A pumpkin seed meal protease hydrolysate zinc chelate and a preparation method and application thereof

Abstract

The present application relates to a pumpkin seed meal protease hydrolysate zinc chelate and a preparation method and application thereof, and belongs to the technical field of plant source nutritional fortifier and functional food ingredient preparation. The method uses pumpkin seed meal or defatted pumpkin seed meal as a raw material to prepare pumpkin seed meal protein isolate, and then performs a chelation reaction with a zinc salt after protease hydrolysis to obtain a pumpkin seed meal protease hydrolysate, and then performs alcohol precipitation, standing, centrifugation and drying treatment to obtain the target zinc chelate. The obtained zinc chelate has good zinc binding capacity, stability and food processing adaptability.

Description

Technical Field

[0001] This invention relates to the field of plant-derived nutrient fortifiers and functional food ingredients, and particularly to a zinc chelate of pumpkin seed meal protein hydrolysate, its preparation method, and its application. Background Technology

[0002] Zinc is an essential trace element for the human body, participating in key life processes such as enzymatic reactions, cell division, immune regulation, and growth and development. Zinc deficiency can lead to problems such as decreased appetite, abnormal taste, weakened immune function, and stunted growth and development. Existing zinc supplements are mainly divided into three categories: inorganic zinc salts, organic acid zinc salts, and amino acid or peptide zinc complexes. Among them, inorganic zinc salts are widely available and inexpensive, but they are highly irritating, have limited bioavailability, and insufficient stability. While organic acid zinc salts improve palatability, their processing stability and compatibility with complex food systems still have room for improvement.

[0003] In recent years, food-derived peptides, rich in coordination sites such as carboxyl and amino groups, have become important carriers for novel organic zinc supplements. The chelates they form with zinc ions can reduce the formation of insoluble precipitates of free zinc ions in the gastrointestinal environment, thus improving zinc stability and utilization efficiency. However, existing peptide-zinc systems exhibit significant performance differences due to variations in protein source, enzymatic hydrolysis, and chelation conditions. There is still a lack of plant-derived products with clearly defined sources, simple processing, suitability for food applications, and good zinc binding capacity and stability.

[0004] Pumpkin seed meal is a byproduct of the deep processing of pumpkin seeds into oil. It has a high protein content and great development potential, but its native protein has limited solubility and dispersibility, making it inefficient as a direct mineral carrier. Enzymatic hydrolysis can release more coordination groups from the protein, which can then chelate with zinc ions, potentially creating a high-value-added plant-based zinc supplement.

[0005] Therefore, developing a zinc chelate from pumpkin seed meal protease hydrolysate and its preparation method is of great significance for improving the utilization level of pumpkin seed meal by-products, expanding the plant-based zinc supplementation system, and developing new functional food ingredients. Summary of the Invention

[0006] To address the limitations of existing plant-based zinc supplements, such as limited zinc binding capacity, insufficient processing stability, and poor food compatibility, this invention provides a zinc chelate from pumpkin seed meal protease hydrolysate, its preparation method, and its applications. This method is simple to operate, low in cost, uses readily available raw materials, and does not require stepwise enzymatic hydrolysis; the entire enzymatic hydrolysis process can be completed with the addition of enzyme only once. The resulting zinc chelate exhibits excellent zinc binding capacity, stability, and food processing compatibility.

[0007] To achieve the above objectives, the present invention provides a method for preparing zinc chelates from pumpkin seed meal protein hydrolysate, comprising the following steps: (1) Using pumpkin seed meal or defatted pumpkin seed meal as raw material, crush it through a 50-200 mesh sieve, add deionized water and mix evenly, adjust the pH to 9.0-11.0, stir for 20-40 min, centrifuge to collect the supernatant, repeat the above steps 1-2 times for the remaining material, combine the supernatants, adjust the pH to 4.0-5.0 to precipitate, centrifuge to collect the precipitate, dialyze and freeze dry to obtain pumpkin seed meal isolated protein; (2) Disperse the pumpkin seed meal protein isolate at a substrate concentration of 3% to 8% in deionized water, adjust the pH to 4.0 to 6.5, add acidic protease, and enzymatically hydrolyze at 45 to 60°C for 2 to 8 hours; (3) Inactivate the enzyme in the enzymatic hydrolysate by boiling water bath at 95-100℃ for 10-20 min, cool to room temperature, centrifuge to collect the supernatant, freeze dry to obtain pumpkin seed meal protein hydrolysate; (4) Disperse the pumpkin seed meal protein hydrolysate in deionized water at a substrate concentration of 1% to 1.5%, adjust the pH to 5.5 to 6.5, add zinc salt to make the peptide-zinc mass ratio 1:(1 to 3), and stir and chelate at 50 to 65°C for 30 to 90 min. (5) Add 2 to 6 times the volume of anhydrous ethanol to the chelation reaction solution for alcohol precipitation, let stand at 0 to 10°C, centrifuge, collect the precipitate and dry it to obtain the zinc chelate of pumpkin seed meal protein hydrolysate.

[0008] Furthermore, the amount of acidic protease added in step (2) is 2000~10000 U / g protein.

[0009] Further, the enzymatic hydrolysis conditions in step (2) are as follows: substrate concentration of 5%, pH adjusted to 4.8, acidic protease of 8000 U / g protein added, and enzymatic hydrolysis at 55℃ for 6 h.

[0010] Further, the zinc salt mentioned in step (4) is selected from one or more of zinc acetate, zinc chloride, zinc lactate, and zinc gluconate.

[0011] Furthermore, the stirring speed for chelation in step (4) is 150-200 r / min.

[0012] Further, the chelation conditions in step (4) are as follows: substrate concentration is 1.2%, pH is adjusted to 6.0, zinc acetate is added to make the peptide-zinc ratio 1:2, and chelation is carried out at 60°C for 60 min with stirring.

[0013] Further, in step (5), the amount of anhydrous ethanol added is 4 times the volume of the reaction liquid, and the mixture is allowed to stand at 4°C and centrifuged at 7000 rpm for 15 min.

[0014] A second aspect of the present invention provides a zinc chelate of pumpkin seed meal protease hydrolysate prepared by the method described in the present invention, which is formed by the chelation of pumpkin seed meal protease hydrolysate and zinc ions through coordination bonds.

[0015] Furthermore, the molecular weight distribution of the pumpkin seed meal protein hydrolysate is 100 Da-6500 Da, accounting for more than 90% of the total protein hydrolysate mass.

[0016] Furthermore, the zinc chelation rate of the zinc chelate from the pumpkin seed meal protein hydrolysate is not less than 25%.

[0017] Furthermore, the zinc retention rate of the pumpkin seed meal protein hydrolysate zinc chelate is not less than 80% under pH 3.0~6.0 conditions.

[0018] Furthermore, the glass transition temperature of the zinc chelate of the pumpkin seed meal protease hydrolysate is not lower than 200°C.

[0019] Furthermore, the DPPH free radical scavenging rate of the zinc chelate of the pumpkin seed meal protein hydrolysate is not less than 65%, and the ABTS free radical scavenging rate is not less than 92%.

[0020] A third aspect of this invention provides the application of the zinc chelate of pumpkin seed meal protease hydrolysate described in this invention in the preparation of zinc-supplemented foods, health foods, or feed additives.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a method for preparing and applying zinc chelates from pumpkin seed meal protease hydrolysates. By enzymatically releasing the potential coordinating groups in pumpkin seed meal protein, the binding capacity with zinc ions is improved, achieving a zinc chelation rate of up to 26.98%. Furthermore, the obtained zinc chelates maintain good stability under certain heat treatment, salt environment, and acid-base conditions, making them suitable for food processing applications. They can be used as plant-based zinc supplements in solid beverages, nutritional powders, plant protein beverages, instant products, meal replacement foods, and other functional food systems. This is of great significance for improving the utilization level of pumpkin seed meal by-products, expanding plant-based zinc supplementation systems, and developing novel functional food ingredients. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1This is a comparison chart of the zinc binding capacity of the chelates obtained in some embodiments and comparative examples of the present invention; Figure 2 This is the ultraviolet absorption spectrum of the electron microscope images of the chelates obtained in this invention and the comparative examples; Figure 3 These are the Fourier transform infrared spectra of the chelates obtained in this invention and the comparative examples; Figure 4 These are scanning electron microscope images of the chelates obtained in this invention and the comparative examples; Figure 5 These are the energy dispersive X-ray spectra of the chelates obtained in this invention and the comparative examples; Figure 6 This is a particle size distribution diagram of the chelates obtained in this invention and comparative examples; Figure 7 This is a Zeta potential diagram of the chelates obtained in this invention and the comparative examples; Figure 8 This is a differential scanning calorimetry curve of the chelates obtained in this invention and the comparative examples; Figure 9 This is a stability graph of the chelates obtained by the present invention and the comparative examples after heating at 100°C for different times; Figure 10 This is a stability diagram of the chelates obtained by the present invention and the comparative examples under pasteurization treatment; Figure 11 This is a stability diagram of the chelates obtained by the present invention and the comparative examples under sodium chloride treatment; Figure 12 This is a stability graph of the chelates obtained by the present invention and the comparative examples under different pH treatments; Figure 13 This is a stability graph of the chelates obtained by the present invention and comparative examples at different temperatures. Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In one embodiment of the present invention, after enzyme inactivation, cooling to room temperature is achieved using a 0-4°C ice-water bath or other rapid cooling methods.

[0026] In one embodiment of the present invention, unless otherwise specified, room temperature generally refers to 15~30°C.

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0029] Example 1 Preparation of zinc chelate from pumpkin seed meal protein hydrolysate (1) Preparation of pumpkin seed meal protein isolate: Weigh defatted pumpkin seed meal, pulverize it through an 80-mesh sieve, add deionized water, and mix thoroughly at a material-to-liquid ratio of 1:10. Adjust the pH of the system to 11.0 using 1 mol / L NaOH, and extract by stirring at room temperature for 30 min. Then, centrifuge at 4 ℃ and 4000 r / min for 20 min, and collect the supernatant. Repeat the extraction twice on the residue, combine the supernatants, and adjust the pH to 4.5 using 1 mol / L HCl to precipitate the protein. Collect the precipitate by centrifugation, wash it with deionized water, and place it in a dialysis bag with a molecular weight cutoff of 6-8 kDa. Dialyze at 4 ℃ for 72 h, and then freeze-dry to obtain isolated pumpkin seed meal protein.

[0030] (2) Preparation of pumpkin seed meal protein hydrolysate: The pumpkin seed meal protein isolate obtained in step (1) was dispersed in deionized water at a substrate concentration of 5%, the pH of the system was adjusted to 4.8, and acidic protease (Shanghai Yuanye Biotechnology Co., Ltd.) was added at an enzyme addition amount of 8000 U / g protein. Enzymatic hydrolysis was carried out at 55 ℃ for 6 h. After the enzymatic hydrolysis was completed, the system was heated to 100 ℃ to inactivate the enzyme for 15 min, cooled to room temperature, centrifuged, the supernatant was collected and freeze-dried to obtain the pumpkin seed meal protease hydrolysate.

[0031] (3) Preparation of zinc chelates: Take the pumpkin seed meal protease hydrolysate obtained in step (2), disperse it in deionized water at a substrate concentration of 1.2%, and adjust the pH of the system to 6.0. Add zinc acetate to the peptide solution to make the peptide-zinc mass ratio 1:2, and stir the reaction at 60 ℃ for 60 min. After the chelation reaction is completed, add 4 volumes of anhydrous ethanol to the reaction solution for alcohol precipitation. After standing at 4 ℃, centrifuge at 7000 rpm for 15 min, collect the precipitate and freeze-dry it to obtain the pumpkin seed meal protease hydrolysate zinc chelate.

[0032] Compare with Example 1 The difference from Example 1 is that a neutral protease (Shanghai Yuanye Biotechnology Co., Ltd., 12000U / g) was used instead of an acidic protease to prepare the pumpkin seed meal hydrolysate, while the remaining enzymatic hydrolysis and chelation steps were the same.

[0033] Compare with Example 2 The difference from Example 1 is that an alkaline protease (Beijing Solarbio Science & Technology Co., Ltd., 20000U / g) was used instead of an acidic protease to prepare the pumpkin seed meal hydrolysate, while the remaining enzymatic hydrolysis and chelation steps were the same.

[0034] Compare with Example 3 The difference from Example 1 is that a complex protease (Shanghai Yuanye Biotechnology Co., Ltd., 10000U / g) was used instead of an acidic protease to prepare pumpkin seed meal protease hydrolysate, while the remaining enzymatic hydrolysis and chelation steps were the same.

[0035] Compare with Example 4 The difference from Example 1 is that papain (Shanghai Yuanye Biotechnology Co., Ltd., 4000U / g) was used instead of acidic protease to prepare pumpkin seed meal hydrolysate, while the remaining enzymatic hydrolysis and chelation steps were the same.

[0036] The present invention also subjected the chelates obtained in the above embodiments and comparative examples to the following tests, and the test methods and results are as follows: 1. Determination of zinc content and chelating ability: Weigh a certain amount of the zinc chelate sample obtained in Example 1 and dissolve it in a quantitative amount of deionized water. Transfer a portion of the sample solution, add ammonia-ammonium chloride buffer and Eriochrome Black T indicator, and titrate with 0.01 mol / L EDTA standard solution. The endpoint is determined when the solution color changes from purple-red to blue.

[0037] The zinc chelation rate can be calculated using the following formula: Zinc chelation rate (%) = (V1 - V0) / (V - V0) × 100% Where V1 is the volume of EDTA solution consumed in titrating the zinc content in the chelated state; V is the volume of EDTA solution consumed to titrate the total zinc content in the reaction solution; V0 is the volume of EDTA solution consumed when deionized water is used instead of the test solution.

[0038] Depend on Figure 1It can be seen that the zinc chelation rates of Example 1 and the control example are between 21% and 27%, with the zinc chelation rate of Example 1 being significantly higher than that of the control example, reaching 26.98%. The acidic protease in Example 1 maintains high activity under low pH conditions (e.g., pH 3.0-5.0), and its endonuclease properties allow for precise cleavage of hydrophobic amino acid sites (e.g., phenylalanine, leucine), exposing more carboxyl groups (-COO). - It contains chelating sites such as amino groups (-NH2). Simultaneously, acidic conditions inhibit competition from metal ions such as calcium and magnesium, promoting the formation of stable coordination structures between zinc and peptide chains, reducing the precipitation of free zinc, and thus improving chelation efficiency.

[0039] 2. Ultraviolet full-wavelength scanning: The ultraviolet absorption spectrum of the 1 mg / mL sample was measured at 25 °C using a UV-Vis spectrophotometer in the range of 190–400 nm, with deionized water as the blank.

[0040] like Figure 2 As shown, the maximum absorption wavelength of the chelate is around 190 nm. The peak of Example 1 is slightly red-shifted (192.4 nm), and the absorbance is slightly lower. Under acidic conditions, the protein partially denatures, exposing hydrophobic groups. Acidic proteases preferentially cleave hydrophobic amino acid sites (such as phenylalanine and leucine) to form short peptide chains. Zinc ions bind to the carboxyl groups at the ends of the peptide chains, but some uncoordinated zinc ions may cause local structural loosening, resulting in slightly lower absorbance. In Control Example 1, a sharp main peak appears at 192.4 nm, with a sharp and symmetrical peak shape. Neutral proteases catalyze under neutral pH conditions, preserving the native conformation of the peptide chain while efficiently cleaving hydrophobic regions, exposing more polypeptide bonds (n→π* transitions). Zinc ions pass through the carboxyl groups (-COO). - The zinc ion forms stable coordination with the amino group (-NH2), reducing peptide chain aggregation and enhancing the absorbance of the free peptide. In Control Example 2, the peak further red-shifted (191.9 nm), and the absorbance decreased. Alkaline conditions led to excessive protein denaturation, exposing basic amino acids such as lysine (ε-NH2) and arginine (guanidinyl), which formed multi-point coordination with zinc. However, excessive cross-linking caused peptide chain aggregation, hindering the n→π* transition of peptide bonds. An alkaline environment may also cause incomplete hydrolysis of some peptide chains, leaving behind large molecular fragments, further reducing absorbance. Control Example 3 exhibited a broad and asymmetrical peak shape. The complex protease possessed both endopeptidation and exopeptidation activities, resulting in shorter and highly compact peptides, leading to reduced peptide bond exposure. Zinc ions formed strong coordination bonds with cysteine ​​(-SH) and histidine (imidazolyl), inhibiting the flexible movement of peptide chains and weakening the UV absorption intensity. The absorption peak of control example 4 is weaker. During papain catalysis, zinc forms stable sulfur-zinc coordination bonds with sulfur-containing amino acids (such as cysteine). However, sulfur coordination increases peptide chain rigidity, inhibiting peptide bond transitions. 2+After forming coordination bonds with the N and O atoms in the polypeptide, changes occur in the chromophores (such as -C=O, -COOH) and the internal spatial structure of the molecule. These results indicate that the acidic protease in the control example formed more short-chain peptides.

[0041] 3. Fourier transform infrared spectroscopy determination: The enzymatic hydrolysate and KBr were dried, then ground and compressed into tablets at a ratio of 5 mg:200 mg. The concentrations were measured at 4000-400 cm⁻¹. - ¹Range spectrum (resolution 4 cm⁻¹) - ¹).

[0042] like Figure 3 As shown, the amide I band mainly originates from the stretching vibration of the C=O bond in the peptide chain (which also includes the coupling of some NH bending vibration and CN stretching vibration), and its characteristic absorption peak is located at 1700-1600 cm⁻¹. -1 Within this range, the characteristic absorption peak position of amide I band shows a significant correlation with the protein secondary structure type. This is particularly evident in the 3750-3000 cm⁻¹ range. -1 The characteristic absorption peaks observed in the high-frequency region are mainly related to NH and OH bond vibrations associated with hydration. (The text abruptly shifts to a seemingly unrelated topic about Zn in Example 1.) 2+ When it coordinates with the peptide chain, the formation of the N-Zn bond leads to the substitution of the NH bond, thereby shifting the characteristic absorption peak to 3300 cm⁻¹. -1 The stretching vibration characteristics of the amide I band are mainly attributed to the vibrational mode of the C=O bond. The characteristic absorption peak appearing at 1100 cm⁻¹ can be attributed to the stretching vibration of the -COO- group. These results indicate that the carbonyl oxygen, carboxyl oxygen, and amino nitrogen atom are the key sites for the interaction in Example 1.

[0043] 4. Microstructure determination Take a small amount of sample, spread it evenly on conductive adhesive, spray gold onto it, and perform SEM scanning at a voltage of 15 kV. Magnify at different magnifications to observe the microscopic morphology.

[0044] like Figure 4As shown, Example 1 has a uniform and smooth surface, with small and tightly packed particles, no obvious pores or agglomeration, and presents as dark, dense particles with clear boundaries. Control Example 1 has larger particles with more irregular shapes, some areas with loose aggregates, increased surface roughness, and a few visible depressions or cracks. Control Example 2 has coarser particles, forming a distinct blocky structure, significantly increased surface complexity, and increased pore distribution. The aggregates are uneven in size, with some areas showing layering or breakage, possibly related to pH regulation or salting out. Control Example 3 has significantly larger particles, presenting a polydisperse system with large and small particles coexisting, a rough surface, and dense aggregates. Fibrous or lamellar structures are visible in some areas, resulting from the interaction between organic components and zinc salts. Control Example 4 has the largest and most widely distributed particles, with a highly irregular surface, containing numerous irregular blocky aggregates and void structures. These results indicate that the pumpkin seed hydrolysate of Example 1 and Zn... 2+ Tight and stable chelation.

[0045] 5. Energy-dispersive X-ray spectroscopy The elemental distribution of the sample was determined by energy-dispersive X-ray spectroscopy.

[0046] like Figure 5 As shown, in Example 1, the Zn peak was the most intense and sharp, while the C and O signals were weak, and there were no obvious impurity peaks. The low impurity signal indicates high specificity of the chelation process, with zinc ions tightly bound to the peptide chain, resulting in the highest zinc content. In Control Example 1, the Zn main peak was broad and of moderate intensity. It was accompanied by obvious C and O signals, with diffuse peak shapes and high background noise. Zinc ions bind tightly to the carboxyl groups (-COO) in the peptide chain. - The zinc nucleus forms a coordination bond with the amino group (-NH2), but it does not completely encapsulate the zinc core, resulting in some residual free zinc (increased peak width). In Control Example 2, the Zn peak intensity is weak and accompanied by high C and O signals, with a sharp peak shape. In Control Example 3, the Zn peak intensity is relatively high, and the peak shape symmetry is the best. In Control Example 4, the Zn peak is extremely weak, C and O signals dominate, and there are no significant peaks for other elements. The above results indicate that in Example 1, the chelation effect of pumpkin seed meal protein hydrolysate with zinc is the best.

[0047] 6. Particle size determination The particle size distribution of the samples was determined using a Malvern Master Sizer 2000 particle size analyzer. Deionized water was used as the dispersion medium, and the samples were equilibrated at 25 °C for 120 s before testing.

[0048] like Figure 6As shown, the particle size distribution of the five chelates is mainly concentrated between 300-1200 nm. The particle size distribution peaks, divided by particle size from largest to smallest, are: Control Example 2 (750-1400 nm), Control Example 1 (550-1200 nm), Control Example 3 (500-1100 nm), Control Example 4 (370-620 nm), and Example 1 (300-500 nm). These results indicate that Example 1 has the smallest particle size. This is because the acidic protease hydrolyzes the peptides more thoroughly, creating more zinc-binding sites, which redisperses the aggregated peptides, thus reducing the particle size.

[0049] 7. Zeta potential measurement The zeta potential of the samples was determined using a Malvern Zetasizer Nano ZS laser particle size analyzer. Test conditions: 25 ℃, refractive index 1.330. Before testing, the samples were prepared as a 0.1 mg / mL solution, ultrasonically mixed, and then transferred to the zeta potential cell for detection.

[0050] The absolute value of the zeta potential represents the electrostatic repulsion between substances. The higher the absolute value of the zeta potential, the greater the electrostatic repulsion between particles, and the more stable the system. For example... Figure 7 As shown, Example 1 (13.9 mV) > Control Example 1 (12.5 mV) > Control Example 2 (11.87 mV) > Control Example 3 (7.21 mV) > Control Example 4 (2.52 mV). These results indicate that Example 1 was the most stable.

[0051] 8. Amino acid analysis Weigh 1.0 g of sample into a hydrolysis tube, add 10 mL of 6 mol / L HCl and 1 g of phenol, seal under nitrogen for 10 min, and hydrolyze at 110 ℃ for 24 h. After cooling, transfer to a 50 mL volumetric flask and dilute to volume with ultrapure water. Take 0.5–1 mL of the hydrolysate into a petri dish, evaporate to dryness in a 60 ℃ water bath, repeat the “evaporation-reconstitution” step twice, and finally reconstitute with 3–5 mL of buffer solution. Vortex to mix, filter through a 0.22 μm filter membrane, and determine the amino acid content in the sample using an automated amino acid analyzer.

[0052] Table 1. Amino acid composition analysis of the examples and control examples. Table 1 shows the amino acid composition of peptide-zinc chelates obtained from different hydrolysates. The chelates are rich in various essential amino acids, possessing the potential to serve as a high-quality source of amino acids. Branched-chain amino acids (BCAAs) include: Leu, Ile, Val. Antioxidant amino acids include: Glu, Try, Val, Tyr. Essential amino acids (EAAs) include: Thr, Cys, Val, Met, Ile, Leu, Tyr, pHe, Lys, His. Among the five chelates, glutamic acid was the most abundant: Example 1 (32.68%), Control Example 1 (31.94%), Control Example 2 (31.48%), Control Example 3 (32.11%), and Control Example 4 (32.01%). Control Example 3 had the highest glutamic acid content, followed by aspartic acid, indicating that glutamic acid and aspartic acid play a crucial role in zinc binding.

[0053] Example 1 showed the highest total content (28.38%) of amino acids with antioxidant activity (including tryptophan, methionine, histidine, lysine, cysteine, arginine, and tyrosine). Furthermore, Example 1 also showed the highest content of amino acids containing acidic side chains (R groups), which readily react with Zn. 2+ A stable peptide-zinc chelate is formed. These results indicate that glutamic acid and aspartic acid in Control Example 3 play a crucial role in the zinc binding process, and these antioxidant amino acids have the highest content, laying an important foundation for further functional studies.

[0054] 9. Thermal Performance Analysis The thermal stability of the sample was determined by differential scanning calorimetry. 5 mg of sample was accurately weighed and placed in a nitrogen atmosphere, and the temperature was programmed to rise from 25 °C to 380 °C at a rate of 10 °C / min.

[0055] like Figure 8 As shown, Example 1 has the highest denaturation temperature of 225.84 °C, and its thermal stability is greater than that of the other four control examples.

[0056] 10. Stability determination (1) Determination of the stability of chelates by heating at 100 °C Five chelates were prepared into 20 mg / mL solutions. The solutions were subjected to gradient heat treatment (100 °C) for 15–75 min in a constant temperature water bath. After high-speed centrifugation (10000 r / min, 10 min), the zinc ion concentrations in the supernatant and the whole solution were determined by EDTA titration to assess the thermal stability of the samples.

[0057] like Figure 9As shown, the zinc retention rate of all samples decreased with increasing heating time. In Example 1, during the entire 75-minute heating process, the Zn content... 2+ The retention rate was significantly higher than that of other samples.

[0058] (2) Stability determination of chelates by pasteurization treatment Five chelates were prepared into 20 mg / mL solutions. Pasteurization conditions were constructed (63 ℃ / 30 min, 69 ℃ / 30 min, 72 ℃ / 16 min, 80 ℃ / 25 min, 90 ℃ / 12 min). After centrifugation (10000 r / min, 10 min), the zinc ion concentrations in the supernatant and whole solution were determined by EDTA titration to assess the sterilization stability of the samples.

[0059] like Figure 10 As shown, the zinc retention rate of all samples decreased with increasing temperature, consistent with the expectation that high temperatures would disrupt the chelate structure. Among them, Example 1 showed the smallest decrease in zinc retention rate, possibly because an acidic environment enhances the stability of the chelate. Example 1 forms a strong coordination bond with zinc, resulting in a more stable molecular structure at high temperatures. These results indicate that Example 1 is more suitable for commercial food production because it maintains a high zinc retention rate and good stability after sterilization.

[0060] (3) Stability determination of NaCl on peptide-zinc chelates Five chelates were prepared into 20 mg / mL solutions. Each solution was placed in a gradient concentration of NaCl solution (2%–10% w / v) and reacted in a constant temperature shaking water bath (37 °C) for 2 h. After centrifugation (10000 r / min, 10 min), the zinc ion concentration in the supernatant and the whole solution was determined by EDTA titration to assess the effect of NaCl concentration on sample stability.

[0061] like Figure 11 As shown, Example 1 still has a zinc retention rate of over 90% at a 2% NaCl concentration, and the zinc retention rate is still over 70% at a 10% concentration. However, in actual production and processing, it is still necessary to avoid contact with NaCl concentrations of 6% or higher.

[0062] (4) Determination of the stability of peptide-zinc chelates by pH A citrate-phosphate buffer system with pH ranges of 3.0–6.0 was prepared. Five chelates were thoroughly mixed with the buffer solution to prepare solutions of 20 mg / mL, and reacted in a constant-temperature shaking water bath (37 °C) for 2 h. After centrifugation (10000 r / min, 10 min), the zinc ion concentrations in the supernatant and the whole solution were determined by EDTA titration to assess the effect of pH on sample stability.

[0063] The pH stability of peptide-zinc chelates directly determines their intestinal absorption efficiency and bioavailability. For example... Figure 12 As shown, Example 1 exhibits the best pH stability, with no effect on Example 1 in the pH range of 3.0-6.0. Example 1 maintains a retention rate of >80% in the pH range of 3.0-6.0, which is attributed to the coordination effect of the carboxylic acid group of the acidic protease and the Cl... - The reduced competitive inhibition is closely related to this. These results indicate that Example 1 exhibits excellent pH stability in acidic environments, which may contribute to its better bioavailability.

[0064] (5) Storage stability test A certain mass of samples were weighed and stored for 15 days under the conditions of room temperature, 4 ℃ refrigeration, and -20 ℃ freezing, respectively. The changes in zinc ion content were monitored to assess the storage stability of the samples.

[0065] like Figure 13 As shown, the chelate stored at room temperature effectively delayed the release of metal ions. This result indicates that the peptide-zinc complex exhibits superior stability in room temperature storage and transportation systems. All samples showed the best performance under room temperature storage conditions, with no significant decrease in zinc content observed in any of them.

[0066] 11. Antioxidant activity assay (1) Determination of DPPH free radical scavenging rate Accurately weigh 0.03943 g of DPPH reagent, dissolve it in anhydrous ethanol, and bring the volume to 500 mL. Take 1 mL of each of the test solutions (1 mg / mL) and incubate with an equal volume of DPPH solution in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader. Two control groups were set up: a blank control group (containing only DPPH solution) and a control group (containing only the sample solution). Calculation formula: DPPH free radical scavenging rate % = 1 - (AC) / B × 100 In the formula: A: absorbance of the experimental group; B: absorbance of the control group; C: absorbance of the decolorized group.

[0067] (2) Determination of ABTS free radical scavenging rate Preparation of ABTS radical working solution: An equal volume of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution was mixed and incubated in the dark for 12 h. The absorbance was adjusted to 0.7 ± 0.02 (734 nm) with 0.1 mol / L PBS (pH 7.4). The sample and working solution were mixed at a ratio of 1:9, and the absorbance at 734 nm was measured after reacting in the dark for 5 min. Calculation formula: ABTS radical scavenging rate % = 1 - (AC) / B × 100 In the formula: A: absorbance of the experimental group; B: absorbance of the control group; C: absorbance of the decolorized group.

[0068] (3) Reducing power measurement Transfer 2.0 mL of sample solution, add 2.0 mL of 0.2 mol / L PBS (pH 6.6) and 2.0 mL of 1% K3[Fe(CN)6] solution sequentially, incubate at 55 ℃ for 20 min, and then cool to room temperature. Add 2.0 mL of 10% TCA solution, centrifuge at 10000 rpm for 10 min, take 2.0 mL of the supernatant and mix with 400 μL of 0.1% FeCl3 solution by vortexing. After developing the solution in the dark for 10 min, measure the absorbance at 700 nm using a UV spectrophotometer to evaluate the total reducing power.

[0069] Table 2 Antioxidant activity of the examples As shown in Table 2, Example 1 exhibits a DPPH radical scavenging rate of 65%, an ABTS radical scavenging rate of 90%, and a high Fe content. 3+ The reducing power indicates that Example 1 has a certain antioxidant effect.

[0070] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing a zinc chelate from pumpkin seed meal protein hydrolysate, characterized in that, Includes the following steps: (1) Using pumpkin seed meal or defatted pumpkin seed meal as raw material, crush it through a 50-200 mesh sieve, add deionized water and mix evenly, adjust the pH to 9.0-11.0, stir for 20-40 min, centrifuge to collect the supernatant, repeat the above steps 1-2 times for the remaining material, combine the supernatants, adjust the pH to 4.0-5.0 to precipitate, centrifuge to collect the precipitate, dialyze and freeze dry to obtain pumpkin seed meal isolated protein; (2) Disperse the pumpkin seed meal protein isolate at a substrate concentration of 3% to 8% in deionized water, adjust the pH to 4.0 to 6.5, add acidic protease, and enzymatically hydrolyze at 45 to 60°C for 2 to 8 hours; (3) Inactivate the enzyme in the enzymatic hydrolysate by boiling water bath at 95-100℃ for 10-20 min, cool to room temperature, centrifuge to collect the supernatant, freeze dry to obtain pumpkin seed meal protein hydrolysate; (4) Disperse the pumpkin seed meal protein hydrolysate in deionized water at a substrate concentration of 1% to 1.5%, adjust the pH to 5.5 to 6.5, add zinc salt to make the peptide-zinc mass ratio 1:(1 to 3), and stir and chelate at 50 to 65°C for 30 to 90 min. (5) Add 2 to 6 times the volume of anhydrous ethanol to the chelation reaction solution for alcohol precipitation, let stand at 0 to 10°C, centrifuge, collect the precipitate and dry it to obtain the zinc chelate of pumpkin seed meal protein hydrolysate.

2. The preparation method according to claim 1, characterized in that, The amount of acidic protease added in step (2) is 2000~10000 U / g protein.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis conditions in step (2) are as follows: substrate concentration of 5%, pH adjusted to 4.8, acidic protease of 8000 U / g protein added, and enzymatic hydrolysis at 55℃ for 6 h.

4. The preparation method according to claim 1, characterized in that, The zinc salt mentioned in step (4) is selected from one or more of zinc acetate, zinc chloride, zinc lactate, and zinc gluconate.

5. The preparation method according to claim 1, characterized in that, The stirring speed for chelation in step (4) is 150-200 r / min.

6. The preparation method according to claim 1, characterized in that, The chelation conditions in step (4) are as follows: substrate concentration is 1.2%, pH is adjusted to 6.0, zinc acetate is added to make the peptide-zinc ratio 1:2, and chelation is carried out at 60°C for 60 min with stirring.

7. The preparation method according to claim 1, characterized in that, The amount of anhydrous ethanol added in step (5) is 4 times the volume of the reaction liquid. The mixture is allowed to stand at 4°C and then centrifuged at 7000 rpm for 15 min.

8. A zinc chelate of pumpkin seed meal protease hydrolysate prepared by the method according to any one of claims 1 to 7, wherein the pumpkin seed meal protease hydrolysate and zinc ions are chelated by coordination bonds.

9. The zinc chelate of pumpkin seed meal protease hydrolysate according to claim 8, characterized in that, The molecular weight distribution of the pumpkin seed meal protein hydrolysate is 100 Da-6500 Da, accounting for more than 90% of the total protein hydrolysate mass; The zinc chelation rate of the zinc chelate from the pumpkin seed meal protein hydrolysate is not less than 25%. The zinc chelate from the pumpkin seed meal protein hydrolysate has a zinc retention rate of not less than 80% under pH 3.0~6.0 conditions; The glass transition temperature of the zinc chelate of the pumpkin seed meal protein hydrolysate is not lower than 200℃; The zinc chelate of the pumpkin seed meal protein hydrolysate has a DPPH free radical scavenging rate of not less than 65% and an ABTS free radical scavenging rate of not less than 92%.

10. The application of the zinc chelate of pumpkin seed meal protease hydrolysate according to claim 8 in the preparation of zinc-supplemented foods, health foods or feed additives.

Images