A pumpkin seed meal low molecular peptide zinc composition, a preparation method, a simulated digestion and absorption evaluation method and application thereof

Abstract

This invention discloses a low-molecular-weight peptide-zinc composition from pumpkin seed meal, its preparation method, a simulated digestion and absorption evaluation method, and its applications, relating to the field of plant-derived mineral element delivery technology. The preparation method of the composition includes: isolating protein from pumpkin seed meal, obtaining low-molecular-weight peptide components with a molecular weight less than 3 kDa through enzymatic hydrolysis and molecular weight fractionation, and then reacting these peptides with edible zinc salts to obtain the final product. This invention also provides the application of this composition in the preparation of peptide-based zinc delivery systems or zinc absorption-enhancing products, and provides a simulated digestion and absorption evaluation method using simulated gastrointestinal digestion and everted intestinal sac models. This invention found that the low-molecular-weight peptide components with a molecular weight less than 3 kDa, when combined with edible zinc salts, exhibit higher zinc chelation capacity in the pumpkin seed meal low-molecular-weight peptide-zinc composition. Furthermore, this composition maintains high soluble zinc levels during simulated intestinal digestion and demonstrates sustained zinc transport characteristics in the everted intestinal sac model, making it suitable for use in zinc supplementation products.

Description

Technical Field

[0001] This invention relates to the fields of plant-derived mineral element delivery, in vitro simulated digestion evaluation, in vitro intestinal transport evaluation, and functional food ingredient preparation technology, specifically to a low molecular weight peptide zinc composition of pumpkin seed meal and its preparation method, simulated digestion and absorption evaluation method, and application. Background Technology

[0002] Zinc is an essential trace element for the human body, participating in processes such as enzyme catalysis, immune regulation, cell proliferation, growth and development, and redox homeostasis. The nutritional effect of dietary zinc depends not only on intake but also on its chemical form, solubility, interaction with food matrices, and intestinal transport capacity in the gastrointestinal tract. Particularly in plant-based food systems, phytic acid, dietary fiber, polyphenols, and other mineral ions can easily affect the solubility of zinc, reducing the proportion of measurable zinc species in the intestinal lumen.

[0003] Existing zinc supplements mainly include inorganic zinc salts, organic acid zinc salts, amino acid zinc, and peptide zinc complexes. Inorganic zinc salts are relatively inexpensive, but they are easily affected by pH changes and food matrix interference in the gastrointestinal environment. Organic acid zinc salts have good water solubility, but their retention and sustained transport after entering the intestines are still limited. Food-derived peptides contain potential coordinating groups such as carboxyl, amino, carbonyl, imidazole, and thiol groups, which can form peptide-zinc binding structures with zinc ions, thereby altering the form of zinc in digestive juices.

[0004] Pumpkin seed meal, a byproduct of pumpkin seed oil processing, contains a high proportion of protein and has the potential to be developed into a plant-derived mineral-bound peptide raw material. Enzymatic hydrolysis with proteases can release low-molecular-weight peptides from the pumpkin seed meal protein and expose more functional groups; further molecular weight fractionation can enrich low-molecular-weight peptide components with high zinc-binding capacity. Constructing peptide-zinc compositions using these low-molecular-weight peptides is expected to improve zinc retention during simulated gastrointestinal digestion and its apparent transport behavior in isolated intestinal models.

[0005] Existing plant-derived zinc peptide technologies primarily focus on raw material enzymatic hydrolysis, chelation rate optimization, structural characterization, and general stability evaluation. However, they lack systematic evaluation of changes in particle size, surface charge, peptide chain hydrolysis, zinc retention, and in vitro intestinal transport after the peptide-zinc composition enters the gastrointestinal digestive environment. Relying solely on the initial chelation rate is insufficient to determine the actual potential of the peptide-zinc composition in intestinal delivery. Therefore, there is an urgent need to establish an evaluation method based on simulated gastrointestinal digestion and everted intestinal sac models, and to use this method to screen low-molecular-weight peptide-zinc intestinal delivery compositions from pumpkin seed meal, providing more direct functional evidence for the development of plant-derived zinc delivery ingredients. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art. To address the issue of prioritizing initial chelation and neglecting the digestion and absorption process in the evaluation of existing plant-derived peptide-zinc systems, this invention provides a low-molecular-weight peptide-zinc composition from pumpkin seed meal, along with its simulated digestion and absorption evaluation method and application. This method uses a low-molecular-weight peptide-zinc composition from pumpkin seed meal as the target, integrating simulated gastrointestinal digestion, zinc retention rate determination, colloidal recombination characterization, and apparent absorption evaluation of everted intestinal sacs into a continuous evaluation process. This allows for the screening of plant-derived peptide-based zinc delivery compositions with intestinal digestion zinc retention capacity and continuous apparent zinc transport capacity.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a low-molecular-weight peptide zinc composition from pumpkin seed meal, comprising the following steps: S1. Prepare pumpkin seed meal isolated protein using pumpkin seed meal or defatted pumpkin seed meal as raw material; S2. The pumpkin seed meal isolated protein is enzymatically hydrolyzed using acidic protease to obtain pumpkin seed meal protein hydrolysate; S3. The pumpkin seed meal protein hydrolysate is fractionated by molecular weight to obtain low molecular weight peptide components with a molecular weight of less than 3 kDa. S4. The low molecular weight peptide component with a molecular weight of less than 3 kDa undergoes a coordination reaction with edible zinc salt to obtain a pumpkin seed meal low molecular weight peptide zinc composition.

[0008] Preferably, S1 includes the following specific steps: pulverizing defatted pumpkin seed meal, adding deionized water, and mixing evenly at a material-to-liquid ratio of 1:8~12; adjusting the pH of the system to 10.5~11.5 using 0.5~1.5 mol / L NaOH, stirring and extracting at room temperature for 25~35 min, then centrifuging at 3~5℃ and 3500~4500 r / min for 15~25 min, and collecting the supernatant; adjusting the pH of the supernatant to 4~5 using 0.5~1.5 mol / L HCl to precipitate the protein; collecting the precipitate by centrifugation, washing with deionized water, and placing it into a dialysis bag with a molecular weight cutoff of 6 to 8 kDa, dialyzing at 3~5℃ for 66~78 h, and freeze-drying to obtain pumpkin seed meal isolated protein.

[0009] Preferably, S2 includes the following specific steps: dispersing pumpkin seed meal protein isolate in deionized water at a substrate concentration of 4-6% w / v, adjusting the pH of the system to 4.6-5.0, adding acidic protease at an enzyme addition amount of 7000-9000 U / g protein, and enzymatically hydrolyzing at 50-60℃ for 2.5-3.5 h; after enzymatic hydrolysis, heating the system to inactivate the enzyme, cooling to room temperature, centrifuging, collecting the supernatant, and freeze-drying to obtain pumpkin seed meal protein hydrolysate.

[0010] Preferably, in step S3, a 3 kDa ultrafiltration membrane is used to fractionate the pumpkin seed meal protein hydrolysate to obtain low molecular weight peptide components with a molecular weight of less than 3 kDa; the zinc binding capacity of the low molecular weight peptide components with a molecular weight of less than 3 kDa is not less than 26%. Preferably, in S4, the edible zinc salt is selected from one or more of zinc acetate, zinc gluconate, zinc lactate, zinc chloride, or zinc sulfate; the mass ratio of the low molecular weight peptide component with a molecular weight less than 3 kDa to the edible zinc salt is 1:1 to 1:3, the pH of the coordination reaction is 5.0 to 7.0, the temperature of the coordination reaction is 45 to 65°C, and the time of the coordination reaction is 30 to 120 min.

[0011] More preferably, in S4, the mass ratio of the low molecular weight peptide component with a molecular weight of less than 3 kDa to the edible zinc salt is 1:2, the coordination pH is 6.0, the coordination temperature is 55°C, and the coordination time is 60 min.

[0012] Secondly, the present invention provides a low molecular weight peptide zinc composition of pumpkin seed meal, which is obtained by the preparation method described above.

[0013] Preferably, the composition comprises a low molecular weight peptide of less than 3 kDa derived from pumpkin seed meal protein and zinc bound to the low molecular weight peptide.

[0014] Furthermore, one or more of the carboxyl, amino, carbonyl, or amide groups in the low molecular weight peptide participate in zinc binding.

[0015] Furthermore, the zinc-binding capacity of the low molecular weight peptide with a molecular weight of less than 3 kDa is not less than 26%.

[0016] Furthermore, the apparent zinc absorption rate of the composition in the everted intestinal sac model is not less than 30% after 60 min.

[0017] Furthermore, the composition retains no less than 40% of zinc at the end of simulated intestinal digestion.

[0018] Thirdly, the present invention provides the application of the aforementioned pumpkin seed meal low molecular weight peptide zinc composition in the preparation of a peptide-based zinc delivery system.

[0019] Fourthly, the present invention provides the application of the aforementioned pumpkin seed meal low molecular weight peptide zinc composition in the preparation of zinc absorption-promoting products.

[0020] Fifthly, the present invention provides the application of the aforementioned pumpkin seed meal low molecular weight peptide zinc composition in the preparation of zinc-supplemented foods, solid beverages, nutritional powders, plant protein beverages, meal replacement foods, special dietary foods, health foods, or feed additives.

[0021] In a sixth aspect, the present invention provides the application of the aforementioned pumpkin seed meal low molecular weight peptide zinc composition in simulated digestion and absorption.

[0022] In a seventh aspect, the present invention provides a method for simulating digestion and absorption evaluation, comprising the following steps: (1) The pumpkin seed meal low molecular weight peptide zinc composition was subjected to simulated gastrointestinal digestion, and the particle size distribution, zeta potential, free amino nitrogen, free amino acids and zinc retention rate were measured during the digestion process; (2) The low molecular weight peptide zinc composition of pumpkin seed meal after simulated gastrointestinal digestion was evaluated by eversion of the intestinal sac, the zinc content on the serosa side was determined, and the apparent zinc absorption rate and apparent zinc transport rate were calculated. (3) The intestinal delivery performance of the pumpkin seed meal low molecular weight peptide zinc composition was determined based on the zinc retention rate in the simulated intestinal digestion stage and the apparent zinc absorption rate in the everted intestinal sac model.

[0023] Preferably, in step (1), the simulated gastrointestinal digestion includes a simulated gastric digestion stage and a simulated intestinal digestion stage; the simulated gastric digestion stage uses simulated gastric juice containing pepsin, and the simulated intestinal digestion stage uses simulated intestinal juice containing pancreatic enzymes and bile salts; Furthermore, during the simulated gastric digestion stage, HCl and pepsin were used to simulate gastric juice. The final concentration of HCl was 0.05~0.2 mol / L, the final concentration of pepsin was 1500~3000 U / mL, and the pH of the simulated gastric juice was 1~2.

[0024] Furthermore, during the intestinal digestion stage, NaHCO3, trypsin, and porcine bile salts were used as simulated intestinal fluid. The final concentration of NaHCO3 was 6-8 g / L, the final concentration of trypsin was 80-120 U / mL, the final concentration of bile salts was 5-10 mmol / L, and the pH of the simulated intestinal fluid was 8.0-8.5.

[0025] Preferably, the simulated gastrointestinal digestion time is 240 minutes, with 0-90 minutes representing the simulated gastric digestion stage and 90-240 minutes representing the simulated intestinal digestion stage. The simulated gastric digestion stage is conducted at a temperature of 37°C for 30 to 90 minutes; the simulated intestinal digestion stage is conducted at a temperature of 37°C for 120 to 240 minutes. Preferably, the mass ratio of the simulated gastrointestinal digestion stage peptide zinc composition to simulated gastric juice is 1:1, and the mass ratio of the simulated gastrointestinal digestion stage peptide zinc composition to simulated intestinal juice is 1:2.

[0026] Preferably, in step (1), the zinc retention rate is calculated using the following formula: Zinc retention rate (%) = Zinc content in the supernatant after digestion / Total zinc content in the digestion system × 100%.

[0027] Preferably, in step (2), the evaluation of the everted intestinal sac adopts the chicken ileal everted intestinal sac model. The pumpkin seed meal low molecular weight peptide zinc composition is added to the mucosal side buffer, incubated in an oxygen-containing gas environment, and the serosal side liquid is collected at 15 min, 30 min, 45 min and 60 min.

[0028] Furthermore, the everted intestinal sac model was developed using 40-50 day old white-feathered chickens weighing 1500-1600 g.

[0029] Furthermore, the white-feathered chickens used were fasted for 12 hours, then injected intravenously with sodium pentobarbital for anesthetic, and the abdominal feathers and skin were removed.

[0030] Furthermore, for the everted intestinal sac model, the abdominal cavity needs to be dissected along the midline of the abdomen, the starting end of the ileocecal valve needs to be located, a 14cm segment of intestine needs to be measured forward, and a 12cm segment of the middle ileum needs to be cut off.

[0031] Furthermore, the everted intestinal sac model requires the injection of 1 mL of Krebs-Ringer buffer containing phenol red indicator from one end for leakage verification.

[0032] Furthermore, the wall thickness of the intestinal sac model needs to be ≥0.3 mm.

[0033] Preferably, in step (2), the apparent zinc absorption rate is calculated using the following formula: apparent zinc absorption rate (%) = zinc content on the slurry side / initial zinc content × 100%.

[0034] The apparent zinc translocation rate is calculated using the following formula: Apparent zinc translocation rate = Change in zinc content on the serosal side / Culture time / Effective length of everted intestinal sac.

[0035] Preferably, the apparent zinc absorption rate of the pumpkin seed meal low molecular weight peptide zinc composition showed a continuous increasing trend at 15 min, 30 min, 45 min and 60 min in the everted intestinal sac model.

[0036] Preferably, the pumpkin seed meal low molecular weight peptide zinc composition undergoes changes in particle size distribution and zeta potential during simulated gastrointestinal digestion, and forms a dispersible zinc binding system during the simulated intestinal digestion stage.

[0037] This invention has at least one of the following beneficial effects: 1. This invention separates, enzymatically hydrolyzes, and fractionates low-molecular-weight peptide components (molecular weight less than 3 kDa) from pumpkin seed meal, and then prepares a low-molecular-weight peptide-zinc composition from pumpkin seed meal by coordinating these peptides with edible zinc salts. Studies have shown that, compared to components with molecular weights of 3-5 kDa and greater than 5 kDa, the low-molecular-weight peptide components (molecular weight less than 3 kDa) exhibit a higher zinc chelating capacity with the pumpkin seed meal low-molecular-weight peptide-zinc composition prepared from edible zinc salts, indicating that the zinc-binding capacity of pumpkin seed peptides is significantly molecular weight dependent. The low-molecular-weight peptide components (molecular weight less than 3 kDa) may have higher diffusion capacity, lower steric hindrance, and more fully exposed potential coordinating groups such as carboxyl, amino, and carbonyl groups, which are beneficial for Zn absorption. 2+ Approaching the binding site and forming a stable coordination.

[0038] 2. This invention also provides the application of low molecular weight peptide-zinc compositions from pumpkin seed meal in simulated digestion and absorption, and provides a method for evaluating simulated digestion and absorption. This invention integrates simulated gastrointestinal digestion and everted intestinal sac models into a peptide-zinc composition evaluation process, which can simultaneously evaluate the structural response during digestion, zinc retention, and apparent absorption behavior in the isolated intestine. Furthermore, this invention uses the zinc retention rate at the end of simulated intestinal digestion and the apparent zinc absorption rate of the everted intestinal sac at 60 min as composition evaluation indicators, which can avoid the limitations of relying solely on the initial chelation rate to evaluate peptide-zinc systems.

[0039] 3. Studies have shown that, compared with edible zinc salts that are not bound to low molecular weight peptide components with a molecular weight of less than 3 kDa, the pumpkin seed meal low molecular weight peptide zinc composition prepared in this invention exhibits controlled peptide hydrolysis and high intestinal zinc retention capacity during simulated gastrointestinal digestion, supporting its potential application value as a peptide-based zinc delivery system. At the same time, the composition obtained in this invention maintains high soluble zinc during intestinal digestion and exhibits continuous apparent zinc transport characteristics in an everted intestinal sac model, making it suitable for development as a plant-based zinc supplement food ingredient. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating the simulated digestion and absorption evaluation of the pumpkin seed meal low molecular weight peptide zinc intestinal delivery composition of the present invention. Figure 2 These are particle size distribution changes in some of the embodiments and control examples during a simulated gastrointestinal digestion process; Figure 3 These are graphs showing the changes in zeta potentials in some of the embodiments and control examples during a simulated gastrointestinal digestion process; Figure 4 These are scanning electron microscope images of some of the embodiments and control examples before and after formation; Figure 5 These are graphs showing the changes in free amino nitrogen in some of the embodiments and control examples during simulated gastrointestinal digestion. Figure 6 These are graphs showing the changes in free amino acids in some of the embodiments and control examples during a simulated gastrointestinal digestion process; Figure 7 This is a graph showing the changes in zinc retention rates in some of the embodiments and control examples during a simulated gastrointestinal digestion process; Figure 8 These are graphs showing the changes in particle size distribution of the serosal fluid after treatment with an everted intestinal sac model in some embodiments and control examples; Figure 9 These are graphs showing the changes in zeta potential of the serosal fluid after treatment in some embodiments and control exceptions of the intestinal sac model; Figure 10 This is a graph showing the changes in apparent zinc absorption rate in the everted intestinal sac model of some embodiments and control examples; Figure 11 This is a graph showing the changes in the apparent zinc transport rate in the everted intestinal sac model of some embodiments and control examples. Detailed Implementation

[0041] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] Example 1 like Figure 1 As shown in the figure, this embodiment provides a method for preparing a low molecular weight peptide zinc composition from pumpkin seed meal, including the following steps: (1) Preparation of pumpkin seed meal protein isolate: Weigh defatted pumpkin seed meal, pulverize it, and pass 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 to 8 kDa. Dialyze at 4℃ for 72 h, and then freeze-dry to obtain isolated pumpkin seed meal protein.

[0043] (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% w / v. The pH of the system was adjusted to 4.8, and acidic protease was added at a concentration of 8000 U / g protein. The mixture was hydrolyzed at 55°C for 3 h. After hydrolysis, the system was heated to 100°C for 15 min to inactivate the enzyme. After cooling to room temperature, the mixture was centrifuged, the supernatant was collected, and freeze-dried to obtain pumpkin seed meal protein hydrolysate (PSPH-Aci).

[0044] (3) Fractionation of low molecular weight peptide components: The PSPH-Aci obtained in step (2) was fractionated using a 3 kDa ultrafiltration membrane to obtain a component with a molecular weight less than 3 kDa, which was labeled as PSP1.

[0045] (4) Preparation of low molecular weight peptide zinc composition from pumpkin seed meal: Weigh 0.3 g of PSP1 and 0.6 g of zinc acetate, add 25 mL of deionized water and dissolve thoroughly. Adjust the pH of the system to 6.0 using 1 mol / L NaOH or 1 mol / L HCl, and react at 55℃ for 60 min. After the reaction is complete, add 4 volumes of anhydrous ethanol to the reaction solution for precipitation. After standing at 4℃, centrifuge, collect the precipitate and freeze-dry to obtain a low molecular weight peptide zinc composition from pumpkin seed meal, denoted as PSP1-Zn.

[0046] Example 2 like Figure 1 As shown, this embodiment provides a method for simulating digestion and absorption evaluation. Specifically, it uses the pumpkin seed meal low molecular weight peptide zinc composition PSP1-Zn prepared in Example 1 to simulate gastrointestinal digestion, including the construction of a simulated gastrointestinal digestion model. The detailed method is as follows: (1) Simulating the gastric digestion stage: Pepsin (3000 U / g) was dissolved in 0.1 mol / L HCl solution, and the pH was adjusted to 1.2. The final concentration of pepsin was 2000 U / mL, which simulated gastric juice.

[0047] 10 g of the pumpkin seed meal low molecular weight peptide zinc composition PSP1-Zn prepared in Example 1 was added to 10 mL of simulated gastric fluid. The system temperature was maintained at 37°C, and samples were taken at 0 min, 30 min, 60 min and 90 min respectively.

[0048] (2) Simulated intestinal digestion stage: Trypsin (250 U / mg) and porcine bile salts were dispersed in a 0.1 mol / L NaHCO3 solution, and the pH was adjusted to 8.0. The final concentration of bile salts was 5 mmol / L, which simulated intestinal fluid.

[0049] 10 g of the component after simulated gastric digestion in step (1) was added to 20 mL of simulated intestinal fluid. The system temperature was maintained at 37°C, and samples were taken at 120 min, 150 min, 180 min, 210 min and 240 min respectively.

[0050] (3) Evaluate the digestion process: The particle size distribution, zeta potential, free amino nitrogen, free amino acids, and zinc retention were determined during digestion.

[0051] Example 3 This embodiment provides a simulated digestion and absorption evaluation method, specifically using the pumpkin seed meal low molecular weight peptide zinc composition PSP1-Zn prepared in Example 1 to evaluate the everted intestinal sac, including the construction of the everted intestinal sac model, the detailed method of which is as follows: (1) Evaluation of everted intestinal sac Using 45-day-old broiler chickens (1500-1600 g), after fasting for 12 hours, intravenous injection of sodium pentobarbital was administered for anesthesia. Abdominal feathers and skin were removed. The abdominal cavity was opened along the midline, the origin of the ileocecal valve was located, and a 14 cm segment of intestine was measured anteriorly, with a 12 cm section of the mid-ileum also removed. The intestinal segment was divided into two equal parts (approximately 5 cm each), and the intestinal contents were flushed with preheated physiological saline at 39-40°C until no residue remained. Using a 2 mm diameter glass rod, the mesentery was gently lifted, and the intestinal sac was rotated along the longitudinal axis of the intestinal wall (mucosal side outward). The intestinal mucosa was then thoroughly infiltrated with Krebs-Ringer buffer. One segment of the intestinal sac was selected, one end was ligated and fixed with surgical suture, and the other end was injected with 1 mL of Krebs-Ringer buffer containing phenol red indicator for leak verification. The opening of the intestinal sac was sealed, excess tissue was removed, and the wet weight was recorded.

[0052] 0.6 g of PSP1-Zn, after simulated gastrointestinal digestion as described in Example 2, was added to 30 mL of Krebs-Ringer buffer to obtain zinc-containing Krebs-Ringer buffer. The intestinal sac was then immersed in 30 mL of this zinc-containing Krebs-Ringer buffer (sample concentration 20 mg / mL). The culture flask was placed in a 39°C constant-temperature shaker, and a 95% O2 / 5% CO2 mixed gas was introduced (30-40 bubbles per minute). After culture, the intestinal sac wall thickness was measured (≥0.3 mm was considered a valid model).

[0053] Liquid from the slurry film was collected at 15 min, 30 min, 45 min, and 60 min, respectively. The absorbance of phenol red in the slurry film at 520, 560, and 600 nm was measured using a UV-Vis spectrophotometer to obtain its concentration. The accurate volume of the slurry film after different incubation times was calculated using the following formula.

[0054] .

[0055] (2) Transshipment evaluation: The zinc content on the slurry film side was determined, and the apparent zinc absorption rate and apparent zinc translocation rate were calculated.

[0056] Compare with Example 1 The difference from Example 1 is that 3 kDa and 5 kDa ultrafiltration membranes were used for fractionation to obtain components with a molecular weight of 3 to 5 kDa, while the other steps were the same.

[0057] Compare with Example 2 The difference from Example 1 is that a 5 kDa ultrafiltration membrane was used for fractionation to obtain components with a molecular weight greater than 5 kDa, while the other steps were the same.

[0058] Compare with Example 3 The difference from Example 1 is that zinc gluconate was used as the control zinc source, i.e. zinc gluconate that was not bound to PSP1. The simulated gastrointestinal digestion evaluation was performed according to the method of Example 2, and the everted intestinal sac evaluation was performed according to the method of Example 3.

[0059] Compare with Example 4 The difference from Example 1 is that zinc acetate was used as the control zinc source, i.e. zinc acetate that was not bound to PSP1. The simulated gastrointestinal digestion evaluation was performed according to the method of Example 2, and the everted intestinal sac evaluation was performed according to the method of Example 3.

[0060] Compare with Example 5 The difference from Example 1 is that, using unbound PSP1 as a control, simulated gastrointestinal digestion was evaluated according to the method of Example 2. Application Example 1 Application in zinc supplementation powder: The low molecular weight peptide zinc composition of pumpkin seed meal prepared in Example 1 was added to the base of a solid beverage and mixed evenly with maltodextrin, oligosaccharides, plant protein powder and flavor powder to obtain a zinc supplementation solid beverage containing the low molecular weight peptide zinc composition of pumpkin seed meal.

[0061] Application Example 2 Application in zinc-supplemented solid beverages: The low molecular weight peptide zinc composition of pumpkin seed meal prepared in Example 1 is added to the nutritional powder system and compounded with cereal powder, whey protein powder or plant protein powder to obtain zinc-supplemented nutritional powder.

[0062] Application Example 3 Application in plant protein foods: The low molecular weight peptide zinc composition of pumpkin seed meal prepared in Example 1 can be added to plant protein beverages, instant plant protein foods or meal replacement foods as a plant-derived peptide zinc fortification ingredient.

[0063] Application Example 4 Application in feed additives: The low molecular weight peptide zinc composition of pumpkin seed meal prepared in Example 1 can be added to feed premix and used as a plant-derived peptide zinc raw material for animal nutrition products.

[0064] test: The present invention also conducted the following tests on the above embodiments and comparative examples, and the test methods and results are as follows: 1. Determination of zinc content and chelating ability: Weigh 0.2 g of the zinc chelate samples obtained in Example 1, Control Example 1, and Control Example 2, and dissolve them in 100 mL of deionized water. Transfer 10 mL of the sample solution, add 1 mL of ammonia-ammonium chloride buffer and two drops of 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.

[0065] 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.

[0066] Table 1 As shown in Table 1, the zinc chelating abilities of Example 1, Control Example 1, and Control Example 2 were 29.11%, 24.50%, and 20.73%, respectively, with significant differences. Among them, Example 1 (<3 kDa) exhibited the highest chelating ability, exceeding that of Control Example 1 (3-5 kDa) by 18.81% and Control Example 2 (>5 kDa) by 40.40%, respectively, indicating that the zinc binding ability of pumpkin seed peptides is significantly molecular weight dependent. The advantage of Example 1 may be related to the higher diffusion capacity, lower steric hindrance, and more complete exposure of potential coordinating groups such as carboxyl, amino, and carbonyl groups in the low molecular weight peptide, characteristics that are beneficial for Zn. 2+ Larger molecular weight components approach the binding site and form stable coordination. Conversely, larger molecular weight components limit the exposure of coordinating groups due to chain entanglement, local aggregation, or reduced conformational flexibility, thereby reducing chelation efficiency.

[0067] 2. Particle size testing during simulated gastrointestinal digestion: The particle size distribution of Examples 1, 3, 4, and 5 treated in Example 2 was tested using a Malvern Master Sizer 2000 particle size analyzer. Deionized water was used as the dispersion medium, and the particles were equilibrated at 25°C for 120 s before testing.

[0068] After entering the gastrointestinal tract, the peptide-zinc complex is simultaneously affected by changes in pH, digestive enzyme hydrolysis, bile salt action, and ionic strength. Its colloidal state may directly influence the retention and subsequent transport of zinc in digestive fluids. Therefore, this invention analyzes the dynamic changes of different zinc systems during simulated gastrointestinal digestion by examining particle size distribution.

[0069] like Figure 2 As shown, simulated digestion significantly altered the particle size distribution of each system. During the gastric digestion stage, all samples exhibited peak shift and broadening of the particle size distribution, indicating that the acidic environment and pepsin activity altered the dispersion state of the system. For Example 1, the relatively broad particle size distribution in the early stages of digestion suggests partial aggregation or structural rearrangement of the peptide zinc assembly, which is related to protonation of the ligands, weakened charge repulsion, and redistribution of peptide zinc interactions. Further changes in particle size distribution occurred during the intestinal digestion stage, indicating that the alkaline environment, pancreatic enzyme hydrolysis, and bile salts jointly drove system reorganization. In Example 1, the particle size remained below 1000 nm at all stages of simulated intestinal digestion, positively impacting its absorption in the intestine.

[0070] 3. Measurement of particle zeta potential during simulated gastrointestinal digestion: Example 1, Control Example 3, Control Example 4, and Control Example 5, after being treated in Example 2, were tested using a laser particle size analyzer (Malvern Zetasizer Nano ZS). Test conditions: 25°C, refractive index 1.330. Before testing, the samples were prepared into 0.1 mg / mL solutions, ultrasonically mixed, and then transferred to a potentiometric cell for detection. Blank subtraction was performed on all samples.

[0071] like Figure 3 As shown, during the gastric digestion stage, the zeta potential of each sample was close to neutral or weakly negative, indicating weak electrostatic repulsion. During the intestinal digestion stage, the zeta potential rapidly shifted to a stronger negative charge, due to the deprotonation of acidic groups, exposure of negatively charged groups after enzymatic hydrolysis, and the introduction of bile salts. Overall, Example 1 formed a dynamic dispersion system composed of peptide fragments, zinc conjugates, and recombinant colloidal particles during gastrointestinal digestion. The strongly negatively charged environment formed during intestinal digestion enhanced the dispersibility of zinc-bound peptide species and facilitated the retention of zinc-related species in the liquid phase. This result provides colloidal structural evidence for subsequent zinc retention rates and sustained intestinal transport behavior.

[0072] 4. Microstructure determination of particles in simulated gastrointestinal digestion stages: A small amount of the powder from Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 that were treated in Example 2 was evenly spread on conductive adhesive, sputtered with gold, and measured using a JSM 6701F field emission scanning electron microscope with energy dispersive spectroscopy at a voltage of 15 kV and magnification at different magnifications to observe the microstructure.

[0073] like Figure 4 As shown, Comparative Example 3 exhibits a heterogeneous, dense aggregate, with stacked and wrinkled structures visible under high magnification; Comparative Example 4 has a relatively smooth surface accompanied by numerous fine particles, appearing as irregular flaky and fragmented crystals under high magnification. Both zinc salts exhibit typical morphological characteristics of inorganic or small-molecule salts. The morphology of Comparative Example 5 is significantly different from the two zinc salts, appearing as dispersed irregular particles or flaky fragments under low magnification, while large particles with smoother surfaces and denser structures are visible under high magnification, indicating that low-molecular-weight peptide components tend to form relatively independent dense particles during the drying process. Compared to Comparative Example 5, Example 1 shows significant structural reorganization, with a rougher surface and the formation of porous, wrinkled, and network-like microstructures. This morphology differs from the smooth, dense particles of Comparative Example 5 and the crystalline structure of the zinc salt, indicating that Zn 2+ Introducing novel assembly behaviors of low molecular weight peptides from pumpkin seed meal.

[0074] The morphological changes in Example 1 originated from Zn 2+ Interactions with potential coordinating groups such as carboxyl, amino, and carbonyl groups in the peptide chain. Zn 2+ It may act as a bridge between peptide chains, promoting local aggregation or cross-linking, thereby forming a rough, porous structure. This result indicates that the formation of Example 1 is accompanied by significant morphological reconstruction.

[0075] 5. Determination of free amino nitrogen content in particles during simulated gastrointestinal digestion: The free amino group content was determined using the o-phthalaldehyde method (OPA method). 2 mL of the prepared OPA reagent was mixed with 100 μL of the sample solution and reacted precisely at room temperature in the dark for 2 min. The absorbance was measured at 340 nm using a UV spectrophotometer (TU-1900, Beijing Purkinje General Instrument Co., Ltd.). A blank was prepared by adding 100 μL of water to the OPA reagent. A standard curve (R0) was constructed using L-leucine. 2 =0.9989, y=0.0152x+0.08069), calculate the free amino concentration.

[0076] The stability of the peptide-zinc complex during digestion depends not only on the initial binding state of zinc to the peptide, but also on the combined effects of the degree of peptide chain hydrolysis and changes in the zinc binding form. This invention further determined the changes in free amino nitrogen during simulated digestion.

[0077] like Figure 5As shown, the free amino nitrogen content in Control Example 5 remained relatively stable during the gastric digestion stage, while that in Example 1 remained at a low level throughout the 0-90 min period. Upon entering the intestinal digestion stage, the free amino nitrogen content in both groups significantly increased, indicating that pancreatic enzymes further promoted peptide chain cleavage. However, the free amino nitrogen content in Example 1 was consistently lower than that in Control Example 5, especially during the intestinal digestion stage, suggesting that zinc coordination can, to some extent, delay peptide chain hydrolysis.

[0078] 6. Determination of free amino acid content during simulated gastrointestinal digestion: Free amino acids were measured using an amino acid (AA) content assay kit (Beijing Solarbio Biotechnology Co., Ltd.) at 570 nm absorbance to calculate the amino acid content. Cysteine ​​standard solution was used as the standard, and the free amino acid content was expressed as concentration in μmol / mL.

[0079] Free amino acid release, such as Figure 6 As shown, the free amino acid content in Control Example 5 increased rapidly during intestinal digestion, while that in Example 1 was released more slowly and at lower levels throughout the digestion process. This indicates that Example 1 was not completely resistant to enzymatic digestion, but its coordination structure may have delayed peptide chain breakage and the release of amino-containing small molecules by reducing the accessibility of enzyme cleavage sites or stabilizing peptide chain assembly structures. This result is consistent with the recombinant microstructure observed by SEM and the digestion-induced colloidal recombination revealed by particle size and zeta potential.

[0080] 7. Determination of zinc retention rate of particles during simulated gastrointestinal digestion: Accurately weigh an appropriate amount of the digestate and dissolve it completely in 100 mL of deionized water. Take 20 mL of this solution, add 5 mL of ammonia-ammonium chloride buffer solution, and then add 2 drops of Eriochrome Black T indicator. Titrate with 0.01 mol / L EDTA standard solution. The titration endpoint is determined when the solution color gradually changes from purplish-red to pure blue.

[0081] Zinc content calculation formula: X(mg / g) = M × c × V × 5 / m Where X: zinc content; M: relative atomic mass of zinc; c: concentration of EDTA standard titrant, mol / L; V: titration volume of EDTA standard titrant, mL; m: mass of chelate weighed, g.

[0082] Zinc retention further reflects the stabilizing effect of low-molecular-weight peptides in pumpkin seed meal on zinc in the digestive environment. For example... Figure 7As shown, Control Examples 3, 4, and Example 1 all maintained high zinc retention rates during the gastric digestion stage. However, upon entering the intestinal digestion stage, the zinc retention rates of all zinc systems decreased, indicating that intestinal pH and digestive components significantly affect the solubility and form of zinc. Notably, Example 1 maintained the highest zinc retention rate at most intestinal digestion time points, with a retention rate at the end of intestinal digestion that was 9.96% and 95.10% higher than zinc gluconate and zinc acetate, respectively. This is attributed to the continuous binding of zinc to peptide ligands during digestion and the increased dispersibility of zinc-bound peptide species after enhanced negative charge. Overall, Example 1 exhibits controlled peptide hydrolysis and high intestinal zinc retention capacity, supporting its potential application value as a peptide-based zinc delivery system.

[0083] 8. Particle size determination after transport of everted intestinal sacs: The particle size distribution of Example 1, Comparative Example 3, and Comparative Example 4 treated with Example 3 was tested using a particle size analyzer (MalvernMaster Sizer 2000). Deionized water was used as the dispersion medium, and the particles were equilibrated at 25°C for 120 s before testing.

[0084] To analyze the liquid phase state of different zinc forms after transintestinal transport, this invention determined the particle size distribution of zinc-related species in the serous membrane fluid of the everted intestinal sac. For example... Figure 8 As shown, the particle size distribution of Control Example 3 was more concentrated after different incubation times, with only a slight shift in the main peak, indicating that it may exist mainly as dissociated zinc ions or small ion aggregates after translocation, forming limited scattering particles. Control Example 4 also showed time-dependent particle size changes, but the overall distribution was narrower than that of Example 1, suggesting its structural heterogeneity. Compared with the two conventional zinc salts, the particle size change of Example 1 in the slurry was more obvious. The wider particle size distribution and multi-peak characteristics after translocation of Example 1 indicate that it may form a complex system composed of small molecule peptides, peptide-zinc complexes, partially dissociated zinc species, and peptide-zinc aggregates. This change may be related to peptide conformational adjustments and Zn 2+ It relates to coordination redistribution and its secondary interactions with the film fluid components.

[0085] 9. Potential measurement of an everted intestinal sac model: Example 1, Control Example 3, and Control Example 4, after being treated in Example 3, were tested using a laser particle size analyzer (Malvern Zetasizer Nano ZS). Test conditions: 25°C, refractive index 1.330. Before testing, the samples were prepared into 0.1 mg / mL solutions, ultrasonically mixed, and then transferred to a potentiometric cell for detection. Blank subtraction was performed on all samples.

[0086] The zeta potential results further revealed the differences in the interfacial properties of the slurry film after different zinc treatments. For example... Figure 9As shown, Example 1 exhibited the largest zeta potential fluctuation. The increased electronegativity in the mid-term may have originated from the exposure of ionizable groups such as carboxyl groups during peptide conformational rearrangement or dissociation. The decreased electronegativity in the later stage may be related to Zn. 2+ This is related to charge shielding, particle aggregation, or adsorption of serous fluid components. Overall, Example 1 showed the most significant changes in particle size and surface charge after intestinal transport, indicating that peptide zinc chelation promotes the formation of more complex zinc-related structures and may help maintain the peptide-bound or peptide-associated form of zinc.

[0087] 10. Determination of zinc absorption rate after transport of everted intestinal sac: To further evaluate the in vitro intestinal transport characteristics of the pumpkin seed meal low molecular weight peptide zinc composition, this invention uses an everted intestinal sac model to determine the dynamic changes in the apparent zinc transport rate and apparent zinc transport volume in the serous fluid. Figure 10 As shown, different zinc sources exhibited differentiated transport patterns. The absorption rates of all three zinc sources increased over time, with Control Example 3 consistently showing the highest rate, reaching approximately 49.68% at 60 min. This is because common zinc supplements such as zinc gluconate and zinc acetate expose zinc ions at their absorption sites, potentially causing irritation to the gastric mucosa and unpleasant odor. In contrast, zinc peptide chelates are absorbed in the form of small peptides, resulting in a lower absorption rate compared to zinc gluconate and zinc acetate. However, zinc peptide chelates are gentle on the gastrointestinal tract, non-irritating, and structurally stable. The absorption rate of Example 1 increased from approximately 16.76% at 15 min to approximately 32.72% at 60 min, higher than Control Example 4. Example 1 showed a 120.54% higher absorption rate than zinc acetate at 60 min.

[0088] 11. Determination of zinc absorption rate after transport of everted intestinal sac: like Figure 11 As shown, the zinc absorption rate results indicate that Control Example 3 reached its peak at 15 min and then gradually decreased, exhibiting rapid transport characteristics; Example 1 maintained a relatively high level between 15 and 60 min, reaching its peak at around 30 min, with overall fluctuations less than zinc gluconate; zinc acetate had the lowest absorption rate and decreased over time. In summary, Example 1 may have delayed the rapid dissociation of zinc through its peptide coordination structure, allowing some zinc to exist as a soluble peptide-zinc complex or peptide-associated zinc, thereby supporting sustained zinc transport. Its transport capacity is higher than that of zinc acetate, and it exhibits sustained absorption characteristics distinct from zinc gluconate.

[0089] The above results demonstrate that the evaluation process for the peptide-zinc composition of this invention can simultaneously evaluate the structural response during digestion, zinc retention, and apparent absorption behavior in the isolated intestinal tract. Furthermore, it avoids the limitations of relying solely on initial chelation rates to evaluate peptide-zinc systems. The resulting low-molecular-weight peptide-zinc composition from pumpkin seed meal maintains high levels of soluble zinc during intestinal digestion and exhibits sustained apparent zinc transport characteristics in an everted intestinal sac model, making it suitable for development as a plant-based zinc supplement ingredient.

[0090] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a low-molecular-weight peptide-zinc composition from pumpkin seed meal, characterized in that, Includes the following steps: S1. Prepare pumpkin seed meal isolated protein using pumpkin seed meal or defatted pumpkin seed meal as raw material; S2. The pumpkin seed meal isolated protein is enzymatically hydrolyzed using acidic protease to obtain pumpkin seed meal protein hydrolysate; S3. The pumpkin seed meal protein hydrolysate is fractionated by molecular weight to obtain low molecular weight peptide components with a molecular weight of less than 3 kDa. S4. The low molecular weight peptide component with a molecular weight of less than 3 kDa undergoes a coordination reaction with edible zinc salt to obtain a pumpkin seed meal low molecular weight peptide zinc composition.

2. The preparation method according to claim 1, characterized in that, S1 includes the following specific steps: The defatted pumpkin seed meal is pulverized, added to deionized water, and mixed evenly at a material-to-liquid ratio of 1:8~12; the pH of the system is adjusted to 10.5~11.5 using 0.5~1.5 mol / L NaOH, and extracted by stirring at room temperature for 25~35 min, followed by centrifugation at 3~5℃ and 3500~4500 r / min for 15~25 min, and the supernatant is collected; the pH of the supernatant is adjusted to 4~5 using 0.5~1.5 mol / L HCl to precipitate the protein. The precipitate was collected by centrifugation, washed with deionized water, and then placed in a dialysis bag with a molecular weight cutoff of 6 to 8 kDa. Dialysis was performed at 3 to 5°C for 66 to 78 h, followed by freeze drying to obtain pumpkin seed meal protein isolate. S2 includes the following specific steps: Pumpkin seed meal protein isolate is dispersed in deionized water at a substrate concentration of 4-6% w / v, the pH of the system is adjusted to 4.6-5.0, acidic protease is added at an enzyme addition amount of 7000-9000 U / g protein, and enzymatic hydrolysis is carried out at 50-60℃ for 2.5-3.5 h; after enzymatic hydrolysis, the system is heated to inactivate the enzyme, cooled to room temperature, centrifuged, the supernatant is collected and freeze-dried to obtain pumpkin seed meal protein hydrolysate.

3. The preparation method according to claim 1, characterized in that, In step S3, a 3 kDa ultrafiltration membrane is used to fractionate the pumpkin seed meal protein hydrolysate by molecular weight to obtain low molecular weight peptide components with a molecular weight of less than 3 kDa; the zinc binding capacity of the low molecular weight peptide components with a molecular weight of less than 3 kDa is not less than 26%. In S4, the edible zinc salt is selected from one or more of zinc acetate, zinc gluconate, zinc lactate, zinc chloride, or zinc sulfate; the mass ratio of the low molecular weight peptide component with a molecular weight of less than 3 kDa to the edible zinc salt is 1:1 to 1:3, the pH of the coordination reaction is 5.0 to 7.0, the temperature of the coordination reaction is 45 to 65°C, and the time of the coordination reaction is 30 to 120 min.

4. A low-molecular-weight peptide-zinc composition from pumpkin seed meal, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 3.

5. The application of the pumpkin seed meal low molecular weight peptide zinc composition according to claim 4 in the preparation of a peptide-based zinc delivery system.

6. The application of the pumpkin seed meal low molecular weight peptide zinc composition according to claim 4 in the preparation of zinc absorption-promoting products.

7. The use of the pumpkin seed meal low molecular weight peptide zinc composition according to claim 4 in the preparation of zinc-supplemented foods, solid beverages, nutritional powders, plant protein beverages, meal replacement foods, special dietary foods, health foods or feed additives.

8. The application of the pumpkin seed meal low molecular weight peptide zinc composition according to claim 4 in simulated digestion and absorption.

9. A method for simulating digestion and absorption evaluation, characterized in that, Includes the following steps: (1) The pumpkin seed meal low molecular weight peptide zinc composition according to claim 4 was subjected to simulated gastrointestinal digestion, and the particle size distribution, zeta potential, free amino nitrogen, free amino acids and zinc retention rate were measured during the digestion process; (2) The low molecular weight peptide zinc composition of pumpkin seed meal after simulated gastrointestinal digestion was evaluated by eversion of the intestinal sac, the zinc content on the serosa side was determined, and the apparent zinc absorption rate and apparent zinc transport rate were calculated. (3) The intestinal delivery performance of the pumpkin seed meal low molecular weight peptide zinc composition was determined based on the zinc retention rate in the simulated intestinal digestion stage and the apparent zinc absorption rate in the everted intestinal sac model.

10. The simulated digestion and absorption evaluation method according to claim 9, characterized in that, In step (1), the simulated gastrointestinal digestion includes a simulated gastric digestion stage and a simulated intestinal digestion stage; the simulated gastric digestion stage uses simulated gastric juice containing pepsin, and the simulated intestinal digestion stage uses simulated intestinal juice containing pancreatic enzymes and bile salts; The simulated gastric digestion stage is conducted at a temperature of 37°C for 30 to 90 minutes; the simulated intestinal digestion stage is conducted at a temperature of 37°C for 120 to 240 minutes. In step (2), the evaluation of the everted intestinal sac was carried out using a chicken ileal everted intestinal sac model. The pumpkin seed meal low molecular weight peptide zinc composition was added to the mucosal buffer and incubated in an oxygen-containing gas environment. The serosal fluid was collected at 15 min, 30 min, 45 min and 60 min.

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