Calcium absorption promoting peptide prepared from defatted egg yolk powder and preparation method and application thereof

Abstract

The present application belongs to the technical field of egg yolk protein deep processing and functional active polypeptide preparation, and relates to the technical field of preparation and separation of calcium absorption promoting peptides, and aims to improve the preparation efficiency and preparation source of food-derived calcium absorption promoting peptides. The present application discloses a method for preparing calcium absorption promoting peptides from defatted egg yolk powder, and the preparation method and application thereof. The method directly uses defatted egg yolk powder as the starting material, and then neutral protease and trypsin are added in sequence for complex enzymolysis. After enzyme inactivation, separation and drying, the product is obtained. Through the step-by-step synergistic effect of double enzymes, the functional peptide segments with phosphoserine modification, VVGS(p)AEAGVDAASVSEEFRADHPF; LGGVGSPVSGGGKQLAS(p); IVLSVNITS(p); NPLS(p)YTSAK, can be efficiently released and enriched, so that the final product has excellent calcium ion chelating capacity and significant osteoblast promoting activity. The product can be directly used as a functional ingredient in various food and health care products related to bone health.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and functional food processing, specifically relating to a method for efficiently preparing calcium absorption-promoting peptides using defatted egg yolk powder as a direct raw material through an optimized compound enzymatic hydrolysis process, as well as its preparation method and application. Background Technology

[0002] Calcium is the most abundant mineral element in the human body, and it is essential for maintaining bone health, nerve conduction, muscle contraction, and cell signaling. Long-term insufficient calcium intake can easily lead to osteoporosis, increased fracture risk, and various metabolic diseases. Currently, commercially available calcium supplements are mainly divided into three categories: inorganic calcium (such as calcium carbonate), organic acid calcium (such as calcium gluconate), and amino acid / peptide chelate calcium. While inorganic calcium has a high calcium content, its absorption rate is low and it can easily cause gastrointestinal discomfort. Organic acid calcium has improved bioavailability, but its calcium content is limited and it may be accompanied by side effects. Peptide calcium chelates, due to their good solubility, high absorption efficiency, and intestinal stability, are considered an important development direction for next-generation high-efficiency calcium supplements. Peptide calcium chelates are usually derived from protein enzymatic hydrolysis products. They form stable complexes with calcium ions through specific amino acid sequences, which not only effectively inhibit calcium from binding and precipitating with phytic acid, phosphate, etc. in the intestine, but also promote the transmembrane absorption of calcium through the peptide transport system.

[0003] Currently, milk-derived proteins (such as casein and whey protein) have been widely used in the development of chelated calcium peptides. For example, the sheep milk casein-derived chelated calcium peptide disclosed in CN116239666A and the fish bone calcium absorption-promoting peptide disclosed in Chinese patent CN119306798B have, to some extent, improved calcium bioavailability. However, the potential of egg-derived protein resources, especially egg yolk protein, which is most closely related to the lives of Chinese people, has not yet been fully explored. Existing egg yolk peptide preparation processes often involve defatting, enzymatic hydrolysis, and ultrafiltration, but often fail to target the separation and purification of products with calcium absorption-promoting activity, resulting in products with low activity or complex compositions.

[0004] In summary, existing technologies generally suffer from narrow raw material adaptability (strong dependence on milk-derived proteins), low process greenness (large amounts of organic solvents), weak modification recognition ability (ignoring key pharmacodynamic groups), and crude separation and purification methods (lacking Ca2+). 2+ Common limitations, such as affinity enrichment strategies, make it difficult to meet the demands of modern functional peptide development that require high activity, traceability, and easy mass production. Therefore, developing a method for efficiently preparing calcium absorption-enhancing peptide monomers with well-defined structures from egg yolks would not only help improve the high-value utilization of egg yolk byproducts but also provide important raw materials and theoretical support for the development of novel calcium supplements. Summary of the Invention

[0005] To address the problems mentioned in the background section, the present invention aims to provide a simple, efficient method for preparing highly active calcium absorption-promoting peptides directly from defatted egg yolk powder. This method overcomes the shortcomings of existing technologies, such as complex pretreatment steps, low enzymatic hydrolysis efficiency, and unclear product functionality. Furthermore, it clarifies the structure and efficacy of novel calcium absorption-promoting peptides, demonstrating economic value and broadening the development scope of active egg yolk polypeptides. It also provides more high-quality raw materials for the development and utilization of egg yolk calcium absorption-promoting peptides.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A calcium absorption-promoting peptide prepared from defatted egg yolk powder, wherein the calcium absorption-promoting peptides are P1, P2, P3, and P4, wherein: P1 has the amino acid sequence VVGS(p)AEAGVDAASVSEEFRADHPF shown in SEQ ID NO.1, P2 has the amino acid sequence LGGVGSPVSGGGKQLAS(p) shown in SEQ ID NO.2, P3 has the amino acid sequence IVLSVNITS(p) shown in SEQ ID NO.3, and P4 has the amino acid sequence NPLS(p)YTSAK shown in SEQ ID NO.4.

[0007] A method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as raw material includes the following steps: A. Raw material pretreatment and dissolution: The defatted egg yolk powder is mixed with water at a ratio of 1:8 to 1:12 (w / v), and stirred evenly to form a suspension.

[0008] B. Enzymatic hydrolysis: In the first stage, neutral protease is used at pH 6.5–7.5 and temperature 45–55℃, with 0.5%–1.5% of the defatted egg yolk powder added for 2–4 hours; in the second stage, at the same pH and temperature, trypsin is added and enzymatic hydrolysis is continued for 1–3 hours at 0.1%–0.5% of the defatted egg yolk powder added to obtain the enzymatic hydrolysate. C. Inactivation and separation of defatted egg yolk powder: The enzymatic hydrolysate was heat-treated at 85–95℃ for 10–30 minutes for inactivation, and the supernatant was collected by centrifugation. The supernatant was then separated by CaCl2-induced ethanol fractionation to obtain the calcium absorption peptide mother liquor. The peptide identification results of the calcium absorption peptide mother liquor are as follows: it includes four active peptides modified with phosphoserine (pS): VVGS(p)AEAGVDAASVSEEFRADHPF (2447.55 Da), LGGVGSPVSGGGKQLAS(p) (1470.60 Da), IVLSVNITS(p) (945.11 Da), and NPLS(p)YTSAK (980.07 Da). D. Drying and powdering: The calcium absorption-promoting peptide mother liquor obtained by separation is spray-dried or freeze-dried to obtain calcium absorption-promoting peptide powder.

[0009] Preferably, the ratio of defatted egg yolk powder to water in step A is 1:10 (w / v).

[0010] Preferably, the amount of neutral protease added in step B is 1.0% of the mass of the defatted egg yolk powder, and the enzymatic hydrolysis time is 3 hours.

[0011] Preferably, the amount of trypsin added in step B is 0.3% of the mass of the defatted egg yolk powder, and the enzymatic hydrolysis time is 2 hours.

[0012] Preferably, the heat inactivation conditions in step C are: 90°C for 20 minutes, CaCl2 reaction time for 1 hour, final ethanol concentration of 50% (v / v), and standing time for 2 hours.

[0013] Preferably, in step C, the amount of CaCl2 added is 0.4% of the mass of the supernatant obtained in step C, and the final concentration of ethanol is controlled at 45%–55% (v / v).

[0014] Preferably, in step C, all peptides were confirmed by UHPLC-LTQ-Orbitrap-Velos Pro high-resolution liquid chromatography-mass spectrometry, and the phosphorylation sites were located by the characteristic peak of neutral loss of 98 Da (H3PO4) in the MS / MS fragment ion spectrum.

[0015] Preferably, the drying method in step D is spray drying, with an inlet air temperature of 180°C and an outlet air temperature of 85°C.

[0016] A calcium absorption-promoting peptide prepared by the above method using defatted egg yolk powder as raw material has the function of promoting intestinal calcium absorption.

[0017] The present invention has the following specific effects: 1. The cost of this invention is controllable. It uses commercially available defatted egg yolk powder as the direct raw material, with a mature supply chain and compatibility with existing spray drying production lines (180℃ inlet air / 85℃ outlet air). No additional ultrafiltration equipment, freeze dryer, or organic solvent recovery system is required. The yield is stable at 76.2±1.4% in ton-scale scale-up verification.

[0018] 2. This invention employs a two-stage sequential enzymatic digestion process using neutral protease and trypsin, with a stable pH (6.5–7.5) and a mild temperature (45–55℃) throughout the process. It also utilizes CaCl2-induced ethanol fractional precipitation to achieve specific enrichment of phosphoserine (pS) peptides.

[0019] 3. This invention confirms four site-specific phosphoserine (pS) modified monomeric peptides (P1–P4) with precise molecular weights (945.11–2447.55 Da). The phosphorylation sites were verified by MS / MS neutral loss 98 Da (H3PO4) spectra. On the UHPLC-LTQ-Orbitrap-Velos Pro platform, the retention time RSD was <1.2%, the peak area RSD was <3.5%, and the batch-to-batch consistency reached 98.6%.

[0020] 4. All peptides of this invention contain pS residues, which can directly react with Ca. 2+ It forms a stable coordination complex and is absorbed via the intestinal epithelial peptide transporter PepT1. All pS sites are confirmed by MS / MS fragment spectroscopy (e.g., pS in P1 is located at position 4, Ser), and the structure is registrable, detectable, and can be used to establish reference materials.

[0021] 5. This invention is green and environmentally friendly, saving energy and reducing emissions: The entire process uses zero organic solvents (no ethanol / ethyl acetate degreasing, no ethanol precipitation dependence), reducing wastewater COD by 92% and VOC emissions close to zero; in contrast, the degreasing and precipitation steps in the comparative paper consume ≥8 times the volume of raw materials in organic solvents, thus eliminating all organic solvent degreasing steps. Simultaneously, it eliminates high-energy-consuming steps such as embryo incubation (35–38℃ constant temperature for 7–15 days), freeze-drying pretreatment, and ultrafiltration centrifugation (7500–8500 rpm), resulting in a reduction of approximately 67% in overall energy consumption per unit product.

[0022] 6. The calcium absorption-promoting peptide of this invention showed ALP activity 2.83 times that of the control group at 100 μg / mL, and OCN secretion of 2.1578 ng / L, which were significantly higher than those of commercially available products (A / B / C / D) and the single enzymatic hydrolysis group (p<0.01); the pS modification retention rate of the peptide fragment after simulated gastrointestinal digestion was >91.3% (determined by HPLC-ICP-MS). Attached Figure Description

[0023] Figure 1 The calcium ion concentration in the supernatant after calcium chelation of the egg yolk calcium absorption peptide powder sample is shown (different letters indicate significant differences in data, p<0.05). Figure 2 This is the total ion chromatogram of hydrolyzed egg yolk powder; Figure 3 This is the first-order mass spectrum of VVGSAEAGVDAASVSEEFRADHPF (z=+3, m / z=843.04065). Figure 4 This is the secondary mass spectrum of VVGSAEAGVDAASVSEEFRADHPF; Figure 5This is the first-order mass spectrum of LGGVGSPVSGGGKQLAS (z=+2, m / z=775.88086). Figure 6 This is a secondary mass spectrum of LGGVGSPVSGGGKQLAS; Figure 7 This is the first-order mass spectrum of IVLSVNITS (z=+2, m / z=513.26685). Figure 8 This is a secondary mass spectrum of IVLSVNITS; Figure 9 This is the first-order mass spectrum of NPLSYTSAK (z=+2, m / z=531.22815). Figure 10 This is a secondary mass spectrum of NPLSYTSAK; Figure 11 The effects of different enzyme hydrolysis products on cellular ALP activity; Figure 12 The effects of different enzyme hydrolysis products on cell OCN activity; Figure 13 The ALP activity of the enzymatic hydrolysis products is compared with that of commercially available products. Figure 14 The enzymatic hydrolysis product is compared with commercially available products to assess OCN activity. Detailed Implementation

[0024] The exemplary embodiments of this disclosure are described below with reference to the accompanying drawings, including various details of the embodiments to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this disclosure. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0025] Example 1: A. Raw material pretreatment and dissolution: Mix defatted egg yolk powder and water at a material-to-liquid ratio of 1:8 (w / v) and stir until a suspension is formed; B. Enzymatic hydrolysis: In the first stage, neutral protease was used at pH 6.5 and temperature 45℃, and enzymatic hydrolysis was carried out for 2 hours at an addition rate of 0.5% of the defatted egg yolk powder. In the second stage, at the same pH and temperature, trypsin was added and enzymatic hydrolysis was continued for 1 hour at an addition rate of 0.1% of the defatted egg yolk powder to obtain the enzymatic hydrolysate. C. Inactivation and separation of defatted egg yolk powder: The enzymatic hydrolysate was heat-treated at 90℃ for 20 minutes to inactivate the enzyme, and the supernatant was collected by centrifugation. CaCl2 was added to the supernatant to a final concentration of 0.4% of the supernatant mass, and the mixture was reacted for 1 hour. Then, ethanol was added to a final concentration of 45% (v / v), and the mixture was allowed to stand for 2 hours. The precipitate was collected by centrifugation, reconstituted, and dialyzed to obtain the calcium absorption peptide mother liquor. The mother liquor was confirmed by UHPLC-LTQ-Orbitrap-Velos Pro high-resolution liquid chromatography-mass spectrometry to contain four phosphoserine-modified peptides: VVGS(p)AEAGVDAASVSEEFRADHPF (2447.55 Da), LGGVGSPVSGGGKQLAS(p) (1470.60 Da), IVLSVNITS(p) (945.11 Da), and NPLS(p)YTSAK (980.07 Da). The phosphorylation sites of these peptides were accurately located by the characteristic peak of 98 Da (H3PO4) in neutral loss in the MS / MS spectrum. D. Drying and powdering: The calcium absorption peptide mother liquor is spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 85℃ to obtain a light yellow powdered calcium absorption peptide product.

[0026] Example 2: A. Raw material pretreatment and dissolution: Mix defatted egg yolk powder and water at a material-to-liquid ratio of 1:10 (w / v) and stir until a suspension is formed; B. Enzymatic hydrolysis: In the first stage, neutral protease was used at pH 7.0 and 50°C for 3 hours at an addition rate of 1.0% of the defatted egg yolk powder. In the second stage, at the same pH and temperature, trypsin was added and enzymatic hydrolysis was continued for 2 hours at an addition rate of 0.3% of the defatted egg yolk powder to obtain the enzymatic hydrolysate. C. Inactivation and separation of defatted egg yolk powder: The enzymatic hydrolysate was heat-treated at 90℃ for 20 minutes to inactivate the enzyme, and the supernatant was collected by centrifugation. CaCl2 was added to the supernatant to a final concentration of 0.4% of the supernatant mass, and the mixture was reacted for 1 hour. Then, ethanol was added to a final concentration of 50% (v / v), and the mixture was allowed to stand for 2 hours. The precipitate was collected by centrifugation, reconstituted, and dialyzed to obtain the calcium absorption peptide mother liquor. The mother liquor was confirmed by UHPLC-LTQ-Orbitrap-Velos Pro high-resolution liquid chromatography-mass spectrometry to contain four phosphoserine-modified peptides: VVGS(p)AEAGVDAASVSEEFRADHPF (2447.55 Da), LGGVGSPVSGGGKQLAS(p) (1470.60 Da), IVLSVNITS(p) (945.11 Da), and NPLS(p)YTSAK (980.07 Da). The phosphorylation sites of these peptides were accurately located by the characteristic peak of 98 Da (H3PO4) in neutral loss in the MS / MS spectrum. D. Drying and powdering: The calcium absorption peptide mother liquor is freeze-dried to obtain a light yellow powdered calcium absorption peptide product.

[0027] Example 3: A. Raw material pretreatment and dissolution: Mix defatted egg yolk powder and water at a material-to-liquid ratio of 1:12 (w / v) and stir until a suspension is formed; B. Enzymatic hydrolysis: In the first stage, neutral protease was used at pH 7.5 and temperature 55℃, and enzymatic hydrolysis was carried out for 4 hours at an addition rate of 1.5% of the defatted egg yolk powder. In the second stage, at the same pH and temperature, trypsin was added and enzymatic hydrolysis was continued for 3 hours at an addition rate of 0.5% of the defatted egg yolk powder to obtain the enzymatic hydrolysate. C. Inactivation and separation of defatted egg yolk powder: The enzymatic hydrolysate was heat-treated at 90℃ for 20 minutes to inactivate the enzyme, and the supernatant was collected by centrifugation. CaCl2 was added to the supernatant to a final concentration of 0.4% of the supernatant mass, and the mixture was reacted for 1 hour. Then, ethanol was added to a final concentration of 55% (v / v), and the mixture was allowed to stand for 2 hours. The precipitate was collected by centrifugation, reconstituted, and dialyzed to obtain the calcium absorption peptide mother liquor. The mother liquor was confirmed by UHPLC-LTQ-Orbitrap-Velos Pro high-resolution liquid chromatography-mass spectrometry to contain four phosphoserine-modified peptides: VVGS(p)AEAGVDAASVSEEFRADHPF (2447.55 Da), LGGVGSPVSGGGKQLAS(p) (1470.60 Da), IVLSVNITS(p) (945.11 Da), and NPLS(p)YTSAK (980.07 Da). The phosphorylation sites of these peptides were accurately located by the characteristic peak of 98 Da (H3PO4) in neutral loss in the MS / MS spectrum. D. Drying and powdering: The calcium absorption peptide mother liquor is spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 85℃ to obtain a light yellow powdered calcium absorption peptide product.

[0028] Comparative Test 1: Comparison of Single Enzymatic Hydrolysis Processes

[0029] Comparative Example 1: Compared with Example 2, the difference is that in step B, only alkaline protease was used for enzymatic hydrolysis for 5 hours, followed by centrifugation at 4000 r / min for 30 min to obtain the hydrolysate.

[0030] Comparative Example 2: Compared with Example 2, the difference is that in step B, only neutral protease was used for enzymatic hydrolysis for 5 hours, followed by centrifugation at 4000 r / min for 30 min to obtain the hydrolysate.

[0031] Comparative Example 3: Compared with Example 2, the difference is that in step B, only pepsin was used for hydrolysis for 5 hours, followed by centrifugation at 4000 r / min for 30 min to obtain the hydrolysate.

[0032] Comparative Example 4: Compared with Example 2, the difference is that in step B, only trypsin hydrolysis was used for 5 hours, followed by centrifugation at 4000 r / min for 30 min to obtain the hydrolysate.

[0033] Comparative Example 5: Compared with Example 2, the difference is that in step B, only papain was used for enzymatic hydrolysis for 5 hours, followed by centrifugation at 4000 r / min for 30 min to obtain the hydrolysate.

[0034] Subsequently, the optimal single enzyme was selected based on a comprehensive evaluation of four indicators: yield, sensory assessment, molecular weight, and degree of hydrolysis.

[0035]

[0036] Table 1 Sensory evaluation of egg yolk powder hydrolyzed by different enzymes Table 1 shows that the neutral protease had the highest sensory evaluation score of 88. The scores were ranked as follows: neutral protease > papain > trypsin > alkaline protease > pepsin. The neutral protease treatment group had the highest overall sensory score (88 points), followed by papain (87 points) and trypsin (85 points), while alkaline protease (79 points) and pepsin (70 points) had relatively lower scores. Significant differences were observed in specific indicators for each enzyme: Color: neutral protease (18 points) and alkaline protease (17 points) were the best, while pepsin (12 points) was significantly lower; Odor: papain (19 points) was the highest, followed by neutral protease (18 points); Taste: neutral protease (26 points) was the best, while pepsin (18 points) was the worst; Texture: trypsin and papain (both 28 points) performed well, followed by neutral protease (26 points), while alkaline protease (20 points) had the lowest score. Overall advantages of neutral proteases: Their neutral pH conditions may be more conducive to maintaining the original color and flavor of egg yolk powder, with fewer bitter peptides in the hydrolysis products, resulting in higher taste acceptability and the best overall performance. Characteristics of papain: It leads in odor score, possibly related to the production of aromatic small molecule peptides or low off-odors during enzymatic hydrolysis, but its color and taste are slightly lower than neutral proteases. Trypsin and tissue morphology: Its specific cleavage of basic amino acid residues may form more uniform peptides, which is beneficial for maintaining tissue morphology, ranking third overall. Reasons for lower scores of alkaline proteases and pepsin: Alkaline proteases suffer greater loss in tissue morphology (20 points), possibly due to vigorous hydrolysis leading to a loose texture; pepsin, under acidic conditions, easily leads to darkening of color (12 points) and deterioration of taste (18 points), but its tissue morphology score is acceptable (27 points), suggesting that its hydrolysis has a smaller impact on texture. Neutral proteases, trypsin, and papain have the greatest application potential in terms of overall sensory balance. Further optimization of the enzymatic hydrolysis process can be achieved by combining physicochemical indicators such as degree of hydrolysis and product composition.

[0037]

[0038] Table 2. Yields of different proteases According to the data in Table 2, the yields of egg yolk hydrolysates prepared by different proteases are shown below: the theoretical yield of the pepsin-treated group was the highest (86.60%), significantly higher than that of other proteases. The theoretical yields of each enzyme, ranked from highest to lowest, are: pepsin (86.60%) > papain (64.71%) > trypsin (49.19%) > neutral protease (36.48%) > alkaline protease (21.35%). Analysis of the hydrolysis mechanism underlying the yield differences: The high yield of pepsin is due to its specific cleavage of aromatic amino acid residues, which may produce more soluble hydrophobic peptides. These peptides are easily soluble in the aqueous phase, resulting in a high solids content in the supernatant. However, its hydrolysis products may contain more bitter peptides, leading to a poorer taste. The low yields of neutral and alkaline proteases are due to their potentially milder hydrolytic processes or the generation of more hydrophilic short peptides, which may not be completely precipitated or extracted under the assay conditions. Alkaline proteases, in particular, can cause excessive protein degradation into volatile ammonia or small-molecule acids, leading to solids loss. Papain, on the other hand, demonstrates excellent performance in both yield (64.71%, second highest) and sensory score (87 points, second highest), showing good potential for comprehensive applications. Its wide operating pH and temperature range may help achieve high recoverable solids and good flavor under mild conditions. In the development of egg yolk protein hydrolysates, yield and sensory quality must be balanced according to the application goals. If the primary goal is to prepare high-yield protein raw materials, pepsin has certain advantages, but subsequent debittering and decolorization treatments are required. If developing high-end flavor enhancers or food ingredients, neutral or papain proteases should be given priority, despite their lower yields, because they have higher product acceptance.

[0039]

[0040] Table 3. Relative content of different molecular weights in hydrolyzed egg yolk powder samples Different enzymes may cleave different fragments of the same target protein, resulting in variations in the molecular weight of the resulting polypeptides. Table 3 shows that polypeptides with molecular weights >10,000 obtained from alkaline protease hydrolysis and papain hydrolysis have relatively higher content. Pepsin hydrolysis yields the most polypeptides with molecular weights <5,000. Neutral protease hydrolysis yields the highest concentrations of polypeptides with molecular weights between 500 and 189, and the lowest concentrations between 3,000 and 2,000. Analysis of variance between groups for different molecular weight ranges revealed significant differences (p<0.05) in the molecular weight ranges >10,000, 10,000–5,000, and <5,000, while no significant difference (p>0.05) was found in the molecular weight range <189.

[0041] Relationship between enzyme cleavage specificity and molecular weight distribution: The molecular weight distribution of the product is directly determined by the specificity of the enzyme's cleavage site. Neutral proteases have a wide range of action sites, tending to generate a large number of small and medium molecular weight peptides (500-189 Da), which makes their 100-5000 Da range the most prevalent, easily meeting specific quality standards. This corresponds to their excellent sensory taste score (26 points), as small molecular weight peptides usually have lower bitterness and milder flavor. Pepsin and trypsin have specific action sites (cleaving the carboxyl terminus of aromatic amino acids and basic amino acids, respectively), easily generating fragments of specific sizes, but due to the cleavage frequency or site distribution, a large number of large molecules (>10000 Da) may remain in the product, thus affecting the proportion of the 100-5000 Da range. Alkaline proteases may cause partial protein denaturation, aggregation, or cross-linking under harsh conditions, leading to an increase in large molecular weight products. This not only results in their lowest proportion of 100-5000 Da products but may also explain their poor tissue morphology score (20 points). The intrinsic relationship between molecular weight distribution and sensory properties and yield: Sensory quality: The success of neutral protease lies in its production of a high proportion of small and medium molecular weight peptides. These peptides have good solubility and low odor, contributing to its highest overall sensory score (88 points). Conversely, although pepsin has the highest yield, its product contains more large molecular fragments, which may be accompanied by more hydrophobic peptides (source of bitterness) and darker color, resulting in its lowest sensory score (70 points). Yield (solids): The high yield of pepsin (86.60%) is directly related to the high proportion of soluble large molecular weight solids it produces. While the yield of neutral protease is only 36.48%, its product contains a large number of small molecular weight active peptides that meet quality requirements, and the product value may be higher. This reveals the difference between "yield" and "effective product yield". Target-oriented selection: If the goal is to produce hydrolyzed egg yolk powder that meets specific molecular weight standards (100-5000 Da ≥ 75%), neutral protease is currently the best choice. If a high yield of protein hydrolysate is desired, and subsequent refining (such as debittering and separation) is acceptable, then pepsin can be considered. A single enzyme is unlikely to simultaneously achieve high yield, ideal molecular weight distribution, and excellent sensory qualities. Future approaches could employ a stepwise enzymatic hydrolysis strategy, such as first using pepsin or alkaline protease for deep hydrolysis to increase yield, and then using neutral or flavor protease for "trimming" to improve molecular weight distribution and reduce bitterness, thereby comprehensively improving product quality.

[0042] According to the approved quality requirements for hydrolyzed egg yolk powder, the proportion of hydrolyzed egg yolk powder with a relative molecular mass of 100-5000 should be ≥75%. From the table above, we can see that the molecular weight of the hydrolysate of different enzymes is 100-5000 Da, and the relative content is inconsistent. Their order of size is: neutral protease > trypsin > pepsin > papain > alkaline protease.

[0043] Depend on Figure 1It was found that the calcium chelating activity of egg yolk polypeptides in all protease-treated groups was higher than that in the untreated control group (CK), confirming that enzymatic hydrolysis is an effective means to enhance the calcium binding capacity of egg yolk powder. Comparative analysis of calcium chelating activity showed no significant difference (P>0.05) among the alkaline protease, neutral protease, trypsin, and papain-treated groups, except for the pepsin-treated group. This indicates that for most enzymes, although the activity values ​​vary, the statistical differences are not significant. The calcium chelating activity of each hydrolysate, from highest to lowest, was: neutral protease > papain > trypsin > pepsin > alkaline protease. Neutral protease showed the best performance.

[0044] The comprehensive advantages of neutral proteases: Their strongest calcium chelating activity is highly correlated with their unique molecular weight distribution characteristics (highest content of small peptides <5000 Da). Small peptides more easily expose calcium binding sites such as carboxyl groups and phosphorylated serine, thus enhancing chelating ability. This forms a logical loop with their excellent sensory taste (26 points) and highest total sensory score (88 points): the enzymatic hydrolysis process produces palatable, low-bitter small peptides while simultaneously generating highly active functional peptides. Their products have the highest proportion of 100-5000 Da molecular weights, fully meeting the target quality requirements. The dilemma of pepsin and alkaline proteases: Both have the lowest calcium chelating activity. For pepsin, its products have a relatively high proportion of large molecular fragments (>10000 Da), and their spatial structure may mask the binding sites; its high yield (86.60%) reflects the total amount of soluble solids, but the proportion of peptides with specific functional structures may not be high. Alkaline proteases, due to their harsh hydrolysis conditions, may disrupt potential calcium-binding sequences or cause amino acid racemization, resulting in low activity, consistent with their lowest yield (21.35%) and worst tissue morphology (20 points). Enzyme cleavage specificity shapes functional activity: calcium chelation activity depends on specific amino acid residues (such as Asp, Glu, Ser-P) and their spatial arrangement on the polypeptide chain. Neutral proteases, with their broader substrate specificity, may more readily release or expose short peptide sequences rich in these residues. Papain and trypsin exhibit mid-range activity, with molecular weight distributions showing high levels of large molecules (>10000 Da), yet still possessing some calcium chelation capacity, suggesting they may have cleaved some peptides with suitable sequences and conformations. Neutral proteases hydrolyzing egg yolk powder demonstrated significant advantages in three core indicators: calcium chelation activity, sensory quality, and molecular weight distribution, revealing their great potential for developing high-value functional egg yolk peptides.

[0045] Comparative Test 2: Comparison of Compound Enzymatic Hydrolysis Processes

[0046] Comparative Example 6: Compared with Example 1, the difference is that in step B, neutral protease + papain are used for secondary enzymatic hydrolysis.

[0047] Comparative Example 7: Compared with Example 2, the difference is that in step B, neutral protease + pepsin are used for secondary enzymatic hydrolysis.

[0048] Comparative Example 8: Compared with Example 2, the difference is that in step B, neutral protease + pepsin are used for combined enzymatic hydrolysis.

[0049] Comparative Example 9: Compared with Example 3, the difference is that in step B, trypsin + neutral protease are used for combined enzymatic hydrolysis.

[0050] Comparative Example 10: Compared with Example 3, the difference is that in step B, trypsin + pepsin are used for secondary enzymatic hydrolysis.

[0051] The yields of enzymatic hydrolysis products varied significantly among different enzyme combinations: the highest yield was achieved with trypsin + neutral protease combined enzymatic hydrolysis (77.80%), which was significantly higher than that of neutral protease + papain (34.98%), neutral protease + pepsin (combined or secondary enzymatic hydrolysis, 35.92% / 45.87%), and other treatment groups.

[0052] Table 4 Comparison of product yields under complex enzymatic hydrolysis

[0053] Performance testing: Test 1: The enzymatic hydrolysates from Examples 1-3 were analyzed using UHPLC-LTQ-Orbitrap-Velos Pro high-resolution mass spectrometry to obtain their total ion chromatograms. Figure 2 ), and based on this, several peptides modified with phosphoserine were screened out. Mass spectrometry analysis (see...) Figure 3-10 The representative monomers obtained are as follows:

[0054] These polypeptides contain phosphoserine structures, consistent with the characteristics of egg yolk phosphopeptides (PPP). Phosphorylated serine residues have strong negative charge and coordination ability, and can form stable coordination structures with calcium ions, often closely related to mineral binding and bone metabolism-related functions.

[0055] Test 2: Evaluation of the efficacy of absorption-enhancing peptides: Figure 11 The effects of different enzyme hydrolysis products on cellular ALP activity; Figure 12 The effects of different enzyme hydrolysis products on cell OCN activity; Figure 13 The ALP activity of the enzymatic hydrolysis products is compared with that of commercially available products. Figure 14 The enzymatic hydrolysis product is compared with commercially available products to assess OCN activity.

[0056] See Figure 11-14,in Figure 13 , 14 In this study, Dispase 1 represents neutral protease, Trypsin represents trypsin, D+T represents a combination of neutral protease and trypsin, and A, B, C, and D represent commercially available hydrolyzed egg yolk powder. Egg yolk calcium absorption-promoting peptides can promote osteoblast proliferation at suitable concentrations: within the concentration range of 25–100 μg / mL, egg yolk calcium absorption-promoting peptides have a significant proliferative effect on osteoblasts, and this effect gradually increases with prolonged culture time. High concentrations, however, exhibit cell-inhibiting effects. All enzymatic hydrolysates can increase ALP activity, with the neutral protease hydrolysate showing a significantly better effect than other hydrolysates and some commercially available products at 100 μg / mL. There was no significant difference between neutral protease and neutral protease + trypsin. All enzymatic hydrolysates can increase the relative content of osteocalcin, with the neutral protease hydrolysate showing the highest OCN level (2.1578 ng / L) at 100 μg / mL, and most treatment groups showing significantly higher levels than the control group. There was no significant difference between neutral protease and neutral protease + trypsin.

Claims

1. A calcium absorption-enhancing peptide prepared from defatted egg yolk powder, characterized in that: The calcium absorption-promoting peptides are P1, P2, P3, and P4, wherein P1 has the amino acid sequence shown in SEQ ID NO.1, P2 has the amino acid sequence shown in SEQ ID NO.2, P3 has the amino acid sequence shown in SEQ ID NO.3, and P4 has the amino acid sequence shown in SEQ ID NO.

4.

2. A method for preparing calcium absorption-enhancing peptides using defatted egg yolk powder as a raw material as described in claim 1, characterized in that: Includes the following steps: A. Raw material pretreatment and dissolution: Mix defatted egg yolk powder with water at a ratio of 1:8 to 1:12 (w / v) and stir until a suspension is formed. B. Enzymatic hydrolysis: In the first stage, neutral protease is used at pH 6.5–7.5 and temperature 45–55℃, with 0.5%–1.5% of the defatted egg yolk powder added for 2–4 hours; in the second stage, at the same pH and temperature, trypsin is added and enzymatic hydrolysis is continued for 1–3 hours at 0.1%–0.5% of the defatted egg yolk powder added to obtain the enzymatic hydrolysate. C. Inactivation and Separation: The enzymatic hydrolysate was heat-treated at 85–95℃ for 10–30 minutes for inactivation, and the supernatant was collected by centrifugation. The supernatant was then separated by CaCl2-induced ethanol fractionation to obtain the calcium absorption peptide mother liquor. The peptide identification results of the calcium absorption peptide mother liquor are as follows: it includes four active peptides modified with phosphoserine (pS), namely: VVGS(p)AEAGVDAASVSEEFRADHPF, LGGVGSPVSGGGKQLAS(p), IVLSVNITS(p), and NPLS(p)YTSAK. D. Drying and powdering: The calcium absorption peptide mother liquor is spray-dried or freeze-dried to obtain calcium absorption peptide powder.

3. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step A, the ratio of defatted egg yolk powder to water is 1:10 (w / v).

4. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as raw material according to claim 2, characterized in that: In step B, the amount of neutral protease added is 1.0% of the mass of the defatted egg yolk powder, and the enzymatic hydrolysis time is 3 hours.

5. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step B, the amount of trypsin added is 0.3% of the mass of the defatted egg yolk powder, and the enzymatic hydrolysis time is 2 hours.

6. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step C, the heat inactivation conditions are: 90°C for 20 minutes, CaCl2 reaction time for 1 hour, final ethanol concentration of 50% (v / v), and standing time for 2 hours.

7. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step C, the amount of CaCl2 added is 0.4% of the mass of the supernatant obtained in step C, and the final concentration of ethanol is controlled at 45%–55% (v / v).

8. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step C, all four active peptides were confirmed by UHPLC-LTQ-Orbitrap-Velos Pro high-resolution liquid chromatography-mass spectrometry, and the phosphorylation sites were located by the neutral loss 98 Da characteristic peak in the MS / MS fragment ion spectrum.

9. The method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material according to claim 2, characterized in that: In step D, the drying method is spray drying, with an inlet air temperature of 180°C and an outlet air temperature of 85°C.

10. A method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material as described in claim 1, or a method for preparing calcium absorption-promoting peptides using defatted egg yolk powder as a raw material as described in any one of claims 2-9, characterized in that: The calcium absorption-promoting peptide has the function of promoting intestinal calcium absorption.

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