pressure homogenized fish scale powder was processed under the enzymatic hydrolysis and separation conditions of Example 1 to obtain 82.7 g of collagen calcium chelate peptide product.

[0090] 4. Yield calculation: Yield = (82.7 g ÷ 100 g) × 100% = 82.7%, which is not significantly different from the yield of tilapia scale raw material group, confirming the adaptability of the process of the present invention to fish scales of different fish.

[0091] Examples 1-3: Process Comparison Experiments (all based on 100 g of dried tilapia scale powder as a uniform standard)

[0092] Two control groups were set up to compare the process effects with those of Example 1 (high-pressure homogenization group). All three groups used 100 g of dried tilapia scale powder as a uniform baseline, and the yield calculation followed the uniform standard of "weight of collagen calcium chelate peptide ÷ weight of 100 g of initial dried fish scale powder × 100%".

[0093] (1) Control group 1 (without high pressure homogenization pretreatment): 100 g of dried tilapia scale powder (starting standard raw material) was taken and directly operated according to the enzymatic hydrolysis conditions of implementation 1 (pH 2.0, 40℃, enzyme-to-substrate ratio 0.1%, substrate concentration 5%, enzymatic hydrolysis for 2 h) without high pressure homogenization. After centrifugation and freeze drying, 63.16 g of collagen calcium chelate peptide product was obtained, and the yield was (63.16 g ÷ 100 g) × 100% = 63.16%.

[0094] (2) Control group 2 (traditional acid decalcification process): 100 g of dried tilapia scale powder (starting standard raw material) was soaked in 5% hydrochloric acid solution for decalcification for 4 h, filtered and washed until neutral and then dried (62 g of decalcified fish scale powder was obtained). Then, the enzymatic hydrolysis and separation conditions of Example 1 were followed to finally obtain 25.0 g of collagen calcium chelate peptide product. The yield was (25.0 g ÷ 100 g) × 100% = 25.0%.

[0095] The core performance indicators of the three processes are compared in Table 1 below:

[0096] Table 1: Comparison of Core Indicators

[0097]

[0098] The comparative results show that the high-pressure homogenization-assisted enzymatic hydrolysis process adopted in this invention has a significantly better product yield than the traditional process under the same benchmark, with a 32.05% improvement compared to the process without homogenization and a 233.6% improvement compared to the acid decalcification process. This fully demonstrates the core advantages of high-pressure homogenization pretreatment in destroying the dense structure of fish scales and improving enzymatic hydrolysis efficiency, and solves the key technical pain points of the traditional process.

[0099] Examples 1-4: Optimization of Enzymatic Hydrolysis Process

[0100] (1) Screening of proteases

[0101] Using high-pressure homogenized fish scale powder as raw material, four different proteases were selected for enzymatic hydrolysis under optimal conditions: papain (pH 6, 55℃), neutral protease (pH 7, 50℃), pepsin (pH 2, 40℃), and trypsin (pH 8, 37℃). Other experimental conditions were fixed as follows: enzyme dosage 0.3%, substrate concentration 5%, and hydrolysis time 2 h. The yield of collagen peptides was determined, and the optimal protease for preparing fish scale collagen calcium chelate peptides was selected.

[0102] Experimental data showed that there were statistically significant differences in the enzymatic hydrolysis effects of the four proteases on high-pressure homogenized fish scale powder (P<0.05), with the hydrolysis efficiency ranked as follows: pepsin > neutral protease > papain > trypsin. Among them, pepsin exhibited the best hydrolysis efficiency, with its collagen protein hydrolysate yield significantly higher than the other three enzymes. Figure 1 ).

[0103] (2) Single-factor experiment of enzymatic hydrolysis process

[0104] Single-factor experiments on enzymatic hydrolysis were conducted by changing one factor while keeping others constant to investigate the effects of substrate concentration, enzyme-to-substrate ratio, pH, temperature, and time on the calcium chelating activity of tilapia scales. The baseline conditions for single-factor hydrolysis were: substrate concentration 3%, enzyme-to-substrate ratio 0.3%, pH 2, hydrolysis temperature 40℃, and hydrolysis time 2 h. The level gradients for each factor were as follows: substrate concentration: 1, 2, 3, 4, 5%; enzyme-to-substrate ratio: 0.1, 0.2, 0.3, 0.4, 0.5%; pH: 1.0, 1.5, 2.0, 2.5, 3.0; hydrolysis temperature: 30, 35, 40, 45, 50℃; hydrolysis time: 1, 2, 3, 4, 5 h.

[0105] Depend on Figure 1 As shown, the highest yield of collagen protein hydrolysate was achieved when the enzyme-to-base ratio was 0.1%. With increasing enzyme-to-base ratio, the yield of collagen protein hydrolysate decreased slightly, but the difference was not statistically significant (P>0.05). Considering factors such as enzymatic hydrolysis effect, production cost, and product quality, 0.1% was determined to be the optimal enzyme-to-base ratio. Enzymatic hydrolysis temperature had a significant regulatory effect on the yield of collagen protein hydrolysate: as the temperature increased from 30℃ to 40℃, the yield showed a continuous upward trend, reaching its maximum at 40℃; when the temperature further increased to 50℃, the yield decreased significantly (P<0.05). Therefore, 40℃ was determined to be the optimal enzymatic hydrolysis temperature.

[0106] (3) Effect of enzymatic hydrolysis time

[0107] Depend on Figure 1As shown, the yield of collagen protein hydrolysate gradually increased as the enzymatic hydrolysis time increased from 1 h to 5 h, but the differences between time points were not statistically significant (P>0.05), with the yield reaching its highest level at 5 h. Considering actual production needs, the yield increase after extending the hydrolysis time to 2 h was less than 5%. Further extending the time could slightly improve the yield, but it would increase energy consumption and production cycle, reducing production efficiency. Taking into account yield, production efficiency, and cost, 2 h was determined to be the optimal hydrolysis time. pH value had a highly significant impact on the yield of collagen protein hydrolysate (P<0.05): as the pH increased from 1.0 to 2.0, the yield increased sharply, reaching a peak at pH 2.0; when the pH continued to increase to 3.0, the yield decreased significantly. Therefore, pH 2.0 was determined to be the optimal hydrolysis condition. The effect of substrate concentration on the yield of collagen protein hydrolysate showed a trend of "rapid increase followed by stabilization": as the substrate concentration increased from 1% to 3%, the yield showed a significant upward trend (P<0.05); when the substrate concentration increased from 3% to 5%, the rate of increase in yield slowed down, reaching its maximum at 5%. Considering both the enzymatic hydrolysis effect and the system flowability, 5% was determined to be the optimal substrate concentration. Based on the above single-factor experimental results, the final process conditions for preparing collagen protein hydrolysate from high-pressure homogenized fish scale powder by pepsin hydrolysis were determined to be: enzyme-to-substrate ratio 0.1%, hydrolysis temperature 40℃, hydrolysis time 2 h, pH 2.0, and substrate concentration 5%.

[0108] Examples 1-5: Product performance testing (using the product from Example 1 as an example)

[0109] The collagen calcium chelating peptide prepared in Example 1 was subjected to system performance testing, and the results are as follows:

[0110] (1) Amino acid composition analysis: According to GB 5009.124-2016 standard, the amino acid analyzer was used to determine that the product contains 18 common amino acids, of which aspartic acid has a relative percentage of 5.63% and glutamic acid has a relative percentage of 9.86%. It is rich in calcium-affinity amino acids, which provide structural support for peptide calcium chelation and absorption promotion activities (Table 2).

[0111] Table 2: Results of amino acid composition analysis

[0112]

[0113] (2) Calcium content determination: According to GB 5009.92-2016 standard, the calcium content of the product was determined by atomic absorption spectrophotometry. The calcium content was 60.32 μg / mg, and the calcium retention rate was 84.16% (calculated based on the calcium content of 100 g of initial dried fish scale powder raw material, the calcium content of tilapia scale powder raw material was 71.67±3.73 μg / mg). Most of the natural calcium in the fish scales was effectively retained. Figure 3 This result fully demonstrates the core advantages of the "in-situ calcium chelation" process of this invention. Traditional fish scale enzymatic hydrolysis processes require pre-decalcification treatment through acid hydrolysis and EDTA chelation, which not only causes the loss of more than 80% of natural calcium but also poses a risk of chemical reagent residue and fails to achieve the synergistic utilization of collagen and natural calcium. In contrast, this invention uses high-pressure homogenization pretreatment to destroy the dense structure of fish scales, combined with optimized acidic enzymatic hydrolysis conditions, so that the natural calcium ions released by the dissolution of hydroxyapatite in the fish scales can immediately chelate with the collagen peptides produced by enzymatic hydrolysis. The carboxyl and amino functional groups in the peptide chains lock the calcium ions firmly in the product through electrostatic interactions and coordination bonds, achieving a one-step synergistic utilization of "efficient enzymatic hydrolysis of collagen - in-situ chelation of natural calcium". This avoids calcium loss and the use of chemical reagents, and converts inorganic calcium into peptide-calcium chelates with higher bioavailability, breaking through the bottleneck of traditional technology in terms of process design.

[0114] (3) Cytotoxicity evaluation: Cytotoxicity experiments were performed according to the method reported by CHEN, with slight modifications. Logarithmic growth phase Caco-2 cells were digested with trypsin and then subjected to 5 × 10⁻⁶ molten iron. 5 Cells were seeded at a density of cells / mL into 96-well plates and cultured for 24 h. After incubation, the complete culture medium was discarded, and 100 μL of collagen calcium chelate peptide hydrolysate solution at different concentrations (25, 50, 100, 250, 500, 1000 μg / mL) was added to each well. DMEM was used as a blank control group, and each concentration was used in 6 replicates. After 24 h of treatment, 100 μL of 0.5 mg / mL MTT solution was added to each well for 4 h. After 4 h, the MTT solution was removed, and 100 μL of DMSO solution was added to each well. The plates were shaken for 15 min, and the absorbance was measured at 490 nm using a microplate reader.

[0115] The results showed that, within the tested concentration range, collagen calcium chelate peptide hydrolysate did not exhibit cytotoxicity against Caco-2 cells. Figure 4 ).

[0116] (4) Detection of intestinal calcium absorption-promoting activity: A Caco-2 cell monolayer model was established (cultured for 21 days, TEER value ≥ 478 Ω·cm). 2 ), with calcium chloride as the control group, in Ca 2+ Calcium transport experiments were conducted at a concentration of 300 μg / mL.

[0117] Establishment of the Caco-2 cell monolayer model: Caco-2 cells in the logarithmic growth phase were digested with trypsin and then subjected to a 4×10⁻⁶ layer of lye per cell. 5Cells were seeded at a density of [number] cells / mL into Transwell 12-well plates, with medium changed every other day, and cultured for 21 days. Transepithelial electrical resistance (TEER) was measured on days 7, 14, and 21. A TEER greater than 280 Ω / cm was considered acceptable. 2 When the cells form a monolayer, they can be used for subsequent calcium transport experiments. (Comprehensive morphological observation) Figure 6 ) and TEER analysis ( Figure 5 This study successfully constructed a Caco-2 cell monolayer model, which can be used for subsequent calcium transport experiments.

[0118] Calcium transport assay: The calcium transport assay was performed according to the method reported by LIAO, with slight modifications. A new 12-well plate was taken, and 1.5 mL of HBSS solution was added to each well. The plate was incubated for 1 h. The culture medium in the Transwell 12-well plate was discarded, and the cells were washed three times with preheated HBSS solution. The HBSS solution was aspirated, and the monolayer of cells was transferred to a new 12-well plate containing 1.5 mL of HBSS solution. 0.5 mL of HBSS solution was added to the top side, and the plate was incubated for 30 min. Subsequently, the HBSS solution on the top side was discarded, and 0.5 mL of a certain concentration of enzyme hydrolysate (Ca) was added to the top side. 2+ The concentration was 300 μg / mL. The control group received only 0.5 mL of CaCl2 solution (Ca...). 2+ (Concentration 300 μg / mL). 1 mL of HBSS solution was collected from the substrate side at 30, 60, 120, and 180 min for the determination of calcium transport (flame atomic absorption spectrophotometry), and an equal volume of HBSS solution was added to maintain system stability.

[0119] The results showed that after 180 min of transport, the calcium transport capacity of the product of this invention was 26.45 μg / well, which was 62.97% higher than that of the calcium chloride control group (16.23 μg / well), demonstrating excellent intestinal calcium absorption promoting activity. Figure 7 ).

[0120] Examples 1-6: Application of the product in calcium supplement tablets

[0121] The collagen calcium chelate peptide prepared in Example 1 was mixed with maltodextrin and lactose in a mass ratio of 1:3:2. Magnesium stearate of 0.5% by total mass was added as a lubricant. The mixture was then compressed into tablets using a rotary tablet press. Each tablet weighed 0.5 g and contained 50 mg of collagen calcium chelate peptide and approximately 3.02 mg of calcium. The tablets had a disintegration time of ≤15 min, a good taste, and better calcium supplementation compliance than traditional calcium carbonate tablets.

[0122] Examples 1-7: Application of the product in oral calcium supplements

[0123] Take 5 g of the collagen calcium chelate peptide prepared in Example 1, add 950 mL of purified water, stir to dissolve, add 5 g of xylitol for flavoring, and then add 0.1 g of xanthan gum as a stabilizer. After homogenization, sterilize at 121℃ for 15 min, and fill to obtain calcium supplement oral solution. Each 100 mL bottle contains 0.5 g of collagen calcium chelate peptide and about 30.16 mg of calcium. The oral solution is clear and transparent and has good stability.

[0124] Example 2: Optimization of the chelation process

[0125] 1. Experimental Methods

[0126] 1.1 High-pressure homogenization pretreatment of fish scales

[0127] Tilapia scales were washed with tap water to remove impurities, dried in a 60℃ oven, and then pulverized through a 60-mesh sieve to obtain fish scale powder. 5% of the fish scale powder was subjected to high-pressure homogenization at 50 MPa for 10 cycles to obtain a fish scale homogenate solution. This solution was then freeze-dried to obtain high-pressure homogenized fish scale powder, which was stored in a -20℃ refrigerator for later use.

[0128] 1.2 Preparation of fish scale collagen calcium chelate peptide hydrolysate

[0129] High-pressure homogenized fish scale powder was used as raw material, and enzymatic hydrolysis with pepsin was performed under the following conditions: enzyme-to-substrate ratio 0.1%, hydrolysis temperature 40℃, hydrolysis time 2 h, pH 2.0, and substrate concentration 5%. The resulting fish scale collagen calcium chelate hydrolysate was obtained by centrifugation and stored at -20℃ for later use. Unhomogenized fish scale powder was used as a control, and hydrolysates were prepared under the same conditions.

[0130] 1.3 Chelation preparation of fish scale collagen calcium peptide chelates

[0131] Take a certain amount of enzymatic hydrolysate, adjust the temperature to (40, 45, 50, 55, 60) ℃ and pH to (6.0, 6.5, 7.0, 7.5, 8.0), and perform the chelation reaction for 1 h, stirring once every 15 minutes. Add 4 volumes of anhydrous ethanol and incubate overnight (10 h) at room temperature or 4 ℃. Centrifuge at 4000 rpm for 10 min, collect the precipitate, dry it in a 60 ℃ oven, and weigh the calcium peptide chelate. Calculate the yield of collagen calcium peptide chelate according to the following formula: Collagen calcium chelate peptide yield % = Dry weight of chelate (g) / Dry weight of raw material (g) × 100%. The method for determining calcium content refers to "GB 5009.92-2016 National Food Safety Standard - Determination of Calcium in Food".

[0132] 1.4 Decalcification of Fish Scale Collagen Calcium Peptide Chelates

[0133] 1 g of calcium peptide chelate was dissolved in 5 mL of deionized water, and 5 mL of saturated disodium EDTA solution was added for decalcification. After reacting at 4°C for 1 day, 5 times the volume of anhydrous ethanol was added, the precipitate was removed by centrifugation, the supernatant was concentrated by rotary evaporation, and then freeze-dried to obtain fish scale collagen calcium-binding peptide. The decalcification rate of the calcium-binding peptide reached 77.36%.

[0134] 1.5 Characterization experiment of fish scale collagen calcium peptide chelate: Refer to Examples 1-5.

[0135] 1.6 Analysis of peptide composition in collagen calcium peptide chelates

[0136] The sample was analyzed by LC-MS / MS equipped with an online nanospray ionization source. The entire system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) with a tandem EASY-nanoLC1200. A total of 5 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated by a gradient over 60 min, with the column flow rate controlled at 300 nL / min, column temperature at 40 °C, and electrospray voltage at 2 kV. The gradient started at 4% B phase, increased non-linearly to 50% within 53 min 40 sec, increased to 95% within 40 sec, and held for 5 min 40 sec. The tandem mass spectra were analyzed using PEAKSStudio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database used was uniprot-Oreochromis mossambicus_2024 (version 2024, 488 entries). Enzymatic digestion was set to None. Search parameters: Fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 10 ppm, variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98, hydroxylation (KP) 16, Ca 40.04. Protein card value: contains at least 1 unique peptide; peptide card value: -10 lgP ≥ 20.

[0137] 1.7 Evaluation of the intestinal calcium transport activity of collagen calcium peptide chelates: Refer to Examples 1-5.

[0138] 1.8 Evaluation of osteogenic activity of collagen calcium peptide chelates

[0139] 1.8.1 MC3T3-E1 Cell Culture: Cells were cultured in α-MEM containing 10% fetal bovine serum and 1% penicillin-dextrose antibody at 37°C in a cell culture incubator with 5% CO2. The medium was changed every three days. When the cells covered approximately 80% of the culture flask, the cells were digested with 0.25% trypsin for 2 min and centrifuged at 1000 r / min for 5 min. The supernatant was discarded, and the cells were resuspended in complete culture medium, mixed thoroughly, transferred to T25 culture flasks, and then placed in a cell culture incubator.

[0140] 1.8.2 Cell proliferation experiment: Cells were inoculated at 5 × 10⁻⁶ cells / day. 3 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured for 24 h, with PBS added to the edge wells. After 24 h of cell adhesion and growth, the culture medium was aspirated, and different concentrations of calcium peptide chelate medium (0, 1, 10, 50, 100, 200 µg / mL) were added for 24 h of further culture. Then, the sample medium was removed, and 100 µL of 0.5 mg / mL MTT solution was added to each well for 4 h. Subsequently, the MTT solution was removed, and 150 µL of DMSO solution was added. The absorbance was measured at 570 nm using a microplate reader.

[0141] 1.8.3 ALP activity assay: Cells were injected at a concentration of 3 × 10⁻⁶ cells / cells. 6 Cells were cultured in 12-well plates at a density of 1 / mL for 24 h. After 24 h of culture, the cells were treated with differentiation medium containing different concentrations of calcium-titanium chelates (0, 1, 10, 50 µg / mL) for 7 days. The medium was changed every two days. After 7 days, the cell supernatant was aspirated, 0.6 mL of cell lysis buffer was added, and the cells were collected in 2 mL centrifuge tubes. Cell protein concentration and ALP activity were measured according to the instructions of the BCA protein assay kit and the ALP assay kit, respectively.

[0142] 1.8.4 Alizarin Red staining: Cells were stained at a concentration of 3 × 10⁻⁶. 6 Cells / mL were cultured in 12-well plates for 24 h. After 24 h, they were cultured in differentiation medium containing different concentrations of calcium peptide chelate (0, 1, 10, 50 µg / mL) for 21 days. The medium was changed every two days. After 21 days of induction, the cells were washed twice with PBS, fixed with 4% paraformaldehyde at 37°C for 30 min, rinsed twice with deionized water, and then rinsed once with PBS to remove non-specific staining. Subsequently, the cells were stained with alizarin red for 5 min, washed three times with distilled water, and photographed under a microscope.

[0143] 1.9 Data Processing: All experiments were performed in triplicate. Excel (2024) was used for calculations. The differences between samples were analyzed using SPSS (IBM, NY, USA) for one-way ANOVA. Data plots were created using Prism 10.

[0144] 2 Results and Analysis

[0145] 2.1 Chelation preparation of fish scale collagen calcium peptide chelates

[0146] 2.1.1 Effect of pH on chelate yield: The results showed that within the pH range of 6.0–7.0, the chelate yield increased significantly with increasing pH (P<0.05); there was no significant difference in yield between pH 7.0 and 8.0 (P>0.05), and the yield reached 82.50% at pH 8.0. Considering both yield and process economy, pH 7.0 was selected as the optimal condition. Figure 8 ).

[0147] 2.1.2 Effect of Temperature on Chelate Yield: The results show that the chelate yield decreases with increasing temperature. 40℃ was selected as the optimal chelation temperature. Figure 8 ).

[0148] 2.1.3 Effect of alcohol precipitation temperature on the yield of calcium peptide chelates: The results showed that the yield of chelates precipitated at 4℃ was significantly higher than that precipitated at room temperature (P<0.05). Figure 9 ).

[0149] 2.1.4 Calcium content determination: Under optimal conditions, the chelate yield reached 81.41%, and the calcium content was 71.44 ± 3.01 μg / mg. The retention rate of natural calcium in fish scales in the scale collagen calcium peptide chelate was as high as 99.68%. Figure 10 ).

[0150] From a resource utilization perspective, fish scales, as a byproduct of aquatic product processing, contain natural calcium resources (mainly in the form of hydroxyapatite) that are tightly bound to collagen. Traditional processes often fail to achieve synergistic utilization of both, leading to significant calcium loss. This study addresses this issue by using high-pressure homogenization pretreatment to disrupt the dense structure of fish scales, fully exposing calcium ions in the hydroxyapatite. Following this, targeted enzymatic decomposition by pepsin releases collagen peptides. The peptide chains form stable coordination bonds with calcium ions through carboxyl oxygen, carbonyl oxygen, and amino nitrogen groups. Finally, low-temperature alcohol precipitation achieves efficient separation of the calcium peptide chelates. The entire process does not introduce exogenous calcium, relying entirely on the natural calcium resources of the fish scales, with a calcium retention rate approaching 100%. This truly achieves "homogeneous synergistic utilization" of collagen and calcium resources, avoiding resource waste and aligning with the industrial trend of green and sustainable development. From a product value perspective, the high calcium retention rate not only reflects the resource utilization efficiency of the process but also ensures the functional value of the product. The calcium content of calcium peptide chelates directly affects their application potential as calcium supplements. In this study, the calcium content of the product was consistently around 71 μg / mg, and it existed in a coordinated state, making it more easily absorbed by the human body compared to traditional inorganic calcium supplements. Simultaneously, the efficient retention of natural calcium resources avoids the introduction of impurities that may result from the addition of exogenous calcium, improving the safety and naturalness of the product and laying a material foundation for the subsequent development of functional calcium supplements. In summary, the calcium content determination results show that the chelation process established in this invention achieves a 99.68% efficient retention of natural calcium resources from fish scales without the addition of exogenous calcium, achieving synergistic utilization of collagen and calcium resources. The process has advantages such as high resource utilization and good product safety.

[0151] 2.2 Structural Feature Analysis of Fish Scale Collagen Calcium Peptide Chelate

[0152] Scanning electron microscopy (SEM) was used to characterize the microstructure of the raw fish scale powder and the target product, calcium peptide chelate. Untreated fish scale powder exhibited a dense, smooth, sheet-like stacked structure. This structure is a natural structure formed by the tight cross-linking of collagen and hydroxyapatite in fish scales through hydrogen bonds, hydrophobic interactions, and ionic bonds. This structure is dense and lacks pores. In contrast, the calcium peptide chelate exhibited significantly different microscopic characteristics: the product showed a loose, porous, flocculent aggregate structure with numerous microspheres and clearly visible pores, contrasting sharply with the dense, sheet-like structure of the raw material. Figure 11 ).

[0153] 2.3 Effect of collagen calcium peptide chelates on the intestinal calcium absorption-promoting activity of Caco-2

[0154] 2.3.1 Cytotoxicity Analysis: The MTT assay was used to assess the toxicity of fish scale collagen calcium peptide chelate to Caco-2 intestinal epithelial cells to verify its biocompatibility. Results showed that within the experimental concentration range of 25–1000 μg / mL, the survival rate of Caco-2 cells treated with the chelate remained above 95%, with no significant difference compared to the blank control group (cell survival rate 100%) (P>0.05). This result indicates that the chelate has no significant toxicity to intestinal epithelial cells within the physiological and potential application concentration range and possesses good biocompatibility. Figure 11 ).

[0155] 2.3.2 Calcium transport experiment: With prolonged transport time, the calcium transport amount in both the chelate group and the CaCl2 control group showed a time-dependent increasing trend, but the transport rate of the chelate group was significantly higher than that of the control group: at 180 min of transport, the calcium transport amount in the chelate group reached 19.60±1.57 μg / well, significantly higher than the 14.67±1.84 μg / well of the CaCl2 control group (P<0.05), and the calcium transport efficiency was 33.61% higher than that of the control group. This result quantitatively confirms that fish scale collagen calcium peptide chelate has significant intestinal calcium transport-promoting activity. Figure 11 ).

[0156] The intracellular calcium ion concentration was visualized using the Fluo-4 AM fluorescent probe Caco-2. This probe has good cell membrane permeability and, after entering the cell, is hydrolyzed by intracellular esterases into Fluo-4. Fluo-4 specifically binds to free calcium ions and produces green fluorescence. The fluorescence intensity is positively correlated with the intracellular calcium ion concentration, making it a classic tool for intuitively reflecting cellular calcium uptake efficiency. Results are as follows: Figure 11 As shown in the figure. Compared with the blank control group, the intracellular fluorescence intensity of both the CaCl2 control group and the calopeptide chelate group was significantly enhanced, with the fluorescence intensity of the calopeptide chelate group being slightly higher than that of the CaCl2 control group.

[0157] 2.4 Osteogenic Activity Analysis of Collagen Calcium Peptide Chelates

[0158] 2.4.1 Effect of collagen calcium peptide chelate on osteoblast proliferation activity: The effect of fish scale collagen calcium peptide chelate on the proliferation of MC3T3-E1 pre-osteoblasts was systematically evaluated using the MTT assay. The results are as follows: Figure 12As shown in the figure. The experimental results showed that the effect of fish scale collagen calcium peptide chelate on the proliferation of MC3T3-E1 cells exhibited a concentration-dependent "first increase then decrease" trend: within the concentration range of 1–50 μg / mL, the cell proliferation rate increased significantly with increasing concentration; when the concentration exceeded 50 μg / mL, the proliferation rate gradually decreased, but was still higher than that of the blank control group. Among them, 10–50 μg / mL was the optimal proliferation concentration range, with a proliferation rate of 127.79%–130.76% at 48 h and maintained at 128.41%–130.47% at 72 h, which was significantly different from the blank control group (P<0.05).

[0159] 2.4.2 Effect of collagen calcium peptide chelate on osteoblast differentiation activity: The changes in ALP activity in MC3T3-E1 cells after treatment with different concentrations of collagen calcium peptide chelate were detected using an ALP activity assay kit. Results are as follows: Figure 12 As shown, ALP activity increased in a concentration-dependent manner with increasing concentration of collagen calcium peptide chelate. At a concentration of 50 μg / mL, ALP activity reached a maximum of 116.34%, significantly higher than that of the control group.

[0160] 2.4.3 Effect of collagen calcium peptide chelate on osteoblast mineralization activity: The effect of fish scale collagen calcium peptide chelate on MC3T3-E1 cell mineralization was evaluated using Alizarin Red staining. The results are as follows: Figure 13 As shown in the figure. Experimental results indicate that fish scale collagen calcium peptide chelate significantly promotes mineralization in MC3T3-E1 cells, exhibiting a clear dose-dependent effect: as the chelate concentration increases from 1 μg / mL to 50 μg / mL, the number and volume of mineralized calcium nodules gradually increase, and the Alizarin Red staining intensity continuously deepens, reaching its optimal mineralization effect at a concentration of 50 μg / mL. In summary, fish scale collagen calcium peptide chelate demonstrates comprehensive osteogenic activity through a continuous action of "promoting proliferation and inducing mineralization," providing solid experimental evidence for its use as a raw material for bone health-related functional products.

[0161] Example 3: Activity of calcium chelating peptides

[0162] 1. Experimental Methods

[0163] 1.1 Enzymatic hydrolysis of fish scale collagen calcium chelate peptides and preparation of calcium peptide chelates

[0164] Take a certain amount of enzymatic hydrolysate, adjust the temperature to 40℃ and pH to 7.0, and perform the chelation reaction for 1 h. Stir every 15 minutes, add 4 times the volume of anhydrous ethanol, and incubate overnight (10 h) at room temperature or 4℃. Centrifuge at 4000 rpm for 10 min, take the precipitate, dry it in an oven at 60℃, weigh the calcium peptide chelate, and calculate the yield of collagen calcium peptide chelate according to the following formula.

[0165] 1.2 Peptide composition analysis and identification

[0166] The calcium peptide chelate sample was dissolved in 0.1% formic acid aqueous solution to prepare a 1 mg / mL solution. After filtration through a 0.22 μm filter membrane, the solution was analyzed using an Orbitrap Fusion Lumos mass spectrometer with EASY-nanoLC 1200 in series. The chromatographic column was an Acclaim PepMap C18 column (75 μm × 25 cm), with a column temperature of 40℃ and a flow rate of 300 nL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution program was as follows: 4% B phase as the initial phase, nonlinearly increasing to 50% B phase within 53 min 40 s, increasing to 95% B phase within 40 s, and maintaining this level for 5 min 40 s. Electrospray ionization was performed at 2 kV in positive ion mode.

[0167] Tandem mass spectra were analyzed using PEAKS Studio 10.6 software. The database was uniprot-Oreochromismossambicus_2024 (version 2024, 488 entries), with the None digestion mode set. Search parameters: parent ion mass tolerance 10 ppm, fragment ion mass tolerance 0.02 Da, and variable modifications including Oxidation (M) 15.99, Deamidation (NQ) 0.98, hydroxylation (KP) 16, and Ca 40.04. Screening criteria: the protein must contain at least one unique peptide, and the peptide card value -10 lg P ≥20.

[0168] 1.3 Peptide Synthesis and Purity Verification

[0169] The two selected peptides were synthesized by Nanjing Peptide Biotechnology Co., Ltd. using solid-phase synthesis. After purification by reversed-phase high-performance liquid chromatography (RP-HPLC), the purity of both peptides was higher than 98%. After mass spectrometry verification that the molecular weight was consistent with the target peptides, they were freeze-dried for later use.

[0170] 1.4 Molecular docking analysis

[0171] The three-dimensional structures of the identified peptides were constructed using Discovery Studio 2019 software, followed by structural optimization and energy minimization. This software was then used to perform molecular docking analysis between the mass spectrometry-identified peptides and the epidermal growth factor receptor (EGFR). The crystal structure of the EGFR-EGF protein-ligand complex (PDB ID: 1IVO) was obtained from the RCSB protein database (https: / / www.rcsb.org / structure / 1ivo). Prior to docking, the complex crystal structure underwent protein cleaning and preparation, including the removal of water molecules and the addition of hydrogen atoms. The active pocket is defined as the original EGF binding site within the 1IVO complex. Molecular docking was performed using the Dock Ligands (CDOCKER) protocol tool, and the binding energy was calculated. The results were evaluated based on the CDOCKER energy score, active site, and interaction type with EGFR.

[0172] 1.5 Activity Evaluation

[0173] For evaluation of osteogenic activity and intestinal calcium transport-promoting activity, refer to Example 2.

[0174] 1.6 Data Processing

[0175] All experiments were repeated three times, and data are expressed as mean ± standard deviation (x ± s). Excel 2024 was used for data processing, and SPSS 26.0 was used for statistical analysis. One-way ANOVA was used to analyze differences between groups, and p < 0.05 was considered statistically significant. Prism 10 and Origin 2024 were used to create graphs and tables.

[0176] 2. Results and Analysis

[0177] 2.1 Identification and Structural Characterization of Fish Scale Collagen Calcium Chelate Peptide

[0178] 2.1.1 Peptide composition and molecular weight distribution

[0179] HPLC-MS / MS analysis of fish scale collagen calcium peptide chelates revealed a total of 5688 peptides. Based on the number of calcium ions bound (≥3) and the characteristics of the acidic amino acid composition, two peptides with high calcium chelation activity potential were further screened (Table 3), and their secondary mass spectra are shown below. Figure 23 .

[0180] 2.1.2 Calcium chelation sites and sequence characteristics

[0181] Table 3 Identification of calcium chelating peptides from fish scale collagen

[0182]

[0183] 2.2 Molecular docking analysis of calcium chelating peptide and EGFR

[0184] 2.2.1 Combining Affinity Evaluation

[0185] The docking results of two calcium chelating peptides with EGFR are shown in Table 4. Figure 22 All peptides were able to successfully bind to the EGFR active pocket.

[0186] Table 4. Results of docking of fish scale collagen calcium chelate peptides with EGFR

[0187]

[0188] 2.2.2 Interaction Modes and Key Sites

[0189] Visualization analysis showed that the binding of both peptides to EGFR involved multiple interaction types, and they shared the core binding site THR 15 with the natural EGFR ligand EGF (Table 5).

[0190] The binding of DLGDDDGF to EGFR involves hydrogen bonds (THR 15, GLY 18), hydrophobic interactions (LEU 14), and electrostatic interactions (LYS 13); EDQEE forms a richer hydrogen bond network with EGFR, involving multiple sites such as ASN 12, THR 15, GLN16, LEU 17, and GLY 18.

[0191] The results indicate that both calcium chelating peptides can activate the EGFR signaling pathway by forming specific interactions with key sites of EGFR, providing a molecular basis for their subsequent osteogenic activity.

[0192] Table 5. Common action sites of calcium chelating peptides and EGF on EGFR

[0193]

[0194] 2.3 Osteogenic Activity Analysis of Calcium Chelating Peptides

[0195] 2.3.1 Effects on MC3T3-E1 cell proliferation

[0196] The effect of two fish scale collagen calcium chelating peptides on the proliferation of MC3T3-E1 cells was detected using the MTT assay. Figure 14 When the treatment time was 24 h and 48 h, within the concentration range of 1-50 μg / mL, both peptides showed varying degrees of cell proliferation promotion compared to the blank control group.

[0197] When the treatment time was 48 h, each peptide showed different optimal concentrations: at a concentration of 10 μg / mL, the cell proliferation rate of DLGDDDGF treatment reached the maximum value of 115.45%; at a concentration of 50 μg / mL, the cell proliferation rate of EDQEE treatment reached the highest value of 131.54%.

[0198] 2.3.2 Effects on MC3T3-E1 cell differentiation

[0199] The results showed that different concentrations of calcium chelating peptide had an effect on ALP activity in MC3T3-E1 cells. Figure 15 Compared with the blank control group, the ALP activity of each peptide treatment group was significantly increased (P<0.05), and showed a concentration-dependent increasing trend in the concentration range of 1-50 μg / mL, reaching a maximum at a concentration of 50 μg / mL. Among them, the ALP activity of treatment group (a) was the highest, which was 37.31% higher than that of the blank control group, and the ALP activity of treatment group (b) was 22.79% higher. Both calcium chelating peptides could significantly increase ALP activity.

[0200] 2.3.3 Effects on MC3T3-E1 cell mineralization

[0201] The effects of two peptides on mineralization in MC3T3-E1 cells were detected using alizarin red staining. Figures 16-17 As shown, with increasing peptide concentration, the number and staining intensity of mineralized nodules in the two calcium chelate peptide treatment groups gradually increased, reaching the optimal mineralization effect at a concentration of 50 μg / mL. Both calcium chelate peptides effectively promoted mineralization in MC3T3-E1 cells, further confirming their complete osteogenic function and regulatory role throughout the entire process from proliferation and differentiation to mineralization, providing solid experimental evidence for their application as a bone health supplement.

[0202] 2.4 Analysis of the intestinal calcium absorption-promoting activity of calcium chelating peptides

[0203] 2.4.1 Effect on Caco-2 cell survival

[0204] The effect of two fish scale collagen calcium chelating peptides on the survival rate of Caco-2 cells was evaluated using the MTT assay. The results are as follows: Figure 18 As shown in the figure, within the concentration range of 25-500 μg / mL, the survival rate of Caco-2 cells treated with the two calcium chelating peptides was higher than 90%, significantly higher than the cytotoxicity threshold (80%), indicating that these peptides had no significant toxicity to intestinal epithelial cells within the experimental concentration range, had good biocompatibility, and could be used for subsequent evaluation of calcium absorption-promoting activity.

[0205] 2.4.2 Effects on calcium transport in Caco-2 cells

[0206] To systematically evaluate the intestinal calcium absorption-promoting activity of the fish scale collagen calcium chelating peptide prepared in this study, a Caco-2 cell monolayer model was established, using calcium chloride, a commonly used calcium supplement, as a control. The absorption-promoting effect of the collagen calcium chelating peptide was quantitatively analyzed by measuring the calcium transport amount at different transport time points. The results are as follows: Figure 19 As shown, the calcium transport capacity of the two calcium chelating peptides gradually increased with prolonged transport time, exhibiting a clear time-dependent effect. At 180 min of transport, compared with the calcium chloride control group (calcium transport capacity: 11.63 μg / well), the calcium transport capacity of each peptide-calcium chelate group was significantly increased (P<0.05). Among them, the calcium transport capacity of the DLGDDDGF and EDQEE groups reached 18.98 and 28.80 μg / well, respectively, which were 63.20% and 147.64% higher than the control group.

[0207] 2.4.3 Intracellular calcium ion fluorescence imaging analysis

[0208] Intracellular calcium ion concentration in Caco-2 cells was visualized using the Fluo-4 AM fluorescent probe. After treating Caco-2 monolayer cells with different samples, fluorescence imaging based on intracellular calcium influx was used for detection. Stronger fluorescence intensity indicated higher intracellular calcium ion concentration and more significant calcium influx. Figures 20-21 As shown, compared with the control group, the fluorescence intensity of the DLGDDDGF and EDQEE groups was significantly increased, by 42.24% and 16.06%, respectively.

[0209] The results of intracellular fluorescence imaging and transmembrane calcium transport quantification experiments showed some discrepancies. This discrepancy may stem from the different principles underlying the two methods: the calcium transport experiment measures the total amount of calcium transported across the cell monolayer, while fluorescence imaging measures the intracellular free calcium ion concentration. Furthermore, different peptide-calcium chelates exhibit different calcium ion release kinetics. Some peptides may release calcium ions slowly within the cell, maintaining a high intracellular free calcium ion concentration, while others may release calcium ions rapidly and be transported extracellularly, resulting in a higher total transport amount but a relatively lower intracellular free calcium ion concentration. In summary, both methods confirm that the two calcium chelating peptides effectively enhance the uptake and transport of calcium ions by intestinal epithelial cells, exhibiting significant activity in promoting intestinal calcium absorption.

[0210] This invention uses fish scales, a byproduct of aquatic product processing, as raw material. Collagen calcium peptide chelates are prepared through enzymatic hydrolysis and chelation enrichment. Two highly active calcium chelating peptides were identified using HPLC-MS / MS technology. This invention systematically clarifies the dual biological activities (promoting calcium absorption and osteogenic activity) of fish scale-derived calcium chelating peptides and their EGFR-mediated osteogenic mechanism. It enriches the database of calcium chelating peptide raw materials and structures, providing high-quality candidate raw materials and scientific basis for the development of novel functional calcium supplements. Further in vivo experiments, in-depth investigation of signaling pathways, and process optimization can be conducted to expand applications.

[0211] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for in situ preparation of calcium-chelating peptides that promote intestinal calcium absorption and osteoblast development from fish scales by high pressure homogenization-assisted enzymatic hydrolysis, characterized by: Includes the following steps: (1) Raw material pretreatment: The fish scales are cleaned and impurities are removed, dried and then crushed and sieved to obtain dried fish scale powder; (2) High pressure homogenization pretreatment: The dried fish scale powder obtained in step (1) is prepared into a suspension with water, and then circulated 8 to 12 times under a pressure of 45 to 55 MPa using a high pressure homogenizer. After freeze drying, high pressure homogenized fish scale powder is obtained. (3) Enzymatic hydrolysis preparation: using pepsin as the hydrolytic enzyme, the high-pressure homogenized fish scale powder obtained in step (2) is placed in the enzymatic hydrolysis system, and the enzymatic hydrolysis conditions are controlled as follows: pH 1.5-2.5, temperature 35-45℃, enzyme-to-substrate ratio 0.05%-0.15%, substrate concentration 3%-7%, and the enzymatic hydrolysis is carried out for 1-3 h to obtain the enzymatic hydrolysate; (4) Product separation: Centrifuge the enzymatic hydrolysate obtained in step (3) to obtain the supernatant, freeze-dry it to obtain collagen calcium chelate peptide hydrolysate.

2. The method for preparing calcium chelating peptides that promote intestinal calcium absorption and osteoblast development from fish scales in situ using high-pressure homogenization-assisted enzymatic hydrolysis according to claim 1, characterized in that: The fish scales mentioned in step (1) are those of tilapia, grass carp, carp, cod, or salmon; The drying process described in step (1) involves drying in an oven at 55–65°C for 4–8 hours. The sieving mentioned in step (1) is sieving through a 50-70 mesh sieve.

3. The method for preparing calcium chelating peptides that promote intestinal calcium absorption and osteoblast development from fish scales in situ using high-pressure homogenization-assisted enzymatic hydrolysis according to claim 1, characterized in that: The concentration of the suspension mentioned in step (2) is 3% to 7% by mass. In step (2), the pressure of the high-pressure homogenization process is 48–52 MPa, and the number of cycles is 9–11. The enzyme activity of pepsin in step (3) is 2.0 x 10 5 ~ 3.0 x 10 5 U / g, and the stirring is performed every 20-40 min during the enzymolysis process, and the stirring time is 1-3 min.

4. The method for preparing calcium chelating peptides that promote intestinal calcium absorption and osteoblast development in situ by high-pressure homogenization-assisted enzymatic hydrolysis of fish scales according to claim 1, characterized in that: In step (2), the pressure of the high-pressure homogenization process is 50 ± 0.5 MPa, and the number of cycles is 10. The enzyme activity of pepsin in step (3) is 2.5 x 10 5 U / g, and the enzymolysis conditions are: pH 2, temperature 40°C, enzyme-substrate ratio 0.1%, substrate concentration 5%, and enzymolysis time 2 h.

5. A fish scale collagen calcium chelate peptide enzymatic hydrolysate, characterized in that: It is obtained by the preparation method described in any one of claims 1-4.

6. The fish scale collagen calcium chelate peptide hydrolysate according to claim 5, characterized in that: The fish scale collagen calcium chelate peptide hydrolysate includes: (1) The peptide Asp-Leu-Gly-Asp-Asp-Asp-Gly-Phe (DLGDDDGF), which binds to 3 calcium ions, has the following binding sites: The fourth aspartic acid, Asp, binds to one calcium ion; The fifth position, aspartic acid (Asp), binds to one calcium ion; The 6th position, aspartic acid (Asp), binds to one calcium ion; (2) The peptide Glu-Asp-Gln-Glu-Glu (EDQEE) which binds to 3 calcium ions has the following binding sites: The fourth glutamate, Glu, binds to one calcium ion; The fifth position is glutamic acid (Glu), which binds to two calcium ions.

7. A calcium chelating peptide, characterized in that: It is an active peptide having any of the following amino acid sequences: Asp-Leu-Gly-Asp-Asp-Asp-Gly-Phe, Glu-Asp-Gln-Glu-Glu.

8. The use of the fish scale collagen calcium chelate peptide hydrolysate according to any one of claims 5-6 or the calcium chelate peptide according to claim 7 in the preparation of products for calcium supplementation, calcium absorption promotion, bone development promotion and / or anti-osteoporosis.

9. The application according to claim 8, characterized in that: The products are functional foods, health products, dietary supplements, foods for special medical purposes, or pharmaceuticals; further, they are foods, health products, or pharmaceuticals that help improve bone density.

10. The application according to claim 9, characterized in that: The amount of fish scale collagen calcium chelate peptide hydrolysate or calcium chelate peptide added to the product is 1% to 20% by mass. The product is available in the form of powder, capsule, tablet, oral liquid, granules, or emulsion.

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