Protein-based composite system for embedding linoleic acid based on physical field as well as preparation method and application of protein-based composite system

Abstract

The invention discloses a protein-based composite system for embedding linoleic acid based on a physical field as well as a preparation method and application of the protein-based composite system, and relates to the field of compositions of high-molecular compounds. According to the method, the protein compound is modified through DHPM or TSE, no organic solvent is used, heating is not needed, and the method is a novel method for obtaining the green and safe functional protein compound and is environmentally friendly and efficient. And meanwhile, a high-pressure emulsion homogenizing technology is adopted to prepare an emulsion loaded with linoleic acid, so that the stability of the linoleic acid is improved, the bioaccessibility of the linoleic acid is improved, and controllable slow release of the linoleic acid in the intestinal tract is realized. The prepared protein-based composite system based on physical field embedded linoleic acid has excellent solubility, thermal stability and oxidation resistance, can resist gastrointestinal tract temperature and is suitable for gastrointestinal tract absorption when people eat the protein-based composite system, and can be applied to edible nutritional factor delivery products.

Description

Technical Field

[0001] The present invention relates to the field of compositions of high molecular compounds, and in particular to a protein-based composite system based on physical field embedded linoleic acid, and a preparation method and application thereof. Background Art

[0002] Elucidating the complex interplay between food, the gut microbiome, and health outcomes is crucial for developing functional foods (such as prebiotics, probiotics, and genetically modified agents) for patients with radiation enteritis. Current research focuses on nanotechnology-driven solutions to overcome the limitations of traditional chemotherapy. Among these, delivery systems based on biopolymers (such as protein-polysaccharide complexes) exhibit unique advantages: these complexes can leverage the surface activity of proteins (such as whey protein and zein) and polysaccharides (such as sodium alginate) to achieve stable loading of functional factors while also enabling targeted delivery by modulating intermolecular forces (such as hydrophobic interactions and hydrogen bonds). This natural, biocompatible carrier offers the potential for developing safe and effective novel nutritional intervention strategies.

[0003] Water-soluble protein (whey protein, abbreviated WPI) offers advantages such as high nutritional value, easy availability, and excellent technical and functional properties. Based on these advantages, nutrient delivery systems based on whey protein have been extensively studied to deliver functional ingredients such as astaxanthin, 3,3'-diindolylmethane, vitamin D3, resveratrol, and β-carotene.

[0004] Zein (abbreviated as Z), the primary storage protein in corn kernels, is poorly water-soluble due to its high percentage of hydrophobic amino acid residues (over 50%), such as proline, leucine, and alanine. It is soluble in 60-90% aqueous ethanol. Zein's unique dissolution properties can be used to prepare nanoparticles for encapsulating non-polar bioactive molecules. In recent years, research on the interactions between proteins and polyphenolic flavonoids has become a hot topic, with the study of non-covalent interactions also becoming a key research topic in the food industry. Most non-covalent interactions are caused by hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces, and are generally reversible. These interactions can cause protein structure to aggregate or unfold, leading to changes in its physicochemical properties, functional characteristics, and nutritional value.

[0005] Sodium alginate (SA) is a linear binary copolymer composed of β-D-mannuronic acid and α-L-guluronic acid residues connected by (1,4) glycosidic bonds. It has good stability and acid and alkali resistance, can form nanoparticles in a variety of pH ranges, and is widely used in the food industry. It is generally recognized as safe (GRAS) food material.

[0006] As people's nutritional and health needs continue to rise, foods must not only provide energy but also possess functional properties to enhance their nutritional value. However, most functional factors in foods suffer from poor solubility, instability, and low bioavailability. Therefore, effective delivery systems are needed to deliver these functional factors. Linoleic acid (LA) is an important long-chain fatty acid and a precursor to fatty acids such as γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid. These fatty acids possess anti-inflammatory properties, lower cholesterol, and help prevent atherosclerosis. However, due to its high reactivity and susceptibility to oxidation and hydrogenation, linoleic acid requires careful storage.

[0007] Currently, most methods for preparing protein complexes use organic solvents, which have certain limitations in their application. Some organic solvents are toxic and irritating and cannot be used on the human body. The pH transfer method, sometimes also called the pH-driven method, has recently been used to incorporate functional factors into different types of edible nanoparticles without the use of organic solvents. The pH conversion method utilizes the fact that the solubility of functional factors in aqueous solutions strongly depends on the pH value. However, in the prior art, the modification of proteins using the pH-driven method usually requires heating the reaction, and too high a temperature will cause the protein to denature and inactivate.

[0008] Therefore, it is necessary to develop a new method that is environmentally friendly, efficient, and can obtain green, safe, and functional protein complexes. Summary of the Invention

[0009] The purpose of the present invention is to propose a protein-based composite system based on physical field encapsulation of linoleic acid, and its preparation method and application. It does not use any organic solvents and does not require heating. It is a new method for obtaining green, safe and functional protein complexes that is environmentally friendly and efficient. At the same time, the prepared protein-based composite system exhibits excellent thermal stability and functionality and can be used in edible nutritional factor delivery products.

[0010] In order to achieve the above object, the technical solution of the present invention is as follows: In a first aspect, the present invention proposes a method for preparing a protein-based composite system for encapsulating linoleic acid based on a physical field. The method comprises: obtaining a ternary composite system of water-soluble protein, zein, and sodium alginate by a pH-driven method, wherein the mass ratio of water-soluble protein, zein, and sodium alginate is 9:1:1; placing the ternary composite system in a dynamic high-pressure microfluidizer (DHPM) device or a micro twin-screw extruder (TSE) device for treatment, wherein the pressure parameters of the DHPM device are set to 250-850 kPa and the temperature parameters are 35°C; the pressure parameters of the TSE device are set to 120-200 rpm and the temperature parameters are 35°C; the ternary composite system after DHPM or TSE treatment is freeze-dried, and the freeze-dried powder is taken and added to deionized water, and stirred with a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; and preparing an emulsion of linoleic acid and the aqueous solution of the protein-based composite system in a volume ratio of 2:1-3:1, i.e., a protein-based composite system for encapsulating linoleic acid.

[0011] Preferably, the method for preparing the above-mentioned protein-based composite system based on physical field embedding of linoleic acid comprises the following steps: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with a 2M NaOH solution and stirred for 2 h under a stirrer. Zein was added to the water-soluble protein solution at a water-soluble protein to zein mass ratio of 9:1 and stirred for 30 min under a stirrer. Sodium alginate was added to the water-soluble protein-zein complex solution at a zein to sodium alginate mass ratio of 1:1 and stirred for 30 min under a stirrer. The complex solution was adjusted to pH 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set as follows: a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into the DHPM equipment for treatment, wherein the pressure parameter of the DHPM equipment is set to 250-850 kPa and the temperature parameter is 35°C; S5. Freeze-dry the ternary composite system after DHPM treatment, take the freeze-dried powder, add it to deionized water, and stir it with a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; prepare an emulsion with linoleic acid and the aqueous solution of the protein-based composite system in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

[0012] Preferably, the method for preparing the above-mentioned protein-based composite system based on physical field embedding of linoleic acid comprises the following steps: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with a 2M NaOH solution and stirred for 2 h under a stirrer. Zein was added to the water-soluble protein solution at a water-soluble protein to zein mass ratio of 9:1 and stirred for 30 min under a stirrer. Sodium alginate was added to the water-soluble protein-zein complex solution at a zein to sodium alginate mass ratio of 1:1 and stirred for 30 min under a stirrer. The complex solution was adjusted to pH 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set as follows: a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into a TSE device for treatment, wherein the pressure parameter of the TSE device is set to 120-200 rpm and the temperature parameter is set to 35°C; S5. The ternary composite system after TSE treatment is freeze-dried, and the freeze-dried powder is taken, added to deionized water, and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; linoleic acid and the aqueous solution of the protein-based composite system are prepared into an emulsion in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

[0013] In a second aspect, the present invention further proposes a protein-based composite system for embedding linoleic acid based on a physical field, which is prepared by any of the methods described above.

[0014] In a third aspect, the present invention further proposes the application of the above-mentioned protein-based composite system based on physical field embedded linoleic acid in the preparation of edible nutritional factor delivery products.

[0015] Compared with the prior art, the technical effects of the present invention are: This invention utilizes dynamic high-pressure microfluidization (DHPM) and micro twin-screw extrusion (TSE) to modify protein complexes, eliminating the need for organic solvents or heating. This method represents a novel, environmentally friendly, and efficient method for obtaining green, safe, and functional protein complexes. High-pressure homogenized emulsion technology is also employed to prepare an LA-loaded emulsion, enhancing LA's stability, improving its bioaccessibility, and enabling controlled intestinal release. The resulting protein-based composite system, based on physical field-encapsulated linoleic acid, exhibits excellent solubility, thermal stability, and antioxidant properties. It withstands gastrointestinal temperatures and is well-suited for gastrointestinal absorption, making it suitable for use in edible nutritional factor delivery products. Compared to untreated protein complexes, the composites treated with physical fields exhibit superior heat resistance and particle size. In addition, the present invention also studies the multi-spectral effects of DHPM and TSE before and after treatment, as well as thermal stability, particle size potential, and antioxidant properties, explores the driving force of different physical fields on the formation of the composite system, and simultaneously analyzes the binding mechanism of different physical fields on the composite system through surface hydrophobicity and free thiol groups, further ensuring its application in the field of functional food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the ultraviolet absorption spectrum of the protein complex after physical field treatment provided in Test Example 1 of the present invention.

[0017] Figure 2 This is the Fourier transform infrared spectrum of the protein complex after physical field treatment provided in Test Example 2 of the present invention.

[0018] Figure 3 This is the DSC graph of the protein complex after physical field treatment provided in Test Example 3 of the present invention.

[0019] Figure 4 This is the particle size potential map of the protein complex after physical field treatment provided in Test Example 4 of the present invention.

[0020] Figure 5 This is the circular dichroism spectrum of the protein complex after physical field treatment provided in Test Example 5 of the present invention.

[0021] Figure 6 This is a graph showing the encapsulation efficiency and loading rate of DHPM-LA and TSE-LA provided in Test Example 6 of the present invention.

[0022] Figure 7 This is a scanning electron microscope image of the protein complex emulsion after physical field treatment provided in Test Example 7 of the present invention.

[0023] Figures 8A-8H The backscattered light intensity change results of emulsions prepared by mixing LA with DHPM850 and TSE200 in different volume ratios within 24 hours are provided in Test Example 8 of the present invention. Figure 8A The ratio of DHPM850 to LA is 3:1. Figure 8B The ratio of TSE200 to LA is 3:1. Figure 8C The ratio of DHPM850 to LA is 2:1. Figure 8D For TSE-LA2:1, Figure 8E The ratio of DHPM850 and LA is 1:1. Figure 8F The ratio of TSE200 and LA is 1:1. Figure 8G The ratio of DHPM850 to LA is 1:2. Figure 8H The ratio of TSE200 to LA is 1:2.

[0024] Figure 9 The Turbiscan stability index (TSI) of DHPM-LA and TSE-LA provided in Test Example 8 of the present invention.

[0025] Figure 10 The thermal stability results of DHPM-LA and TSE-LA are provided in Test Example 8 of the present invention.

[0026] Figure 11 The freeze-thaw stability results of DHPM-LA and TSE-LA are provided in Test Example 8 of the present invention.

[0027] Figure 12 The centrifugation stability results of DHPM-LA and TSE-LA provided in Test Example 8 of the present invention.

[0028] Figure 13 The lipid oxidation stability results of DHPM-LA and TSE-LA provided in Test Example 8 of the present invention.

[0029] Figure 14 The free radical scavenging ability stability results of DHPM-LA and TSE-LA provided in Test Example 8 of the present invention.

[0030] Figure 15 The results of in vitro digestion simulation protein release characteristics of DHPM-LA and TSE-LA provided in Test Example 9 of the present invention.

[0031] Figure 16 The results of the free fatty acid release characteristics of DHPM-LA and TSE-LA simulated by in vitro digestion are provided in Test Example 9 of the present invention. DETAILED DESCRIPTION

[0032] To help those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0034] Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Sodium alginate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., zein was purchased from Beijing Bailingwei Technology Co., Ltd., and water-soluble protein (whey protein) was purchased from Yingxin Laboratory.

[0035] The present invention proposes a method for preparing a protein-based composite system for embedding linoleic acid, wherein a water-soluble protein (WPI) solution, zein (Z) and sodium alginate (SA) are mixed by a pH-driven method to obtain a ternary composite system (WPI-Z-SA), wherein the mass ratio of zein, zein and sodium alginate is 9:1:1. The ternary composite system was placed in a DHPM apparatus with pressures set to 0, 250, 400, 550, 700, and 850 kPa, respectively, and a temperature parameter of 35°C. Samples named DHPM0, DHPM250, DHPM400, DHPM550, DHPM700, and DHPM850 were obtained. The ternary composite system was placed in a TSE apparatus with pressures set to 0, 120, 140, 160, 180, and 200 rpm, respectively, and a temperature parameter of 35°C. Samples named TSE0, TSE120, TSE140, TSE160, TSE180, and TSE200 were obtained. The WPI-Z-SA ternary composite system, treated with DHPM at 850 kPa and TSE at 200 rpm, was lyophilized. 2.0 g of the lyophilized powder was added to 100.0 mL of deionized water and stirred at 1200 rpm in a high-pressure homogenizer for 2 min. Linoleic acid (LA) and a protein-based composite system aqueous solution are prepared into an emulsion in a volume ratio of 1:2 to 3:1, namely, a protein-based composite system encapsulating linoleic acid.

[0036] Example 1 Using deionized water as the solvent, 0.045 g of water-soluble protein was added to 50 mL of deionized water and hydrated overnight at 4°C. The pH was adjusted to 12 with 2 M NaOH and the mixture was stirred thoroughly at room temperature (25 ± 2°C) for 2 h. 0.005 g of zein was added to the water-soluble protein solution and rapidly stirred for 30 min. 0.005 g of sodium alginate was added to the water-soluble protein-zein complex solution and rapidly stirred for 30 min. The pH was adjusted to 7 with 1 M HCl to obtain a water-soluble protein-zein-sodium alginate ternary complex, designated WPI-Z-SA. The stirring parameters were: stirring speed 800 r / min, ambient temperature 25 ± 2°C.

[0037] The ternary composite system WPI-Z-SA was placed in a DHPM apparatus with the pressure set to 250 kPa and the temperature set to 35°C, yielding a sample named DHPM250. The ternary composite system WPI-Z-SA was placed in a TSE apparatus with the pressure set to 120 rpm and the temperature set to 35°C, yielding a sample named TSE120.

[0038] Example 2 The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure set to 400 kPa and the temperature set to 35°C, to obtain a sample named DHPM400. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the pressure set to 140 rpm and the temperature set to 35°C, to obtain a sample named TSE140.

[0039] Example 3 The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure set to 550 kPa and the temperature to 35°C, to obtain a sample named DHPM550. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the pressure set to 160 rpm and the temperature to 35°C, to obtain a sample named TSE160.

[0040] Example 4 The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure set to 700 kPa and the temperature set to 35°C, to obtain a sample named DHPM700. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the pressure set to 180 rpm and the temperature set to 35°C, to obtain a sample named TSE180.

[0041] Example 5 The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure parameters set to 850 kPa and the temperature parameters set to 35°C, to obtain a sample named DHPM850. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the pressure parameters set to 200 rpm and the temperature parameters set to 35°C, to obtain samples named TSE200.

[0042] Example 6 The ternary composite system WPI-Z-SA (denoted as DHPM850 and TSE200) was freeze-dried after treatment with DHPM (850 kPa) and TSE (200 rpm). 2.0 g of each freeze-dried powder was added to 100.0 mL of deionized water and stirred at 1200 rpm with a high-pressure homogenizer for 2 minutes to obtain an aqueous solution of the protein-based composite system. LA was then prepared with DHPM850 and TSE200 at volume ratios of 1:2, 1:1, 2:1, and 3:1, respectively. Multiple photodetection revealed that the optimal ratio was 3:1, and these emulsions were designated DHPM-LA and TSE-LA, respectively.

[0043] Comparative Example 1 The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure parameters set to 0 kPa and the temperature parameters set to 35°C, to obtain a sample named DHPM0. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the pressure parameters set to 0 rpm and the temperature parameters set to 35°C, to obtain samples named TSE0.

[0044] Test Example 1 Ultraviolet absorption spectroscopy: The freeze-dried solids of the protein complexes prepared in Examples 1-5 and Comparative Example 1 were ground into a fine powder and dissolved in deionized water to prepare a 0.2 mg / mL solution. The sample solution was transferred to a quartz cuvette and scanned across the entire UV range of 200-400 nm using a UV-visible spectrophotometer to record the sample's absorption characteristics. Deionized water was used for baseline correction before scanning.

[0045] The UV spectrum changes of the WPI-Z-SA composite system before and after treatment with different pressure parameters of DHPM and TSE are shown in Figure 2. Figure 1 As shown. Figure 1Both physical treatments resulted in a significant increase in the system's absorbance, with TSE reaching its maximum absorbance at 200 rpm and DHPM at 850 kPa. WPI, Z, and SA primarily form a non-covalent complex through hydrogen bonding and hydrophobic interactions. The introduction of SA induces conformational changes in the protein, promoting molecular decomposition, and the intensity of intermolecular interactions exceeds the effects of the physical field treatment. Comparing the two treatment methods, the increase in UV absorbance after DHPM treatment is more pronounced. This phenomenon may be related to the cavitation effect and microfluidic mechanism of DHPM, which more effectively promote the unfolding of protein molecules, exposing internal hydrophobic regions, thereby enhancing the binding between WPI, Z, and SA, ultimately manifesting as a significant increase in the UV absorption characteristics of the complex system.

[0046] Test Example 2 Fourier transform infrared spectroscopy: The protein complexes prepared in Examples 1-5 and Comparative Example 1 were freeze-dried and ground into fine powders. The sample powders were mixed with KBr at a ratio of 1:100. The sample and KBr were then ground into an even finer powder using a mortar and pestle. The mixture was uniformly mixed and the film was pressed for 1 minute before being placed in the instrument for measurement. The scanning range was 4000-400 cm -1 , with a resolution of 4 cm -1 , scanned 32 times. Potassium bromide tablets were used as blank control during the measurement, and the obtained infrared spectra were analyzed using Origin software.

[0047] The infrared spectrum results of WPI-Z-SA composite system under DHPM treatment at 250-850 kPa are as follows Figure 2 After high pressure treatment, the characteristic absorption peak of the composite system shifted significantly, and the OH stretching vibration peak shifted from 3395 cm -1 Shifted to lower wavenumbers to 3384 cm -1 , indicating that high pressure treatment promotes the formation of intermolecular hydrogen bonds. -1 ) and amide II band (1543 cm -1 ) relative to the absorption peak position of the untreated sample (1635 cm -1 and 1540 cm -1 ) shifted significantly, indicating that high-pressure microfluidization treatment enhanced the vibrational intensities of the C=O, C-N, and N-H bonds in peptide bonds. Despite increasing treatment pressure, the positions of the OH stretching vibrations of the amide A band and the characteristic peaks of amide regions I and II remained relatively stable, showing no significant shifts. The infrared spectra of the WPI-Z-SA composite system treated with TSE at 120-200 rpm showed that TSE had less effect on functional group stretching vibrations than DHPM.

[0048] Test Example 3 Thermal Stability Determination: The protein complexes prepared in Examples 1-5 and Comparative Example 1 were freeze-dried and then ground into a fine powder. A 3-5 mg sample of the freeze-dried powder was placed in an aluminum crucible. After capping, the crucibles containing the samples and an empty crucible were placed inside the instrument. The empty crucible, serving as a control, remained in the instrument until the measurement was completed. Test conditions were: starting temperature 20°C, heating rate 10°C / min, reaction end temperature 200°C, and nitrogen flow rate 50 mL / min. After the test, the thermal properties of the samples were analyzed using analysis software compatible with the instrument.

[0049] The DSC curves of the WPI-Z-SA composite system after DHPM and TSE treatment are shown in Figure 2. Figure 3 The thermal stability of the composite system after DHPM and TSE treatment is lower than that of the untreated composite system, indicating that under the action of DHPM and TSE, the composite system unfolds, the structure becomes loose and more disordered, and the thermal stability decreases. However, the denaturation temperature (T d ) As the pressure continues to increase, the thermal stability of the WPI-Z-SA composite system gradually increases. When treated with 850 kpa DHPM and 160 rpm TSE, the denaturation temperature of the WPI-Z-SA composite system rises to 119°C and 121°C, respectively. This process describes that the samples treated with TSE show different changes. As the mechanical shear force increases, the T of the composite system increases. d This phenomenon indicates that under high shear strength conditions, the interaction between Zein molecules is destroyed, resulting in the unfolding of its tertiary structure, which ultimately leads to a decrease in the thermal stability of the composite system.

[0050] Test Example 4 Particle Size Potential Determination: The prepared samples were diluted to 1 mg / mL with ultrapure water. The zeta potential and particle size of the ternary composite nanoparticles treated with DHPM and TSE at different pressure parameters were measured using a Nano-ZS laser particle size analyzer. The particle size and zeta potential of the samples were calculated using the Stokes-Einstein equation and the Smoluchowski model, respectively. All samples were measured in triplicate, and the average value was calculated.

[0051] The results are as follows Figure 4As shown in the figure, the average particle size of the WPI-Z-SA composite system without DHPM treatment is 389.66 nm and the PDI is 0.23. After DHPM treatment, the protein particle size and PDI have a significant decreasing trend. When the DHPM pressure is 250 kPa, the particle size and PDI of the composite system are 217.116 nm and 0.62, respectively. With the increase of pressure, the protein particle size and PDI gradually decrease. When the high pressure is 850 kPa, the particle size reaches 145.697 nm and the PDI reaches 0.45 ( Figure 4 The particle size and PDI of the WPI-Z-SA composite system treated with TSE were larger than those treated with DHPM. As the TSE pressure increased, the particle size and PDI of the composite system decreased. When the TSE speed was 200 rpm, the particle size and PDI of the composite system were 150.100 nm and 0.45 ( Figure 4 (B) This indicates that high pressure treatment reduces the average particle size and PDI of proteins because the cavitation effect and shear force caused by high pressure transform the protein from macromolecular particles into smaller particles, making the protein more uniform in the dispersion system.

[0052] Zeta potential of protein is another important indicator to characterize the stability of dispersed systems. Figure 4 Figure C in the middle shows the Zeta potential of the protein before and after DHPM. It can be seen that the Zeta potential of all samples is negative, indicating that the surface of the protein is mostly negatively charged amino acids. The Zeta potential value of the protein without DHPM treatment is -30.190 mV. The absolute value of the Zeta potential of the protein after DHPM treatment increases. As the high pressure increases, the absolute value of the Zeta potential shows a decreasing trend. At DHPM 850 kpa, the minimum Zeta potential is -38.388 mV. This shows that the cavitation effect generated during the high pressure process causes the protein structure to unfold, more negatively charged amino acid groups are exposed, and the charge on the molecular surface increases. The absolute value of the Zeta potential of TSE has an even more obvious trend of increasing. At TSE 200 rpm, the minimum Zeta potential is -49.150 mV (as shown in Figure 2). Figure 4 This indicates that the protein structure has become looser and the number of negatively charged amino acids on the surface has increased, which is consistent with the results of electron microscopy and particle size analysis.

[0053] Test Example 5 Circular dichroism (CD) spectroscopy: Using a CD spectrometer equipped with a 0.1 cm pathlength quartz cuvette and deionized water as the reference solution, the far-UV CD signals of the WPI-Z-SA composite system before and after DHPM and TSE treatment were measured in the 190-260 nm wavelength range. The sample concentration was maintained at 0.2 mg / mL during the test, and nitrogen was continuously purged to eliminate air interference. The following formula was used to express the residual ellipticity (MRE) in deg·cm 2 ·d·mol −1 CD results:

[0054] Where: Cp represents the molar concentration of protein, n represents the number of amino acid residues in zein (266), l represents the path length (0.1 cm), and the average residue ellipticity value at 208 nm can be used to calculate the α-helix content of the sample as follows:

[0055] The specific analysis results are as follows Figure 5 As shown, with increasing treatment intensity, the characteristic peak at 208 nm of the composite system exhibited a positive shift. The α-helix content of the untreated composite system was 6%, which increased to 9-10% after DHPM treatment and to 9-12% after TSE treatment. This result confirms that physical field treatment induces a conformational shift from a compact to a loose state. This suggests that while SA can partially maintain the helical structure, it cannot completely inhibit pressure-induced conformational changes. Further analysis revealed that DHPM treatment resulted in an increase in α-helix and β-sheet content and a decrease in β-turn angles, indicating that the treatment reduced protein disorder and promoted unfolding. The reduction in β-sheets enhances molecular flexibility, with weakening of hydrogen bonding being the primary factor driving the changes in secondary structure. This process demonstrates that within the pressure range (250-850 kPa), the secondary structure content exhibited no significant pressure-dependent changes. Comparing the two treatment methods, the α-helix retention rate was slightly higher in the 200 rpm TSE treatment group than in the DHPM treatment group, indicating that mechanical shearing induces a lower degree of protein folding, likely due to the partial dissociation of protein aggregates promoted by twin-screw extrusion. Overall, DHPM treatment showed a more significant effect in regulating protein secondary structure and also effectively improved the solubility properties of the complex system. These structural changes may be due to pressure-induced conformational rearrangements and exposure of specific protein groups, thereby enhancing their ability to interact with other molecules.

[0056] Test Example 6 Determination of DHPM-LA and TSE-LA Encapsulation and Loading Efficiency: The encapsulation efficiency (EE) and loading rate (LC) of linoleic acid in the DHPM-LA and TSE-LA emulsions were determined by the following method. The LA-loaded emulsions DHPM-LA and TSE-LA were centrifuged at 10,000 rpm for 10 min to remove the upper cream layer. After repeating this process twice, the fraction obtained at the bottom of the tube was mixed with ethyl acetate (4:1, v / v), and the ethyl acetate was collected to measure the amount of unencapsulated LA. A standard curve for LA (y = 0.0728x + 0.0161; R 2 = 0.9995, x represents the concentration of LA (μg / mL), and y represents the absorbance at 234 nm as described in our previous study). The encapsulation efficiency (EE) and loading rate (LC) of the emulsion were calculated as follows:

[0057] In the above equation, M1 represents the total amount of linoleic acid, M0 represents the amount of unencapsulated linoleic acid, and M2 represents the amount of emulsion.

[0058] The results of DHPM-LA and TSE-LA embedding and loading rates are as follows Figure 6 As shown in the figure, the entrapment efficiency of DHPM-LA was 23.4% and the loading efficiency was 72.8%, while the entrapment efficiency of TSE-LA was 30.2% and the loading efficiency was 68.9%. This may be because the internal voids of TSE-LA are larger due to extrusion, which allows a larger number of LA to be filled in the voids.

[0059] Test Example 7 Scanning electron microscopy measurement: The protein complex samples DHPM-LA and TSE-LA that had undergone physical field treatment and the protein complex sample WPI-Z-SA that had not undergone physical field treatment were placed in a freeze dryer to obtain a protein complex solid system, which was then ground into a fine powder, sprayed with gold, and placed under a scanning electron microscope to observe the microscopic morphology.

[0060] Scanning electron microscopy images of DHPM-LA and TSE-LA emulsions in different states are shown in Figure 2. Figure 7 As shown. TSE-LA has a rough surface and a flaky structure. Compared with TSE-LA, DHPM-LA emulsions are adhered to each other and have a smooth structure. DHPM-LA shows a uniform particle size and a denser structure, which is due to the close aggregation of the three substances of DHPM. Both have a compact structure, which is attributed to the fact that the insoluble protein complex system weakens the electrostatic force under strong pH, thereby reducing the aggregation and nucleation, opening the structure and gradually forming larger low aggregates to obtain a soluble complex system. In addition, as Figure 7As shown in the figure, scanning electron micrographs of freeze-dried and non-freeze-dried emulsions DHPM-LA and TSE-LA can be seen. No obvious difference was found in the comparison of the two structures, indicating that freeze-drying has no effect on the structure of the emulsion.

[0061] Test Example 8 1. Determination of storage stability of DHPM-LA and TSE-LA Emulsions were prepared by mixing LA with aqueous solutions of a protein-based composite system treated with DHPM (850 kPa) and TSE (200 rpm) at volume ratios of 3:1, 2:1, 1:1, and 1:2. The physical stability of the emulsion samples was quantitatively assessed by analyzing the changes in backscattered light intensity over 24 hours; greater overlap in the spectral curves indicates better stability. A 20.0 mL sample of each emulsion was placed in the scanning cell of a Turbiscan Lab multiple light scattering instrument in multiple scan mode with a temperature set to (25.0±0.5)°C. Scans were performed every 30 minutes for a total of 24 hours. The stability of the emulsion samples over a 24-hour period at room temperature was determined by analyzing the changes in the instability kinetics curves.

[0062] The results are as follows Figures 8A-8H As shown, the spectral curves of the protein-based composite aqueous solution treated with LA and DHPM at 850 kPa or TSE at 200 rpm at volume ratios of 2:1, 1:1, and 1:2 show significant differences, indicating varying degrees of stability. However, the spectral curves of the TSE-LA 3:1 sample almost completely overlap, indicating uniform particle dispersion and no significant aggregation or phase separation during the test, demonstrating excellent stability. In contrast, the backscattered light signal at the top of the DHPM-LA 3:1 sample gradually decreases, suggesting particle sedimentation and relatively poor stability.

[0063] Further analysis of the Turbiscan Stability Index (TSI) Figure 9 A quantitative comparison was conducted. This index combines the changing characteristics of the transmitted and backscattered light signals, with larger values indicating a more unstable system. 24-hour monitoring data showed that the TSI value of the TSE-LA sample remained stable, confirming its optimal storage stability; whereas the TSI value of the DHPM-LA sample increased significantly, indicating its poorest stability.

[0064] 2. Determination of thermal stability and freeze-thaw stability of DHPM-LA and TSE-LA The DHPM-LA and TSE-LA emulsions were placed in glass bottles and heated at 90°C for 1 hour. Their thermal stability was assessed by oil release and stratification on the emulsion surface. The emulsions were frozen at -20°C for 24 hours and then placed in a 30°C water bath for 5 hours. The freeze-thaw state of the emulsions was recorded and photographed. Their freeze-thaw stability was assessed by phase separation and oil precipitation.

[0065] Thermal stability of DHPM-LA and TSE-LA emulsions Figure 10 As shown. No obvious oiling phenomenon was observed in both DHPM-LA and TSE-LA emulsions. In general, the emulsions stabilized by the protein compound have good thermal stability. SA in the compound plays a steric hindrance role in preventing the droplets from approaching during heating, thereby preventing their heat-induced aggregation. The effects of heating on DHPM-LA and TSE-LA emulsions are different. The emulsion stabilized by DHPM-LA did not show obvious oiling phenomenon, while the emulsion stabilized by TSE-LA showed oiling phenomenon after heating. Figure 10 It can be seen that WPI-Z-SA is primarily adsorbed at the oil-water interface of this emulsion. At high temperatures, protein molecules denature and unfold, exposing more hidden hydrophobic groups on the surface. This increases hydrophobic interactions between protein molecules and leads to protein aggregation, thus deteriorating the thermal stability of the emulsion. Furthermore, the network structure of TSE-LA is inferior to that of DHPM-LA. This is primarily due to the weaker network structure and thinner interface layer, which causes the emulsion droplets to aggregate under thermal influence, reducing the thermal stability of the emulsion.

[0066] The freeze-thaw stability analysis results of DHPM-LA and TSE-LA emulsions are shown in Figure 2. Figure 11As shown in Figure 3 . After freeze-thaw cycles, both DHPM-LA and TSE-LA emulsion systems exhibited phase separation, manifested as oily layer precipitation and demixing. This instability is primarily due to the collision and aggregation of droplets caused by crystallization of the oil and water phases during freezing. Upon warming, the heterogeneous aggregates formed during freezing fuse to form large oil droplets, ultimately triggering emulsion breakdown. The freeze-thaw stability of an emulsion is primarily influenced by its composition and structural properties. Generally speaking, emulsions with high viscosity and a strong interfacial membrane effectively resist mechanical damage from ice crystals, thereby reducing the risk of flocculation. In this study, the interfacial layer of the emulsion stabilized by WPI-Z-SA was relatively thin, which may be one of the reasons for its poor freeze-thaw stability. Furthermore, low-temperature-induced protein conformational changes may also weaken the stability of the interfacial membrane. Furthermore, all treatments retained a partial emulsion layer after freeze-thaw, indicating that the WPI-Z-SA composite system increases the unfrozen water content in the continuous phase, thereby inhibiting the proximity of droplets. Comparing the different treatments, the DHPM-LA emulsion exhibited relatively good freeze-thaw stability. This is attributed to the fact that the three-dimensional network structure it forms can effectively block the growth of ice crystals, and at the same time the thicker interface layer can provide better mechanical protection, preventing the crystals formed during the freezing process from destroying the oil droplet structure, and ultimately reducing the droplet coalescence phenomenon after thawing.

[0067] 3. Determination of centrifugal stability of DHPM-LA and TSE-LA Transfer 15.0 mL of each DHPM-LA and TSE-LA emulsion into a 50.0 mL centrifuge tube and centrifuge at 4000 rpm for 20 min. Take photos to record the phase separation and evaluate the centrifugal stability by comparing the height of the emulsion layer.

[0068] Centrifugal stability of DHPM-LA and TSE-LA emulsions Figure 12 As shown. Under the action of centrifugal force, the emulsion droplets aggregate and creaming and stratification occur. After centrifugation, the higher the emulsification layer of the emulsion, the more evenly distributed and stable the emulsion layer in the emulsion system is. The centrifugal stability of DHPM-LA and TSE-LA emulsions is different. After centrifugation, water phase precipitates from the two emulsions. The emulsion stabilized by the TSE-LA compound has the worst centrifugal stability, while the emulsion stabilized by the DHPM-LA compound has better centrifugal stability, which is mainly related to the internal structure of the emulsion. Combined with the electron microscopy results, the emulsion stabilized by the DHPM-LA compound has a relatively strong network structure and viscosity. The strong interactions between the emulsion droplets, between the protein molecular chains, and between the droplets and the protein molecular chains effectively resist the centrifugal force and hinder the aggregation of oil droplets, thereby ensuring the centrifugal stability of the emulsion. It can be found that the results of centrifugal stability are similar to those of static storage stability.

[0069] 4. Determination of lipid oxidation stability of DHPM-LA and TSE-LA emulsions 10.0 mL of each DHPM-LA and TSE-LA emulsion was placed in a glass bottle and randomly placed in a dark oven at 50°C for 14 days. Samples were collected and analyzed for primary and secondary oxidation products on days 0, 1, 3, 5, 7, 10, and 14. The following experimental procedure was used to determine the primary lipid oxidation products: 0.3 mL of the emulsion sample was accurately placed in a 5 mL centrifuge tube, and 1.5 mL of extraction solvent A, prepared from a 3:1 (v / v) ratio of isooctane to isopropanol, was added. The sample was vortexed (30 s, with 10 s intervals) and then centrifuged at 4000 rpm for 2 min to achieve phase separation. Mix 200 μL of the upper organic phase with 2.8 mL of a methanol / n-butanol mixture (2:1, v / v). Then, add 50 μL of ammonium thiocyanate solution and 50 μL of a ferrous ion solution (containing an equal volume mixture of 0.132 mol / L BaCl₂ and 0.144 mol / L FeSO₄) in sequence. Incubate the mixture in the dark for 2 minutes, then measure absorbance at 510 nm. Quantify the peroxide content in the sample using a hydrogen peroxide standard working curve.

[0070] 5. Determination of secondary oxidation products of DHPM-LA and TSE-LA emulsions 0.2 mL of each DHPM-LA and TSE-LA emulsion was diluted to 1.0 mL with distilled water, followed by the addition of 2.0 mL of TBA solution (15.0 g of trichloroacetic acid (TCA) and 0.375 g of 2-thiobarbituric acid (TBA) dissolved in 100.0 mL of 0.25 mol / L HCl). The samples were then boiled in boiling water for 20 minutes, cooled to room temperature, and centrifuged at 4000 rpm for 30 minutes. The supernatant was then measured for absorbance at 532 nm. The MDA content in the samples was calculated using a standard curve using 1,1,3,3-tetramethoxypropane.

[0071] The results of the lipid oxidation stability study of DHPM-LA and TSE-LA emulsions are as follows Figure 13 The oxidative stability of different emulsion systems was evaluated by measuring the changes in the content of primary oxidation products (lipid hydroperoxides, LH) and secondary oxidation products (malondialdehyde, MDA) during accelerated oxidation. Figure 13, during the 14-day storage period, the LH content of all samples increased significantly: the TSE-LA group increased from the initial 6.13 mmol / kg to 79.9 mmol / kg, and the DHPM-LA group increased from 7.13 mmol / kg to 83.99 mmol / kg. Moreover, the DHPM-LA emulsion showed the best oxidative stability. The lipid oxidation reaction in the emulsion system is mainly affected by the following factors: First, the large interface area between the dispersed phase and the continuous phase promotes the contact between the oxidant and the lipid; and the composition and structural characteristics of the interface layer have a decisive effect on the oxidation rate. In this study, the three-dimensional network structure and dense interface layer formed by the DHPM-LA emulsion played a dual protection mechanism: on the one hand, it restricted the diffusion of oxygen through physical barriers, and on the other hand, it interfered with the propagation of free radical chain reactions. As Figure 13 As shown in the figure, during the oxidation process, the MDA content of all samples showed an upward trend, and its change pattern was highly consistent with the LH content. This indicates that the primary oxidation products continue to decompose to produce aldehydes and ketones as secondary oxidation products, further confirming the cascade nature of the oxidation reaction.

[0072] 6. Determination of antioxidant activity of DHPM-LA and TSE-LA emulsions The antioxidant properties of the WPI-Z-SA composite system before and after physical field treatment were evaluated by DPPH and ABTS free radical scavenging methods, respectively. The sample solution was uniformly prepared at a concentration of 2.0 mg / mL. The sample supernatant was mixed with 0.1 mM DPPH solution at a ratio of 1:30 (v / v), incubated at 25°C in the dark for 15 minutes, and the absorbance was measured at a wavelength of 517 nm. Preparation of ABTS free radicals: 2.45 mmol / L potassium persulfate and 7.0 mmol / L ABTS solution were mixed in equal volumes and reacted at 25°C in the dark for 12-16 hours to generate ABTS free radical stock solution. During the determination, the sample was mixed with ABTS working solution at a ratio of 1:20 (v / v), reacted in the dark for 15 minutes, and the absorbance at 734 nm was measured. The free radical scavenging rate was calculated according to the following formula: The DPPH free radical scavenging ability of TSE-LA and DHPM-LA is as follows Figure 14As shown, both the TSE-LA and DHPM-LA emulsion systems exhibited significant DPPH radical scavenging activity (p < 0.05). The scavenging rate of the DHPM-LA emulsion reached 93.4%, approximately double that of the pure LA sample; the TSE-LA emulsion achieved a scavenging rate of 90.3%, also demonstrating significant antioxidant activity. This enhanced effect is likely due to the structural changes induced by high-pressure treatment in the WPI-Z-SA, which expose sulfhydryl groups and aromatic amino acid residues that effectively block free radical chain reactions. The significant enhancement of LA antioxidant activity by high-pressure-treated WPI-Z-SA is likely due to a pH-dependent reaction process. Reducing ketones, melanoidins, and volatile heterocyclic compounds generated during this process enhance antioxidant activity. This explains why the DHPM-LA emulsion exhibited the best antioxidant performance.

[0073] The scavenging ability of TSE-LA and DHPM-LA emulsions on ABTS cationic free radicals is as follows: Figure 14 As shown. The DHPM-LA and TSE-LA emulsion systems exhibited scavenging rates of 91.30% and 88.68%, respectively, both showing statistically significant differences compared to LA (p<0.05). The DHPM-LA group exhibited the best scavenging effect. This result confirms that stabilizing LA in an emulsion system significantly enhances its antioxidant properties, and dynamic high-pressure microfluidization (DHPM) treatment further strengthens this effect. Furthermore, this trend is highly consistent with the results of the DPPH free radical scavenging test, indicating that the conclusions drawn from the two evaluation methods are mutually validated.

[0074] Test Example 9 Determination of TSE-LA and DHPM-LA emulsion digestion experiments Digestive fluid was prepared and simulated in vitro digestion analysis was performed.

[0075] Artificial saliva: Prepare 15.1 mL of 0.5 mol / L potassium chloride solution, 3.7 mL of 0.5 mol / L potassium dihydrogen phosphate solution, 6.8 mL of 1 mol / L sodium chloride solution, 0.5 mL of 0.15 mol / L magnesium chloride hexahydrate solution, 0.06 mL of 0.5 mol / L ammonium carbonate solution, 0.09 mL of 6 mol / L hydrochloric acid solution, and 0.025 mL of 0.3 mol / L calcium chloride dihydrate solution. Mix thoroughly and adjust the pH to 7.0.

[0076] Artificial gastric fluid: Prepare 6.9 mL of 0.5 mol / L potassium chloride solution, 0.9 mL of 0.5 mol / L potassium dihydrogen phosphate solution, 12.5 mL of 1.0 mol / L sodium chloride solution, 11.8 mL of 2.0 mol / L sodium chloride solution, 0.4 mL of 0.15 mol / L magnesium chloride hexahydrate solution, 0.5 mL of 0.5 mol / L ammonium carbonate solution, 1.3 mL of 6.0 mol / L hydrochloric acid solution, and 0.005 mL of 0.3 mol / L calcium chloride dihydrate solution, mix the above solutions thoroughly and set aside.

[0077] Artificial intestinal fluid: Prepare 6.8 mL of 0.5 mol / L potassium chloride solution, 0.8 mL of 0.5 mol / L potassium dihydrogen phosphate solution, 42.5 mL of 1.0 mol / L sodium chloride solution, 9.6 mL of 2.0 mol / L sodium chloride solution, 1.1 mL of 0.15 mol / L magnesium chloride hexahydrate solution, 0.09 mL of 6.0 mol / L hydrochloric acid solution, and 0.04 mL of 0.3 mol / L calcium chloride dihydrate solution, mix the above solutions evenly and set aside.

[0078] 1. Determination of protein release To 8.0 mL of artificial saliva, add 0.1 g of sample, 0.025 mL of 0.3 mol / L calcium chloride dihydrate solution, and distilled water to 10.0 mL. Incubate in a 37°C water bath for 2 min. After oral digestion is complete, add 8.0 mL of prepared artificial gastric fluid, 0.005 mL of 0.3 mol / L calcium chloride dihydrate solution, and distilled water to 20.0 mL. Adjust the pH to 3.0, add 13.3 mg of pepsin, and incubate in a shaker at 37°C for 2 h. 2.0 mL of sample was collected at 0, 30, 60, 90, and 120 min. Immediately after the reaction, the sample was frozen in ice. Freeze-inactivation was performed on each sample during the digestion process. After gastric digestion is complete, add 8.0 mL of the artificial intestinal fluid prepared in 4.3.8.3 to the remaining 10.0 mL of gastric digestate, along with 0.02 mL of 0.3 mol / L calcium chloride dihydrate and 300.0 mg of bile salts. Add distilled water to 20.0 mL, adjust the pH to 7.0, and add 8.0 mg of trypsin. Incubate in a shaking water bath at 37°C for 2 h. During the reaction, take 2.0 mL samples at 0, 30, 60, 90, and 120 min. Immediately after the reaction, freeze-inactivate the samples by placing them in ice. Freeze-inactivate each sample during the digestion process. After the samples have returned to room temperature (25°C), add an equal volume of 15.0% trichloroacetic acid to each sample. Centrifuge at 10,000 rpm for 10 min, and determine the protein content of the supernatant. The in vitro digestibility is calculated using the following formula: Protein digestion (%) = protein content in supernatant ⁄ (sample weight × sample protein content) × 100 The combination of protein and small molecule organic matter will affect the microstructure of protein, and DHPM and TSE will also affect the internal group distribution of protein and complex system, thus affecting the digestibility of protein emulsion. In order to explore the applicability of TSE-LA and DHPM-LA emulsions as nutrient delivery systems, the present invention investigated their protein release characteristics in a simulated gastrointestinal environment. The results are as follows Figure 15 As shown in Figure 2, at 37°C, the two emulsion systems exhibited different release kinetics. Figure 15 As shown in Figure A, the release curves of the two samples are similar, both showing a slow release rate. This slow release behavior may be attributed to the acidic environment of the simulated gastric fluid causing the molecular chain of sodium alginate to break, thereby promoting the gradual release of WPI and Z into the medium. Figure 15 As shown in Figure B, the release rate is slower because WPI and Z are gradually released as the composite particles further swell and dissolve.

[0079] 2. Determination of linoleic acid release rate The pH of the sample after gastric digestion was adjusted to neutral (7.0). Then, 1.5 mL of 2.7 mg / mL calcium chloride solution, 3.5 mL of bile salt solution (117.8 mg / mL, prepared in 10.0 mmol / L PBS buffer, pH 7.0), and 2.5 mL of a mixed enzyme solution containing lipase and pancreatin (both at a concentration of 24.0 mg / mL) in 10.0 mmol / L PBS buffer, pH 7.0, were added sequentially. After the pH of the mixture was adjusted to 7.0, the mixture was shaken in a 37°C water bath at 150 rpm for 2 hours to simulate the digestive environment of the small intestine. Throughout the digestion process, a constant pH was maintained using 1.0 mol / L sodium hydroxide solution, and the sodium hydroxide consumption was recorded at 30, 60, 90, and 120 minutes. LA release from the emulsion samples was quantitatively assessed by measuring the amount of free fatty acids released from the reaction system. The amount of free fatty acids released was calculated according to the following formula:

[0080] Wherein, Moil is the average molecular weight of oil, CNaOH is the concentration of NaOH solution, VNaOH(t) is the volume of NaOH solution consumed at different digestion times, and Woil is the total mass of camellia oil in the digestive fluid.

[0081] Free fatty acid (FFA) release from different emulsions TSE-LA and DHPM-LA in simulated in vitro digestion Figure 16As shown in the figure, FFA release increases rapidly within the first 30 minutes of the intestinal stage. This is because lipase rapidly adsorbs to the droplet surface during the initial phase, breaking down triglycerides into fatty acids. The final FFA release rates of TSE-LA and DHPM-LA emulsions differ. The higher FFA release rate of TSE-LA compared to DHPM-LA is likely due to the increased steric hindrance and intermolecular repulsion caused by DHPM, which contributes to the emulsion's greater strength and viscoelasticity, inhibiting the migration of lipase to the oil droplets and, consequently, the release of fatty acids. Furthermore, proteins fill the gaps between the droplets, making it difficult for bile salts to adsorb to the interface and replace the emulsifier on the droplet surface, inhibiting lipase adsorption to the lipid droplet surface. This further inhibits lipid digestion and reduces FFA release. Moreover, although the WPI-stabilized emulsion has a smaller droplet size that can provide a larger specific surface area to increase the number of bile salt and lipase adsorption sites, and the weaker strength is conducive to the adsorption of bile salts and lipase, the actual WPI-stabilized emulsion does not maximize the release of FFA. This is because the emulsion is extremely susceptible to pepsin and acidic conditions during the gastric digestion stage, and the emulsion droplets aggregate to form large oil droplets, which reduces the contactable specific surface area and is not conducive to the action of lipase during the intestinal digestion stage.

[0082] Based on the above experimental results, the following conclusions are drawn: (1) The results of multispectral analysis show that DHPM treatment can effectively induce the decomposition of protein molecular conformation through its unique cavitation effect and microfluidic mechanism, promote the exposure of internal hydrophobic regions, and thus enhance the interaction between WPI, Z and SA. This process is directly manifested in the significant enhancement of the ultraviolet absorption characteristics of the composite system. Fourier transform infrared spectroscopy analysis shows that DHPM treatment strengthens the vibration modes of the carbonyl (C=O), CN bond and NH bond in the amide bond. At 250-850 kPa, the OH stretching vibration of the amide A band and the characteristic peak positions of the amide I and II regions remain relatively stable. Compared with TSE, DHPM shows a more significant effect in regulating the secondary structure of proteins, and can also effectively improve the solubility properties of the composite system.

[0083] (2) The results of thermodynamic analysis and particle size potential measurement show that the denaturation temperature (Td) of the WPI-Z-SA composite system shows a gradual downward trend with the increase of DHPM treatment pressure. This phenomenon reveals the significant effect of high-pressure treatment on protein structure: under the action of the continuously increasing mechanical force field, the three-dimensional conformation of the composite system gradually unfolds, the molecular arrangement tends to be disordered, and ultimately leads to the continuous weakening of its thermal stability. With the increase of pressure, the protein particle size and PDI gradually decrease. When the high pressure is 850 kPa, the particle size reaches 145.697 nm and the PDI reaches 0.45. When the TSE speed is 200 rpm, the particle size and PDI of the composite system are 150.100 nm and 0.45, respectively. The zeta potential of the protein without DHPM treatment was -30.190 mV. After DHPM treatment, the absolute value of the zeta potential increased. However, with increasing high pressure, the absolute value of the zeta potential decreased, reaching a minimum of -38.388 mV at DHPM 850 kPa. The absolute value of the zeta potential increased even more significantly with TSE, reaching a minimum of -49.150 mV at TSE 200 rpm. Based on these studies, we selected DHPM 850 kPa and TSE 200 rpm for subsequent experimental studies. Emulsions were prepared using different volume ratios of LA to aqueous solutions of the protein-based composite system treated with DHPM 850 kPa and TSE 200 rpm, respectively. Multiple photodetection revealed that the optimal ratio was 3:1. These emulsions were designated DHPM-LA and TSE-LA, respectively.

[0084] (3) The results of stability and antioxidant analysis showed that the TSE-LA and DHPM-LA emulsion systems showed significant differences. Multiple light scattering data showed that the backscattering spectrum curves of the TSE-LA sample within 24 hours were highly overlapping, indicating that it had good colloidal stability and no obvious particle aggregation; while the backscattering signal in the upper area of the DHPM-LA sample showed a gradual decrease, suggesting the presence of particle sedimentation behavior. Turbiscan stability index (TSI) quantitative analysis further confirmed that TSE-LA maintained a stable TSI value during storage, showing the best physical stability, while the TSI value of DHPM-LA increased significantly. Thermal stability tests showed that the two emulsions responded differently to heat treatment: DHPM-LA did not show phase separation, while TSE-LA showed obvious creaming behavior. Oxidative stability tests showed that after 14 days of storage, the lipid hydroperoxide (LH) content of both emulsions increased significantly, with DHPM-LA increasing from 7.13 mmol / kg to 83.99 mmol / kg and TSE-LA from 6.13 mmol / kg to 79.9 mmol / kg, demonstrating superior oxidative stability. Antioxidant activity assessment revealed that both emulsion systems significantly enhanced the free radical scavenging capacity of LA (p<0.05). Specifically, DHPM-LA achieved scavenging rates of 93.4% and 91.3% for DPPH and ABTS radicals, respectively, approximately double that of free LA. TSE-LA also demonstrated significant antioxidant activity, with scavenging rates of 90.3% and 88.68%, respectively. DHPM-LA demonstrated the best overall performance across all indicators.

[0085] (4) In vitro digestion results showed that there were significant differences in FFA release rate and bioaccessibility between DHPM-LA and TSE-LA. DHPM-LA showed a lower FFA release rate, which may be due to the fact that high pressure treatment improved the mechanical strength and viscoelasticity of the emulsion.

[0086] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A method for preparing a protein-based composite system for embedding linoleic acid based on a physical field, characterized in that: A ternary composite system was obtained by using a pH-driven method with water-soluble protein, zein and sodium alginate, wherein the mass ratio of water-soluble protein, zein and sodium alginate was 9:1:1; the ternary composite system was placed in a DHPM device or a TSE device for treatment, wherein the pressure parameters of the DHPM device were set to 250-850 kPa and the temperature parameters were 35°C; the pressure parameters of the TSE device were set to 120-200 rpm and the temperature parameters were 35°C; the ternary composite system after DHPM or TSE treatment was freeze-dried, and the freeze-dried powder was taken and added to deionized water, and stirred using a high-pressure homogenizer to obtain a protein-based composite system aqueous solution; linoleic acid and the protein-based composite system aqueous solution were prepared into an emulsion in a volume ratio of 2:1-3:1, i.e., a protein-based composite system encapsulating linoleic acid.

2. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The pH-driven method is used to prepare the ternary composite system, which includes the following steps: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution and stirred for 2 h under a stirrer. Zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min under a stirrer. Sodium alginate was added to the water-soluble protein-zein composite solution at a mass ratio of 1:1, and stirred for 30 min under a stirrer. The composite solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex. The parameters of the stirrer were set as follows: stirring speed of 800 r / min and ambient temperature of 25±2°C.

3. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into the DHPM equipment for treatment, with the pressure parameters of the DHPM equipment set to 250~850kPa and the temperature parameter to 35°C; S5. Freeze-dry the ternary composite system after DHPM treatment, take the freeze-dried powder, add it to deionized water, and stir it with a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; prepare an emulsion with linoleic acid and the aqueous solution of the protein-based composite system in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

4. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into a TSE device for treatment, wherein the pressure parameter of the TSE device is set to 120-200 rpm and the temperature parameter is set to 35°C; S5. The ternary composite system after TSE treatment is freeze-dried, and the freeze-dried powder is taken, added to deionized water, and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; linoleic acid and the aqueous solution of the protein-based composite system are prepared into an emulsion in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

5. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into the DHPM equipment for treatment, with the pressure parameters of the DHPM equipment set to 850 kPa and the temperature parameters to 35°C; S5. Freeze-dry the ternary composite system after DHPM treatment, take the freeze-dried powder, add it to deionized water, and stir it with a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; prepare an emulsion with linoleic acid and the aqueous solution of the protein-based composite system in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

6. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into a TSE device for treatment, with the pressure parameter of the TSE device set to 160 rpm and the temperature parameter to 35°C; S5. The ternary composite system after TSE treatment is freeze-dried, and the freeze-dried powder is taken, added to deionized water, and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; linoleic acid and the aqueous solution of the protein-based composite system are prepared into an emulsion in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

7. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into a TSE device for treatment, with the pressure parameter of the TSE device set to 180 rpm and the temperature parameter to 35°C; S5. The ternary composite system after TSE treatment is freeze-dried, and the freeze-dried powder is taken, added to deionized water, and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; linoleic acid and the aqueous solution of the protein-based composite system are prepared into an emulsion in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

8. The method for preparing a protein-based composite system based on physical field embedding of linoleic acid according to claim 1, characterized in that: The following steps are involved: S1: Using deionized water as the solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9 and incubate overnight to obtain a water-soluble protein solution; S2: Prepare 2 M NaOH solution and 1 M HCl solution using deionized water as solvent; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, and zein was added to the water-soluble protein solution at a mass ratio of 9:1, and stirred for 30 min. Sodium alginate was added to a water-soluble protein-zein complex solution in a mass ratio of zein to sodium alginate of 1:1, and the mixture was stirred for 30 minutes under a stirrer. The complex solution was adjusted to 7 with a 1M HCl solution to obtain a ternary functional protein complex. The stirrer parameters were set at a stirring speed of 800 r / min and an ambient temperature of 25±2°C. S4, placing the ternary composite system into a TSE device for treatment, wherein the pressure parameter of the TSE device is set to 200 rpm and the temperature parameter is set to 35°C; S5. The ternary composite system after TSE treatment is freeze-dried, and the freeze-dried powder is taken, added to deionized water, and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system; linoleic acid and the aqueous solution of the protein-based composite system are prepared into an emulsion in a volume ratio of 3:1, i.e., a protein-based composite system encapsulating linoleic acid.

9. A protein-based composite system based on physical field embedding of linoleic acid, characterized in that: The method according to any one of claims 1 to 8 is used for preparation.

10. Use of the protein-based composite system based on physical field embedding of linoleic acid according to claim 9 in the preparation of edible nutritional factor delivery products.

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