Preparation method and application of pure plant protein structured oil with adjustable rheological properties and freeze-thaw resistance

Abstract

The application discloses a preparation method and application of a pure plant protein structured oil with adjustable rheological properties and freeze-thaw resistance. The method comprises the following steps: taking plant protein as raw material, preparing plant protein particles through pH driving technology, further preparing high internal phase emulsion by taking the plant protein particles as structural units and oil phase, and forming structured oil through glutamine transaminase catalysis cross-linking. The structured oil does not contain exogenous structural enhancers such as xanthan gum and pectin, and by adjusting the plant protein type, plant protein particle concentration and cross-linking degree, the structured oil system with different viscosity, yield stress and gel strength can be obtained, and the rheological property adjustment is realized. Meanwhile, the system can effectively inhibit the liquid drop coalescence, liquid separation and structure collapse in the freeze-thaw process, and improve the freeze-thaw stability of the product. The preparation process is simple, the formula composition is simple, and the application can be widely applied to the fields of plant-based food, frozen food, fat-replaced food and other foods requiring structured oil.

Description

Technical Field

[0001] This invention relates to the fields of food science and food processing technology, specifically to a method for preparing pure plant protein structured oils with adjustable rheological properties and freeze-thaw resistance, and its application. Background Technology

[0002] With the rapid development of the plant-based food, fat substitute, and structured food industries, constructing structured oil systems from plant-derived raw materials that possess excellent textural properties, stability, and nutritional value has become an important research direction in food science and the food industry. Structured oils refer to the transformation of liquid vegetable oils into structured systems with semi-solid or solid characteristics through physical, chemical, or biological methods. This allows them to maintain a high content of unsaturated fatty acids while possessing rheological properties and processing performance similar to traditional solid fats. Therefore, structured oils have broad application prospects in plant-based meat, vegetable shortening, ice cream, frozen baked goods, pre-prepared foods, and fat substitutes.

[0003] In recent years, high internal phase emulsions (HIPEs) have been widely used in the construction of structured greases due to their typically high oil phase volume fraction (above 74%), solid-like characteristics, and good structural designability. Compared to traditional emulsion systems, the oil droplets in HIPEs are in a highly compacted state, which can endow the system with higher viscoelasticity and structural stability. Therefore, it is considered one of the important technical routes for constructing novel structured greases.

[0004] To improve the stability of high internal phase emulsions and structured oils, existing technologies have employed plant protein particles, protein microgels, natural oil bodies, and protein-polysaccharide complexes as interface stabilizers or network building units. For example, Chinese patent CN111905642B discloses a method for constructing Pickering emulsions using mung bean extract particles as stabilizers, which improves emulsion stability by forming a protective layer through particle adsorption at the oil-water interface; Chinese patent CN113812609A discloses a method for preparing protein microgels using pea protein isolate or mung bean protein isolate as raw materials, and further constructing gelled fat substitutes; Chinese patent CN115226784B discloses a method for constructing plant-based meat-specific fats using microalgae protein nanoparticles; and Chinese patents CN115349556B and CN116458550B respectively disclose methods for constructing structured plant oils using peanut oil-polysaccharide systems and peanut protein nanoparticle-konjac gum systems.

[0005] However, the aforementioned technologies primarily focus on the formation of emulsions or structured oils and their stability during room temperature storage. Effective solutions are still lacking for maintaining the structure of plant protein particle-stabilized high-internal-phase emulsions during freeze-thaw cycles. High-internal-phase emulsions typically have an oil phase volume fraction of at least 74%, with droplets in a highly compacted state and very limited continuous aqueous phase space. During freeze-thaw cycles, ice crystal growth and oil crystallization easily lead to damage to the droplet interface, droplet coalescence, and network structure collapse. Therefore, achieving long-term freeze-thaw stability with high-internal-phase emulsions is more challenging compared to ordinary emulsions or systems with lower oil phases.

[0006] To further improve the freeze-thaw stability of emulsion systems, especially those with high internal phase stability, existing technologies typically employ exogenous structural enhancers such as xanthan gum, pectin, κ-carrageenan, alginate, konjac gum, cellulose, and their derivatives to construct a continuous phase network, or enhance interfacial film strength and system stability through protein glycosylation, Maillard reactions, and composite gels. For example, Chinese patent CN113040369A discloses a method for constructing a high freeze-thaw stability high internal phase emulsion based on glycosylated proteins; Chinese patent CN116636612A discloses a method for constructing a high freeze-thaw stability high internal phase emulsion using a glycosylated protein-reducing sugar composite system.

[0007] While the above technologies can improve the stability of emulsion systems or achieve the structuring of liquid oils, they still have the following shortcomings: (1) Complex system composition. Existing structured oil systems usually require the addition of polysaccharides, cellulose, natural waxes or other exogenous structural enhancers to construct a continuous phase network, which increases the complexity of product formulation and production costs, and is not conducive to meeting consumers' demand for clean label food; (2) Freeze-thaw stability still needs to be improved. Frozen foods inevitably undergo freezing, thawing and temperature fluctuations during production, storage and transportation. Ice crystal formation and freeze-thaw concentration effects can easily lead to droplet aggregation, network structure destruction, liquid separation and phase separation, which can cause a decline in product quality. Existing high internal phase emulsions and structured oil systems mostly rely on non-covalent interactions to maintain structural stability, and are still prone to structural degradation under long-term freeze-thaw conditions; (3) Rheological properties are highly dependent on exogenous structural enhancers, and their controllability is limited. Existing high internal phase emulsion and structured oil systems mainly adjust the strength and stability of the system through exogenous structural enhancers such as xanthan gum, pectin, κ-carrageenan, konjac gum, cellulose, and natural waxes. Their rheological properties, such as viscosity, yield stress, and gel strength, largely originate from the exogenous network structure. Research on utilizing the structural characteristics of plant proteins to regulate the rheological properties of structured oils is relatively insufficient, making it difficult to obtain structured oil products with different textural levels while maintaining a simple system composition.

[0008] Therefore, developing a structured lipid system that does not require the addition of exogenous structural enhancers such as polysaccharides, cellulose, and natural waxes, but relies solely on plant proteins to construct a structured lipid system, while also possessing good rheological properties and freeze-thaw stability, is of great significance for promoting the development of the plant-based food and frozen food industries. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing pure plant protein structured oils with adjustable rheological properties and freeze-thaw resistance, and its application, so as to solve the problems of existing plant protein high internal phase emulsion systems that rely on exogenous colloidal stabilizers, have limited ability to regulate rheological properties, and have insufficient freeze-thaw stability.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A structured oil made from pure plant protein with adjustable rheological properties and freeze-thaw resistance is disclosed. The structured oil is prepared by using plant protein particles prepared by a pH-driven method as the sole emulsifying structural unit to form a high internal phase emulsion with the oil phase, followed by cross-linking catalyzed by transglutaminase to form the structured oil. The plant protein particles are single-type plant protein particles or composite particles of multiple plant proteins prepared by the pH-driven method. The mass fraction of the plant protein particles in the aqueous phase of the structured oil is 0.5%–10%. The volume fraction of the oil phase in the high internal phase emulsion is 74%–90% (preferably 74%–85%). The structured oil maintains a stable gel structure without significant precipitation or phase separation after at least five freeze-thaw cycles.

[0011] Furthermore, the plant protein is derived from one or more of legume protein, cereal protein, oilseed protein, or microalgae protein, preferably one or more of soybean protein, pea protein, mung bean protein, broad bean protein, chickpea protein, lentil protein, rapeseed protein, and zein.

[0012] Furthermore, the single-species plant protein particles are prepared by dispersing plant protein in an aqueous phase, adjusting the pH to 10–13, stirring for 10–120 min, and then placing it at 4°C for 2–12 h. Subsequently, the mixture is brought back to room temperature, the pH is adjusted to 6.5–7.5, stirred for 10–120 min, and then centrifuged at 1000 g for 3 min. The resulting supernatant is the plant protein particles.

[0013] Furthermore, the method for preparing the composite particles includes the following steps: (a) Prepare a protein suspension with a mass fraction of 3-7% by the plant protein, stir for 1-3 hours, adjust the pH to 6.5-7.5, let it stand overnight at 2-8°C, then return it to room temperature, stir for 0.5-1.5 hours, centrifuge and collect the supernatant to obtain the soluble component of the plant protein. (b) Mix the soluble components of plant protein with zein at a mass ratio of (1-20):1, adjust the pH to 11.0-13, dissolve the protein, and then adjust the pH back to 6.5-7.5. Stir to form a composite particle dispersion.

[0014] Furthermore, the average particle size of the plant protein particles is 50–2000 nm, preferably 100–1000 nm.

[0015] Furthermore, the amount of glutamine transaminase added is 1–100 U / g protein, preferably 6–60 U / g protein.

[0016] Furthermore, the oil phase is selected from one or more of camellia oil, sunflower seed oil, coconut oil, olive oil, rapeseed oil, soybean oil, corn oil, flaxseed oil, walnut oil, and medium-chain triglyceride oil.

[0017] The present invention also provides a method for preparing the above-mentioned pure plant protein structured oil, comprising the following steps: firstly, plant protein particles are prepared by pH-driven method, then the plant protein particles are dispersed in an aqueous phase to form a continuous phase, an oil phase is added, and an emulsion with high internal phase is formed by emulsification, and then transglutaminase is added for enzymatic crosslinking, and finally pure plant protein structured oil with adjustable rheological properties and freeze-thaw resistance is obtained.

[0018] Furthermore, the emulsification is carried out using one or more of the following methods: high-speed homogenization, high-speed shearing, rotor-stator emulsification, or ultrasonic emulsification. The emulsification speed is 3000–30000 rpm, and the emulsification time is 0.5–10 min.

[0019] Furthermore, the temperature for the enzymatic cross-linking is 35–50°C; the cross-linking time is 0.5–24 h, preferably 1–6 h.

[0020] The present invention also provides the application of the above-mentioned pure plant protein structured oil in frozen foods, plant-based foods, fat substitute foods and structured foods, characterized in that: the structured oil is used to improve the freeze-thaw stability and storage stability of the food, and the structured oil is used to replace animal fat or partially hydrogenated oil.

[0021] This invention discovers that different plant proteins, when formed through pH-driven processes, exhibit different particle sizes, interfacial adsorption capacities, and interparticle interaction behaviors. By adjusting the source of the plant protein, the concentration of the plant protein particles, and the amount of transglutaminase added, the structured oil storage modulus (G'), loss modulus (G''), yield stress, apparent viscosity, and gel strength can be controlled, thereby meeting the textural and processing performance requirements of different food systems.

[0022] Furthermore, plant protein particles form a stable particle layer at the oil-water interface, enhancing interfacial mechanical strength and steric hindrance; simultaneously, transglutaminase further catalyzes the formation of a continuous covalent cross-linked network. The synergistic effect of these two factors effectively inhibits ice crystal-induced droplet coalescence, network collapse, and liquid separation during freeze-thaw cycles, thereby improving the freeze-thaw stability and storage stability of structured oils.

[0023] Compared with the prior art, the present invention has the following advantages: (1) Constructing a pure plant protein structured lipid system without the need for exogenous colloidal stabilizers Existing plant protein high-internal-phase emulsions and structured oil systems typically require the addition of exogenous structural enhancers such as xanthan gum, pectin, κ-carrageenan, alginate, and cellulose to improve system stability and gel strength. This leads to complex formulations, increased costs, and is not conducive to the development of clean-label foods. In contrast, this invention utilizes pH-driven plant protein particles as the sole structural building block, combined with glutamine transaminase cross-linking to form a stable network structure. A stable structured oil system can be obtained without the addition of exogenous polysaccharides, colloidal stabilizers, or fibrous materials, facilitating simplified formulations and the development of clean-label foods.

[0024] (2) To achieve the regulation of the rheological properties of plant protein structured lipids Most existing plant protein emulsion systems primarily aim to improve emulsion stability, offering limited control over rheological properties and failing to meet the diverse textural and processing performance requirements of various food products. This invention discovers that different plant proteins, when formed through pH-driven processes, exhibit differences in particle size, interfacial adsorption behavior, and interparticle interactions. By adjusting the plant protein source, particle concentration, and the amount of transglutaminase added, the storage modulus (G'), loss modulus (G''), yield stress, apparent viscosity, and gel strength of structured oils can be controlled, thereby constructing structured oil systems with different textural properties. Therefore, this invention not only yields stable structured oils but also enables the targeted design of rheological properties according to the specific needs of different food systems.

[0025] (3) Significantly improves the freeze-thaw stability of structured oils Existing high internal phase emulsion systems are susceptible to ice crystal formation and freeze-thaw concentration during cryopreservation, leading to problems such as droplet coalescence, network collapse, liquid separation, and phase separation, thus causing a decline in product quality. In this invention, plant protein particles can form a stable particle layer at the oil-water interface, improving interfacial mechanical strength and steric hindrance; simultaneously, transglutaminase further catalyzes the formation of a continuous covalently cross-linked network of protein molecules. The synergistic effect of interfacial stabilization and continuous phase network effectively inhibits droplet coalescence, network collapse, and liquid separation during freeze-thaw cycles, thereby significantly improving the freeze-thaw stability and storage stability of structured oils.

[0026] (4) It has a wide range of applicability to plant proteins. This invention is applicable to a variety of plant-derived proteins, including legume proteins, cereal proteins, and oilseed proteins. Different plant proteins can all form interfacially active protein particles through pH-driven processes, and further construct structured lipid systems, thus demonstrating good versatility and potential for widespread application.

[0027] (5) Applicable to various oil systems and food application scenarios This invention is applicable to camellia oil, sunflower seed oil, coconut oil, olive oil, rapeseed oil, soybean oil, and other vegetable oil systems. It can construct structured oils with different rheological properties and structural characteristics based on the different characteristics of these oils. The resulting structured oils can be widely used in ice cream, vegetable shortening, frozen mousse, frozen baked goods, pre-made foods, fat substitutes, and other plant-based foods, showing promising prospects for industrial application.

[0028] (6) It has both nutritional value and processing performance advantages. This invention uses plant protein and plant oil as the main raw materials, which can reduce the use of traditional solid fats, increase the content of unsaturated fatty acids and plant protein in the system, and improve the nutritional value of the product while maintaining good processing performance, which is in line with the development trend of health food and plant-based food. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the preparation process of the pure plant protein structured oil of the present invention.

[0030] Figure 2 The particle size distribution diagrams are shown for mung bean protein particles (Mwhole), mung bean protein soluble components (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ).

[0031] Figure 3Optical micrographs and macroscopic appearance images of whey protein (WPI, control protein), mung bean protein particles (Mwhole), mung bean protein soluble fraction (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ) stabilizing high internal phase emulsions of different vegetable oils at different mass fractions.

[0032] Figure 4 Figure 4,3 shows the droplet size distribution and volume average particle size D of whey protein (WPI, control protein), mung bean protein particles (Mwhole), mung bean protein soluble component (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ) in stable high internal phase emulsions in different vegetable oil systems.

[0033] Figure 5 Rheological properties of whey protein (WPI, control protein), mung bean protein particles (Mwhole), soluble mung bean protein component (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ) in different vegetable oil systems.

[0034] Figure 6 Rheological properties of whey protein (WPI, control protein), mung bean protein particles (Mwhole), mung bean protein soluble component (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ) stable high internal phase emulsions after enzymatic cross-linking to form structured oils under different amounts of transglutaminase added.

[0035] Figure 7 Macroscopic appearance of structured oils formed by whey protein (WPI, control protein), mung bean protein particles (Mwhole), mung bean protein soluble component (Msup), and mung bean protein-zein complex particles (9MsupZ and 4MsupZ) after one freeze-thaw cycle, under no addition and different addition amounts of transglutaminase treatment.

[0036] Figure 8 Macroscopic appearance of structured lipids formed in a 6% protein / particle system with different amounts of transglutaminase added after multiple freeze-thaw cycles.

[0037] Figure 9 This is a particle size distribution diagram of broad bean protein particles (Fwhole), mung bean protein particles (Mwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole).

[0038] Figure 10Comparison of droplet size distribution and volume average particle size D(4,3) of high internal phase emulsions stabilized by broad bean protein particles (Fwhole), mung bean protein particles (Mwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole) and the structured oils formed by cross-linking with transglutaminase.

[0039] Figure 11 This is a rheological diagram showing the structured oils formed by broad bean protein particles (Fwhole), mung bean protein particles (Mwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole) at different mass fractions before and after cross-linking with transglutaminase.

[0040] Figure 12 Comparative images of the macroscopic appearance of high internal phase emulsions stabilized by broad bean protein particles (Fwhole), mung bean protein particles (Mwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole) and the structured oils formed before and after cross-linking with transglutaminase before and after freeze-thaw cycles. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0042] The preparation process of the pure plant protein structured oil of this invention is as follows: Figure 1 As shown: Plant protein isolate is used as raw material to prepare plant protein particles through pH-driven treatment, or the soluble protein components are further compounded with zein and then assembled into composite protein particles through pH-driven assembly; vegetable oil is added to the particle dispersion as the continuous phase, and a high internal phase emulsion is prepared by high-speed shear emulsification. Finally, a three-dimensional covalent network structure is formed by enzymatic cross-linking of transglutaminase, resulting in pure plant protein structured oil with adjustable rheological properties and freeze-thaw resistance.

[0043] Unless otherwise specified, all raw materials used in this embodiment can be obtained through conventional commercial channels.

[0044] Example 1: Preparation of mung bean protein granules (Mwhole), soluble mung bean protein component (Msup), and zein complex granules.

[0045] (1) Preparation of mung bean protein particles Weigh out mung bean protein isolate (Yantai Shuangta Food Co., Ltd.), add deionized water to prepare a 5% (w / w) protein suspension, and magnetically stir at room temperature for 2 h to ensure complete protein hydration. Adjust the pH of the system to 12.0 using 2 mol / L NaOH solution and incubate at 4 ℃ for 12 h. Then, return to room temperature and adjust the pH of the system to 7.0 using 2 mol / L HCl solution, and stir at 800 rpm for 1 h. Finally, centrifuge the obtained sample at 1000 g for 3 min, collect the supernatant to obtain a pH-driven mung bean protein particle dispersion, denoted as Mwhole.

[0046] (2) Preparation of soluble components of mung bean protein and its zein complex particles Weigh out mung bean protein isolate and add deionized water to prepare a 5% (w / w) protein suspension. Stir magnetically at room temperature for 2 h to ensure complete protein hydration. Adjust the pH of the system to 7.0 using 2 mol / L HCl and NaOH solutions and incubate overnight at 4 °C. Then, return to room temperature and stir at 800 rpm for 1 h. Finally, centrifuge the obtained sample at 1000 g for 3 min, collect the supernatant to obtain the soluble fraction of mung bean protein, denoted as Msup.

[0047] Msup and zein were mixed at mass ratios of 9:1 and 4:1, respectively, to obtain mixed protein solutions. The pH of the system was adjusted to 12.0 using 2 mol / L NaOH solution to ensure complete dissolution of both proteins. Subsequently, the pH of the system was adjusted to 7.0 using 2 mol / L HCl solution, and the mixture was stirred at 800 rpm for 1 h to form mung bean protein-zein composite particle dispersions, denoted as 9MsupZ and 4MsupZ, respectively.

[0048] (3) Comparison of protein and particle size Using whey protein isolate (WPI) as a control, the Dv(50) values ​​of WPI, Msup, Mwhole, 9MsupZ, and 4MsupZ were measured to be 4.25 nm, 8.10 nm, 82.37 nm, 86.06 nm, and 160.63 nm, respectively. Figure 2 ).

[0049] Depend on Figure 2 It is evident that WPI and Msup mainly exist in molecular or small-scale aggregated states, while Mwhole, 9MsupZ, and 4MsupZ form larger particle structures. As the proportion of zein increases, the composite particle Dv(50) increases from 86.06 nm to 160.63 nm, indicating that the introduction of zein can induce the formation of composite particles from the soluble components of mung bean protein, and the particle size can be regulated by adjusting the mass ratio of the two proteins.

[0050] Example 2: Preparation of stabilized structured oils from mung bean protein particles and mung bean protein-zein composite particles in different vegetable oil systems.

[0051] Mung bean protein granules (Mwhole), soluble mung bean protein component (Msup), and mung bean protein-zein complex granules (9MsupZ and 4MsupZ) were prepared according to the method in Example 1.

[0052] Whey protein isolate (WPI, control protein), Msup, 9MsupZ, 4MsupZ, and Mwhole were used as emulsifying stabilizers, and sunflower oil, camellia oil, and coconut oil were used as the oil phase, respectively. The volume fraction of the oil phase was controlled at 75%, and the mass fractions of the emulsifying stabilizers were 0.5%, 1%, 3%, and 6%, respectively. High internal phase emulsions were prepared by shear emulsification at 20,000 rpm for 1 min using a high-speed shear emulsifier.

[0053] like Figure 3 As shown, WPI, Msup, 9MsupZ, 4MsupZ, and Mwhole can all form high internal phase emulsions in sunflower seed oil, camellia oil, and coconut oil systems. However, their microscopic droplet morphology and macroscopic self-supporting ability are jointly influenced by the protein structure, mass fraction, and oil phase type. As the mass fraction increases from 0.5% to 6%, the emulsion droplets tend to become finer and more densely packed, and the macroscopic structural stability is correspondingly enhanced. The WPI system mainly exhibits obvious self-supporting characteristics when the mass fraction reaches 3% and 6%, while mung bean protein and its particle system exhibit different degrees of interfacial stability and continuous phase network support under the same conditions.

[0054] like Figure 4 As shown, the overall D(4,3) values ​​of each system in the three vegetable oils were approximately 10-50 μm. With the increase of the mass fraction of the emulsifying stabilizer from 0.5% to higher levels, the overall D(4,3) decreased by approximately 40%-75%, indicating that increasing the stabilizer dosage can improve the oil-water interface coverage, reduce droplet re-agglomeration during emulsification, and promote the formation of finer, more uniform droplet structures. The droplet size and the rate of decrease varied among different oil phases. In most systems, the particle size in sunflower seed oil and camellia oil showed a relatively continuous decreasing trend with increasing mass fraction, while some coconut oil systems exhibited broadened particle size distribution or D(4,3) fluctuations at higher mass fractions. This suggests that the viscosity, interfacial properties, and crystallization characteristics of the oil phase further influence droplet breakup, particle interface adsorption, and droplet network reorganization processes.

[0055] Figure 5 The rheological data shown indicate that all fresh high internal phase emulsions exhibit significant shear-thinning behavior. In all systems tested, the 50 s...-1 The apparent viscosity ranged from 266.73 to 2654.13 mPa·s, and the yield stress ranged from 0.35 to 23.89 Pa. As the mass fraction of the emulsifying stabilizer increased from 0.5% to 6%, the apparent viscosity range of different systems increased from 266.73-873.62 mPa·s to 628.98-2654.13 mPa·s, and the yield stress range increased from 0.35-13.81 Pa to 2.51-23.89 Pa. This indicates that a higher mass fraction is beneficial for the formation of stronger droplet stacking and a continuous phase support network.

[0056] Significant differences exist between different protein or particle systems. The apparent viscosity and yield stress of the WPI system are 289.83-1026.67 mPa·s and 0.35-3.76 Pa, respectively; those of the Msup system are 552.16-2139.70 mPa·s and 3.69-15.72 Pa, respectively; those of the 9MsupZ system are 266.73-1076.00 mPa·s and 1.19-8.44 Pa, respectively; those of the 4MsupZ system are 282.04-867.94 mPa·s and 1.65-14.41 Pa, respectively; and those of the Mwhole system are 562.94-2654.13 mPa·s and 7.21-23.89 Pa, respectively.

[0057] The apparent viscosities of 6% Mwhole in sunflower oil, camellia oil, and coconut oil systems were 2424.87, 2654.13, and 2194.07 mPa·s, respectively, and the yield stresses were 21.56, 22.00, and 23.89 Pa, respectively. These values ​​were generally higher than those of WPI and composite particle systems under the same conditions, indicating that Mwhole possesses a strong ability to construct continuous phase networks. The apparent viscosities of the 6% Msup system ranged from 1754.47 to 2139.70 mPa·s, and the yield stresses ranged from 8.16 to 15.72 Pa, also demonstrating strong rheological support capabilities.

[0058] The above results indicate that the mung bean protein particles and mung bean protein-zein complex particles described in this invention are suitable for different vegetable oil systems such as sunflower seed oil, camellia oil, and coconut oil. Furthermore, by adjusting the protein structure, particle composition, mass fraction, and oil phase type, the structured oil droplet size, flow resistance, and deformation resistance can be directionally controlled.

[0059] Example 3: Effect of different amounts of transglutaminase added on the rheological properties of structured lipids in mung bean protein particles and their composite particles

[0060] Following the method in Example 2, high internal phase emulsions with an oil phase volume fraction of 75% were prepared using WPI, Msup, 9MsupZ, 4MsupZ, and Mwhole as emulsifying stabilizers, and sunflower seed oil, camellia oil, and coconut oil as the oil phase. The protein or particle mass fractions were selected as 0.5% and 6%, respectively. The high internal phase emulsions were prepared by shear emulsification at 20,000 rpm for 1 min using a high-speed shear emulsifier.

[0061] Transglutaminase was added to the obtained high internal phase emulsion at concentrations of 6 U / g protein, 12 U / g protein, and 24 U / g protein, respectively, and cross-linked at 50 °C for 1 h. Immediately after the reaction was completed, the enzyme was inactivated by treatment at 85 °C for 10 min to obtain structured oils with different degrees of cross-linking.

[0062] like Figure 6 As shown, transglutaminase treatment significantly altered the rheological properties of each system, but the degree of enhancement was influenced by the protein or particle type, mass fraction, oil phase type, and enzyme addition amount.

[0063] At a mass fraction of 0.5%, the apparent viscosity of fresh high internal phase emulsion ranged from 266.73 to 873.62 mPa·s. After treatment with 6, 12, and 24 U / g protein glutamine transaminase, the apparent viscosities were 372.72–1825.55, 359.18–2066.13, and 426.21–2078.65 mPa·s, respectively. The corresponding yield stresses changed from 0.35–13.81 Pa for the fresh emulsion to 0.13–44.57, 0.14–44.70, and 0.25–39.72 Pa.

[0064] At a mass fraction of 6%, the apparent viscosity of fresh high internal phase emulsion ranged from 628.98 to 2654.13 mPa·s. Treatment with 6, 12, and 24 U / g protein transglutaminase increased the apparent viscosity to 1233.10–6784.45, 1319.20–7245.95, and 1500.40–7329.85 mPa·s, respectively; the yield stress changed from 2.51–23.89 Pa to 3.07–67.94, 3.53–55.68, and 3.56–60.37 Pa. These results indicate that a high mass fraction system provides more crosslinkable protein substrates and particle contact sites, thus facilitating the formation of interconnected three-dimensional networks.

[0065] The Mwhole system exhibited a significant response to transglutaminase treatment. At a 0.5% mass fraction, the apparent viscosities of fresh Mwhole emulsions in sunflower oil, camellia oil, and coconut oil were 873.62, 746.29, and 562.94 mPa·s, respectively; after treatment with 12 U / g protein transglutaminase, these viscosities increased to 1779.20, 1630.80, and 1158.60 mPa·s, respectively. The corresponding yield stresses increased from 13.81, 12.92, and 7.21 Pa to 30.39, 33.72, and 44.70 Pa, respectively.

[0066] At a mass fraction of 6%, the apparent viscosity of the three oil phase fresh emulsions of Mwhole ranged from 2194.07 to 2654.13 mPa·s. After treatment with 6, 12, and 24 U / g protein transglutaminase, the apparent viscosities reached 4482.95-5790.00, 5499.00-6463.45, and 4922.30-7216.20 mPa·s, respectively. The yield stress increased from 21.56-23.89 Pa to 54.27-61.84, 53.95-55.68, and 51.04-57.02 Pa, respectively, indicating that transglutaminase can significantly enhance the network connectivity of the Mwhole system.

[0067] The Msup system also exhibited a strong enzymatic cross-linking response at 6%. After treatment with 6, 12, and 24 U / g protein glutamine transaminase, the highest apparent viscosities in the three oil phases reached 6784.45, 7245.95, and 7329.85 mPa·s, respectively, and the highest yield stresses were 67.94, 53.61, and 60.37 Pa, respectively.

[0068] It should be noted that when the enzyme addition increased from 6 U / g protein to 24 U / g protein, the various indicators did not show a strictly monotonic increase in all systems. For example, some Msup, 9MsupZ, 4MsupZ, and Mwhole systems showed plateaus or fluctuations in viscosity or yield stress at 12 U / g protein or 24 U / g protein. This indicates that enhanced cross-linking depends not only on the enzyme amount but also on factors such as the degree of exposure of reactive sites, particle spatial arrangement, oil phase properties, and network rearrangement after excessive cross-linking.

[0069] Therefore, within the protein range of 6-24 U / g tested in this invention, adjusting the amount of transglutaminase added can significantly alter the viscosity and anti-flow ability of structured oils. However, the appropriate amount of enzyme should be selected based on the protein or particle type, mass fraction, and oil phase type, rather than simply assuming that the highest addition amount is the optimal condition in all systems.

[0070] Example 4: Effect of different amounts of transglutaminase added on the freeze-thaw stability of mung bean protein particles and their composite particles.

[0071] Structured lipids with added glutamine transaminase levels of 6 U / g protein, 12 U / g protein, and 24 U / g protein were prepared according to the method in Example 3. Each system was cross-linked at 50 °C for 1 h, and immediately after the reaction was completed, it was treated at 85 °C for 10 min to inactivate the enzyme.

[0072] The obtained samples were frozen at -20 °C for 24 h, and then thawed in a 37 °C water bath for 30 min to complete one freeze-thaw cycle. After freeze-thaw, the macroscopic appearance, gel integrity, liquid precipitation, and oil precipitation of the samples were observed.

[0073] like Figure 7 As shown, systems without transglutaminase treatment or with weak cross-linking networks exhibited varying degrees of liquid precipitation, oil precipitation, loose structure, or gel collapse after freeze-thaw cycles; after cross-linking with transglutaminase, most samples were able to maintain a basic gel shape, and the degree of macroscopic phase separation decreased.

[0074] Under 0.5% mass fraction conditions, after treatment with 6 U / g protein, 12 U / g protein, and 24 U / g protein-glutamine transaminase, most mung bean protein particles and the mung bean protein-zein complex particle system maintained a basic gel morphology, while the WPI system still showed significant precipitation and structural collapse, with certain differences between different oil phases. Among them, the Mwhole, Msup, 9MsupZ, and 4MsupZ systems all showed good macroscopic stability, with only a small amount of precipitation or slight structural shrinkage observed, indicating that glutamine transaminase cross-linking can effectively improve the macroscopic structural stability of the system.

[0075] When the protein fraction was increased to 6%, all systems treated with the three amounts of transglutaminase exhibited a more complete and denser gel appearance, with further reductions in liquid and oil separation. The Mwhole system maintained a relatively intact self-supporting structure throughout, while the Msup, 9MsupZ, and 4MsupZ systems also showed good macroscopic stability. Although the WPI system showed improvement compared to 0.5%, some structural damage was still observed. These results indicate that increasing the protein particle mass fraction increases the contact and cross-linking sites between protein particles, promoting the formation of a more continuous three-dimensional covalent network, thereby improving the macroscopic stability and freeze-thaw resistance of structured lipids.

[0076] The effect of glutamine transaminase addition on freeze-thaw stability is not a simple linear relationship under different protein structures and oil phase conditions. Some systems, despite having high initial structural strength at higher enzyme levels, may still exhibit localized network breakage, droplet aggregation, or redispersibility of structural units after freeze-thaw cycles.

[0077] The above results indicate that the covalent cross-linked network induced by transglutaminase can improve the ability of structured oils to resist the mechanical damage of aqueous phase freezing and oil phase crystallization; its freeze-thaw stability is determined by the amount of enzyme added, protein or particle structure, mass fraction and oil phase properties.

[0078] To further evaluate long-term freeze-thaw stability, the 6% protein / particle system was subjected to multiple freeze-thaw cycles in two different vegetable oils.

[0079] like Figure 8 As shown, the 12 U / g protein treatment group generally showed good stability after repeated freeze-thaw cycles, and most samples were able to maintain a relatively intact gel morphology after repeated freeze-thaw cycles; the 6 U / g protein treatment group showed obvious stratification, liquid separation or loose structure in some systems due to insufficient cross-linking; the 24 U / g protein treatment group did not show further improvement, and some samples still showed structural shrinkage or local phase separation.

[0080] Among different protein / particle systems, 9MsupZ exhibited superior long-term freeze-thaw stability, maintaining a relatively intact and uniform gel appearance even after multiple freeze-thaw cycles. In contrast, the WPI system was more prone to delamination and structural damage. This indicates that appropriate TG enzyme addition and composite particle structure are beneficial for achieving a balance between network strength and structural toughness, thereby improving the repeated freeze-thaw tolerance of structured lipids.

[0081] Example 5: Preparation of structured oils from broad bean protein particles (Fwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole).

[0082] Except for replacing the mung bean protein isolate in step (1) of Example 1 with broad bean protein isolate (Yantai Shuangta Food Co., Ltd.), soybean protein isolate (Wandefu Biotechnology Co., Ltd.), and pea protein isolate (Yantai Shuangta Food Co., Ltd.), the other particle preparation conditions are the same as in step (1) of Example 1. The resulting protein particles are broad bean protein particle dispersion (Fwhole), soybean protein particle dispersion (Swhole), and pea protein particle dispersion (Pwhole), respectively.

[0083] The measured Dv(50) of Fwhole was 329.67 nm, that of Swhole was 24.62 nm, and that of Pwhole was 232.80 nm. Figure 9As shown, Fwhole has the largest particle size among the four types of legume protein particles, indicating that broad bean protein formed relatively large particle aggregates after pH-driven treatment. Swhole has the smallest Dv(50) among the four types of legume protein particles, indicating that soybean protein can form smaller protein particles after pH-driven treatment. Pwhole forms protein particles with a scale of hundreds of nanometers, and its particle size is between that of Swhole and Fwhole.

[0084] Fwhole, Swhole, and Pwhole particle dispersions were used as continuous phases, with protein particle mass fractions adjusted to 0.5% and 3%, respectively. Coconut oil was used as the oil phase, with an oil phase volume fraction controlled at 75%. High internal phase emulsions were prepared by shearing and emulsifying at 20,000 rpm for 1 min using a high-speed shear emulsifier. Subsequently, 12 U / g protein transglutaminase was added to the obtained high internal phase emulsion, and crosslinking was performed at 50 °C for 1 h. Immediately after the reaction was completed, the enzyme was inactivated by treatment at 85 °C for 10 min, yielding structured lipids from broad bean protein particles, soybean protein particles, and pea protein particles, respectively.

[0085] like Figure 10 As shown, at a mass fraction of 0.5%, the D(4,3) of the Fwhole-stable high-internal-phase emulsion before TG enzyme crosslinking was 29.1 μm, increasing to 67.0 μm after crosslinking; at a mass fraction of 3%, D(4,3) increased from 23.2 μm to 114 μm. The increase in D(4,3) measured after TG enzyme crosslinking mainly reflects the formation of larger-scale crosslinked structural units between protein particles, continuous phase proteins, and emulsion droplets, and should not be simply interpreted as droplet aggregation. At a mass fraction of 0.5%, the D(4,3) of the Swhole-stable high-internal-phase emulsion before TG enzyme crosslinking was 27.5 μm, increasing to 93.2 μm after crosslinking; at a mass fraction of 3%, D(4,3) increased from 15.3 μm to 187 μm. The significant increase in apparent structural units measured after crosslinking under high mass fraction conditions indicates that the higher protein content provides more substrate and interparticle contact sites for the TG enzyme reaction, promoting the formation of trans-droplet crosslinking networks. At a mass fraction of 0.5%, the D(4,3) of the Pwhole-stabilized high-internal-phase emulsion was 28.5 μm before TG enzyme crosslinking, increasing to 115 μm after crosslinking; at a mass fraction of 3%, the D(4,3) increased from 23.0 μm to 188 μm. Similar to the Swhole system, Pwhole formed larger-scale crosslinked structural units after TG enzyme crosslinking under 3% conditions, indicating that increasing the mass fraction of protein particles is beneficial for promoting the extension and connectivity of the continuous phase network.

[0086] like Figure 11As shown, at a mass fraction of 0.5%, the apparent viscosity of the Fwhole system increased from 381.01 mPa·s before crosslinking to 765.87 mPa·s, and the yield stress slightly increased from 6.21 Pa to 6.57 Pa; at a mass fraction of 3%, the apparent viscosity increased from 612.27 mPa·s to 858.63 mPa·s, and the yield stress increased from 7.12 Pa to 9.88 Pa. Similarly, at a mass fraction of 0.5%, the apparent viscosity of the Swhole system increased from 552.71 mPa·s before crosslinking to 1320.90 mPa·s, and the yield stress increased from 7.88 Pa to 11.23 Pa; at a mass fraction of 3%, the apparent viscosity increased from 1258.90 mPa·s to 2580.90 mPa·s, and the yield stress increased from 9.17 Pa to 30.08 Pa. At a mass fraction of 0.5%, the apparent viscosity of the Pwhole system increased from 276.36 mPa·s before crosslinking to 874.22 mPa·s, and the yield stress increased from 7.11 Pa to 8.14 Pa; at a mass fraction of 3%, the apparent viscosity increased from 866.72 mPa·s to 1915.70 mPa·s, and the yield stress increased from 8.65 Pa to 27.95 Pa.

[0087] The above results indicate that TG enzyme crosslinking can increase the apparent viscosity and yield stress of the Fwhole system, enabling it to form a structured oil with a certain degree of self-support. However, compared with the Mwhole, Swhole, and Pwhole systems, the rheological enhancement of Fwhole after increasing its mass fraction and TG enzyme crosslinking is relatively limited, suggesting that its larger initial particle size may limit the effective access of protein reaction sites and the continuity of the crosslinked network. Smaller-sized soybean protein particles can form stable high-internal-phase emulsions and, under sufficient protein substrate conditions, form highly interconnected three-dimensional crosslinked networks through TG enzyme action. Pea protein particles can effectively stabilize coconut oil high-internal-phase emulsions and, after TG enzyme crosslinking, form structured oils with self-support. Compared with the 0.5% system, the 3% Pwhole system exhibits a more significant increase in apparent viscosity and yield stress after crosslinking, indicating that increasing the mass fraction of protein particles can enhance the continuity of the crosslinked network and the overall structural strength.

[0088] Example 6: Effects of different plant protein particles and protein mass fractions on the freeze-thaw stability of structured oils in a coconut oil system

[0089] Dispersions of mung bean protein particles (Mwhole), broad bean protein particles (Fwhole), soybean protein particles (Swhole), and pea protein particles (Pwhole) were prepared according to the methods of Examples 1 and 5, respectively. The particle systems were formulated into dispersions with protein mass fractions of 0.5% and 3%, respectively. Coconut oil was used as the oil phase, with the oil phase volume fraction controlled at 75%. The mixtures were sheared and emulsified at 20,000 rpm for 1 min using a high-speed shear emulsifier to prepare high internal phase emulsions.

[0090] Transglutaminase was added to each emulsion at a concentration of 12 U / g protein, and cross-linked at 50 °C for 1 h. Immediately after the reaction was completed, the enzyme was inactivated by treatment at 85 °C for 10 min to obtain structured lipids.

[0091] The high internal phase emulsion without transglutaminase crosslinking and the structured oil formed by transglutaminase crosslinking were respectively frozen at -20 °C for 24 h, and then thawed in a 37 °C water bath for 30 min to complete one freeze-thaw cycle. After freeze-thaw, the macroscopic appearance, structural integrity, liquid precipitation and oil precipitation of the samples were observed.

[0092] like Figure 12 As shown, Fwhole, Mwhole, Swhole, and Pwhole high internal phase emulsions that were not cross-linked with transglutaminase all exhibited varying degrees of demulsification, oil separation, aqueous phase separation, or structural collapse after one freeze-thaw cycle. This indicates that the physical network formed solely by interfacial adsorption and non-covalent interactions of plant protein particles is insufficient to fully resist the mechanical damage caused by the combined effects of aqueous phase freezing and coconut oil crystallization.

[0093] After cross-linking with transglutaminase, the macroscopic freeze-thaw stability of each system was improved to varying degrees, but the degree of improvement was affected by the plant protein source and protein mass fraction.

[0094] When the protein content was 0.5%, the Mwhole system maintained a basic gel morphology after freeze-thaw cycles, and no obvious macroscopic oil-water separation was observed. The Swhole and Pwhole systems showed varying degrees of liquid precipitation or loose structure. The Fwhole system showed more obvious structural collapse and oil precipitation.

[0095] When the protein content was increased to 3%, the Mwhole, Swhole, and Pwhole systems were able to maintain a relatively intact macroscopic gel structure after freeze-thaw cycles. Among them, the Mwhole system showed good structural integrity and no obvious oil-water separation was observed. Although the Swhole and Pwhole systems maintained a basic gel morphology, some degree of softening or localized loosening of the structure was observed. The freeze-thaw stability of the Fwhole system was relatively weak, and some degree of oil separation or structural deterioration was still observed.

[0096] The above results indicate that increasing the mass fraction of plant protein particles can increase the cross-linking substrate of glutamine transaminase and the contact sites between particles, promoting the formation of a more continuous covalent cross-linked network, thereby improving the macroscopic structure retention ability of structured oils after freeze-thaw cycles. Different plant protein particles exhibit varying cross-linking abilities and network connectivity; among them, the mung bean protein particle system showed better freeze-thaw stability under the conditions of this embodiment.

[0097] This embodiment demonstrates that by selecting plant protein particle sources, adjusting protein mass fraction, and cross-linking with transglutaminase, the macroscopic stability of coconut oil high internal phase emulsion during freeze-thaw processes can be effectively improved, providing a basis for the application of this structured oil in frozen foods, plant-based foods, and cold chain stored and transported foods.

[0098] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A structured oil made from pure plant protein with adjustable rheological properties and freeze-thaw resistance, characterized in that: The structured oil uses plant protein particles prepared by pH-driven method as the only emulsifying structural unit to prepare a high internal phase emulsion with the oil phase, and then cross-links it with transglutaminase to form the structured oil. The plant protein particles are single plant protein particles or composite particles of multiple plant proteins prepared by pH-driven method; the mass fraction of the plant protein particles in the structured oil aqueous phase is 0.5% to 10%; the oil phase volume fraction of the high internal phase emulsion is 74% to 90%; the structured oil maintains a stable gel structure and shows no obvious precipitation or phase separation after undergoing at least 5 freeze-thaw cycles.

2. The pure plant protein structured oil according to claim 1, characterized in that: The plant protein is derived from one or more plant proteins such as soybean protein, pea protein, mung bean protein, broad bean protein, chickpea protein, lentil protein, rapeseed protein, and zein.

3. The pure plant protein structured oil according to claim 1, characterized in that: The single plant protein particles are obtained by dispersing plant protein in an aqueous phase, adjusting the pH to 10-12, stirring for 10-120 min, placing at 4°C for 2-12 h, then restoring to room temperature, adjusting the pH to 6.5-7.5, stirring for 10-120 min, and centrifuging at 1000 g for 3 min. The resulting supernatant is the plant protein particles.

4. The pure plant protein structured oil according to claim 1, characterized in that: The method for preparing the composite particles includes the following steps: (a) Prepare a protein suspension with a mass fraction of 3-7% by the plant protein, stir for 1-3 h, adjust the pH to 6.5-7.5, let stand overnight at 2-8℃, then return to room temperature, stir for 0.5-1.5 h, centrifuge and take the supernatant to obtain the soluble component of plant protein; (b) Mix the soluble components of plant protein with zein at a mass ratio of (1-20):1, adjust the pH to 11.0-13, dissolve the protein, and then adjust the pH back to 6.5-7.

5. Stir to form a composite particle dispersion.

5. The pure plant protein structured oil according to claim 1, characterized in that: The amount of transglutaminase added is 6–60 U / g protein.

6. The pure plant protein structured oil according to claim 1, characterized in that: The oil phase is selected from one or more of camellia oil, sunflower seed oil, coconut oil, olive oil, rapeseed oil, soybean oil, corn oil, flaxseed oil, walnut oil, and medium-chain triglyceride oil.

7. The method for preparing the pure plant protein structured oil according to any one of claims 1 to 6, characterized in that: Includes the following steps: First, plant protein particles were prepared using a pH-driven method. Then, the plant protein particles were dispersed in an aqueous phase to form a continuous phase. An oil phase was added, and the mixture was emulsified to form a high internal phase emulsion. Finally, transglutaminase was added for enzymatic cross-linking, and a pure plant protein structured oil with adjustable rheological properties and freeze-thaw resistance was obtained.

8. The preparation method according to claim 7, characterized in that: The emulsification is carried out using one or more of the following methods: high-speed homogenization, high-speed shearing, rotor-stator emulsification, or ultrasonic emulsification. The emulsification speed is 3000–30000 rpm, and the emulsification time is 0.5–10 min.

9. The preparation method according to claim 7, characterized in that: The temperature for the enzymatic cross-linking is 35–50°C; the cross-linking time is 1–6 h.

10. The application of the pure plant protein structured oil as described in any one of claims 1 to 6 in frozen foods, plant-based foods, fat substitute foods, and structured foods, characterized in that: The structured oils are used to improve the freeze-thaw stability and storage stability of food, and are used to replace animal fats or partially hydrogenated oils.

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