Preparation method and application of peanut protein microgel high internal phase emulsion with high oxidation stability
By preparing microgel particles from peanut protein isolate through deamidation treatment and combining them with resveratrol, the problem of easy oxidation of emulsions was solved, achieving high antioxidant stability and 3D printing suitability, which is suitable for the development of functional foods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-27
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, the interfacial stability of natural proteins is limited, emulsions are easily oxidized, the interfacial enrichment capacity of natural antioxidants is insufficient, and the antioxidant efficiency is low.
Peanut protein isolate was deamidated to prepare microgel particles, which were then combined with resveratrol to form a complex system. This enabled the efficient delivery of resveratrol to the oil-water interface, enhancing the stability and antioxidant properties of the interfacial membrane.
It significantly improves the antioxidant properties of the emulsion, delays the quality deterioration of the emulsion system during storage, and enhances the suitability for 3D printing. The product has a fine structure, good self-support, and complies with the concepts of clean labeling and sustainable development.
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Figure CN122096404A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically to a method for preparing a peanut protein microgel with high internal phase emulsion and high antioxidant stability. Background Technology
[0002] In recent years, with the increasing demand from consumers for healthy and functional foods, oil-rich emulsion foods have received widespread attention in areas such as nutritional fortification, fat substitution, and structured food design. Among these, high internal phase Pickering emulsions, due to their advantages such as not requiring traditional surfactants, good stability, and the ability to construct complex structures, have shown promising application prospects in food 3D printing and functional food development. However, in practical applications, the oils in emulsion systems are highly susceptible to oxidation, leading to flavor degradation, decreased nutritional value, and shortened shelf life. Therefore, improving the antioxidant stability of emulsion systems has become one of the important research directions.
[0003] Natural plant proteins are widely used in constructing Pickering emulsion systems due to their excellent biocompatibility and emulsifying properties. Among them, peanut protein isolate shows great potential for application in emulsion stabilization systems due to its wide availability, high nutritional value, and good interfacial activity. However, the interfacial film structure formed by natural peanut protein isolate in high oil-phase systems is relatively loose, making it difficult to maintain the emulsion structure for a long time. Therefore, improving its interfacial stability through structural modification has become an important strategy for enhancing emulsion performance. Studies have shown that deamidation modification can change the molecular conformation of proteins, increasing the flexibility of protein molecules and surface charge, making it easier to form a stable adsorption layer at the oil-water interface, thereby improving the physical stability of the emulsion system. Based on this, introducing functional components with natural antioxidant activity has become an important means to further improve emulsion stability. Resveratrol, as a typical natural polyphenol compound, is widely found in plants such as grapes, peanuts, and blueberries. It has excellent free radical scavenging ability and metal ion reducing ability, and therefore has received widespread attention in food antioxidant research. However, due to its poor water solubility and limited interfacial accumulation capacity in emulsion systems, the antioxidant function of resveratrol is often limited in complex food systems. Therefore, forming a complex system with protein particles to enrich it at the oil-water interface is an important way to improve its antioxidant efficiency. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing peanut protein microgel high internal phase emulsion with high antioxidant stability, so as to solve the following problems existing in the prior art: the natural protein interface has limited stability, the emulsion is easily oxidized, the natural antioxidant interface enrichment capacity is insufficient, and the antioxidant efficiency is low.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A method for preparing a peanut protein microgel high internal phase emulsion with high antioxidant stability, characterized by comprising the following steps:
[0007] (1) Deamidation treatment: The obtained peanut protein isolate was dissolved in deionized water to prepare a solution of 5-15 mg / mL. After stirring at 200 rpm / min for 4 h with a magnetic stirrer, 20-80 U of protein glutaminase was added. The solution was stirred at 37℃ for enzymatic deamidation treatment. After enzymatic hydrolysis for 10-50 min, the enzyme was inactivated and the solution was freeze-dried to obtain peanut protein isolate with a deamidation degree of 10-30%.
[0008] (2) Preparation of microgel particles: The lyophilized peanut protein isolate powder with a deamidation degree of 20% obtained in (1) was dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25 °C. It was then stored overnight at 4 °C to ensure complete hydration, resulting in a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90 °C for 30 min before cooling to 25 °C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12000 rpm / min for 5 min using a homogenizer. Subsequently, it was homogenized 1-5 times using a high-pressure homogenizer at 10-50 MPa to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 1-5 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4 °C and partially lyophilized for subsequent applications.
[0009] (3) Preparation of resveratrol-stabilized high internal phase Pickering emulsion: Resveratrol powder was dissolved in anhydrous ethanol solution to obtain five resveratrol solutions of different concentrations (0.1-0.2 mg / mL). The deamidated peanut protein isolate microgel particle solution obtained in (2) was mixed with the resveratrol solution at a volume ratio of 1:1 and incubated in a water bath at 25℃ for 2 h. Then, the ethanol was removed by rotary evaporation at 37℃. 10-30 mL of the deamidated peanut protein isolate microgel particle solution was mixed with 20-50 mL of peanut oil solution. The mixture was then homogenized at 12000 rpm / min for 2 min at 25℃ using an IKA homogenizer. Finally, a resveratrol-deamidated peanut protein isolate microgel particle stabilized high internal phase Pickering emulsion was obtained.
[0010] Preferably, in step (1), the concentration of the peanut protein isolate solution is 10 mg / mL, and the total amount of protein glutaminase added is 50 U.
[0011] Preferably, the enzymatic hydrolysis time in step (1) is 30 min, and the degree of deamidation of the peanut protein isolate is 20%.
[0012] Preferably, in step (2), peanut protein isolate lyophilized powder with a deamidation degree of 20% is dissolved in deionized water, stirred at 200 rpm / min for 4 h at 25°C, and stored overnight at 4°C to ensure complete hydration, so as to obtain a deamidated peanut protein isolate solution with a concentration of 10 wt%.
[0013] Preferably, in step (2), the obtained gel is mixed with deionized water at a ratio of 1:4, homogenized at 12000 rpm / min for 5 min using a homogenizer, and then homogenized three times using a high-pressure homogenizer at 30 MPa to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%.
[0014] Preferably, in step (3), resveratrol powder with different contents is dissolved in anhydrous ethanol solution, and the concentrations of the resveratrol solutions obtained are 0.0125, 0.025, 0.05, 0.1 and 0.2 mg / mL, respectively.
[0015] Preferably, in step (3), 10 mL of deamidated peanut protein isolate microgel particle solution is mixed with 40 mL of peanut oil solution.
[0016] This technical solution has the following beneficial technical effects:
[0017] (1) Peanut protein isolate microgel particles were prepared by deamidation modification, and then formed a complex system with resveratrol. Resveratrol was efficiently delivered to the oil-water interface through the complex action, effectively exerting its free radical scavenging and metal ion reduction capabilities, significantly improving the antioxidant performance of the emulsion, thereby effectively inhibiting oil oxidation and delaying the quality deterioration of the emulsion system during storage.
[0018] (2) The high internal phase Pickering emulsion prepared by this invention exhibits excellent 3D printing suitability. The printed products have fine structures, clear outlines, and good self-support, providing a new material for the development of personalized and nutritionally fortified foods.
[0019] (3) All raw materials used in this invention are food-grade, and the preparation process is mild and green, in line with the concepts of clean labeling and sustainable development. The resulting products can be used directly as fat substitutes, bioactive substance delivery carriers, or widely applied in the field of 3D food printing, and have extremely high industrialization and application value. Attached Figure Description
[0020] Appendix Figure 1Peroxide value evaluation results of high internal phase Pickering emulsions with different amounts of resveratrol added and deamidated peanut protein isolate microgel particles-resveratrol composite stability;
[0021] Appendix Figure 2 Thiobarbituric acid value evaluation of high internal phase Pickering emulsions with different amounts of resveratrol added, characterized by deamidated peanut protein isolate microgel particles-resveratrol composites.
[0022] Appendix Figure 3 Evaluation results of the DPPH free radical scavenging ability of resveratrol-deamidated peanut protein isolate microgel particle complex under different resveratrol addition amounts;
[0023] Appendix Figure 4 Evaluation results of the effect of different amounts of resveratrol on the ABTS free radical scavenging ability of the resveratrol-deamidated peanut protein isolate microgel particle complex;
[0024] Appendix Figure 5 Evaluation results of the effect of different amounts of resveratrol addition on the iron ion reducing capacity of the resveratrol-deamidated peanut protein isolate microgel particle complex;
[0025] Appendix Figure 6 The particle size distribution and average particle size of high internal phase Pickering emulsions formed by deamidated peanut protein isolate microgel particles-resveratrol composites with different amounts of resveratrol were evaluated.
[0026] Appendix Figure 7 3D printing imaging evaluation results of deamidated peanut protein isolate microgel particles-resveratrol composite stable high internal phase Pickering emulsion formed by different amounts of resveratrol addition; Detailed Implementation
[0027] A clear and complete description of the technical solution is provided below for a specific embodiment of the present invention. It should be noted that the embodiment described represents only some, not all, implementations of the present invention. The specific method is as follows:
[0028] 1. Peroxide value test:
[0029] 0.1 g of high internal phase Pickering was completely dissolved in a mixture of 1.5 mL dichloromethane and 95% ethanol (volume ratio: 3:2). Then, 100 μL of ammonium sulfate hexahydrate (5 mM, 100 μM), 200 μL of methanol-sulfuric acid (0.25 M), and 200 μL of methanol-dimethylphenol orange tetrasodium salt (0.1 M) were added and thoroughly mixed. After incubation in the dark for 30 min, 1 mL of deionized water was added to the reaction mixture. The mixture was then centrifuged at 4000 g for 5 min. The upper layer (200 μL) was collected, and the absorbance was measured at 560 nm. The peroxide value (mg / kg peanut oil) was calculated after plotting a standard curve for isopropylbenzene hydroperoxide.
[0030] 2. Thiobarbituric acid value test:
[0031] Secondary oxidation reactions in emulsions were assessed using the thiobarbiturate reactive substance assay. Absorbance was measured at 531 nm using a UV-Vis spectrophotometer. Results are expressed as milligrams of malondialdehyde per kilogram of emulsion (mg MDA / kg emulsion). The calculation formula is as follows:
[0032]
[0033] Where A is the absorbance of the sample; m is the weight (mg) of the sample being analyzed.
[0034] 3. DPPH free radical scavenging activity test
[0035] Mix 1 mL of resveratrol-deamidated peanut protein isolate microgel particle composite solution with 3 mL of DPPH solution. Incubate the reaction mixture at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm. Calculate the DPPH radical scavenging content using the following formula:
[0036]
[0037] In the formula A 样品 It is the absorbance of the sample solution, A DPPH It is the absorbance of the DPPH solution.
[0038] 4. ABTS Free Radical Scavenging Activity Test
[0039] 0.2 mL of resveratrol-deamidated peanut protein isolate microgel particle composite solution was mixed with 3.9 mL of ABTS solution. The mixture was then incubated in the dark for 10 min. The absorbance was measured at 751 nm. The ABTS radical scavenging activity was calculated using the following equation:
[0040]
[0041] In the formula A 样品 It is the absorbance of the sample solution, A ABTS It is the absorbance of the ABTS solution.
[0042] 5. Iron ion reducing ability test
[0043] The ability of the resveratrol-deamidated peanut protein isolate microgel particle complex to reduce ferric ions was further determined by mixing the resveratrol-deamidated peanut protein isolate microgel particle complex sample with FRAP reagent and incubating at 37°C for 2 h. Absorbance was measured at 593 nm.
[0044] 6. Emulsion particle size test
[0045] Before measurement, dilute the emulsion 100 times. It is worth noting that the dilution was performed using a phosphate buffer solution (100 mM) that matched the pH of the emulsion.
[0046] 7. 3D printing of resveratrol-stabilized high internal phase Pickering emulsions:
[0047] A high internal phase Pickering emulsion with resveratrol-deamidated peanut protein isolate microgel particles was printed using a 3D printer.
[0048] The relevant printing parameters are as follows: nozzle height 0.8 mm, nozzle diameter 0.74 mm, and printing speed 20 mm / s. The shapes printed in this test included a three-dimensional spade (40 mm × 40 mm × 10 mm) and a hollow cross (40 mm × 40 mm × 20 mm). The 3D printed products were recorded and photographed.
[0049] Example 1
[0050] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm / min for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. Resveratrol powder was dissolved in anhydrous ethanol to obtain a resveratrol solution with a concentration of 0.0125 mg / mL. The deamidated peanut protein isolate microgel particle solution and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25°C for 2 h. The ethanol was then removed by rotary evaporation at 37°C. The resveratrol-deamidated peanut protein isolate microgel particle mixture (10 mL) was then mixed with peanut oil (40 mL). Finally, it was homogenized at 25°C using an IKA homogenizer at 12,000 rpm / min for 2 min. Finally, a high-inner-phase Pickering emulsion (DD20%-RESV) containing 0.0125 mg / mL resveratrol-deamidated peanut protein isolate microgel particles was obtained. 0.0125 ).
[0051] Example 2
[0052] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. Resveratrol powder was dissolved in anhydrous ethanol to obtain a resveratrol solution with a concentration of 0.025 mg / mL. The deamidated peanut protein isolate microgel particle solution and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25°C for 2 h. The ethanol was then removed by rotary evaporation at 37°C. The resveratrol-deamidated peanut protein isolate microgel particle mixture (10 mL) was then mixed with peanut oil (40 mL). Finally, it was homogenized at 25°C using an IKA homogenizer at 12,000 rpm for 2 min. Finally, a high-inner-phase Pickering emulsion (DD20%-RESV) containing 0.025 mg / mL resveratrol-deamidated peanut protein isolate microgel particles was obtained. 0.025 ).
[0053] Example 3
[0054] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. Resveratrol powder was dissolved in anhydrous ethanol to obtain a resveratrol solution with a concentration of 0.05 mg / mL. The deamidated peanut protein isolate microgel particle solution and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25°C for 2 h. The ethanol was then removed by rotary evaporation at 37°C. The resveratrol-deamidated peanut protein isolate microgel particle mixture (10 mL) was then mixed with peanut oil (40 mL). Finally, it was homogenized at 25°C using an IKA homogenizer at 12,000 rpm for 2 min. Finally, a high-inner-phase Pickering emulsion (DD20%-RESV) containing 0.05 mg / mL resveratrol-deamidated peanut protein isolate microgel particles was obtained. 0.05 ).
[0055] Example 4
[0056] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm / min for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. Resveratrol powder was dissolved in anhydrous ethanol to obtain a resveratrol solution with a concentration of 0.1 mg / mL. The deamidated peanut protein isolate microgel particle solution and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25°C for 2 h. The ethanol was then removed by rotary evaporation at 37°C. The resveratrol-deamidated peanut protein isolate microgel particle mixture (10 mL) was then mixed with peanut oil (40 mL). Finally, it was homogenized at 25°C using an IKA homogenizer at 12,000 rpm / min for 2 min. The final product was a highly stable internal phase Pickering emulsion (DD20%-RESV) containing 0.1 mg / mL of resveratrol-deamidated peanut protein isolate microgel particles. 0.1 ).
[0057] Example 5
[0058] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm / min for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. Resveratrol powder was dissolved in anhydrous ethanol to obtain a resveratrol solution with a concentration of 0.2 mg / mL. The deamidated peanut protein isolate microgel particle solution and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25°C for 2 h. The ethanol was then removed by rotary evaporation at 37°C. The resveratrol-deamidated peanut protein isolate microgel particle mixture (10 mL) was then mixed with peanut oil (40 mL). Finally, it was homogenized at 25°C using an IKA homogenizer at 12,000 rpm / min for 2 min. Finally, a high-internal-phase Pickering emulsion (DD20%-RESV) containing 0.2 mg / mL resveratrol-deamidated peanut protein isolate microgel particles was obtained. 0.2 ).
[0059] Comparative Example 1
[0060] The extracted peanut protein isolate was dissolved in deionized water (10 mg / mL) and stirred at 200 rpm / min for 30 min using a magnetic stirrer. 50 U of protein glutaminase was added, and the mixture was stirred at 37°C to facilitate enzymatic hydrolysis. The enzyme was then inactivated and lyophilized to obtain a 20% deamidated peanut protein isolate lyophilized powder. This lyophilized powder was then dissolved in deionized water and magnetically stirred at 200 rpm / min for 4 h at 25°C, followed by overnight storage at 4°C to ensure complete hydration, yielding a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90°C for 30 min before cooling to 25°C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm / min for 5 min using a homogenizer. This was followed by three cycles of homogenization at 30 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4°C and partially lyophilized for later use. A solution of deamidated peanut protein isolate microgel particles (10 mL) and peanut oil (40 mL) was mixed. Then, the mixture was homogenized at 25°C using an IKA homogenizer at 12,000 rpm / min for 2 min. Finally, a high internal phase Pickering emulsion (DD20%) stable by deamidated peanut protein isolate microgel particles was obtained.
[0061] The performance of the peanut protein microgel high internal phase emulsions prepared in Examples 1-5 and Comparative Example 1 was tested, as follows:
[0062] 1. Peroxide value assessment results of high internal phase Pickering emulsions with different amounts of resveratrol-added deamidated peanut protein isolate microgel particles-resveratrol composites.
[0063] Result statement: From Figure 1As can be seen, the lipid hydrogen peroxide content in all examples and comparative examples increased significantly with increasing storage time, indicating that peanut oil in high internal phase Pickering emulsions is oxidized to some extent during storage. The examples showed better oxidative stability than the comparative examples. Furthermore, the oxidative stability of the five examples gradually increased with increasing resveratrol content. Examples 1-5 containing resveratrol showed higher oxidative stability than Comparative Example 1, possibly due to resveratrol's free radical scavenging and metal ion reducing capabilities. It may also be due to the enhanced interfacial properties of the deamidated peanut protein isolate microgel particles combined with resveratrol, forming a denser interfacial film on the peanut oil surface, thus preventing the contact of pro-oxidants in the aqueous phase with the peanut oil. Oxidation of peanut oil shortens the shelf life of high internal phase Pickering emulsions. The shelf life of high internal phase Pickering emulsions is affected by their high peanut oil content and unsaturated fatty acid content, which are highly susceptible to auto-oxidation. Therefore, the addition of resveratrol, which has high antioxidant properties, plays a very important role in the stability of high internal phase Pickering emulsions.
[0064] 2. Thiobarbituric acid value evaluation results of high internal phase Pickering emulsions with different amounts of resveratrol-added deamidated peanut protein isolate microgel particles-resveratrol composites
[0065] Result description: such as Figure 2 As shown, the thiobarbituric acid value of the high internal phase Pickering emulsion exhibited a significant upward trend with prolonged storage time. Compared to the comparative examples, the rate of increase in thiobarbituric acid value was significantly slowed in Examples 1-5 with added resveratrol, indicating that the addition of resveratrol has a significant effect on inhibiting the formation of lipid secondary oxidation products. Notably, the thiobarbituric acid value of the emulsion showed a downward trend with increasing resveratrol addition. The results indicate that the addition of resveratrol significantly enhances the antioxidant properties of the deamidated peanut isolate-stabilized high internal phase Pickering emulsion. More importantly, it confirms that the antioxidant effect of resveratrol is dose-dependent, meaning that the antioxidant properties of the high internal phase Pickering emulsion can be targeted and regulated by precisely controlling the amount of resveratrol added.
[0066] 3. Evaluation results of the DPPH free radical scavenging ability of resveratrol-deamidated peanut protein isolate microgel particle complex under different resveratrol addition amounts
[0067] Result statement: From Figure 3It can be seen that Comparative Example 1 itself has a certain ability to scavenge free radicals. This may be because certain amino acids exposed on the surface of the peanut protein microgel particles can react with DPPH free radicals, thus providing a good scavenging effect. Furthermore, the DPPH scavenging ability of the examples is concentration-dependent; with the increase of resveratrol addition, the DPPH free radical scavenging ability of the examples also increases significantly. This is because resveratrol is a strong antioxidant that can neutralize DPPH free radicals by providing hydrogen atoms or electrons, thereby exhibiting free radical scavenging ability. Moreover, the higher the resveratrol concentration, the more molecules in the complex system can react with DPPH free radicals, and the scavenging ability is enhanced accordingly. In addition, the deamidated peanut protein isolate microgel particles may enhance the dispersibility and stability of resveratrol through their structure, making it more effectively contacted and reacted with DPPH free radicals.
[0068] 4. Evaluation results of the effect of different amounts of resveratrol on the ABTS free radical scavenging ability of the resveratrol-deamidated peanut protein isolate microgel particle complex.
[0069] Result statement: From Figure 4 It can be seen that Comparative Example 1 itself exhibits a certain degree of ABTS radical scavenging ability, which may be due to the presence of some negatively charged amino acid residues on the protein surface. These residues can interact with ABTS radicals, thereby causing ABTS radical decolorization. Furthermore, with the increase of resveratrol addition, the ABTS radical scavenging ability of the examples significantly increased in a dose-dependent manner. This may be because the phenolic hydroxyl groups in resveratrol become negatively charged after losing hydrogen ions, enabling them to interact electrostatically with ABTS radicals, thus further enhancing their ABTS radical scavenging effect.
[0070] 5. Evaluation results of the effect of different amounts of resveratrol on the iron ion reducing capacity of the resveratrol-deamidated peanut protein isolate microgel particle complex.
[0071] Result statement: From Figure 5 As can be seen, the iron-reducing ability of the examples significantly increased after the addition of resveratrol, and this increase was proportional to the amount of resveratrol added. Resveratrol is a natural polyphenol compound containing multiple phenolic hydroxyl groups in its molecule. These phenolic hydroxyl groups have strong reducing power and can reduce iron ions by donating hydrogen atoms or electrons. 3+ Reduced to Fe 2+ Furthermore, the resveratrol itself possesses a certain reducing ability, possibly stemming from the negatively charged amino acid residues on its surface. The addition of resveratrol not only provides additional reducing active sites but may also bind to the protein through electrostatic interactions or other intermolecular interactions, thereby further enhancing the overall reducing ability of the complex.
[0072] 6. Evaluation results of particle size distribution and average particle size of deamidated peanut protein isolate microgel particles-resveratrol composite stable high internal phase Pickering emulsions formed with different amounts of resveratrol.
[0073] Result description: such as Figure 6 As shown, all high internal phase Pickering emulsions exhibited a unimodal distribution, indicating a relatively uniform droplet size distribution. The droplet size gradually decreased with increasing resveratrol addition. This phenomenon may be related to the chemical structure of resveratrol: the phenolic hydroxyl groups in the resveratrol molecule interact with the amino side chains of the deamidated peanut protein isolate microgel particles, enhancing the surface activity of the resveratrol-deamidated peanut protein isolate microgel particle complex and promoting its adsorption at the oil-water interface. Therefore, the non-covalent interaction between resveratrol and deamidated peanut protein isolate microgel particles helps reduce the droplet size of the emulsion. Furthermore, resveratrol itself has good interfacial adsorption properties, forming a protective film at the oil-water interface, thereby improving emulsion stability and further reducing droplet size. Smaller droplet size means a larger interfacial area and a stronger interfacial layer, which not only improves the physical stability of the emulsion but also contributes to the uniform distribution of resveratrol, enhancing its contact area with oxidizing substances, and thus improving the antioxidant performance of high internal phase Pickering emulsions. At the same time, a stronger interfacial film provides better protection against pro-oxidants, which further enhances the antioxidant properties of the emulsion.
[0074] 7. 3D printing imaging evaluation results of deamidated peanut protein isolate microgel particles-resveratrol composite stable high internal phase Pickering emulsions formed with different amounts of resveratrol.
[0075] Result statement: From Figure 7 It can be seen that all embodiments and comparative examples successfully constructed complex three-dimensional structures, including hollow cross structures and spade structures. However, there are significant differences in molding quality and structural stability between Comparative Example 1 and Examples 1-5. Although Comparative Example 1 can complete basic printing, the molding quality has defects: the hollow cross structure exhibits flocculent and uneven texture inside, with a rough surface and unfused areas; the spade structure has slight edge collapse, and insufficient resolution and pattern fineness. Examples 1-5, on the other hand, demonstrate excellent molding quality: clear structural outlines and high resolution; uniform and consistent lines and fine patterns; good support, maintaining a stable three-dimensional structure, and the improvement in printing performance is positively correlated with the concentration of resveratrol.
Claims
1. A method for preparing a peanut protein microgel with high internal phase emulsion and high antioxidant stability, characterized in that, Includes the following steps: (1) Deamidation treatment: The obtained peanut protein isolate was dissolved in deionized water to prepare a solution of 5-15 mg / mL. After stirring at 200 rpm / min for 4 h with a magnetic stirrer, 20-80 U of protein glutaminase was added. The solution was stirred at 37℃ for enzymatic deamidation treatment. After enzymatic hydrolysis for 10-50 min, the enzyme was inactivated and the solution was freeze-dried to obtain peanut protein isolate with a deamidation degree of 10-30%. (2) Preparation of microgel particles: The lyophilized peanut protein isolate powder with a deamidation degree of 20% obtained in (1) was dissolved in deionized water, magnetically stirred at 200 rpm / min for 4 h at 25 °C, and stored overnight at 4 °C to ensure complete hydration, to obtain a 10 wt% deamidated peanut protein isolate solution. The pH of the solution was adjusted to 7.0 with 2 mol / L NaOH, and then incubated at 90 °C for 30 min and cooled to 25 °C. The obtained gel was mixed with deionized water at a ratio of 1:4 and homogenized at 12000 rpm / min for 5 min using a homogenizer, followed by 1-5 cycles of homogenization at 10-50 MPa using a high-pressure homogenizer to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 1-5 wt%. The obtained deamidated peanut protein isolate microgel particles were stored at 4 °C and partially lyophilized for subsequent applications; (3) Preparation of resveratrol-stabilized high internal phase Pickering emulsion: Resveratrol powder was dissolved in anhydrous ethanol solution to obtain five resveratrol solutions of different concentrations (0.1-0.2 mg / mL). The deamidated peanut protein isolate microgel particle solution obtained in (2) and the resveratrol solution were mixed at a volume ratio of 1:1 and incubated in a water bath at 25℃ for 2 h. Then, the ethanol was removed by rotary evaporation at 37℃. 10-30 mL of the deamidated peanut protein isolate microgel particle solution was mixed with 20-50 mL of peanut oil solution. The mixture was then homogenized at 12000 rpm / min for 2 min at 25℃ using an IKA homogenizer. Finally, a resveratrol-deamidated peanut protein isolate microgel particle stabilized high internal phase Pickering emulsion was obtained.
2. The method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (1), the concentration of the peanut protein isolate solution is 10 mg / mL, and the total amount of protein glutaminase added is 50 U.
3. The method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (1), the enzymatic hydrolysis time is 30 min, and the degree of deamidation of the peanut protein isolate is 20%.
4. The method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (2), peanut protein isolate lyophilized powder with a deamidation degree of 20% was dissolved in deionized water, stirred at 200 rpm / min for 4 h at 25°C, and stored overnight at 4°C to ensure complete hydration, resulting in a deamidated peanut protein isolate solution with a concentration of 10 wt%.
5. The method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (2), the obtained gel is mixed with deionized water at a ratio of 1:4 and homogenized at 12,000 rpm / min for 5 min using a homogenizer. Then, it is homogenized three times in a high-pressure homogenizer at 30 MPa to obtain deamidated peanut protein isolate microgel particles with a protein concentration of 2 wt%.
6. A method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (3), resveratrol powder with different contents was dissolved in anhydrous ethanol solution, and the concentrations of the resveratrol solutions obtained were 0.0125, 0.025, 0.05, 0.1 and 0.2 mg / mL, respectively.
7. A method for preparing a peanut protein microgel with high antioxidant stability high internal phase emulsion according to claim 1, characterized in that: In step (3), 10 mL of deamidated peanut protein isolate microgel particle solution is mixed with 40 mL of peanut oil solution.