Folate fortified double emulsion substitute fat, its preparation method and application

Abstract

This invention belongs to the field of fat substitute technology, specifically relating to a folic acid-fortified dual emulsion substitute fat, its preparation method, and its application. The preparation method provided by this invention first constructs a lactoglobulin-ascorbic acid-folic acid ternary complex, utilizing the synergistic effect of lactoglobulin and ascorbic acid to enhance the stability of folic acid under various conditions. Based on this, the ternary complex is embedded in the inner aqueous phase of a high-internal-phase dual emulsion, and a two-step emulsification method combined with high-pressure microfluidic technology is used to construct the high-internal-phase dual emulsion. Finally, experiments demonstrate that the high-internal-phase dual emulsion, as a fat substitute, has good application effects in low-fat cream emulsions and low-fat whipped cream, thereby achieving the dual goals of fat reduction and folic acid fortification. Therefore, this invention not only provides a new theoretical basis and technical path for the development of nutritional foods with both fat substitution and nutritional fortification functions, but also has broad application prospects in the preparation of fat substitute fats.

Description

Technical Field

[0001] This invention belongs to the field of fat substitute technology, specifically relating to a folic acid-fortified dual emulsion fat substitute, its preparation method, and its application. Background Technology

[0002] As a nutrient, fat provides the body with energy and essential fatty acids. Furthermore, as a crucial component of food, fat plays a vital role in imparting good texture, rich flavor, and a smooth, delicate mouthfeel. However, excessive fat intake can increase the risk of obesity, type 2 diabetes, and cardiovascular diseases. Currently, while simply reducing fat content can help control fat intake, it often leads to poor food texture, decreased palatability, and reduced consumer acceptance. Therefore, how to reduce fat intake while maintaining good food quality and meeting the body's need for essential fatty acids has become an urgent problem to be solved.

[0003] Fat substitutes refer to technologies that reduce fat intake by partially or completely replacing fat in food, while mimicking the structural, functional, and sensory properties of fat in the food system (such as texture, lubricity, and flavor components). In recent years, the development of fat substitutes has become an important way to reduce dietary fat intake. Currently, although fat substitutes have been applied in many types of food (such as dairy products and baked goods), their applicability and functional performance in different food systems still vary considerably. In particular, existing fat substitutes mostly focus on mimicking the structure of fat, and still have shortcomings in terms of active ingredient loading and delivery, making it difficult to achieve nutritional fortification and functional enhancement while reducing fat content. Meanwhile, folic acid, as an essential micronutrient, can increase the health risks of neural tube defects, cardiovascular disease, and some cancers if intake is insufficient. However, folic acid is sensitive to environmental factors such as light, heat, and pH, is easily degraded, and has poor stability, thus limiting its effective utilization in food systems.

[0004] Existing research has shown that the formation of complexes between proteins and active ingredients through intermolecular interactions is an effective way to improve the stability of active ingredients. Currently, while the construction of fat substitute systems and protein-active ingredient stabilization systems has become a research hotspot, most studies focus on simple regulation of single functions, lacking integrated research that combines the construction of fat substitutes with the stable delivery of active ingredients. Therefore, developing fat substitute systems that combine the ability to mimic fat structure with the ability to deliver functional components, thereby achieving synergistic optimization of lipid reduction and nutritional fortification, has become a key research direction. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a folic acid-fortified dual emulsion alternative lipid, its preparation method, and its applications. The folic acid-fortified dual emulsion alternative lipid provided by this invention achieves integrated fat substitution and stable folic acid delivery.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a folic acid-fortified dual emulsion alternative lipid includes the following steps:

[0008] S1. Prepare a β-lactoglobulin solution from β-lactoglobulin; first add L-ascorbic acid to the β-lactoglobulin solution and stir to react, then add folic acid and stir to react; after the reaction is completed, adjust the pH of the resulting reaction solution to 6-8, then dialyze and dry to obtain a lactoglobulin-ascorbic acid-folic acid ternary complex.

[0009] S2. Prepare a ternary complex solution from the lactoglobulin-ascorbic acid-folic acid ternary complex as the inner aqueous phase; prepare a pectin solution from pectin as the outer aqueous phase; mix medium-chain triglycerides, vegetable oil and polyglycerol ricinoleate as the oil phase;

[0010] S3. The inner aqueous phase is added to the oil phase and subjected to high-speed shearing to obtain W1 / O primary emulsion; the W1 / O primary emulsion is mixed with the outer aqueous phase and subjected to high-speed shearing to obtain W1 / O / W2 crude emulsion; the W1 / O / W2 crude emulsion is subjected to high-pressure microjets to obtain folic acid-enhanced dual emulsion substitute lipid.

[0011] Preferably, in step S1, the concentration of the β-lactoglobulin solution is 0.5~2% w / v.

[0012] Preferably, in step S1, the mass ratio of β-lactoglobulin, L-ascorbic acid, and folic acid is 1:(0.05~0.2):(0.4~0.6).

[0013] Preferably, in step S1, the stirring reaction time is 0.5~2h; the dialysis temperature is 2~8℃, the time is 30~60h, and the molecular weight cutoff is 2800~3500Da; the drying is freeze drying.

[0014] Preferably, in step S2, the concentration of the ternary complex solution is 0.0005~0.005%w / w; and the concentration of the pectin solution is 4~6%w / w.

[0015] Preferably, in step S2, the vegetable oil is one or more of olive oil, flaxseed oil, coconut oil, sunflower seed oil, soybean oil, corn oil, and peanut oil; the mass ratio of the medium-chain triglyceride to the vegetable oil is 1:(1.5~2.5); and the mass of the polyglycerol ricinoleate is 1~2% w / w of the total mass of the medium-chain triglyceride and the vegetable oil.

[0016] Preferably, in step S3, the amount of the inner aqueous phase is 18-22 parts by mass, the amount of the oil phase is 54-70 parts, and the amount of the outer aqueous phase is 10-26 parts. More preferably, the amount of the inner aqueous phase is 20 parts, the amount of the oil phase is 66 parts, and the amount of the outer aqueous phase is 14 parts. This invention introduces an outer aqueous phase on top of the inner aqueous phase, which can further enhance the protective effect on the ternary complex in the inner aqueous phase.

[0017] Preferably, in step S3, the high-speed shearing rotation speed is 8000~12000 rpm, and the time is 1~5 min; the pressure of the high-pressure microjets is 100M~200MPa. In this step, the two-step emulsification process of high-speed shearing is used to construct a multilayer structure of the dual emulsion, while the microjets further optimize the droplet size and interfacial characteristics, thereby providing technical support for constructing a dual emulsion system that combines structural stability and efficient delivery performance.

[0018] A folic acid-fortified dual emulsion substitute lipid prepared by the method described above.

[0019] An application of the folic acid-fortified double emulsion substitute as described above, wherein the application is in the preparation of cream emulsions or whipped cream; the cream emulsion or whipped cream is mainly prepared from an oil phase and an aqueous phase; the oil phase comprises a base oil and the folic acid-fortified double emulsion substitute; the mass ratio of the folic acid-fortified double emulsion substitute to the base oil is (10~30):(70~90), more preferably 20:80. This ratio helps to optimize the structural stability and performance of the cream emulsion or whipped cream.

[0020] The technical solution of the present invention has the following beneficial effects:

[0021] This invention first constructs a ternary complex of lactoglobulin-ascorbic acid-folic acid, utilizing the synergistic effect of lactoglobulin and ascorbic acid to enhance the stability of folic acid under light, heat treatment, and different pH conditions. Based on this, the ternary complex is embedded in the inner aqueous phase of a high-internal-phase dual emulsion. A two-step emulsification method and high-pressure microfluidic technology are then combined to construct the high-internal-phase dual emulsion, thereby achieving synergistic regulation of the construction of the multilayer structure of the dual emulsion and the improvement of structural stability and encapsulation efficiency, further enhancing the protective effect on folic acid. Finally, using the high-internal-phase dual emulsion as a fat substitute, with cream as a model system, it is demonstrated that the high-internal-phase dual emulsion has good application effects as a fat substitute in low-fat cream emulsions and low-fat whipped cream, thus achieving the dual goals of product fat reduction and folic acid fortification.

[0022] Therefore, this invention achieves synergistic enhancement in terms of the binding, stability and functional regulation of active ingredients, effectively improving the stability and delivery efficiency of folic acid. It can not only provide new theoretical basis and technical path for the development of nutritional foods or functional drugs with both fat substitution and nutritional fortification functions, but also has broad application prospects in the preparation of fat substitute lipids. Attached Figure Description

[0023] Figure 1 The results show the particle size and zeta potential of β-Lg and its binary and ternary complexes; where a is the particle size and b is the zeta potential.

[0024] Figure 2 The binding efficiency and binding capacity of β-Lg and its binary and ternary complexes;

[0025] Figure 3 Scanning electron microscope images of β-Lg and its binary and ternary complexes;

[0026] Figure 4 Fourier transform infrared spectra of β-Lg and its binary and ternary complexes;

[0027] Figure 5 The values ​​represent the circular dichroism chromatograms and secondary structure contents of β-Lg and its binary and ternary complexes; where a is the circular dichroism chromatogram and b is the secondary structure content.

[0028] Figure 6 The intrinsic fluorescence spectra of β-Lg and its binary and ternary complexes are shown.

[0029] Figure 7 The ultraviolet absorption spectra of β-Lg and its binary and ternary complexes are shown.

[0030] Figure 8 The results show the determination of the surface hydrophobicity of β-Lg and its binary and ternary complexes;

[0031] Figure 9Differential scanning calorimetry (DSC) spectra of β-Lg and its binary and ternary complexes;

[0032] Figure 10 The results show the antioxidant properties of β-Lg and its binary and ternary complexes; where a represents ABTS radical scavenging ability, b represents DPPH radical scavenging ability, and c represents iron ion reduction antioxidant ability.

[0033] Figure 11 denoted as emulsifying activity index and emulsifying stability index for β-Lg and its binary and ternary complexes; where a is the emulsifying activity index and b is the emulsifying stability index.

[0034] Figure 12 The foaming capacity and foam stability of β-Lg and its binary and ternary complexes are given; where a is the foaming capacity and b is the foam stability.

[0035] Figure 13 The changes in FA retention rates of free FA and its binary complex β-Lg-FA and ternary complex β-Lg-LAA-FA under 254 nm and 365 nm UV irradiation, different heating conditions, and pH conditions are shown. Among them, a represents 254 nm UV irradiation, b represents 365 nm UV irradiation, c represents different heating conditions, and d represents different pH conditions.

[0036] Figure 14 Micrographs and appearance images of high internal phase double emulsions with different oil phase contents;

[0037] Figure 15 Results of encapsulation efficiency and loading of high internal phase dual emulsions with different oil phase contents;

[0038] Figure 16 The results show the particle size and Zeta potential of high internal phase dual emulsions with different oil phase contents; where a is the particle size and b is the Zeta potential.

[0039] Figure 17 The particle size changes of high internal phase dual emulsions with different oil phase contents during storage at different temperatures are shown; where a is the particle size at 4℃ and b is the particle size at 25℃.

[0040] Figure 18 The results show the changes in POV in high internal phase double emulsions with different oil phase contents during 28 days of storage at 4℃ and 25℃; where a represents the POV change at 4℃ and b represents the POV change at 25℃.

[0041] Figure 19 The results show the changes in MDA content in high internal phase double emulsions with different oil phase contents during 28 days of storage at 4℃ and 25℃; where a represents the MDA change at 4℃ and b represents the MDA change at 25℃.

[0042] Figure 20 The encapsulation efficiency of high internal phase double emulsions with different oil phase contents was shown during 28 days of storage at 4℃ and 25℃; where a represents the encapsulation efficiency at 4℃ and b represents the encapsulation efficiency at 25℃.

[0043] Figure 21 The images show the particle size and appearance of high internal phase dual emulsions with different oil phase contents after one freeze-thaw cycle; where a is the particle size and b is the appearance.

[0044] Figure 22 The images show the particle size and appearance of high internal phase dual emulsions with different oil phase contents after heat treatment; where a is the particle size and b is the appearance.

[0045] Figure 23 Particle size, appearance, and emulsion index of high internal phase double emulsions with different oil phase contents after centrifugation; where a is particle size, b is appearance, and c is emulsion index.

[0046] Figure 24 The storage modulus, loss modulus, and apparent viscosity of high internal phase dual emulsions with different oil phase contents are shown in the test results; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity.

[0047] Figure 25 Appearance of cream emulsion samples obtained with different high internal phase dual emulsion substitution ratios;

[0048] Figure 26 Particle size of cream emulsion samples obtained with different high internal phase dual emulsion substitution ratios;

[0049] Figure 27 TSI stability index of cream emulsion samples with different internal phase dual emulsion substitution ratios;

[0050] Figure 28 The aggregation rate of the fat fraction in cream emulsion samples obtained with different high internal phase dual emulsion substitution ratios;

[0051] Figure 29 The storage modulus, loss modulus, and apparent viscosity of cream emulsion samples obtained with different internal phase dual emulsion substitution ratios are shown in the figure; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity.

[0052] Figure 30 Appearance of whipped cream samples obtained with different high internal phase dual emulsion substitution ratios;

[0053] Figure 31 Whipping yield of whipped cream samples with different high internal phase dual emulsion substitution ratios;

[0054] Figure 32The textural test results are for whipped cream samples obtained with different high internal phase dual emulsion substitution ratios; where a is hardness, b is elasticity, c is cohesiveness, d is chewiness, e is resilience, and f is adhesiveness.

[0055] Figure 33 The storage modulus, loss modulus, and apparent viscosity of whipped cream samples obtained with different internal phase dual emulsion substitution ratios are shown in the figure; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments. The following experiments were conducted at least three times, and the mean and standard deviation were calculated. Data were analyzed using SPSS one-way ANOVA. A p-value < 0.05 was considered significant, and different letters indicated significant differences between groups.

[0057] In the following embodiments of this invention, folic acid (purity ≥97%), medium-chain triglycerides (analytical grade), polyglycerol ricinoleate (analytical grade), pectin (purity 65%), and 8-aniline-1-naphthalenesulfonic acid (analytical grade) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. β-lactoglobulin (purity >95%) was obtained from Aladdin Biochemical Technology Co., Ltd. L-ascorbic acid (purity ≥90%) was obtained from Tianjin Tianli Chemical Reagent Co., Ltd. Phosphate buffer (analytical grade) was obtained from Beijing Solarbio Science & Technology Co., Ltd. 1,1-diphenyl-2-picrylhydrazine radical (analytical grade) and 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid (analytical grade) were obtained from Sigma-Aldrich, USA. Flaxseed oil, olive oil, sodium caseinate, sucrose, xanthan gum, citric acid, and olive oil were all food-grade raw materials, while Nile Red, Nile Blue, Oil Red O, and hydrochloric acid were all analytical grade raw materials obtained from conventional commercial channels. The malondialdehyde detection kit was obtained from Nanjing Jiancheng Bioengineering Institute. The high-speed shear emulsifier is specifically the IKA high-speed homogenizer from IKA Instruments GmbH, Germany, and the high-pressure microjet equipment is specifically the DNH-340 nano high-pressure microjet homogenizer from Shenzhen Deheng Nanotechnology Co., Ltd.

[0058] Example 1: Preparation, structural characterization, and functional property analysis of the complex

[0059] I. Preparation of the complex

[0060] This embodiment provides a lactoglobulin-ascorbic acid-folic acid ternary complex, the preparation method of which includes the following steps:

[0061] β-lactoglobulin (β-Lg) was dissolved in 0.01 M, pH 7.0 phosphate buffer, and magnetically stirred at 600 rpm for 2 h at room temperature (25±5℃). Then, it was hydrated overnight (10~18 h) at 4℃ to obtain a 1% w / v β-Lg solution.

[0062] Subsequently, LAA was added to the β-Lg solution at a mass ratio of 10:1 (w / w) to L-ascorbic acid (LAA), and the mixture was reacted at room temperature for 1 h under magnetic stirring at 750 rpm to obtain the β-Lg-LAA binary complex.

[0063] Further, FA was added to the β-Lg-LAA binary complex at a mass ratio of 2:1 (w / w) of β-Lg to folic acid (FA), and the reaction was carried out at room temperature for 1 h under the same magnetic stirring conditions. After the reaction was completed, the pH of the resulting reaction solution was adjusted to 7.0. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed at 4 °C for 48 h to remove unbound small molecules. After dialysis, the resulting sample was freeze-dried to obtain a lyophilized powder, which is the lactoglobulin-ascorbic acid-folic acid ternary complex, denoted as β-Lg-LAA-FA, for subsequent analysis.

[0064] Similarly, this embodiment also provides a lactoglobulin-folate binary complex, the preparation method of which includes the following steps: A 1% w / v β-Lg solution is prepared according to the aforementioned method. Then, FA is added to the β-Lg solution at a mass ratio of β-Lg to folic acid (FA) of 2:1 (w / w), and the reaction is carried out under magnetic stirring for 1 h. After the reaction is complete, the resulting reaction solution is subjected to pH adjustment, dialyzing, and freeze-drying according to the post-processing procedure used in the preparation of β-Lg-LAA-FA. The resulting freeze-dried powder is the lactoglobulin-folate binary complex, denoted as β-Lg-FA.

[0065] Similarly, this embodiment also provides a lactoglobulin-ascorbic acid binary complex, the preparation method of which includes the following steps: A 1% w / v β-Lg solution is prepared according to the aforementioned method. Then, LAA is added to the β-Lg solution at a mass ratio of β-Lg to L-ascorbic acid (LAA) of 10:1 (w / w), and the reaction is carried out under magnetic stirring for 1 h. After the reaction is complete, the resulting reaction solution is subjected to pH adjustment, dialyzing, and freeze-drying according to the post-processing procedure used in the preparation of β-Lg-LAA-FA. The resulting freeze-dried powder is the lactoglobulin-ascorbic acid binary complex, denoted as β-Lg-LAA.

[0066] II. Structural Characterization of the Complex

[0067] (1) Particle size and Zeta potential

[0068] The particle size and zeta potential (protein concentration 0.2 mg / mL) of four samples—β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex—were determined using a particle size and potential analyzer. The parameters were set as follows: stable temperature 25℃, equilibration time 1 min, and 10–100 scans per cycle. Figure 1 The results show the particle size and zeta potential of β-Lg and its binary and ternary complexes; where a is the particle size and b is the zeta potential.

[0069] Depend on Figure 1 As shown in 'a', there are significant differences in particle size among the complexes (p < 0.05). The particle size of the binary complexes β-Lg, β-Lg-LAA, and β-Lg-FA, and the ternary complex β-Lg-LAA-FA show a gradually increasing trend. The ternary complex β-Lg-LAA-FA has the largest particle size, indicating that the introduction of LAA and FA leads to an increase in the particle size of the complex, suggesting that the binding of small molecules affects the molecular conformation of β-Lg. Figure 1 As shown in b, the Zeta potentials of all complexes are negative. Compared to β-Lg, the absolute values ​​of the Zeta potentials of all complexes are significantly increased (p < 0.05), indicating that the introduction of small molecules alters the surface charge distribution of the protein. The β-Lg-LAA-FA ternary complex has the largest absolute value of Zeta potential, indicating that it has the highest surface negative charge density and exhibits higher surface negative charge, thereby enhancing the electrostatic repulsion between particles.

[0070] (2) Folic acid binding efficiency and binding capacity

[0071] Fatty acid (FA) was used as an essential nutrient for the growth of the standard Lactobacillus strain (L. rhamnosus ATCC 7469). The binding efficiency (BE) and binding capacity (BC) of FA with β-Lg were evaluated by assessing cell growth. During testing, the β-Lg-FA binary complex and the β-Lg-LAA-FA ternary complex were subjected to ultrafiltration, and the retentate and permeate were collected. Each sample was diluted to an appropriate concentration and inoculated into ATCC 7469, then incubated at 37°C for 40 h. After incubation, the absorbance at 540 nm was measured, and the total FA and free FA content in the samples were calculated according to the standard curve. The BE and BC of FA were calculated using the following formulas: Binding efficiency BE (%) = (bound FA / total FA) × 100%; Binding capacity BC = bound FA / (β-Lg). Wherein, “bound FA” is the difference between total FA and free FA; “total FA” is the total amount of FA added; and “β-Lg” is the mass of β-Lg used to form the complex (FA is in μg and β-Lg is in mg). Figure 2The binding efficiency and binding capacity of β-Lg and its binary and ternary complexes are shown.

[0072] Figure 2 The results showed that β-Lg exhibited good binding performance to FA, with the binding efficiency of the β-Lg-FA binary complex reaching 46.18%, while the β-Lg-LAA-FA ternary complex significantly improved to 88.04% (p < 0.05). In terms of binding capacity, the β-Lg-FA binary complex and the β-Lg-LAA-FA ternary complex had binding values ​​of 167.51 and 257.40 μg / mg, respectively. This indicates that the introduction of LAA significantly promoted the binding of FA to β-Lg (p < 0.05), which is beneficial for the formation of non-covalent complexes.

[0073] (3) Scanning electron microscope

[0074] The microstructure of the samples was observed using a scanning electron microscope at an accelerating voltage of 5 kV. During testing, four samples—β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex—were fixed on the sample stage, sputtered with gold, and observed at 5000× magnification. Figure 3 Scanning electron microscope (SEM) images of β-Lg and its binary and ternary complexes.

[0075] like Figure 3 As shown, the β-Lg sample exhibits a relatively intact aggregate morphology. After the addition of LAA, the complex shows localized depressions or pores, indicating that the introduction of small molecules affects the protein surface morphology. In contrast, the β-Lg-FA complex maintains a relatively dense overall structure, but exhibits more pronounced morphological inhomogeneity and localized collapse. Compared to the binary complex, the β-Lg-LAA-FA ternary complex displays a more irregular aggregate structure, indicating more significant changes in its microstructure. Overall, the introduction of both LAA and FA can alter the microstructure characteristics of β-Lg.

[0076] (4) Fourier transform infrared spectrum

[0077] Fourier transform infrared (FTIR) spectroscopy was used to analyze the infrared spectra of four samples: β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex. The samples were mixed with dry potassium bromide and ground homogeneously, and transparent thin films were prepared using a pelleting method. The samples were then placed under FTIR spectrometry at 4000–400 cm⁻¹. -1 The wavelength range was scanned 16 times, with a resolution of 4 cm⁻¹. -1 . Figure 4Fourier transform infrared spectra of β-Lg and its binary and ternary complexes.

[0078] like Figure 4 As shown, amide A, amide I, amide II, and amide III are the four main characteristic peaks in the β-Lg spectrum. The amide A band primarily corresponds to the stretching vibration of NH4+. (3283.38 cm⁻¹) -1 Compared to the previous comparison, all complexes showed a blue shift in the amide A band to 3292.24 cm⁻¹. -1 This indicates that the introduction of small molecules alters the NH-related microenvironment, and that non-covalent interactions such as hydrogen bonding may participate in the formation of the complex. The amide I band mainly corresponds to the stretching vibration of C=O. β-Lg is measured from 1647.46 cm⁻¹. -1 The redshifted values ​​are 1643.19, 1641.77, and 1641.77 cm. -1 This indicates a change in the C=O-related structural environment, with a more pronounced shift in complexes containing FA. The amide II band primarily corresponds to the NH bending vibration and CN stretching vibration, with β-Lg shifting from 1533.54 cm⁻¹. -1 Blue shift. The amide III band shifted from 1262.97 cm⁻¹. -1 The red shift further reflects adjustments in the local structural environment of the protein. Overall, the introduction of LAA and FA did not lead to the formation of new covalent bonds, but it altered the characteristic peak positions of the β-Lg amide band through non-covalent interactions, indicating that they have a regulatory effect on the local structural environment of the protein. Compared with the binary complex, the β-Lg-LAA-FA ternary complex exhibited more pronounced peak position changes in some characteristic bands, suggesting that the co-existence of LAA and FA may have a synergistic regulatory effect on the local structural environment of β-Lg.

[0079] (5) Circular dichroism

[0080] Circular dichroism chromatography (CD) assesses structural changes in proteins after binding to small molecules by analyzing their secondary structure. The secondary structure of proteins in the complex was analyzed using a CD spectrometer. At room temperature, with phosphate buffer as a baseline, the sample (protein concentration 0.1 mg / mL) was scanned within a wavelength range of 190–260 nm to analyze the secondary structure content of the protein. Figure 5 The values ​​represent the circular dichroism chromatograms and secondary structure contents of β-Lg and its binary and ternary complexes; where a is the circular dichroism chromatogram and b is the secondary structure content.

[0081] like Figure 5 As shown in a, β-Lg exhibits a characteristic negative peak near 215 nm, indicating that its structure is predominantly β-sheet. Compared to β-Lg, the positions and intensities of the characteristic peaks in the 190-260 nm range of each complex changed, indicating that the introduction of LAA and FA affected the protein conformation. Figure 5 The effects of LAA and FA on the secondary structure composition of β-Lg were analyzed in section b. The contents of α-helix, β-sheet, β-turn, and random coil of β-Lg were 12.2%, 36.0%, 21.6%, and 30.2%, respectively. After the addition of LAA, the α-helix content decreased by 2.7%, and the β-sheet content increased by 1.5%, indicating that the addition of LAA affected the local structure of the protein. After the addition of FA, the α-helix content decreased by 5.2%, and the β-sheet content increased by 4%, indicating that FA had a greater regulatory effect on the secondary structure of β-Lg than LAA. In the β-Lg-LAA-FA ternary complex, the α-helix content decreased to 5.5%, and the β-sheet content reached 42%, indicating that the secondary structure of β-Lg was adjusted under the combined action of the two small molecules. It can be seen that the introduction of both LAA and FA can change the secondary structure composition of β-Lg, and the structural changes are more obvious in the ternary complex. This suggests that the introduction of small molecules may affect the conformational stability of proteins through non-covalent interactions, causing a certain degree of structural remodeling.

[0082] (6) Intrinsic fluorescence spectrum

[0083] The intrinsic fluorescence spectra of the samples were determined using a fluorescence spectrometer. At room temperature, β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex (protein concentration 0.1 mg / mL) were placed in 10 × 10 mm quartz cuvettes for measurement. The excitation wavelength was set to 280 nm, and the emission spectral scanning range was 350–450 nm. The excitation and emission slit widths were both 5 nm, the scan rate was 600 nm / min, and the data interval was 1.0 nm. Figure 6 The intrinsic fluorescence spectra of β-Lg and its binary and ternary complexes are shown.

[0084] like Figure 6 As shown, under 280 nm excitation conditions, the maximum emission peaks of β-Lg and its complexes are both located at approximately 375 nm, indicating that the fluorescence mainly originates from tryptophan residues. Compared to β-Lg, the fluorescence intensity of the complexes significantly decreased upon the addition of LAA or FA, indicating an interaction between the small molecule and the protein, leading to quenching of intrinsic fluorescence. Specifically, the fluorescence intensity decrease was greater in the β-Lg-FA binary complex than in the β-Lg-LAA binary complex, indicating a greater influence of FA on the protein's intrinsic fluorescence environment. In the β-Lg-LAA-FA ternary complex, the fluorescence intensity further decreased, indicating enhanced interactions under multi-component coexistence conditions. This demonstrates that the introduction of small molecules affects the conformational state of β-Lg and alters its fluorescence properties through non-covalent interactions.

[0085] (7) Ultraviolet absorption spectrum

[0086] The absorption spectra of the samples were determined using a UV-Vis spectrophotometer. β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex were dissolved in deionized water (protein concentration 0.1 mg / mL), and the wavelengths were scanned in the range of 260–400 nm. Figure 7 The UV absorption spectra of β-Lg and its binary and ternary complexes are shown.

[0087] like Figure 7 As shown, β-Lg and its complexes exhibit characteristic absorption peaks at approximately 280 nm in the near-ultraviolet region, primarily originating from aromatic residues such as tryptophan and tyrosine. Compared to β-Lg, the absorption intensities of each complex changed significantly, with the β-Lg-LAA-FA ternary complex showing the highest absorption intensity, followed by β-Lg-FA and β-Lg-LAA. This indicates that the introduction of different small molecules has varying effects on the molecular microenvironment of the aromatic residues in the protein. Furthermore, each complex showed a redshift compared to β-Lg, suggesting a change in the polarity of the environment surrounding the aromatic residues. The combined results suggest that the introduction of LAA and FA may influence the conformational state of β-Lg through non-covalent interactions, thereby altering the spectral characteristics of its aromatic residues.

[0088] (8) Surface hydrophobicity

[0089] Surface hydrophobicity analysis further reflects changes in the β-Lg surface microenvironment. During testing, solutions of β-Lg, the β-Lg-LAA binary complex, the β-Lg-FA binary complex, and the β-Lg-LAA-FA ternary complex were diluted to protein concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL, respectively. Four mL of each concentration sample was taken and 20 μL of 8 mM 8-aniline-1-naphthalenesulfonic acid (ANS) was added. The reaction was carried out in the dark for 15 min. The fluorescence intensity of the samples was measured using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. Surface hydrophobicity was characterized by the linear regression slope (H0) of protein concentration versus fluorescence intensity. Figure 8 The results show the determination of the surface hydrophobicity of β-Lg and its binary and ternary complexes.

[0090] like Figure 8As shown, compared with β-Lg, the H0 values ​​of all complexes were significantly reduced (p < 0.05), exhibiting a decreasing trend of β-Lg > β-Lg-LAA > β-Lg-FA > β-Lg-LAA-FA, indicating that the introduction of LAA and FA reduced the exposure of hydrophobic sites on the protein surface. In contrast, the H0 value of the FA-treated group decreased more significantly, indicating a higher degree of influence on the protein surface microenvironment. This suggests that the exposure of hydrophobic regions on the protein surface is further reduced under the combined action of multiple components, and the surface microenvironment undergoes more significant changes.

[0091] (9) Thermal stability analysis

[0092] The thermal stability of β-Lg and its complexes was characterized by differential scanning calorimetry. Figure 9 Differential scanning calorimetry (DSC) spectra of β-Lg and its binary and ternary complexes.

[0093] like Figure 9 As shown, all samples exhibited significant endothermic peaks in the 70-80℃ range, indicating thermal denaturation of β-Lg. The thermal denaturation temperature (Tp) of β-Lg without the addition of small molecules was 71.59℃. With the introduction of LAA or FA, Tp increased to 72.08℃ and 72.43℃, respectively; in the β-Lg-LAA-FA ternary complex, Tp further increased to 73.40℃, indicating a gradual increase in the thermal stability of the complex. In summary, the coexistence of LAA and FA, through a synergistic effect, contributes to the higher thermal stability of the ternary complex.

[0094] III. Functional Characteristics Analysis of the Complex

[0095] (1) Antioxidant properties

[0096] The antioxidant properties of each complex sample were evaluated by assessing its ABTS radical scavenging ability, DPPH radical scavenging ability, and iron ion reducing ability. Specifically, ① the DPPH radical scavenging ability was determined as follows: DPPH was dissolved in methanol, and the concentration was adjusted to achieve an absorbance of 0.7 at 517 nm, which served as the DPPH working solution. A certain volume of solutions (1 mg / mL) of the β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex were mixed with 0.2 mM DPPH working solution and reacted at room temperature in the dark for 30 min. After the reaction, the absorbance of the mixed solution was measured at 517 nm and recorded as A. The absorbance obtained by replacing the DPPH working solution with methanol, with the other procedures remaining the same, was recorded as A1 (control group). The absorbance obtained by mixing the DPPH working solution with phosphate buffer was recorded as A0 (blank group). The DPPH free radical scavenging capacity was calculated using the following formula: DPPH free radical scavenging capacity (%) = (1 - (A - A1) / A0) × 100%. Where A, A1, and A0 represent the absorbance values ​​of the sample group, control group, and blank group at a wavelength of 517 nm, respectively. ② The ABTS free radical scavenging capacity was determined as follows: 7 mM ABTS stock solution was mixed with an equal volume of 2.45 mM potassium persulfate solution and reacted for 12 h under light-protected conditions to obtain the ABTS working solution. The absorbance of the ABTS working solution at 734 nm was adjusted to 0.7 ± 0.02. 50 μL of solutions of β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex (10 mg / mL) were mixed with 150 μL of the ABTS working solution, shaken thoroughly, and reacted for 10 min under light-protected conditions. The absorbance was measured at 734 nm and recorded as A. The sample solution was replaced with phosphate buffer, and the rest of the operation was the same. The absorbance was measured and recorded as A0. The ABTS free radical scavenging ability was calculated according to the following formula: ABTS free radical scavenging ability (%) = (1-A / A0) × 100%. A and A0 represent the absorbance values ​​of the sample group and the control group at a wavelength of 734 nm, respectively. ③ The method for determining the iron ion reducing power (FRAP) was as follows: The FRAP working solution was prepared by mixing 10 mM 2,4,6-tris(2-pyridyl)-1,3,5-triazine solution (dissolved in 40 mM HCl), 20 mM FeCl3 solution and 300 mM acetate buffer (pH 3.6) in a volume ratio of 10:1:1 (v / v / v). Take 5 μL of β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex and β-Lg-LAA-FA ternary complex solutions (10 mg / mL) and mix them with 180 μL of FRAP working solution. After thorough shaking, react at 37℃ for 5 min and measure the absorbance at 593 nm.A standard curve was plotted using FeSO4·7H2O as the standard. FRAP results are expressed as Fe per gram of sample dry weight. 2+ Equivalent, expressed in μmol FeSO4·7H2O / g. Figure 10 The results show the antioxidant properties of β-Lg and its binary and ternary complexes; where a represents ABTS radical scavenging ability, b represents DPPH radical scavenging ability, and c represents iron ion reduction antioxidant ability.

[0097] like Figure 10 As shown in figures a~c, the ABTS radical scavenging ability results indicate that, compared with β-Lg (17.46%), the β-Lg-LAA binary complex (64.81%), and the β-Lg-FA binary complex (23.42%), the β-Lg-LAA-FA ternary complex (78.87%) showed a significantly increased antioxidant activity (p < 0.05), consistent with the DPPH radical scavenging ability and ferric ion reducing ability. The further enhanced antioxidant capacity of the ternary complex suggests a synergistic effect between LAA and FA, resulting in superior antioxidant properties in the β-Lg-LAA-FA ternary complex.

[0098] (2) Emulsifying properties

[0099] The emulsifying properties of the samples were evaluated using the Emulsifying Activity Index (EAI) and the Emulsifying Stability Index (ESI). EAI reflects the adsorption capacity of proteins at the oil-water interface, while ESI characterizes the emulsion's resistance to stratification. During testing, 6 mL of each of the following compounds (6 mL, 0.5 mg / mL): β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex, were mixed with 2 mL of olive oil and homogenized at 10000 rpm for 1 min. At 0 min and 10 min after homogenization, 50 μL of the emulsion was rapidly added to 5 mL of 0.1% (w / v) sodium dodecyl sulfate solution for dilution, and the absorbance was measured at 500 nm. The absorbance values ​​measured at 0 min and 10 min were recorded as A0 and A1, respectively. 10 EAI and ESI are calculated using the following formulas: EAI(m 2 / g)=(2×2.303×DF×A0) / (c×0.25×10000); ESI(min)=A0 / (A0-A 10 )×10. Where 2.303 is the constant T, DF is the dilution factor, and A0 and A 10 The values ​​represent the absorbance of the emulsion at the initial moment and after standing for 10 min, respectively. c is the sample concentration (g / mL), and 0.25 is the volume fraction of the oil phase. Figure 11The results show the emulsifying activity index and emulsifying stability index of β-Lg and its binary and ternary complexes; where a is the emulsifying activity index and b is the emulsifying stability index.

[0100] like Figure 11 As shown in a and b, the introduction of FA significantly affected the emulsifying behavior of β-Lg (p < 0.05). Regarding EAI, the EAI of the β-Lg, β-Lg-LAA binary complex, β-Lg-FA binary complex, and β-Lg-LAA-FA ternary complex were 29.81, 65.69, 62.39, and 69.53 m² / g, respectively. Compared with β-Lg, the EAI of each complex was significantly increased, with the β-Lg-LAA-FA ternary complex showing the highest value, indicating that the introduction of LAA and FA enhances the protein's adsorption capacity at the oil-water interface. Regarding ESI, compared with β-Lg, the ESI of the β-Lg-LAA binary complex decreased, while the ESI of the β-Lg-LAA-FA ternary complex was higher. However, the ESI of the β-Lg-LAA binary complex was not significantly different from that of β-Lg (p > 0.05). Among them, the β-Lg-LAA-FA ternary complex exhibited the highest ESI, indicating that different small molecules have different effects on emulsion stability. The change in ESI may be related to the regulation of the β-Lg interface structure by LAA and FA, and show a synergistic effect in the ternary system.

[0101] (3) Foaming properties

[0102] Foaming ability is an important indicator for evaluating protein functional properties. The foaming capacity (FC) and foam stability (FS) of the samples were evaluated. 10 mL of the sample (V0, protein concentration 1 mg / mL) was homogenized at 10000 rpm for 1 min. The foaming properties at rest (V0) were observed after 0 min. T ) and 10 minutes later (V t The sample volume, FC, and FS are calculated using the following formula: FC(%) = (V T -V0) / V0 ×100%;FS(%)=(V t -V0) / (V T -V0) ×100%. Where V0 is the initial volume of the sample before homogenization, V T V is the total volume of the system measured immediately after homogenization. t The total volume is the volume after standing for 10 minutes. Figure 12 The foaming capacity and foam stability of β-Lg and its binary and ternary complexes are given by α, where a represents foaming capacity and b represents foam stability.

[0103] like Figure 12As shown in figures a and b, the introduction of LAA and FA significantly affected the foaming behavior of β-Lg. Regarding FC (foaming capacity), the FC values ​​of the binary complexes β-Lg, β-Lg-LAA, and β-Lg-FA, and the ternary complex β-Lg-LAA-FA were 39.67%, 23.67%, 25.33%, and 32.67%, respectively. Compared to β-Lg, the FC of all complex systems decreased, with the β-Lg-LAA binary complex showing the most significant decrease, indicating that the introduction of small molecules reduced the protein's adsorption and spreading ability at the gas-liquid interface to some extent. Regarding FS (foaming efficiency), all complex systems showed a significant overall increasing trend. Compared to β-Lg, the FS of the binary complexes β-Lg-LAA, β-Lg-FA, and β-Lg-LAA-FA increased by 6.00%, 12.78%, and 21.94%, respectively, with the ternary complex showing the most significant increase (p < 0.05). Overall, the introduction of LAA and FA reduced the FC of β-Lg but increased the FS, with the ternary complex showing better performance in stabilizing the foam structure.

[0104] (4) Folic acid stability

[0105] Fatty acid (FA) is unstable in nature, and its main influencing factors include light, heat, and pH. Therefore, this study systematically evaluated the FA retention rates of free FA, the β-Lg-FA binary complex, and the β-Lg-LAA-FA ternary complex under different UV irradiation, heating time, and pH conditions to investigate the effect of the complexes on FA stability. Specifically, the FA content in the β-Lg-FA binary complex and the β-Lg-LAA-FA ternary complex was determined under different treatment conditions, following the same procedures as described above, to evaluate the stability of FA in the complexes. Simultaneously, the content of free FA was determined under the same conditions using phosphate buffer instead of β-Lg as a control group. The specific treatment conditions for each sample are as follows: ① Photostability. Under dark, room temperature conditions, the β-Lg-FA binary complex, the β-Lg-LAA-FA ternary complex, and the free FA sample were irradiated under UV lamps (8 W) at 365 nm and 254 nm, respectively, with a distance of 9 cm between the UV light source and the front surface of the sample vial. Samples were taken and measured at 0, 15, 30, 60, 120, 240, and 360 min, respectively. ② Thermal stability. Under dark conditions, the β-Lg-FA binary complex, β-Lg-LAA-FA ternary complex, and free FA samples were placed in a 90℃ water bath for 0, 20, 40, 60, 80, 100, and 120 min, respectively, and then samples were taken and measured. ③ Acid-base stability. Under dark room temperature conditions, the β-Lg-FA binary complex, β-Lg-LAA-FA ternary complex, and free FA sample solutions were adjusted to different pH values ​​(3.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 11.0) using 1.0 M NaOH and 1.0 M HCl, and samples were taken and measured after equilibration for 2 h. The retention rate of FA after measurement was calculated using the following formula: Retention rate (%) = (C t / C0) ×100%. Where, C t C0 represents the concentration of FA in the treated sample, and C0 represents the initial concentration of FA. Figure 13 The changes in FA retention rates of free FA and its binary complex β-Lg-FA and ternary complex β-Lg-LAA-FA under 254 nm and 365 nm UV irradiation, different heating conditions, and pH conditions are shown. Among them, a represents 254 nm UV irradiation, b represents 365 nm UV irradiation, c represents heating conditions, and d represents pH conditions.

[0106] like Figure 13As shown in figures a and b, under 254 nm UV light, the retention rate of free FA was close to 0 after 120 min, while the retention rate of the β-Lg-FA binary complex also decreased to close to 0 after 6 h of extended irradiation. In contrast, under 365 nm UV light, the retention rate of free FA decreased to below 10% after 6 h of irradiation, while the retention rate of the β-Lg-FA binary complex was 55.76%. Furthermore, the β-Lg-LAA-FA ternary complex maintained a FA retention rate of over 45% after continuous irradiation with 254 nm and 365 nm UV light for 6 h, both higher than that of free FA and the binary complex. This indicates that the combined effect of β-Lg and LAA can effectively improve the photostability of FA. Figure 13 As shown in Figure c, the thermal stability of FA varied significantly among different systems under a 90℃ heating condition. Free FA degraded the fastest, with a retention rate below 50% after 60 min and near-complete degradation after 100 min. In contrast, the β-Lg-FA binary complex maintained a retention rate of 68.32% after 120 min of heating, indicating that the introduction of β-Lg helps improve the thermal stability of FA. The β-Lg-LAA-FA ternary complex exhibited superior protective effect, achieving an FA retention rate of 85.42% under the same treatment conditions, higher than that of free FA and the binary complex. This suggests that the addition of LAA enhances the thermal protection of FA by the complex. Figure 13 As shown in d, the pH stability results indicate that the stability of FA is affected by the acidity or alkalinity of the environment. Overall, FA exhibits lower stability under acidic conditions and higher stability under neutral and alkaline conditions. At pH 3.0, the retention rate of free FA is 54.43%, indicating that it is prone to degradation under acidic conditions. In contrast, the retention rates of the β-Lg-FA binary complex and the β-Lg-LAA-FA ternary complex are 67.12% and 70.84%, respectively. With increasing pH, the FA retention rate in all systems shows an upward trend, with the ternary complex consistently maintaining a higher level. Within the pH range of 7.0-11.0, the FA retention rate of the β-Lg-LAA-FA ternary complex is 80.93%-98.88%, while that of free FA and the β-Lg-FA binary complex is 76.78%-89.68% and 78.48%-92.23%, respectively. At pH 11.0, the retention rate of the ternary complex reached 98.88%, demonstrating good adaptability to alkaline environments. The results indicate that the formation of the complex helps improve the stability of fatty acids (FAs) under different pH conditions.

[0107] The stability results under UV light, heating, and pH conditions indicate that the β-Lg-LAA-FA ternary complex exhibits superior protection for fatty acids (FA). The introduction of β-Lg helps slow down the degradation of FA under external environmental conditions, while the addition of LAA enhances the protective effect of the system. The combined effect of these two factors results in a high FA retention rate for the ternary complex under various treatment conditions.

[0108] Example 2: Preparation, structural characterization and stability analysis of high internal phase dual emulsion

[0109] I. Preparation of High Internal Phase Dual Emulsions

[0110] High internal phase dual emulsions (TBLF-HDEs) loaded with the β-Lg-LAA-FA ternary complex were prepared using a two-step emulsification method. The internal aqueous phase (W1) was prepared by dissolving the β-Lg-LAA-FA ternary complex from Example 1 in distilled water (0.001%, w / w) under light-protected conditions and hydrating overnight at 4°C. The oil phase (O) mainly consisted of medium-chain triglycerides, olive oil, and linseed oil in a 1:1:1 mass ratio, with 1.5% (w / w) of polyglycerol ricinoleate (PGPR) added to the total oil. The external aqueous phase (W2) consisted of a 5% (w / w) aqueous pectin solution.

[0111] In various emulsion systems, the internal aqueous phase accounted for 20% (w / w), and the oil phase addition amounts were 54%, 58%, 62%, 66%, and 70% (w / w), respectively. The internal aqueous phase was slowly added to the corresponding mass ratio of the oil phase, and the mixture was sheared at 10,000 rpm for 2 min in a high-speed shear emulsifier to obtain the W1 / O primary emulsion. The obtained W1 / O primary emulsion was mixed with the external aqueous phase according to the mass composition of each system, with the external aqueous phase accounting for the remaining mass ratio of the system excluding the internal aqueous and oil phases (i.e., 26%, 22%, 18%, 14%, and 10%, w / w). Subsequently, the mixture was sheared at 10,000 rpm for 2 min in a high-speed shear emulsifier to form the W1 / O / W2 crude emulsion. Finally, the crude emulsion was treated three times at 150 MPa using a high-pressure microjet apparatus to obtain high internal phase dual emulsions of the β-Lg-LAA-FA ternary complex supported on oil phase with contents of 54%, 58%, 62%, 66% and 70% (w / w), respectively, denoted as TBLF-HDEs.

[0112] II. Structural Characterization of High Internal Phase Dual Emulsions

[0113] (1) Optical microscope and appearance

[0114] The microstructure of five TBLF-HDEs with different oil phase contents was observed using an optical microscope, and the appearance of TBLF-HDEs with different oil phase contents was recorded. Figure 14Microscopic and visual images of high internal phase double emulsions with different oil phase contents.

[0115] like Figure 14 As shown, TBLF-HDEs systems with different oil phase contents (54%, 58%, 62%, 66%, and 70%) were prepared using microfluidic technology. Optical micrographs revealed that, except for the system with 54% oil phase content, the other emulsions exhibited varying degrees of droplet bridging and flocculation, with the 62% and 70% samples showing more pronounced effects. In contrast, when the oil phase content was 66%, droplet bridging and flocculation were significantly reduced, with no obvious bridging or large-scale flocculation observed, and the emulsion structure was the most homogeneous, indicating that droplets could form a more stable and ordered spatial arrangement under this condition. Visual results showed that all emulsions were milky white, and no obvious phase separation was observed, indicating good initial dispersibility. With increasing oil phase content, the opacity of the samples gradually increased, with the 66% sample exhibiting the most uniform appearance.

[0116] (2) Laser confocal scanning microscope

[0117] Laser confocal scanning microscopy (LCSMS) can clearly characterize the spatial distribution and interfacial structure of emulsion droplets. The microstructure of TBLF-HDEs with different oil phase contents was characterized using LCSMS. First, five TBLF-HDEs with different oil phase contents were diluted at a mass ratio of 1:4. 400 μL of the sample was added to 1.6 mL of ultrapure water to obtain a homogeneous dispersion. Then, a mixed staining solution of Nile Red (0.1%, w / v) and Nile Blue (1%, w / v) was prepared. 1 mL of the diluted sample was added to 200 μL of the mixed staining solution, gently shaken to disperse the dye, and allowed to stand in the dark for 30 min. Subsequently, 8 μL of the stained sample was placed on a clean glass slide, carefully covered with a coverslip, and observed using a LCSMS.

[0118] The stained emulsion system was observed using laser confocal scanning microscopy. The oil phase stained with Nile Red appeared red, while the protein stained with Nile Blue appeared green. All continuous phases on the outside of the emulsion were green, while the internal structure exhibited a multilayered distribution of red oil droplets encasing a green aqueous phase, indicating a typical W / O / W dual emulsion structure. Structurally, when the oil phase content increased from 54% to 62%, the oil droplet size did not change significantly; however, when the oil phase content increased to 66% and 70%, the oil droplet size increased, indicating enhanced inter-droplet interactions and structural reorganization under high oil phase conditions.

[0119] (3) Encapsulation efficiency and loading of folic acid

[0120] To evaluate the encapsulation performance of TBLF-HDEs for fatty acids (FA), the encapsulation efficiency (EE) and loading (LC) of the emulsion system were determined, with FA mainly distributed in the inner aqueous phase. Specifically, taking advantage of FA's essential nutrient status for the growth of *L. rhamnosus* ATCC 7469, the EE and LC of FA in the emulsion were determined based on its growth status. Five TBLF-HDEs with different oil phase contents were mixed with anhydrous ethanol and stirred. The mixtures were centrifuged at 2500 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.22 μm organic filter membrane, diluted to appropriate concentrations, and then inoculated into ATCC 7469. The mixtures were incubated at 37°C for 40 h. After incubation, the absorbance was measured at 540 nm, and the FA content in the sample was calculated based on the established standard curve. The EE and LC of the emulsion were calculated using the following formula: EE (%) = ((C total -C outer ) / C total )×100%;LC%= ( (C total -C outer ) / C phase )×100%. Where, C total The initial amount of FA added to the aqueous phase of the double emulsion, C outer C refers to the amount of unencapsulated fatty acids (FA) in the aqueous phase of a double emulsion. phase This refers to the amount of the inner aqueous phase in a double emulsion. Figure 15 The encapsulation efficiency and loading of high internal phase dual emulsions with different oil phase contents are presented.

[0121] like Figure 15 As shown, the encapsulation efficiencies of TBLF-HDEs with oil phase contents of 54%, 58%, 62%, 66%, and 70% were 75.05%, 81.26%, 84.22%, 90.46%, and 77.97% (p < 0.05), respectively, showing an overall trend of first increasing and then decreasing, with EE and LC showing the same trend. Within the oil phase content range of 54%-66%, EE and LC gradually increased with increasing oil phase content, indicating that appropriately increasing the oil phase content is beneficial to improving the system's ability to retain FA. When the oil phase content was 66%, EE and LC reached their maximum values ​​of 90.46% and 0.00023%, respectively. This demonstrates that the emulsion system of the present invention exhibits high encapsulation performance.

[0122] (4) Particle size and Zeta potential

[0123] Particle size in emulsion samples serves as an indicator of droplet aggregation and significantly impacts emulsion stability. The particle size and zeta potential of five TBLF-HDEs with different oil phase contents were determined using a laser particle size and potential analyzer. The volume-weighted average particle size (D...) was... 4,3 Calculate using the following formula: D 4,3 =∑n i d i 4 / ∑n i d i 3 Where n i d represents the number of droplets. i denoted as the droplet diameter. Figure 16 The results show the particle size and Zeta potential of high internal phase dual emulsions with different oil phase contents; where a is the particle size and b is the Zeta potential.

[0124] like Figure 16 As shown in Figure a, within the oil phase content range of 54%-62%, the droplet size gradually increases with increasing oil phase content. When the oil phase content is 66%, the droplet size decreases significantly (p < 0.05) and reaches a relatively small level. Figure 16 As shown in b, the absolute values ​​of the zeta potentials of each emulsion system all exceed 50 mV, indicating that the systems all possess good stability. As the oil phase content increases from 54% to 70%, the absolute value of the zeta potential shows a trend of first increasing, then decreasing, and then increasing again, reaching its maximum value (-61.01 mV) at 66%. Combining the particle size and zeta potential results, it can be concluded that an oil phase content of 66% exhibits superior stability.

[0125] III. Stability Analysis of High Internal Phase Dual Emulsions

[0126] (1) Structural stability

[0127] Five TBLF-HDEs with different oil phase contents were placed in glass sample vials and stored at 4℃ and 25℃ for 28 days, respectively, to evaluate the effect of different oil phase contents on the structural stability of TBLF-HDEs during storage. The particle size of TBLF-HDEs was measured at different time points (0, 7, 14, 21, and 28 days), and the appearance of the emulsion was observed. Figure 17 The particle size changes of high internal phase dual emulsions with different oil phase contents during storage at different temperatures are shown; where a is the particle size at 4℃ and b is the particle size at 25℃.

[0128] like Figure 17As shown in a and b, after 28 days of storage at 4℃ and 25℃, the particle size of the emulsion only increased to varying degrees. Further comparison of different storage temperatures shows that although there are some differences in particle size under 4℃ and 25℃ conditions, their changing trends are consistent and the growth rates are similar, indicating that TBLF-HDEs can maintain a relatively stable structural state under different storage conditions.

[0129] Further analysis of the emulsion's appearance revealed that its microstructure remained intact after storage at 4°C and 25°C, with no obvious droplet aggregation or structural damage observed. This indicates that the emulsion did not undergo significant aggregation or demulsification during storage and maintained good overall stability. In summary, these results show that under conditions of 4°C and 25°C, the droplets in TBLF-HDEs can maintain an intact double emulsion structure for at least 28 days, demonstrating the excellent storage stability of TBLF-HDEs.

[0130] (2) Oxidative stability

[0131] Peroxide value (POV) characterizes the degree of primary lipid oxidation, while malondialdehyde (MDA), as a typical secondary oxidation product, is used to evaluate the level of further lipid oxidation. Five TBLF-HDEs with different oil phase contents and pure oil phases were stored at 4℃ and 25℃ for 28 days, respectively. The POV and MDA contents of the emulsions were measured at different time points (0, 7, 14, 21, and 28 days). The POV determination procedure was as follows: 0.50 g of emulsion was dissolved in 5 mL of distilled water. 10 mL of a 3:1 (v / v) mixture of isooctane and isopropanol was added to 0.1 mL of the diluted emulsion. After vortexing and centrifugation at 3500 rpm for 5 min, 1 mL of the supernatant was added to 2.8 mL of a 2:1 (v / v) mixture of n-butanol and methanol. Then, 15 μL of NH4SCN solution and 15 μL of Fe were added sequentially. 2+ Mix thoroughly and react in the dark for 20 min. Measure the absorbance at 510 nm and calculate the POV based on the standard curve. MDA content was determined using a malondialdehyde (MDA) test kit according to the manufacturer's instructions. The results were compared with those obtained from the pure oil phase test.

[0132] Figure 18 The results show the changes in POV in high internal phase double emulsions with different oil phase contents during 28 days of storage at 4℃ and 25℃; where a represents the POV change at 4℃ and b represents the POV change at 25℃. Figure 19 The results show the changes in MDA content in high internal phase double emulsions with different oil phase contents during 28 days of storage at 4℃ and 25℃; where a represents the MDA change at 4℃ and b represents the MDA change at 25℃.

[0133] like Figure 18 a, b and Figure 19 As shown in Figures a and b, under storage conditions of 4℃ and 25℃, the POV and MDA content of TBLF-HDEs were lower than those of the pure oil phase, indicating that the dual emulsion system can effectively delay lipid oxidation and reduce the effect of pro-oxidative factors on the oil phase. Combined with the storage stability results, it can be seen that during the 28-day storage period, TBLF-HDEs maintained good droplet structure integrity at both 4℃ and 25℃, thus helping to reduce the degree of oxidation. This confirms that the encapsulation of the oil phase in TBLF-HDEs can delay the lipid oxidation process. At the end of storage, the POV of the pure oil phase at 4℃ was approximately three times that of the dual emulsion, and the difference further widened at 25℃. The MDA content results also showed a similar trend, indicating that the dual emulsion has a significant inhibitory effect on lipid oxidation. Furthermore, the oxidative stability of TBLF-HDEs mainly depends on the droplet structure and the integrity of the interfacial film. Appropriate oil phase content helps to form an effective interfacial barrier, thereby reducing the degree of lipid oxidation.

[0134] (3) Changes in folic acid encapsulation rate

[0135] Five TBLF-HDEs with different oil phase contents were stored at 4℃ and 25℃, respectively. The EE of FA was measured on days 0, 7, 14, 21 and 28. The EE changes of FA in the emulsion at the two storage temperatures were plotted. The EE measurement method was the same as that in the emulsion structure characterization section. Figure 20 The encapsulation efficiency of high internal phase double emulsions with different oil phase contents was shown during 28 days of storage at 4℃ and 25℃; where a represents the encapsulation efficiency at 4℃ and b represents the encapsulation efficiency at 25℃.

[0136] like Figure 20 As shown, the FA encapsulation efficiency gradually decreased in samples with different oil phase contents as storage time increased. Since the overall emulsion structure remained intact during storage, this indicates that FA loss mainly stemmed from chemical degradation rather than large-scale structural damage. Taking the TBLF-HDEs with the best encapsulation efficiency (66% oil phase content) as an example, its initial FA encapsulation efficiency was 90.33%, which decreased to 81.03% and 74.92% after 28 days of storage at 4℃ and 25℃, respectively. This demonstrates that the system can maintain a high encapsulation level under different storage conditions. Therefore, TBLF-HDEs can maintain the integrity of the dual emulsion structure for at least 28 days under refrigerated and room temperature conditions, but the FA encapsulation efficiency gradually decreases with storage time and is significantly affected by temperature.

[0137] (4) Freeze-thaw stability

[0138] Freeze-thaw stability is a key factor affecting the quality of frozen foods. This experiment evaluated the freeze-thaw stability of TBLF-HDEs with five different oil phase contents. The emulsions were placed in glass bottles and placed at -20°C for 22 h, followed by thawing in a 37°C water bath for 2 h. Changes in emulsion particle size before and after freeze-thaw were recorded, and the appearance of the samples before and after freeze-thaw was photographed. Figure 21 The images show the particle size and appearance of high internal phase dual emulsions with different oil phase contents after one freeze-thaw cycle; where a is the particle size and b is the appearance.

[0139] like Figure 21 As shown in a and b, all five TBLF-HDEs maintained a uniform appearance after one freeze-thaw cycle, with no obvious phase separation observed. The particle size of each sample increased only slightly before and after freeze-thaw, indicating that the system as a whole still possesses good structural stability. There were some differences among samples with different oil phase contents; the 66% oil phase system showed the smallest particle size change, while the 54% oil phase sample showed the largest increase, indicating that a higher oil phase content contributes to maintaining structural stability to some extent. Furthermore, the TBLF-HDEs of this invention maintained a relatively uniform structure after freeze-thaw, indicating good freeze-thaw stability. Good freeze-thaw stability helps avoid leakage of the internal aqueous phase due to ice crystal breakage, thereby reducing FA loss and improving the system's application potential in frozen foods.

[0140] (5) Thermal stability

[0141] This experiment evaluated the thermal stability of TBLF-HDEs with five different oil phase contents. The emulsions were placed in glass bottles and in a 90°C water bath for 30 min, followed by rapid cooling in an ice-water bath. Changes in emulsion particle size before and after heating were recorded, and the appearance of the samples before and after heating was photographed. Figure 22 The images show the particle size and appearance of high internal phase dual emulsions with different oil phase contents after heat treatment; where a represents the particle size and b represents the appearance.

[0142] like Figure 22 As shown in figures a and b, the appearance of each group of TBLF-HDEs did not change significantly after heating to 90℃, with only slight stratification. No thermal instability phenomena such as oil phase floating, internal water phase leakage, or interface rupture were observed, indicating that the system can maintain structural integrity under high-temperature conditions. Particle size determination results showed that the droplet size of all samples did not increase significantly, and no obvious aggregation or flocculation was observed. The TBLF-HDEs with 66% oil phase content showed the smallest particle size change, indicating that the oil phase interface layer formed by this oil phase content has high interfacial strength and thermal resistance. In addition, the particle size did not increase significantly after heating, indicating that the droplets did not undergo significant agglomeration or structural differentiation, proving its good thermal stability. Therefore, TBLF-HDEs with 66% oil phase content are beneficial for constructing high internal phase dual emulsions with strong heat resistance and have certain thermal processing applicability.

[0143] (6) Centrifugal stability

[0144] This experiment evaluated the centrifugal stability of TBLF-HDEs by accelerating the emulsion separation process through centrifugation. The emulsion was centrifuged at 10000g for 30 min, and the emulsion separation index (CI) was measured after centrifugation. Changes in emulsion particle size before and after centrifugation were recorded, and the appearance of the centrifuged sample was photographed. The CI determination method was as follows: after centrifugation, the sample was allowed to stand until clear stratification occurred, and the height H of the upper emulsion layer was measured. e And measure the total height H of the emulsion. t CI is calculated using the following formula: CI(%) = (H e / H t ) ×100%. Where, H e It is the height of the emulsion layer, H t It is the total height of the emulsion. Figure 23 Particle size, appearance, and emulsion index of high internal phase double emulsions with different oil phase contents after centrifugation; where a is particle size, b is appearance, and c is emulsion index.

[0145] like Figure 23 As shown in figures a~c, after centrifugation, a certain degree of oil phase precipitation appeared in the upper layer of each group of samples, indicating that the system exhibited varying degrees of demulsification under strong external forces. With increasing oil phase content, the CI value gradually decreased, indicating that a high oil phase content helps improve the system's resistance to centrifugal force, resulting in higher centrifugal stability of the emulsion. Particle size analysis results showed that the droplet size of each group of TBLF-HDEs shifted towards larger sizes after centrifugation, with the largest increase observed in the 54% oil phase content group, while the 66% oil phase content group exhibited the smallest particle size, indicating that the lower oil phase system is more prone to droplet aggregation under centrifugation. Therefore, TBLF-HDEs with higher oil phase content exhibited better centrifugal stability, indicating stronger interfacial resistance to damage and the ability to reduce the risk of FA leakage under adverse conditions.

[0146] (7) Rheological testing

[0147] The rheological testing method was as follows: The rheological properties of five TBLF-HDEs with different oil phase contents were evaluated using a MARS40 rotational rheometer. All tests were conducted at 25°C. Strain scanning was performed on the emulsions at a frequency of 1 Hz and a strain range of 0.01%–100%. Based on the test results, suitable strain values ​​within the linear viscoelastic region were determined for subsequent frequency scanning and shear testing to obtain the changes in storage modulus (G′) and loss modulus (G″) with frequency. The viscoelasticity of the emulsions was also determined by frequency scanning, with a test frequency range of 1–100 Hz. Finally, shear testing was performed at 1 Hz to determine the apparent viscosity of the emulsions, with a shear rate range of 0.01–100 s⁻¹. -1 . Figure 24 The results show the storage modulus, loss modulus, and apparent viscosity of high internal phase dual emulsions with different oil phase contents; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity.

[0148] like Figure 24 As shown in a~c, the G′ values ​​of all samples were higher than G″, and both G′ and G″ gradually increased with increasing frequency, indicating that the emulsion system was dominated by elastic behavior and exhibited obvious viscoelastic characteristics, which helped maintain system stability. Comparison of different samples revealed that the 66% group showed the highest G′ and G″ values ​​across the entire frequency range, indicating higher structural strength. Apparent viscosity results showed that all emulsion samples decreased significantly with increasing shear rate, exhibiting typical shear-thinning behavior. Further comparison of different samples revealed that the 66% group showed the highest apparent viscosity across the entire shear rate range, implying stronger droplet interactions and better system stability. Therefore, the oil phase content affects the rheological properties of the emulsion, with the 66% group exhibiting a stronger viscoelastic structure and superior system stability, demonstrating the best rheological performance.

[0149] Example 3: Preparation and Characterization of Cream Emulsion and Whipped Cream

[0150] I. Preparation of Cream Emulsion and Whipped Cream

[0151] The preparation method of the cream emulsion is as follows: ① Based on the total mass of the cream emulsion as 100% w / v, sodium caseinate (1%) was dispersed in deionized water (52.85%), and stirred and heated at 85°C for 20 min to fully hydrate, obtaining a sodium caseinate solution. Sucrose (10%) and xanthan gum (0.2%) were added to the above sodium caseinate solution, and stirring was continued at 85°C for 20 min to form a stable viscoelastic system in the aqueous phase; sodium citrate (0.1%) and a water-soluble emulsifier (specifically glyceryl monostearate, 0.05%) were further added, and stirring was carried out at 85°C for 15 min to obtain a homogeneous aqueous phase. ② The oil phase consisted of butter and a high internal phase dual emulsion (TBLF-HDEs) with an oil phase content of 66% prepared in Example 2, with 0.8% PGPR added, and the oil phase was fully mixed at 85°C. ③ Using a fixed oil phase content of 35%, seven groups of oil phase samples were set up. The control group consisted of 35% butter, while the other samples had TBLF-HDEs replacing a portion of the butter at substitution ratios of 0%, 10%, 20%, 30%, 40%, 50%, and 60%, respectively. This resulted in seven cream emulsion systems with different fat substitution levels. Taking the preparation of 100 g of cream emulsion system as an example, Table 1 lists the specific amounts of TBLF-HDEs emulsion and butter added at different substitution ratios.

[0152] Table 1. Substitution ratio and specific amount of cream added to the samples

[0153] In the emulsion preparation process, the preheated oil phase was slowly added to the aqueous phase at a constant rate under 85°C, and stirred for 30 min to complete the initial emulsification. Subsequently, homogenization was performed by shearing at 10,000 r / min for 5 min using an IKA high-speed shear mill. The resulting emulsion was immediately cooled and aged at 4°C for 24 h to form a structurally stable cream emulsion sample.

[0154] The method for preparing whipped cream is as follows: Whipped cream is prepared under low temperature conditions, with the sample temperature controlled below 10℃. The seven groups of cream emulsions prepared above are placed in a mixer and whipped at high speed for 1 minute to form a preliminary structure, and then the speed is reduced to low and whipped for 25 seconds to obtain seven stable whipped cream samples.

[0155] II. Characterization of the properties of cream emulsion

[0156] (1) Analysis of appearance and theoretical fat reduction

[0157] Take 10 mL of freshly prepared cream emulsion in a petri dish and photograph its appearance parallel to the dish. Furthermore, the theoretical fat reduction for samples with different substitution ratios was calculated based on the portion of butter replaced by TBLF-HDEs. Using the total fat content of the control group as a baseline, the theoretical fat reduction for each group was calculated. The theoretical fat reduction for each group was calculated using the following formula: Theoretical fat reduction (g) = Mr × (1 - 0.66). Where Mr is the mass (g) of the added emulsion, and 0.66 is the percentage of the oil phase content of TBLF-HDEs. Figure 25 The appearance of cream emulsion samples obtained with different high internal phase double emulsion substitution ratios is shown in Table 2. Table 2 shows the theoretical calculated values ​​of fat reduction in cream emulsion samples at different substitution ratios.

[0158] Table 2. Theoretical calculations of fat reduction in cream emulsion samples with different substitution ratios.

[0159] like Figure 25 As shown, all samples exhibited a uniform and fine emulsion state, with good fluidity and no obvious clumping or phase separation observed. The T0 control group displayed a typical yellowish-white appearance. As the substitution ratio of TBLF-HDEs increased, the yellow color of the samples gradually weakened, and the overall color of the emulsion gradually tended towards milky white. Meanwhile, the theoretical calculations in Table 2 show that the fat content of the samples gradually decreased with increasing substitution ratio. Taking a 100 g cream emulsion system as an example, the theoretical reduction in fat in groups T1-T6 were 1.19, 2.38, 3.57, 4.76, 5.95, and 7.14 g, respectively, indicating that TBLF-HDEs can achieve different degrees of fat substitution while maintaining the basic appearance characteristics of the system.

[0160] (2) Particle size

[0161] Use a pipette to take 1 mL of cream emulsion with 7 different substitution ratios and measure their particle size. Figure 26 The particle size of cream emulsion samples obtained with different high internal phase dual emulsion substitution ratios is shown. Figure 26 As shown, the D4,3 of the emulsion generally increases with the increase of the TBLF-HDEs substitution ratio. When the substitution ratio is increased to T4-T6, D4,3 continues to increase, with T6 reaching the maximum value of 68.15 μm.

[0162] (3) Overall stability

[0163] The overall stability of seven cream emulsions with different substitution ratios was evaluated using a multiple light scattering analyzer. 20 mL of freshly prepared cream emulsion was placed in a dedicated glass sample vial. The testing conditions were: 40°C, standing for 10 min, scanning every 2 min for 2 h. The instrument analyzed the stability of the system by measuring the changes in the intensity of backscattered and transmitted light after the sample was illuminated, and calculated the Turbiscan stability index (TSI). A lower TSI value indicated better overall emulsion stability. Figure 27 TSI stability index of cream emulsion samples with different internal phase double emulsion substitution ratios.

[0164] Depend on Figure 27 It can be seen that there are significant differences in the stability of each sample. Compared with the control group (T0), the TSI of T1 decreased significantly after the introduction of an appropriate amount of TBLF-HDEs, indicating that the system stability was improved. Among them, T1 had the lowest TSI value (4.448), indicating that the system stability was better than T0 under appropriate substitution conditions, showing the best stability, while T5 had the highest TSI value (7.234), indicating that its stability was the worst. This shows that TBLF-HDEs can effectively improve the stability of emulsions at appropriate addition levels.

[0165] (4) Adipose tissue aggregation rate

[0166] Partial aggregation of fat globules is a crucial process in the formation of structure in whipped cream and a key factor in the system's solid-like properties after whipping. 0.0075 g of Oil Red O pigment was added to 500 g of corn oil and stirred at room temperature for at least 12 hours under dark conditions until completely dissolved. The solution was then transferred to a brown bottle. 12 g of cream emulsion and 6 g of Oil Red O solution were mixed thoroughly and centrifuged at 25°C and 10,000 rpm for 30 min. After centrifugation, the mixture was allowed to stand until the layers stabilized, and the clear upper oil phase was used for subsequent measurements. The absorbance of the original Oil Red O solution and the supernatant Oil Red O solution after centrifugation was measured at 520 nm using a microplate reader. The absorbance of the corn oil was also measured as a blank control. The partial aggregation rate of fat globules was: φ d (%) = ((m0 (A1 / A2 -1)) / (m e φ))×100%. Where φ d The percentage of fat aggregation is given by m0, where m is the mass of Oil Red O solution added. e The mass of cream emulsion added is φ, the mass fraction of fat in the emulsion is φ, A1 is the absorbance of the Oil Red O solution before centrifugation, and A2 is the absorbance of the Oil Red O solution after centrifugation. Figure 28 The aggregation rate of the fat fraction in cream emulsion samples obtained with different internal phase double emulsion substitution ratios.

[0167] like Figure 28As shown, the degree of partial aggregation of fat globules gradually decreased with increasing TBLF-HDEs substitution ratio. The T0 group showed the highest value at 28.72%, while the T6 group showed the lowest value at 3.76%, representing an overall decrease of 24.96%. Therefore, although the particle size increased with increasing substitution ratio, it mainly reflects the change in the structural size of the TBLF-HDEs composite droplets, rather than the enhancement of the fat crystal network, and does not necessarily promote partial aggregation.

[0168] (5) Rheological testing

[0169] The rheological testing method is the same as in Example 2. Figure 29 The storage modulus (G′), loss modulus (G″), and apparent viscosity of cream emulsion samples obtained with different internal phase dual emulsion substitution ratios are shown in the test results; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity.

[0170] Depend on Figure 29 As shown in a~c, with increasing frequency, the G′ and G″ of each sample all show an upward trend, and G′ is always higher than G″ throughout the entire frequency range, indicating that the system is mainly elastic. In terms of modulus, T2 exhibits high G′ and G″ throughout the entire frequency range, while T3 also shows relatively high and stable modulus changes, indicating that its system can form a relatively stable structure. Furthermore, the apparent viscosity of all samples decreases significantly with increasing shear rate, exhibiting typical shear thinning characteristics. There are differences in apparent viscosity among different samples. Overall, T0 and T1 have high initial viscosity at low shear rates, but decrease rapidly with increasing shear rate, indicating that their structures are easily destroyed under shear. In contrast, T2 maintains high viscosity throughout the entire shear rate range, especially at medium to high shear rates, still possessing a certain degree of anti-flow ability, indicating that its structure is more stable. T6 has high viscosity at low shear rates but decreases rapidly, while T5 has low overall viscosity, reflecting its relatively loose structure. In summary, T2 performs better in terms of viscosity retention and shear resistance structural stability.

[0171] III. Characterization of Whipped Cream

[0172] (1) Appearance

[0173] Seven sets of whipped cream with different substitution ratios were put into piping bags and piped at room temperature. The gloss and clarity of the texture on the surface of the cream were observed. Figure 30 Appearance of whipped cream samples obtained with different internal phase dual emulsion substitution ratios.

[0174] like Figure 30As shown, the appearance characteristics of samples with different substitution ratios differ significantly. In T0, T1, and T2, a relatively complete spiral structure can be formed after piping, with high surface gloss, clear texture, and good peak morphology integrity, indicating that the system still possesses strong structural support capabilities within this substitution range. Compared to T0, the introduction of an appropriate amount of TBLF-HDEs did not have a significant adverse effect on the piping structure. As the substitution ratio increases to T3, the sample can still form a basic piping shape, but the peak integrity decreases, and slight collapse occurs in some local textures, indicating a weakening of the system's structural support capabilities. When the substitution ratio further increases to T4-T6, the sample struggles to form a typical piping structure, the peaks are not obvious, and it easily spreads after extrusion, presenting a relatively smooth surface, indicating that the system's structural support for the overall morphology is further reduced. Overall, as the TBLF-HDEs substitution ratio increases, the system's morphology retention capability decreases, but it can still maintain good piping performance within an appropriate substitution range.

[0175] (2) Optical microscope

[0176] The microstructure of whipped cream with seven different substitution ratios was observed using an optical microscope. Samples were diluted five times with distilled water, and 10 μL was spread onto a glass slide and covered with a coverslip to create a thin sample. The sample was then observed under a 20× objective lens. Optical microscopy revealed numerous large bubbles in the unsubstituted sample T0 and the low substitution ratio samples T1 and T2. Obvious fat aggregation structures were observed around the bubble interfaces, indicating that the fat globules underwent sufficient partial aggregation and formed a continuous support network. This suggests that the introduction of TBLF-HDEs did not significantly affect the construction of the fat network. As the substitution ratio increased to T3, the bubble size decreased, and the system structure became more refined. When the substitution ratio was further increased to T4-T6, large bubbles significantly decreased, and the system was dominated by smaller, evenly distributed bubbles, while the fat aggregation structures also decreased. Overall, at appropriate substitution ratios, the system maintained a relatively intact structure without significantly adversely affecting the foam structure.

[0177] (3) Dispensing rate

[0178] Place the cream emulsion and whipped cream into identical plastic containers, ensuring consistent volume and avoiding compaction during the filling process. Weigh each sample separately. Calculate the whipping rate of the cream using the following formula: Whipping rate (%) = ((M0 - M1) / M1) × 100%; where M0 is the mass of the cream emulsion and M1 is the mass of the whipped cream. Figure 31 The whipping rate results are shown for whipped cream samples with different internal phase double emulsion substitution ratios.

[0179] like Figure 31As shown, T0 exhibits the highest whipping rate at 170.45%, indicating strong air-introduction capability. T1's whipping rate, at 142.68%, while slightly lower, remains at a relatively high level. With increasing substitution ratios to T2 and T3, the whipping rates further decrease to 110.66% and 108.57%, respectively, indicating a gradual reduction in the system's air-introduction capability. When the substitution ratio continues to increase to T4-T6, the whipping rate decreases significantly (p < 0.05), to 17.55%, 10.71%, and 6.24%, respectively, indicating difficulty in forming a stable foam structure. Overall, within an appropriate substitution ratio range, the introduction of TBLF-HDEs has a relatively small impact on the system's whipping performance, while higher substitution ratios reduce the system's whipping rate.

[0180] (4) Texture characteristics

[0181] Texture properties were determined using a texture analyzer. Seven groups of whipped cream with different substitution ratios were placed in molds, and measurements were performed using a P / 36R probe. The parameters were set as follows: pre-measurement rate 1.0 mm / s, measurement rate 1.0 mm / s, post-measurement rate 10 mm / s, descent distance 6 mm, trigger force 10 g, and room temperature. Figure 32 The textural test results are for whipped cream samples obtained with different internal phase dual emulsion substitution ratios; where a is hardness, b is elasticity, c is cohesiveness, d is chewiness, e is resilience, and f is adhesiveness.

[0182] like Figure 32 As shown in figures a~f, the textural properties of the system change with the increase of the TBLF-HDEs substitution ratio. Hardness, adhesiveness, and chewiness show a gradual decreasing trend. In T1 and T2, the sample hardness is similar to that of the unsubstituted sample, indicating that the system still possesses a certain structural support capacity. When the substitution ratio increases to T3, the hardness decreases slightly but still maintains a certain strength. When the substitution ratio increases to T4-T6, the hardness decreases significantly (p < 0.05), indicating that the system's ability to resist external deformation weakens. The changing trends of adhesiveness and chewiness are basically consistent with hardness, reflecting a decrease in the structural strength of the system. Elasticity and resilience reflect the deformation recovery characteristics of the sample after being subjected to force. With the increase of the substitution ratio, the sample elasticity shows an increasing trend, indicating that the system has a certain deformation recovery capacity at higher substitution ratios; while resilience is higher at low substitution ratios and then decreases slightly, indicating that the system's recovery capacity changes with the substitution level. The overall change in cohesiveness is relatively small, indicating that the system still maintains a certain degree of structural continuity. In summary, within an appropriate substitution ratio range (such as T1 and T2), the introduction of TBLF-HDEs can maintain the system's textural properties to a certain extent.

[0183] (5) Rheological testing

[0184] The rheological testing method is the same as in Example 2. Figure 33 The storage modulus (G′), loss modulus (G″), and apparent viscosity of whipped cream samples obtained with different internal phase dual emulsion substitution ratios are shown in the figure; where a is the storage modulus, b is the loss modulus, and c is the apparent viscosity.

[0185] like Figure 33 As shown in a~c, all samples exhibit a higher G′ than G″, indicating that the system is primarily elastic, exhibiting solid-like behavior and forming a certain degree of structural network. Differences in modulus exist among the samples, with T0 and T1 generally having higher moduli, T3-T6 relatively lower moduli, while T2 maintains a high and stable modulus level across the entire frequency range, indicating a relatively balanced characteristic between structural support and deformability. Furthermore, the apparent viscosity of all samples decreases significantly with increasing shear rate, exhibiting typical shear-thinning behavior. Differences in viscosity exist among the samples. At low shear rates, T2 has a significantly higher apparent viscosity than other samples, indicating a relatively dense internal structure; T1 and T4 are next, while T3, T5, and T6 are lower. Therefore, sample T2 exhibits relatively stable viscoelastic characteristics, thus demonstrating relatively superior rheological properties.

[0186] In summary, the present invention constructs a β-Lg-LAA-FA ternary complex and embeds it in the aqueous phase of a high internal phase dual emulsion. A two-step emulsification method combined with high-pressure microfluidic technology is used to construct the delivery system TBLF-HDEs. Finally, TBLF-HDEs are applied to cream emulsions and whipped cream, and their effects on the structure, stability, and whipping performance are systematically analyzed. This effectively demonstrates the application effect and potential of TBLF-HDEs as a fat substitute in actual food products.

Claims

1. A method for preparing a folic acid-fortified dual emulsion alternative lipid, characterized in that, Includes the following steps: S1. Prepare a β-lactoglobulin solution from β-lactoglobulin; first add L-ascorbic acid to the β-lactoglobulin solution and stir to react, then add folic acid and stir to react; after the reaction is completed, adjust the pH of the resulting reaction solution to 6-8, then dialyze and dry to obtain a lactoglobulin-ascorbic acid-folic acid ternary complex. S2. Prepare a ternary complex solution from the lactoglobulin-ascorbic acid-folic acid ternary complex as the inner aqueous phase; prepare a pectin solution from pectin as the outer aqueous phase; mix medium-chain triglycerides, vegetable oil and polyglycerol ricinoleate as the oil phase; S3. The inner aqueous phase is added to the oil phase and subjected to high-speed shearing to obtain W1 / O primary emulsion; the W1 / O primary emulsion is mixed with the outer aqueous phase and subjected to high-speed shearing to obtain W1 / O / W2 crude emulsion; the W1 / O / W2 crude emulsion is subjected to high-pressure microjets to obtain folic acid-enhanced dual emulsion substitute lipid.

2. The method for preparing the folic acid-fortified dual emulsion alternative lipid according to claim 1, characterized in that, In step S1, the concentration of the β-lactoglobulin solution is 0.5~2% w / v.

3. The method for preparing the folic acid-fortified dual emulsion alternative lipid according to claim 1, characterized in that, In step S1, the mass ratio of β-lactoglobulin, L-ascorbic acid, and folic acid is 1:(0.05~0.2):(0.4~0.6).

4. The method for preparing the folic acid-fortified dual emulsion alternative lipid according to claim 1, characterized in that, In step S1, the stirring reaction time is 0.5~2h; the dialysis temperature is 2~8℃, the time is 30~60h, and the molecular weight cutoff is 2800~3500Da; the drying is freeze drying.

5. The method for preparing the folic acid-fortified dual emulsion alternative lipid according to any one of claims 1 to 4, characterized in that, In step S2, the concentration of the ternary complex solution is 0.0005~0.005%w / w; the concentration of the pectin solution is 4~6%w / w.

6. The method for preparing folic acid-fortified dual emulsion alternative lipids according to any one of claims 1 to 4, characterized in that, In step S2, the vegetable oil is one or more of olive oil, flaxseed oil, coconut oil, sunflower seed oil, soybean oil, corn oil, and peanut oil; the mass ratio of the medium-chain triglyceride to the vegetable oil is 1:(1.5~2.5); and the mass of the polyglycerol ricinoleate is 1~2% w / w of the total mass of the medium-chain triglyceride and the vegetable oil.

7. The method for preparing the folic acid-fortified dual emulsion alternative lipid according to any one of claims 1 to 4, characterized in that, In step S3, the amount of the inner aqueous phase is 18-22 parts by mass, the amount of the oil phase is 54-70 parts, and the amount of the outer aqueous phase is 10-26 parts.

8. The method for preparing folic acid-fortified dual emulsion alternative lipids according to any one of claims 1 to 4, characterized in that, In step S3, the high-speed shearing speed is 8000~12000 rpm and the time is 1~5 min; the pressure of the high-pressure microjet treatment is 100M~200MPa.

9. A folic acid-fortified dual emulsion substitute lipid prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the folic acid-fortified dual emulsion alternative lipid as described in claim 9, characterized in that, The application is the use of folic acid-fortified double emulsion substitute lipids in the preparation of cream emulsions or whipped cream; the cream emulsions or whipped creams are mainly prepared from an oil phase and an aqueous phase; the oil phase contains base oil and folic acid-fortified double emulsion substitute lipids; the mass ratio of the folic acid-fortified double emulsion substitute lipids to the base oil is (10~30):(70~90).

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