Protein-based composite system embedding linoleic acid based on physical field and preparation method and application thereof

Abstract

The application discloses a protein-based composite system embedding linoleic acid based on a physical field and a preparation method and application thereof, and relates to the field of high polymer compound compositions. The application modifies the protein composite through DHPM or TSE, does not use any organic solvent, and does not need heating, and is a new method for obtaining green and safe functional protein composite while being environmentally friendly and efficient. Meanwhile, the high-pressure homogenization emulsion technology is adopted to prepare the linoleic acid-loaded emulsion, the stability of the linoleic acid is increased, the bioavailability of the linoleic acid is improved, and the intestinal controllable slow release of the linoleic acid is realized. The prepared protein-based composite system embedding linoleic acid based on a physical field has excellent solubility, thermal stability and antioxidant property, can resist the temperature of the gastrointestinal tract and be suitable for the absorption of the gastrointestinal tract when people eat, and can be applied to edible nutrition factor delivery products.

Description

Technical Field

[0001] This invention relates to the field of polymer compound compositions, and more particularly to protein-based composite systems based on physical field-encapsulated linoleic acid, their preparation methods, and applications. Background Technology

[0002] Elucidating the complex interactions between food, gut microbiota, and health outcomes is crucial for developing functional foods (such as prebiotics, probiotics, and genetically modified preparations) 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 utilize the surface-active properties of proteins (such as whey protein and zein) and polysaccharides (such as sodium alginate) to achieve stable loading of functional factors, and can also achieve targeted delivery by regulating intermolecular forces (hydrophobic interactions, hydrogen bonds, etc.). This natural biocompatible carrier offers the possibility of developing safe and efficient novel nutritional intervention strategies.

[0003] Water-soluble proteins (whey proteins, abbreviated WPIs) have advantages such as high nutritional value, easy availability, and excellent technical and functional properties. Based on these advantages, nutrient delivery systems constructed with water-soluble proteins (whey proteins) as the main component have been extensively studied to deliver functional components, such as astaxanthin, 3,3'-diindolemethane, vitamin D3, resveratrol, and beta-carotene.

[0004] Zein (Z), the main storage protein in corn kernels, has poor water solubility due to its high percentage (over 50%) of hydrophobic amino acid residues, such as proline, leucine, and alanine. It is soluble in 60-90% aqueous ethanol solutions. The unique dissolution properties of zein can be used to prepare nanoparticles for encapsulating nonpolar bioactive molecules. In recent years, research on the interaction between proteins and polyphenols and flavonoids has become a hot topic, with non-covalent interactions being a key focus in the food industry. Most non-covalent interactions are caused by hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces, and are usually reversible. These interactions can cause protein aggregation or unfolding, leading to changes in their 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 linked 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 recognized as a GRAS (Generally Recognized As Safe) food material.

[0006] As people's demands for nutrition and health continue to increase, food, in addition to providing energy, must also possess functional properties to provide nutritional value. However, most food functional ingredients suffer from drawbacks such as poor solubility, poor stability, and low bioavailability. Therefore, an effective delivery system is needed to transport functional ingredients. Linoleic acid (LA) is an important long-chain fatty acid and a precursor to fatty acids such as gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid. These fatty acids have anti-inflammatory properties, lower cholesterol, and help prevent atherosclerosis. Due to its high reactivity, linoleic acid is prone to oxidation and hydrogenation reactions, requiring careful storage.

[0007] Currently, most methods for preparing protein complexes rely on organic solvents, which limits their application. Some organic solvents are toxic and irritating and unsuitable for human use. pH-transfer methods, sometimes called pH-driven methods, have recently been used to incorporate functional factors into various types of edible nanoparticles without the use of organic solvents. These methods utilize the fact that the solubility of functional factors in aqueous solutions is strongly dependent on pH. However, existing techniques for modifying proteins using pH-driven methods typically require heating, and excessively high temperatures can lead to protein denaturation and inactivation.

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

[0009] The purpose of this invention is to propose a protein-based complex system based on physical field encapsulation of linoleic acid, its preparation method and application. It does not use any organic solvents and does not require heating. It is a new method to obtain green, safe and functional protein complexes that is both environmentally friendly and efficient. The protein-based complex system prepared also exhibits excellent thermal stability and function and can be applied to edible nutrient delivery products.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows:

[0011] In the first aspect, the present invention proposes a method for preparing a protein-based composite system based on physical field-encapsulated linoleic acid. A ternary composite system is obtained by using a pH-driven method with water-soluble protein, zein, and sodium alginate, in a mass ratio of 9:1:1. The ternary composite system is then processed in a dynamic high-pressure microfluidic (DHPM) device or a micro twin-screw extruder (TSE) device. The pressure parameters of the DHPM device are set to 250–850 kPa and the temperature parameter to 35°C. The rotation speed parameters of the TSE device are set to 120–200 rpm and the temperature parameter to 35°C. The ternary composite system after DHPM or TSE treatment is freeze-dried, and the freeze-dried powder is 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 then mixed in a volume ratio of 2:1 to 3:1 to prepare an emulsion, i.e., a protein-based composite system encapsulated with linoleic acid.

[0012] Preferably, the preparation method of the above-mentioned protein-based complex system based on physical field-encapsulated linoleic acid includes the following steps:

[0013] S1: Using deionized water as solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9, and let it stand overnight to obtain a water-soluble protein solution.

[0014] S2: Using deionized water as a solvent, prepare 2M NaOH solution and 1M HCl solution;

[0015] S3: Adjust the pH of the overnight water-soluble protein solution to 12 using 2M NaOH solution, stir for 2 hours, add zein to the water-soluble protein solution at a mass ratio of 9:1 (water-soluble protein to zein), and stir for 30 minutes; add sodium alginate to the water-soluble protein-zein composite solution at a mass ratio of 1:1 (zein to sodium alginate), and stir for 30 minutes; adjust the composite solution to pH 7 using 1M HCl solution to obtain a ternary functional protein complex; the stirrer parameters are set as follows: stirring speed 800 r / min, ambient temperature 25±2℃.

[0016] S4. Place the ternary composite system into the DHPM equipment for processing. The pressure parameters of the DHPM equipment are set to 250-850 kPa and the temperature parameters are 35℃.

[0017] 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 a protein-based composite system aqueous solution; prepare an emulsion by mixing linoleic acid and the protein-based composite system aqueous solution in a volume ratio of 3:1, that is, a protein-based composite system encapsulating linoleic acid.

[0018] Preferably, the preparation method of the above-mentioned protein-based complex system based on physical field-encapsulated linoleic acid includes the following steps:

[0019] S1: Using deionized water as solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10:9, and let it stand overnight to obtain a water-soluble protein solution.

[0020] S2: Using deionized water as a solvent, prepare 2M NaOH solution and 1M HCl solution;

[0021] S3: Adjust the pH of the overnight water-soluble protein solution to 12 using 2M NaOH solution, stir for 2 hours, add zein to the water-soluble protein solution at a mass ratio of 9:1 (water-soluble protein to zein), and stir for 30 minutes; add sodium alginate to the water-soluble protein-zein composite solution at a mass ratio of 1:1 (zein to sodium alginate), and stir for 30 minutes; adjust the composite solution to pH 7 using 1M HCl solution to obtain a ternary functional protein complex; the stirrer parameters are set as follows: stirring speed 800 r / min, ambient temperature 25±2℃.

[0022] S4. Place the ternary composite system into the TSE equipment for processing. Set the rotation speed parameters of the TSE equipment to 120-200 rpm and the temperature parameters to 35℃.

[0023] S5. Freeze-dry the ternary composite system after TSE treatment, take the freeze-dried powder, add it to deionized water, and stir it with a high-pressure homogenizer to obtain a protein-based composite system aqueous solution; prepare an emulsion by mixing linoleic acid and the protein-based composite system aqueous solution in a volume ratio of 3:1, that is, a protein-based composite system encapsulating linoleic acid.

[0024] Secondly, the present invention also proposes a protein-based composite system based on physical field encapsulation of linoleic acid, which is prepared by any of the methods described above.

[0025] Thirdly, the present invention also proposes the application of the above-mentioned protein-based composite system based on physical field-encapsulated linoleic acid in the preparation of edible nutrient delivery products.

[0026] Compared with the prior art, the technical effects of the present invention are as follows:

[0027] This invention modifies protein complexes using dynamic high-pressure microfluidic (DHPM) and micro-twin-screw extrusion (TSE), without using any organic solvents or requiring heating. This represents a novel, environmentally friendly, and highly efficient method for obtaining green, safe, and functional protein complexes. Simultaneously, high-pressure homogenized emulsion technology is employed to prepare linoleic acid (LA)-loaded emulsions, increasing LA stability, improving its bioavailability, and achieving controlled, sustained release of LA into the gut. The resulting protein-based complex system, based on linoleic acid encapsulated in a physical field, exhibits excellent solubility, thermal stability, and antioxidant properties. When consumed, it can withstand gastrointestinal temperatures and is suitable for gastrointestinal absorption, making it applicable to edible nutrient delivery products. Compared to untreated protein complexes, the physically field-treated complexes demonstrate superior heat resistance and particle size characteristics. Furthermore, this invention also studied the effects of DHPM and TSE on multispectral properties, thermal stability, particle size potential, and antioxidant properties before and after treatment, respectively, and explored the driving force of different physical fields on the formation of the composite system. Moreover, it was also able to analyze the binding mechanism of different physical fields on the composite system through surface hydrophobicity and free thiol groups, further ensuring its application in the fields of functional foods and medicines. Attached Figure Description

[0028] Figure 1 The ultraviolet absorption spectrum of the protein complex after physical field treatment provided in Test Example 1 of the present invention.

[0029] Figure 2 The Fourier transform infrared spectrum of the protein complex after physical field treatment provided in Test Example 2 of the present invention.

[0030] Figure 3 The image shows a DSC diagram of the protein complex after physical field treatment, as provided in Test Example 3 of this invention.

[0031] Figure 4 The particle size potential diagram of the protein complex after physical field treatment provided in Test Example 4 of the present invention.

[0032] Figure 5 The circular dichroism chromatogram of the protein complex after physical field treatment provided in Test Example 5 of the present invention.

[0033] Figure 6 The encapsulation efficiency and loading rate diagrams for DHPM-LA and TSE-LA provided in Test Example 6 of this invention are shown.

[0034] Figure 7 The image is a scanning electron microscope (SEM) image of the protein complex emulsion after physical field treatment provided in Test Example 7 of this invention.

[0035] Figures 8A-8H For Test Example 8 of the present invention, LA was respectively compared with DHPM850 and

[0036] Results of backscattered light intensity changes over 24 hours for emulsions prepared by TSE200 at different volume ratios. 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 to LA is 1:1. Figure 8F The ratio of TSE200 to 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.

[0037] Figure 9 The Turbiscan stability index (TSI) of DHPM-LA and TSE-LA provided in Test Example 8 of this invention.

[0038] Figure 10 The thermal stability results of DHPM-LA and TSE-LA provided in Test Example 8 of this invention are shown.

[0039] Figure 11 The freeze-thaw stability results of DHPM-LA and TSE-LA provided in Test Example 8 of this invention are shown.

[0040] Figure 12 The centrifugation stability results of DHPM-LA and TSE-LA provided in Test Example 8 of this invention are shown.

[0041] Figure 13 The results show the lipid oxidation stability of DHPM-LA and TSE-LA provided in Test Example 8 of this invention.

[0042] Figure 14 The results show the stability of the free radical scavenging ability of DHPM-LA and TSE-LA provided in Test Example 8 of this invention.

[0043] Figure 15 The results show the in vitro digestion-simulated protein release characteristics of DHPM-LA and TSE-LA provided in Test Example 9 of this invention.

[0044] Figure 16 The results show the in vitro digestion simulation of free fatty acid release characteristics of DHPM-LA and TSE-LA provided in Test Example 9 of this invention. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0047] Unless otherwise specified, all 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.

[0048] The present invention proposes a method for preparing a protein-based complex system encapsulated with linoleic acid. A ternary complex system (WPI-Z-SA) is obtained by using a pH-driven method with a water-soluble protein (WPI) solution, zein (Z), and sodium alginate (SA). The mass ratio of zein, zein, and sodium alginate is 9:1:1. The ternary complex system is placed in a DHPM apparatus, with pressure parameters set to 0, 250, 400, 550, 700, and 850 kPa, and a temperature parameter of 35°C, yielding the following products:

[0049] Samples DHPM0, DHPM250, DHPM400, DHPM550, DHPM700, and DHPM850 were prepared. The ternary composite system was placed in a TSE apparatus with rotational speeds set to 0, 120, 140, 160, 180, and 200 rpm, and a temperature of 35℃, resulting in samples named TSE0, TSE120, TSE140, TSE160, TSE180, and TSE200, respectively. The WPI-Z-SA ternary composite system treated at DHPM 850 kPa and TSE 200 rpm was freeze-dried. 2.0 g of the freeze-dried powder was added to 100.0 mL of deionized water and homogenized at 1200 rpm for 2 min using a high-pressure homogenizer. Linoleic acid (LA) and the protein-based composite system aqueous solution were prepared into an emulsion at a volume ratio of 1:2 to 3:1, thus creating a protein-based composite system encapsulating linoleic acid.

[0050] Example 1

[0051] 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 stirred rapidly for 30 min. 0.005 g of sodium alginate was added to the water-soluble protein-zein composite solution and stirred rapidly for 30 min. The pH was adjusted to 7 with 1 M HCl to obtain the water-soluble protein-zein-sodium alginate ternary composite system, denoted as WPI-Z-SA. The stirrer parameters were: stirring speed 800 r / min, ambient temperature 25 ± 2 °C.

[0052] The ternary composite system WPI-Z-SA was placed in the DHPM apparatus, with the pressure parameter set to 250 kPa and the temperature parameter set to 35℃, resulting in a sample named DHPM250. The ternary composite system WPI-Z-SA was placed in the TSE apparatus, with the rotation speed parameter set to 120 rpm and the temperature parameter set to 35℃, resulting in a sample named TSE120.

[0053] Example 2

[0054] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in the DHPM device, with the pressure parameter set to 400 kPa and the temperature parameter set to 35 °C, to obtain a sample named DHPM400.

[0055] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE device with the rotation speed set at 140 rpm and the temperature set at 35°C, resulting in a sample named TSE140.

[0056] Example 3

[0057] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in the DHPM device, with the pressure parameter set to 550 kPa and the temperature parameter set to 35 °C, to obtain a sample named DHPM550.

[0058] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE device with the rotation speed set at 160 rpm and the temperature set at 35°C, resulting in a sample named TSE160.

[0059] Example 4

[0060] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in the DHPM device, with the pressure parameter set to 700 kPa and the temperature parameter set to 35 °C, to obtain a sample named DHPM700.

[0061] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE device with the rotation speed set at 180 rpm and the temperature set at 35°C, resulting in a sample named TSE180.

[0062] Example 5

[0063] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in the DHPM device, with the pressure parameter set to 850 kPa and the temperature parameter set to 35 °C, to obtain a sample named DHPM850.

[0064] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in the TSE device, with the rotation speed set to 200 rpm and the temperature set to 35°C, resulting in samples named TSE200.

[0065] Example 6

[0066] The ternary composite systems WPI-Z-SA (denoted as DHPM850 and TSE200) treated with DHPM at 850 kPa and TSE at 200 rpm were freeze-dried separately. 2.0 g of each freeze-dried powder was added to 100.0 mL of deionized water and homogenized at 1200 rpm for 2 min using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system. Emulsions were prepared by mixing LA with DHPM850 and TSE200 at volume ratios of 1:2, 1:1, 2:1, and 3:1, respectively. Multiplex optical emission testing determined that the 3:1 ratio was optimal, and these were named DHPM-LA and TSE-LA, respectively.

[0067] Comparative Example 1

[0068] The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a DHPM apparatus, with the pressure parameter set to 0 kPa and the temperature parameter set to 35°C, resulting in a sample named DHPM0. The ternary composite system WPI-Z-SA prepared in Example 1 was placed in a TSE apparatus, with the rotation speed parameter set to 0 rpm and the temperature parameter set to 35°C, resulting in a sample named TSE0.

[0069] Test Example 1

[0070] Ultraviolet absorption spectroscopy: The lyophilized solids of the protein complex systems prepared in Examples 1-5 and Comparative Example 1 were ground into fine powder and dissolved in deionized water to prepare a complex system solution with a concentration of 0.2 mg / mL. The sample solution was transferred to a quartz cuvette, and a UV-Vis spectrophotometer was used to perform a full-wavelength scan in the 200-400 nm ultraviolet region to record the light absorption characteristics of the sample. Baseline correction was performed using deionized water before scanning.

[0071] The UV spectral changes of the WPI-Z-SA composite system before and after treatment with different pressure parameters of DHPM and TSE are as follows: Figure 1 As shown. From Figure 1 Both physical treatments significantly enhanced the absorbance of the system, with TSE reaching its maximum absorbance at 200 rpm and DHPM at 850 kPa. WPI, Z, and SA mainly form a non-covalent complex system through hydrogen bonding and hydrophobic interactions. The introduction of SA induced conformational changes in the protein, promoting molecular structure decomposition, and the strength of intermolecular interactions exceeded the effect of the physical field treatment. Comparing the two treatment methods, the increase in UV absorbance after DHPM treatment was more significant. This phenomenon may be due to the fact that the cavitation effect and microfluidic mechanism of DHPM can more effectively promote the unfolding of protein molecules, exposing internal hydrophobic regions, thereby enhancing the binding interaction between WPI, Z, and SA, ultimately resulting in a significant increase in the UV absorption characteristics of the complex system.

[0072] Test Example 2

[0073] Fourier transform infrared spectroscopy determination: The protein complexes prepared in Examples 1-5 and Comparative Example 1 were freeze-dried and then ground into fine powder. The sample powders were mixed with KBr at a ratio of 1:100, and then ground together with KBr in a mortar and pestle into even finer powders until homogeneous. After pressing into a film for 1 minute, the film was placed in the instrument for measurement. The scanning range was 4000–400 cm⁻¹. -1 Its resolution is 4cm -1 The infrared spectra were scanned 32 times. Potassium bromide pellets were used as a blank control during the measurements, and the obtained infrared spectra were analyzed using Origin software.

[0074] The infrared spectra of the WPI-Z-SA composite system under DHPM treatment at 250-850 kPa are as follows: Figure 2 As shown. After high-pressure treatment, the characteristic absorption peaks of the composite system shifted significantly, with the OH stretching vibration peak shifting from 3395 cm⁻¹. -1 Shift to lower wavenumbers to 3384cm -1 This indicates that high-pressure treatment promotes the formation of intermolecular hydrogen bonds. Meanwhile, the amide I band (1645 cm⁻¹) -1 ) and amide II band (1543cm) -1 The absorption peak position of ) is relative to that of the untreated sample (1635 cm⁻¹). -1 and 1540cm -1A significant shift occurred, indicating that high-pressure microfluidic treatment enhanced the vibrational intensity of C=O, CN, and NH bonds in the peptide bonds. Although the treatment pressure gradually increased, the OH stretching vibration of amide A and the characteristic peak positions of amide I and II regions remained relatively stable, without significant shift. TSE treatment at 120-200 rpm...

[0075] Compared to DHPM, TSE has a smaller impact on the stretching vibrations of functional groups in the infrared spectral changes of the WPI-Z-SA composite system.

[0076] Test Example 3

[0077] Determination of thermal stability: The protein complexes prepared in Examples 1-5 and Comparative Example 1 were freeze-dried and then ground into fine powder. 3-5 mg of the freeze-dried powder was placed in an aluminum crucible, capped, and the crucible containing the sample and an empty crucible were placed inside the instrument. The empty crucible served as a control and remained in the instrument until the measurement was completed. The test conditions were: initial temperature 20℃, heating rate 10℃ / min, reaction termination temperature 200℃, and nitrogen flow rate 50 mL / min. After the test, the thermal properties of the sample were analyzed using analysis software compatible with the instrument.

[0078] The DSC curve results of the WPI-Z-SA composite system after DHPM and TSE treatment are as follows: Figure 3 As shown, the composite systems treated with DHPM and TSE all exhibited lower thermal stability than the untreated composite system. This indicates that under the influence of DHPM and TSE, the composite system expands, becomes more loose and disordered, and its thermal stability decreases. However, the WPI-Z-SA composite system shows a significantly lower densities (T0). d As the pressure continued to increase, the thermal stability of the WPI-Z-SA composite system gradually increased. When treated with 850 kPa DHPM and 160 rpm TSE, the denaturation temperature of the WPI-Z-SA composite system rose to 119℃ and 121℃, respectively. This process describes the different changes observed in the TSE-treated samples; with increasing mechanical shear force, the TSE of the composite system... d The trend shows a decrease. This phenomenon indicates that under high shear strength conditions, the intermolecular forces of Zein molecules are disrupted, leading to the unfolding of its tertiary structure and ultimately resulting in a decrease in the thermal stability of the composite system.

[0079] Test Example 4

[0080] Particle size potential determination: The prepared sample was diluted to 1 mg / mL with ultrapure water, and then...

[0081] The zeta potential and particle size of ternary composite nanoparticles treated with different pressure parameters under DHPM and TSE conditions were determined 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 taken.

[0082] The results are as follows Figure 4 As shown, the average particle size of the WPI-Z-SA composite system without DHPM treatment was 389.66 nm, and the PDI was 0.23. After DHPM treatment, both the protein particle size and PDI showed a significant decreasing trend. At a DHPM pressure of 250 kPa, the composite system's particle size and PDI were 217.116 nm and 0.62, respectively. With increasing pressure, both the protein particle size and PDI gradually decreased, reaching 145.697 nm and 0.45 at a high pressure of 850 kPa. Figure 4 middle

[0083] A). Compared with DHPM-treated WPI-Z-SA, the WPI-Z-SA composite system treated with TSE has a larger particle size and PDI. During the TSE pressure enhancement process, the particle size and PDI of the composite system decrease accordingly. When the TSE rotation speed is 200 rpm, the particle size and PDI of the composite system are 150.100 nm and 0.45 nm, respectively. 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 break down large protein molecules into smaller particles, resulting in a more uniform protein dispersion in the system.

[0084] The zeta potential of a protein is another important indicator for characterizing the stability of a dispersion system. Figure 4 Figure C shows the Zeta potential of proteins before and after DHPM. It can be seen that the Zeta potential of all samples is negative, indicating that the protein surface is dominated by negatively charged amino acids. The Zeta potential of the untreated protein is -30.190 mV. The absolute value of the Zeta potential of the protein after DHPM treatment increases, and with increasing high voltage, the absolute value of the Zeta potential tends to decrease, reaching a minimum of -38.388 mV at DHPM 850 kPa. This indicates that the cavitation effect generated during high voltage causes the protein structure to unfold, exposing more negatively charged amino acid groups and increasing the charge on the molecular surface. The trend of increasing absolute value of the Zeta potential is even more pronounced in TSE.

[0085] The minimum Zeta potential at TSE 200 rpm is -49.150 mV (e.g. Figure 4(D). This indicates that the protein structure has become looser and there are more negatively charged amino acids on the surface, which is consistent with the results of electron microscopy and particle size analysis.

[0086] Test Example 5

[0087] Circular dichroism (CBD) determination: Using a circular dichroism chromatograph equipped with a 0.1 cm path length quartz cuvette, and with deionized water as the reference solution, the far-ultraviolet CBD signal of the WPI-Z-SA composite system before and after DHPM and TSE treatment was measured in the wavelength range of 190-260 nm. The sample concentration was maintained at 0.2 mg / mL during the test, and nitrogen gas was continuously purged to eliminate air interference. The CBD signal was expressed using the following formula based on residue ellipticity (MRE) deg·cm. 2 ·d·mol -1 CD results:

[0088] MRE 208 =observed CD(millidegree) / C p nl×10

[0089] In the formula: Cp represents the molar concentration of the 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 shown in the following formula:

[0090] α-helix(%)=-[MRE 208 -4000] / 33000-4000×100

[0091] Specific analysis results are as follows: Figure 5As shown, with increasing treatment intensity, the characteristic peak at 208 nm of the complex system showed a positive shift. The α-helix content of the untreated complex system was 6%, which increased to 9-10% after DHPM treatment and reached 9-12% after TSE treatment. This result confirms that physical field treatment promotes the transformation of protein conformation from a compact to a loose state. This indicates that although SA can partially maintain the helical structure, it cannot completely inhibit the conformational changes caused by pressure. Further analysis revealed that DHPM treatment led to an increase in α-helix and β-sheet content and a decrease in β-turn, indicating that the treatment reduced protein disorder and promoted unfolding. The reduction in β-sheet enhanced molecular flexibility, with the weakening of hydrogen bonding being the main factor causing changes in secondary structure. This process describes that within the pressure range (250-850 kPa), the secondary structure content did not show a significant pressure-dependent change. Comparing the two treatment methods, the α-helix retention rate of the 200 rpm TSE treatment group was slightly higher than that of the DHPM treatment group, indicating that the degree of protein folding caused by mechanical shearing was lower. This may be because twin-screw extrusion promoted the partial dissociation of protein aggregates. In summary, DHPM treatment showed a more significant effect in regulating protein secondary structure and also effectively improved the solubility of the complex system. These structural changes may be due to stress-induced conformational rearrangement and exposure of specific protein groups, thereby enhancing their interaction with other molecules.

[0092] Test Example 6

[0093] Determination of encapsulation efficiency and loading rate of DHPM-LA and TSE-LA emulsions: DHPM-LA and

[0094] The encapsulation efficiency (EE) and loading rate (LC) of linoleic acid in TSE-LA were determined by the following method. LA-loaded emulsions DHPM-LA and TSE-LA were centrifuged at 10,000 rpm for 10 min to remove the supernatant cream layer. After repeating the above 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. The standard curve of LA was determined on a spectrophotometer (y = 0.0728x + 0.0161; R0). 2 =0.9995, where 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:

[0095] EE (%) = M1 - M0 / M1 × 100%

[0096] LC (%) = M1 - M0 / M2 × 100%

[0097] 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.

[0098] The embedding and loading rate results for DHPM-LA and TSE-LA are as follows: Figure 6 As shown, DHPM-LA has an embedding rate of 23.4% and a loading rate of 72.8%; TSE-LA has an embedding rate of 30.2% and a loading rate of 68.9%. This may be because TSE-LA is produced by extrusion, resulting in larger internal voids and a greater number of LAs filling the voids.

[0099] Test Example 7

[0100] Scanning electron microscopy determination: The protein complex samples DHPM-LA and TSE-LA, which have undergone physical field treatment, and the protein complex sample WPI-Z-SA, which have not undergone physical field treatment, were placed in a freeze dryer to obtain a solid protein complex system. The solid was then ground into a fine powder, sputtered with gold, and observed under a scanning electron microscope to determine its microstructure.

[0101] Scanning electron micrographs of DHPM-LA and TSE-LA emulsions in different states are shown below. Figure 7 As shown. TSE-LA has a rough, sheet-like surface. Compared to TSE-LA, DHPM-LA emulsions are more tightly bound together, with a smooth structure. DHPM-LA exhibits a more uniform particle size and a denser structure, due to the tight aggregation of the three substances in DHPM. Both exhibit a tight structure, attributed to the weakening of electrostatic forces in the insoluble protein complex system under strong pH, resulting in reduced aggregation and nucleation. The structure opens up and gradually forms larger oligomers, leading to a soluble complex system. Furthermore, as... Figure 7 As shown, the scanning electron microscope images of the DHPM-LA and TSE-LA emulsions after and without lyophilization can be seen. No significant difference was found in the structure of the two, indicating that lyophilization does not affect the structure of the emulsion.

[0102] Test Example 8

[0103] 1. Determination of storage stability of DHPM-LA and TSE-LA

[0104] Emulsions were prepared by mixing LA with aqueous solutions of protein-based complex systems 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 emulsions was quantitatively assessed by analyzing the changes in backscattered light intensity over 24 hours; greater overlap in the spectral curves indicated better stability. 20.0 mL of each emulsion was then placed in…

[0105] In the scanning cell of the Turbiscan Lab multiple light scattering instrument, multiple scans were performed at a temperature of (25.0±0.5)℃, with scans occurring every 30 minutes for a total of 24 hours. The stability changes of the emulsion sample within 24 hours of standing at room temperature were obtained by analyzing the changes in the unstable kinetic curves.

[0106] The results are as follows Figures 8A-8H As shown, the spectral curves of protein-based composite aqueous solutions treated with LA at DHPM 850 kPa or TSE 200 rpm at volume ratios of 2:1, 1:1, and 1:2 differed significantly, indicating varying degrees of stability. In contrast, the spectral curves of the TSE-LA3:1 sample almost completely overlapped, indicating uniform particle dispersion and no significant aggregation or phase separation during the test, demonstrating excellent stability. The backscattered light signal at the top of the DHPM-LA3:1 sample showed a gradual decrease, suggesting particle sedimentation and relatively poor stability.

[0107] Further, using the Turbiscan stability index (TSI) as... Figure 9 Quantitative comparisons were conducted, and this index integrates the changes in transmitted and backscattered light signals; a higher value indicates greater instability of the system. 24-hour monitoring data showed that the TSI value of the TSE-LA sample remained stable, confirming its optimal storage stability; while the TSI value of the DHPM-LA sample increased significantly, indicating its worst stability.

[0108] 2. Determination of thermal stability and freeze-thaw stability of DHPM-LA and TSE-LA

[0109] The emulsions DHPM-LA and TSE-LA were placed in glass bottles and heated at 90°C for 1 hour. Their thermal stability was assessed by observing oil emergence and stratification on the emulsion surface. The emulsions were then frozen at -20°C for 24 hours and thawed in a 30°C water bath for 5 hours. The state of the emulsions after freeze-thaw was photographed and recorded. Their freeze-thaw stability was assessed by observing phase separation and oil separation.

[0110] The thermal stability of DHPM-LA and TSE-LA emulsions is as follows: Figure 10 As shown. No significant oil separation was observed in either DHPM-LA or TSE-LA emulsions. Overall, the protein complex-stabilized emulsions exhibited good thermal stability. SA in the complex acted as a steric hindrance, preventing droplets from approaching each other during heating and thus preventing their thermally induced aggregation. Heating had different effects on DHPM-LA and TSE-LA emulsions; the DHPM-LA-stabilized emulsion did not show significant oil separation, while the TSE-LA-stabilized emulsion did show oil separation after heating. Figure 10It can be seen that the main adsorbed component at the oil-water interface of this emulsion is WPI-Z-SA. At high temperatures, protein molecules denature and unfold, exposing more hidden hydrophobic groups on the surface. This increases hydrophobic interactions between protein molecules, leading to protein aggregation and thus reducing the emulsion's thermal stability. Furthermore, the TSE-LA network structure is inferior to DHPM-LA, primarily because the weaker network structure and thinner interfacial layer cause emulsion droplets to aggregate under thermal conditions, reducing the emulsion's thermal stability.

[0111] The freeze-thaw stability analysis results of DHPM-LA and TSE-LA emulsions are as follows: Figure 11 As shown, after freeze-thaw cycles, both the DHPM-LA and TSE-LA emulsion systems exhibited phase separation, manifested as oil layer precipitation and system stratification. This instability is mainly due to droplet collision and aggregation caused by crystallization of the oil and water phases during freezing. When the temperature rises, the heterogeneous aggregates formed during freezing fuse to form large oil droplets, ultimately leading to emulsion disruption. The freeze-thaw stability of emulsions is mainly affected by their composition and structural characteristics. Generally, emulsion systems with high viscosity and robust interfacial films can effectively resist mechanical damage from ice crystals, thereby reducing the risk of flocculation. In this study, the interfacial layer of the stable WPI-Z-SA emulsion 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 film. Moreover, all treatment groups retained a portion of the emulsion layer after freeze-thaw cycles, indicating that the WPI-Z-SA composite system can increase the content of unfrozen water in the continuous phase, thereby inhibiting droplet approach. Compared with different treatment groups, the DHPM-LA emulsion showed relatively better freeze-thaw stability. This is attributed to the fact that its three-dimensional network structure can effectively block ice crystal growth, and at the same time, the thicker interface layer can provide better mechanical protection, preventing crystals formed during freezing from damaging the oil droplet structure, and ultimately reducing droplet coalescence after thawing.

[0112] 3. Determination of centrifugal stability of DHPM-LA and TSE-LA

[0113] Take 15.0 mL of each of the DHPM-LA and TSE-LA emulsions into 50.0 mL centrifuge tubes and centrifuge at 4000 rpm for 20 min. Take pictures to record the phase separation status, and evaluate the centrifugal stability by comparing the height of the emulsion layer.

[0114] The centrifugal stability of DHPM-LA and TSE-LA emulsions is as follows: Figure 12As shown. Under the action of centrifugation, the emulsion droplets aggregate, resulting in emulsion separation and stratification. After centrifugation, the higher the emulsion layer, the more uniform and stable the distribution of the emulsion layer in the emulsion system. The centrifugal stability of DHPM-LA and TSE-LA emulsions differs; both emulsions show aqueous phase precipitation after centrifugation. The TSE-LA compound-stabilized emulsion exhibits the worst centrifugal stability, while the DHPM-LA compound-stabilized emulsion shows better centrifugal stability. This is mainly related to the internal structure of the emulsion. Combined with electron microscopy results, the DHPM-LA compound-stabilized emulsion has a relatively strong network structure and viscosity. The strong interactions between emulsion droplets, between protein molecular chains, and between droplets and protein molecular chains effectively resist centrifugal force and prevent oil droplet aggregation, thus ensuring the centrifugal stability of the emulsion. Furthermore, the results of centrifugal stability are similar to those of stability during static storage.

[0115] 4. Determination of lipid oxidation stability of DHPM-LA and TSE-LA emulsions

[0116] 10.0 mL of each of the DHPM-LA and TSE-LA emulsions were placed in glass bottles and randomly stored in a 50°C oven protected from light for 14 days. Primary and secondary oxidation products were analyzed on days 0, 1, 3, 5, 7, 10, and 14. The determination of primary lipid oxidation products followed this procedure: 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 by mixing isooctane and isopropanol in a 3:1 (v / v) ratio, was added. After vortexing (30 s, 10 s intervals), the sample was centrifuged at 4000 rpm for 2 min to achieve phase separation. 200 μL of the upper organic phase was mixed with 2.8 mL of a methanol / n-butanol mixture (2:1, v / v), followed by the addition of 50 μL of ammonium thiocyanate solution and 50 μL of ferrous ion solution (containing equal volumes of 0.132 mol / L BaCl2 and 0.144 mol / L FeSO4). The mixture was allowed to react in the dark for 2 min, and the absorbance was measured at 510 nm. The peroxide content in the sample was then quantitatively calculated based on the hydrogen peroxide standard curve.

[0117] 5. Determination of secondary oxidation products in DHPM-LA and TSE-LA emulsions

[0118] Take 0.2 mL of DHPM-LA and TSE-LA emulsions respectively, dilute to 1.0 mL with distilled water, and add 2.0 mL of TBA solution (15.0 g of TCA (trichloroacetic acid) and 0.375 g of TBA (2-thiobarbituric acid) dissolved in 100.0 mL of 0.25 mol / L HCl). Then, boil the samples in boiling water for 20 min, cool at room temperature, and centrifuge at 4000 rpm for 30 min. Measure the absorbance of the supernatant at 532 nm. Finally, calculate the MDA content in the samples based on the standard curve of 1,1,3,3-tetramethoxypropane.

[0119] The results of lipid oxidation stability studies of DHPM-LA and TSE-LA emulsions are as follows: Figure 13 As shown. The oxidative stability of different emulsion systems was evaluated by measuring the changes in the content of primary oxidation products (lipid hydroperoxide, 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 an 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. Furthermore, the DHPM-LA emulsion exhibited the best oxidative stability. Lipid oxidation in the emulsion system is mainly influenced by the following factors: firstly, the large interfacial area between the dispersed and continuous phases promotes the contact between the oxidant and lipids; and secondly, the composition and structural characteristics of the interfacial layer play a decisive role in the oxidation rate. In this study, the three-dimensional network structure and dense interfacial layer formed by the DHPM-LA emulsion played a dual protective mechanism: on the one hand, limiting oxygen diffusion through physical barrier action, and on the other hand, interfering with the propagation of free radical chain reactions. Figure 13 As shown, during the oxidation process, the MDA content of all samples exhibited an increasing trend, and its variation pattern was highly consistent with that of the LH content. This indicates that the primary oxidation products continued to decompose, producing secondary oxidation products such as aldehydes and ketones, further confirming the cascade nature of the oxidation reaction.

[0120] 6. Determination of antioxidant activity of DHPM-LA and TSE-LA emulsions

[0121] The antioxidant properties of the WPI-Z-SA composite system before and after physical field treatment were evaluated using DPPH and ABTS free radical scavenging methods, respectively. Sample solutions were uniformly prepared to 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 min, and the absorbance was measured at 517 nm. Preparation of ABTS free radicals: An equal volume of 2.45 mmol / L potassium persulfate and 7.0 mmol / L ABTS solution was mixed and reacted at 25°C in the dark for 12-16 h to generate an ABTS free radical stock solution. For measurement, the sample was mixed with ABTS working solution at a ratio of 1:20 (v / v), reacted in the dark for 15 min, and the absorbance was measured at 734 nm. The free radical scavenging rate was calculated using the following formula:

[0122] Free radical scavenging capacity (%) = A 对照组 -A 样品组 / A 对照组 ×100

[0123] The DPPH radical scavenging abilities of TSE-LA and DHPM-LA are as follows: Figure 14 As shown, both the TSE-LA and DHPM-LA emulsion systems exhibited significant DPPH radical scavenging capabilities (p<0.05). The DHPM-LA emulsion achieved a scavenging rate of 93.4%, approximately twice that of the pure LA sample; the TSE-LA emulsion achieved a scavenging rate of 90.3%, also demonstrating significant antioxidant activity. This enhancement effect may be due to the structural changes in WPI-Z-SA induced by high-pressure treatment, which allows exposed thiol groups and aromatic amino acid residues to effectively block free radical chain reactions. The significant enhancement of LA's antioxidant activity by WPI-Z-SA after high-pressure treatment is likely due to the pH-dependent reaction process. The reductive ketones, melanoidins, and volatile heterocyclic compounds generated during this process collectively enhance the antioxidant activity. This explains why the DHPM-LA emulsion exhibits the best antioxidant performance.

[0124] The ability of TSE-LA and DHPM-LA emulsions to scavenge 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 demonstrated the best scavenging effect. This result confirms that stabilizing LA through an emulsion system significantly enhances its antioxidant properties, and dynamic high-pressure microfluidic (DHPM) treatment further strengthens this effect. Furthermore, this trend is highly consistent with the results of the DPPH free radical scavenging experiment, indicating that the conclusions from the two evaluation methods mutually validate each other.

[0125] Test Example 9

[0126] Determination of TSE-LA and DHPM-LA emulsion digestion assay

[0127] Prepare digestive solutions and perform simulated in vitro digestion analysis.

[0128] Artificial saliva: Take 15.1 mL of potassium chloride solution with a molar concentration of 0.5 mol / L, 3.7 mL of potassium dihydrogen phosphate solution with a molar concentration of 0.5 mol / L, 6.8 mL of sodium chloride solution with a molar concentration of 1 mol / L, 0.5 mL of magnesium chloride hexahydrate solution with a molar concentration of 0.15 mol / L, 0.06 mL of ammonium carbonate solution with a molar concentration of 0.5 mol / L, 0.09 mL of hydrochloric acid solution with a molar concentration of 6 mol / L, and 0.025 mL of calcium chloride dihydrate solution with a molar concentration of 0.3 mol / L. Mix the above solutions thoroughly and adjust the pH to 7.0.

[0129] Artificial gastric fluid: Take 6.9 mL of potassium chloride solution with a molar concentration of 0.5 mol / L, 0.9 mL of potassium dihydrogen phosphate solution with a molar concentration of 0.5 mol / L, 12.5 mL of sodium chloride solution with a molar concentration of 1.0 mol / L, 11.8 mL of sodium chloride solution with a molar concentration of 2.0 mol / L, 0.4 mL of magnesium chloride hexahydrate solution with a molar concentration of 0.15 mol / L, 0.5 mL of ammonium carbonate solution with a molar concentration of 0.5 mol / L, 1.3 mL of hydrochloric acid solution with a molar concentration of 6.0 mol / L, and 0.005 mL of calcium chloride dihydrate solution with a molar concentration of 0.3 mol / L. Mix the above solutions thoroughly and set aside.

[0130] Artificial intestinal fluid: Take 6.8 mL of potassium chloride solution with a molar concentration of 0.5 mol / L, 0.8 mL of potassium dihydrogen phosphate solution with a molar concentration of 0.5 mol / L, 42.5 mL of sodium chloride solution with a molar concentration of 1.0 mol / L, 9.6 mL of sodium chloride solution with a molar concentration of 2.0 mol / L, 1.1 mL of magnesium chloride hexahydrate solution with a molar concentration of 0.15 mol / L, 0.09 mL of hydrochloric acid solution with a molar concentration of 6.0 mol / L, and 0.04 mL of calcium chloride dihydrate solution with a molar concentration of 0.3 mol / L. Mix the above solutions thoroughly and set aside.

[0131] 1. Determination of protein release

[0132] Take 8.0 mL of artificial saliva, add 0.1 g of sample, add 0.025 mL of 0.3 mol / L calcium chloride dihydrate solution, and add distilled water to a final volume of 10.0 mL. React in a 37°C water bath for 2 min. After partial digestion in the oral cavity, add 8.0 mL of prepared artificial gastric juice, add 0.005 mL of 0.3 mol / L calcium chloride dihydrate solution, and add distilled water to a final volume of 20.0 mL. Adjust the pH to 3.0, add 13.3 mg of pepsin, and react thoroughly in a 37°C water bath shaker for 2 h. During the reaction, take 2.0 mL samples at 0, 30, 60, 90, and 120 min. Immediately after the reaction, freeze the samples in prepared ice blocks for inactivation. Each sample taken during the digestion process must undergo freeze-inactivation. After gastric digestion is complete, add 8.0 mL of the artificial intestinal fluid prepared in section 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 solution and 300.0 mg of bile salts. Add distilled water to a final volume of 20.0 mL, adjust the pH to 7.0, add 8.0 mg of trypsin, and incubate at 37°C in a water bath for 2 hours. During the reaction, take 2.0 mL samples at 0, 30, 60, 90, and 120 min. Immediately after the reaction, freeze the samples in prepared ice packs for inactivation. Each sample obtained during digestion should undergo freeze-inactivation. After the samples have returned to room temperature (25°C), add an equal volume of 15.0% trichloroacetic acid to each sample, centrifuge at 10000 rpm for 10 min, and determine the protein content of the supernatant. The in vitro digestibility is calculated using the following formula:

[0133] Protein digestion (%) = Protein content in supernatant / (Sample weight × Sample protein content) × 100

[0134] The binding of proteins to small organic molecules affects their microstructure, and DHPM and TSE also influence the distribution of functional groups within the protein and complex system, thus affecting the digestibility of protein emulsions. To investigate the suitability of TSE-LA and DHPM-LA emulsions as nutrient delivery systems, this invention examined their protein release characteristics in a simulated gastrointestinal environment. The results are as follows: Figure 15 As shown, at 37°C, the two emulsion systems exhibited different release kinetics. The gastric phase release test is shown below. Figure 15 As shown in Figure A, the release curves of the two samples are similar, both exhibiting slow release rates. This sustained-release behavior may be attributed to the acidic environment of simulated gastric juice causing the sodium alginate molecular chains to break, thereby promoting the gradual release of WPI and Z into the medium. In simulated intestinal fluid, as... 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.

[0135] 2. Determination of linoleic acid release rate

[0136] After gastric digestion, the pH of the sample was adjusted to neutral (7.0). Then, 1.5 mL of a 2.7 mg / mL calcium chloride solution, 3.5 mL of a bile salt solution (117.8 mg / mL, prepared with 10.0 mmol / L PBS buffer, pH 7.0) was added sequentially, along with 2.5 mL of a mixed enzyme solution containing lipase and trypsin (both at 24.0 mg / mL), prepared with 10.0 mmol / L PBS buffer, pH 7.0.

[0137] 7.0). After adjusting the pH of the above mixture to 7.0, it was placed in a 37℃ constant temperature water bath shaker and reacted at 150 r / min for 2 hours to simulate the small intestine digestion environment. Throughout the digestion process, the pH was maintained constant using 1.0 mol / L sodium hydroxide solution, and the amount of sodium hydroxide solution consumed at 30, 60, 90, and 120 minutes was recorded. The release of LA from the emulsion sample was quantitatively assessed by measuring the release of FFA in the reaction system. The release of free fatty acids was calculated according to the following formula:

[0138] FFA release (%) = C NaOH ×V NaOH (t)×M 油 / 2W 油 ×100

[0139] Where M_oil is the average molecular weight of the oil, C_NaOH is the concentration of the NaOH solution, V_NaOH(t) is the volume of NaOH solution consumed at different digestion times, and W_oil is the total mass of camellia seed oil in the digestion solution.

[0140] Release of free fatty acids (FFA) from different emulsions, TSE-LA and DHPM-LA, during simulated in vitro digestion, such as Figure 16 As shown. During the first 30 minutes of the intestinal phase, FFA release increases rapidly because lipases quickly adsorb onto the droplet surface in the initial stage, breaking down triglycerides into fatty acids. The final FFA release rates of TSE-LA and DHPM-LA emulsions differ. TSE-LA has a higher FFA release rate than DHPM-LA, possibly because DHPM increases steric hindrance and intermolecular repulsion, resulting in a higher emulsion strength and viscoelasticity, which inhibits lipase movement towards the oil droplets, thus suppressing fatty acid release. Additionally, proteins fill the gaps between droplets, making it difficult for bile salts to adsorb onto the interface and replace the emulsifier on the droplet surface, inhibiting lipase adsorption onto the lipid droplet surface, thereby further inhibiting lipid digestion and reducing FFA release. Furthermore, in the process of…

[0141] Although WPI-stabilized emulsions have smaller droplet sizes that provide a larger specific surface area, increasing the number of adsorption sites for bile salts and lipases, and their weaker strength is conducive to the adsorption of bile salts and lipases, in reality, WPI-stabilized emulsions do not result in the maximum release of FFAs. This is because emulsions are highly susceptible to the effects of pepsin and acidic conditions during gastric digestion. Emulsion breakdown causes droplets to aggregate and form large oil droplets, reducing the contact surface area and hindering the action of lipases during intestinal digestion.

[0142] Based on the above experimental results, the following conclusions can be drawn:

[0143] (1) Multispectral analysis results showed that DHPM treatment, through its unique cavitation effect and microfluidic mechanism, can effectively induce conformational decomposition of protein molecules, exposing internal hydrophobic regions and thus enhancing the interaction between WPI, Z, and SA. This process is directly manifested as a significant enhancement of the UV absorption characteristics of the composite system. Fourier transform infrared spectroscopy analysis showed that DHPM treatment strengthened the vibrational modes of carbonyl (C=O), CN, and NH bonds in the amide bond. In the range of 250-850 kPa, the OH stretching vibration of the amide A band and the characteristic peak positions of amide I and II regions remained relatively stable. Compared with TSE, DHPM showed a more significant effect in regulating protein secondary structure and could also effectively improve the solubility of the composite system.

[0144] (2) Thermodynamic analysis and particle size potential (PDI) measurements showed that the denaturation temperature (Td) of the WPI-Z-SA composite system gradually decreased with increasing DHPM treatment pressure. This phenomenon reveals the significant effect of high-pressure treatment on protein structure: under the action of continuously increasing mechanical force, the three-dimensional conformation of the composite system gradually unfolds, the molecular arrangement tends to become disordered, and ultimately, its thermal stability is continuously weakened. With increasing pressure, both the protein particle size and PDI gradually decrease. At a high pressure of 850 kPa, the particle size reaches 145.697 nm, and the PDI reaches 0.45. At a TSE rotation speed of 200 rpm, the particle size and PDI of the composite system are 150.100 nm and 0.45, respectively. The zeta potential of untreated protein was -30.190 mV. After DHPM treatment, the absolute value of the zeta potential increased, but with increasing voltage, the absolute value of the zeta potential tended to decrease, reaching a minimum of -38.388 mV at DHPM 850 kPa. The trend of increasing absolute value of the zeta potential was even more pronounced with TSE, reaching a minimum of -49.150 mV at TSE 200 rpm. Based on these findings, we selected DHPM 850 kPa and TSE 200 rpm for subsequent experimental studies. Emulsions were prepared by mixing LA with aqueous solutions of protein-based complexes treated with DHPM 850 kPa and TSE 200 rpm at different volume ratios. Multiplex optical analysis revealed that a 3:1 ratio was optimal, and these were named DHPM-LA and TSE-LA, respectively.

[0145] (3) Stability and antioxidant analysis results showed significant differences between the TSE-LA and DHPM-LA emulsion systems. Multiple light scattering data showed that the backscattering spectra of the TSE-LA sample highly overlapped within 24 hours, indicating good colloidal stability and no obvious particle aggregation. In contrast, the backscattering signal in the upper region of the DHPM-LA sample gradually decreased, suggesting particle sedimentation. Quantitative Turbiscan stability index (TSI) analysis further confirmed that TSE-LA maintained a stable TSI value during storage, exhibiting optimal 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 exhibit phase separation, while TSE-LA showed obvious emulsification 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, indicating that the former exhibited superior oxidative stability. Antioxidant activity assessment showed that both emulsion systems significantly enhanced the free radical scavenging capacity of LA (p<0.05). Specifically, DHPM-LA showed increased free radical scavenging capacity against DPPH and...

[0146] The scavenging rates of ABTS free radicals reached 93.4% and 91.3%, respectively, approximately twice that of free LA; the scavenging rates of TSE-LA were 90.3% and 88.68%, respectively, also demonstrating significant antioxidant effects. Considering all indicators, DHPM-LA exhibited the best overall performance.

[0147] (4) In vitro digestion results showed that DHPM-LA and TSE-LA differed significantly in terms of FFA release rate and bioavailability. DHPM-LA exhibited a lower FFA release rate, which may be due to the high-pressure treatment improving the mechanical strength and viscoelasticity of the emulsion.

[0148] The foregoing descriptions have only illustrated certain exemplary embodiments of the present invention. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of the claims of the present invention.

Claims

1. A method for preparing a protein-based complex system based on physical field-encapsulated linoleic acid, characterized in that, A ternary composite system was obtained by pH-driven method using water-soluble protein, zein, and sodium alginate, with a mass ratio of 9:1:

1. The ternary composite system was then processed in a DHPM or TSE device. The pressure parameters of the DHPM device were set to 850 kPa and the temperature parameters to 35°C. The rotation speed parameters of the TSE device were set to 200 rpm and the temperature parameters to 35°C. The DHPM or TSE-treated ternary composite system was freeze-dried, and the freeze-dried powder was added to deionized water and stirred using a high-pressure homogenizer to obtain an aqueous solution of the protein-based composite system. Linoleic acid was mixed with the aqueous solution of the protein-based composite system at a volume ratio of 3:1 to prepare an emulsion, i.e., a protein-based composite system encapsulating linoleic acid.

2. The method for preparing the protein-based composite system based on physical field-encapsulated linoleic acid according to claim 1, characterized in that, The preparation of a ternary composite system using a pH-driven method includes the following steps: S1: Using deionized water as solvent, add water-soluble protein to deionized water at a volume-to-mass ratio of 10L:9g, and let it stand overnight to obtain a water-soluble protein solution. S2: Using deionized water as a solvent, prepare 2M NaOH solution and 1M HCl solution; S3: Adjust the pH of the overnight water-soluble protein solution to 12 using 2M NaOH solution, stir for 2 hours, add zein to the water-soluble protein solution at a mass ratio of 9:1 (water-soluble protein to zein), and stir for 30 minutes; add sodium alginate to the water-soluble protein-zein composite solution at a mass ratio of 1:1 (zein to sodium alginate), and stir for 30 minutes; adjust the composite solution to pH 7 using 1M HCl solution to obtain a ternary functional protein complex; the stirrer parameters are set as follows: stirring speed 800 r / min, ambient temperature 25±2℃.

3. A protein-based composite system based on linoleic acid encapsulated in a physical field, characterized in that, It is prepared by the method described in any one of claims 1 to 2.

4. The application of the protein-based composite system based on physical field-encapsulated linoleic acid as described in claim 3 in the preparation of edible nutrient delivery products.

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