Preparation method and application of myofibrillar protein emulsion based on enzymatic modification

Abstract

The invention discloses a preparation method and application of a myofibrillar protein emulsion based on enzymatic modification. According to the technology, a myofibrillar protein aqueous suspension is subjected to enzymatic deamidation and glycosylation reaction through protein glutaminase and glucan, a protein-polysaccharide compound with good emulsifying performance is constructed, soybean oil serves as an oil phase, and emulsion systems with different oil phase volume fractions are constructed through a controllable homogenizing technology. The obtained emulsion has the characteristics of natural degradability, excellent rheological property, uniform particle size distribution, long-term physical stability and the like. Compared with a traditional chemical modification technology, the technology adopts a whole bio-based raw material combined enzyme method green technology, the problems of organic solvent residues and the like are effectively solved, and the production cost is reduced while the product safety is ensured. The emulsion has remarkable application advantages in the fields of functional food active ingredient delivery, drug controlled release carriers, cosmetic transdermal absorption systems and the like, and a new thought is provided for development of a biomacromolecule-based delivery system.

Description

Technical Field

[0001] This invention relates to a method for preparing an enzymatically modified myofibrillar protein emulsion, belonging to the field of food processing technology. Background Technology

[0002] Emulsions have a wide and important application value in the field of food science. From everyday drinks like milk and cream to mayonnaise, cheese, salad dressings, and various meat products such as sausages, all rely on emulsification technology. Essentially, an emulsion is a homogeneous dispersion system composed of two immiscible liquid phases, where the dispersed phase is uniformly distributed in the form of micron or nanometer-sized droplets within a continuous phase. Based on the oil phase volume fraction, emulsions can be classified into different types, such as high internal phase emulsions (HIPEs) and medium-to-low oil emulsions, each with unique characteristics and applications. High internal phase emulsions are known for their significant oil phase volume fraction (φ ≥ 0.74), a unique structure that allows them to effectively mimic the texture and mouthfeel of fat, thus finding wide application in the food industry, cosmetics, and pharmaceuticals. Emulsions with a medium oil phase content (0.3-0.7%) show great potential in the delivery and release of functional food ingredients. Low-oil emulsions with an oil phase volume fraction of less than 0.3% are widely used in the production of low-fat foods such as ice cream, sausages, yogurt, and tofu due to their healthier properties.

[0003] Food emulsions, as highly efficient and easily digestible nutrient delivery carriers, not only effectively improve patients' appetite due to their excellent palatability and easy absorption, but also significantly enhance their acceptance and adherence to specific dietary plans. This nutrient delivery system, with its superior digestibility and absorption characteristics, has significant application value in clinical nutritional support. This emulsion system can significantly improve the textural properties of traditional foods; for example, it can eliminate oil floating and maintain clarity and transparency in plant protein beverages, and achieve a delicate and smooth taste experience in emulsified condiments. Addressing the specific needs of functional factor delivery, this technology encapsulates and protects easily degradable active ingredients such as vitamins and polyphenols by constructing a stable oil core-water shell structure.

[0004] It is worth noting that oil-in-water (O / W) emulsions, as one of the most common emulsion forms, are widely used, but their inherent thermodynamic instability makes them prone to phase separation and droplet aggregation during long-term storage, which limits their practical application to some extent. Therefore, improving the stability of various emulsions, especially those with different oil-to-water ratios and structural characteristics, has become an important research topic in food science and technology. From low-oil emulsions to high-internal-phase emulsions, the stability mechanism of each system requires targeted research and optimization to meet the dual demands of the food processing industry for quality and functionality. Summary of the Invention

[0005] The purpose of this invention is to provide a novel method for preparing myofibrillar protein emulsions. This method innovatively employs a combined modification technique of dextran and protein glutaminase to prepare an aqueous emulsifier, and uses soybean oil of different volume fractions as the oil phase to successfully construct various emulsion systems, including high internal phase emulsions (HIPEs) and medium-low oil emulsions. The prepared emulsions not only exhibit excellent stability but also demonstrate good rheological properties, providing a new technical approach for the development of functional emulsions and possessing significant application value.

[0006] This invention includes the following steps: (1) Myofibrillar protein was uniformly dispersed in water to obtain a myofibrillar protein suspension; (2) The myofibrillar protein suspension was mixed with protein glutaminase and dextran aqueous solution, and heated and stirred in a water bath at 37°C to carry out glycosylation and enzymatic deamidation reactions to obtain the modified myofibrillar protein suspension; (3) Soybean oil was added to the modified myofibrillar protein suspension and homogenized at 12,000 rpm for 2 min to obtain the modified myofibrillar protein emulsion.

[0007] The present invention also includes the following features: In step (2), the concentration of the myofibrillar protein suspension is 80 mg / mL; the mass of added protein glutaminase is 3.2 times the protein mass; the concentration of the dextran aqueous solution is 40 mg / mL, and the molecular weight of the dextran in the dextran aqueous solution is 70 kDa; the volume ratio of the myofibrillar protein suspension to the dextran aqueous solution is 2:1, and the heating time is 8 h; The volume fractions of soybean oil added in (3) are 25%, 33%, 50%, 66%, and 75%, respectively; The properties of the myofibrillar protein emulsion of the present invention were characterized by particle size, emulsification performance, microstructure and rheological properties.

[0008] The present invention has the following advantages: (1) This invention innovatively employs enzymatic modification technology to treat myofibrillar protein. All raw materials are derived from natural ingredients and contain no chemically synthesized additives, fully complying with the current development concepts of clean labeling and green food. This technical approach not only responds to modern consumers' pursuit of healthy and natural foods but also provides a safe and reliable raw material solution for developing a new generation of functional foods; (2) By adjusting the volume fraction of soybean oil, this invention can flexibly prepare various types of emulsions, such as high internal phase emulsions (HIPEs) and medium-low oil emulsions, to meet the differentiated needs of different food systems for texture and rheological properties; (3) This invention, through the optimized design of a mild preparation process combining water bath heating, stirring, and homogenization, maximizes the preservation of the nutritional value of proteins. It achieves a balance between ease of operation, process controllability, and low energy consumption, providing a reliable technical guarantee for industrial-scale production. (4) The myofibrillar protein emulsion prepared by this invention can be used as a delivery carrier for bioactive substances, providing a new way to develop a new generation of foods with precise nutritional functions, and has broad application prospects in the fields of health foods and special medical purpose formula foods. Attached Figure Description

[0009] Figure 1 The centrifugal stability of emulsions prepared by enzymatic modification of myofibrillar protein with soybean oil of different oil phase volume fractions is shown in the figure.

[0010] Figure 2 The effect of emulsions prepared from enzymatically modified myofibrillar protein and soybean oil with different oil phase volume fractions on particle size; Figure 3 The effect of emulsions prepared from enzymatically modified myofibrillar protein and soybean oil with different oil phase volume fractions on potential; Figure 4 Confocal microscopy images of emulsions prepared from enzymatically modified myofibrillar protein and soybean oil with different oil phase volume fractions; Figure 5 The effect of emulsions prepared from enzymatically modified myofibrillar protein and soybean oil with different oil phase volume fractions on emulsifying activity and emulsion stability; Figure 6 The effect of emulsions prepared from enzymatically modified myofibrillar protein and soybean oil with different oil phase volume fractions on apparent viscosity. Detailed Implementation Example 1:

[0011] A method for preparing myofibrillar protein emulsion based on enzymatic modification includes the following steps: Step 1: Disperse myofibrillar protein evenly in water to obtain a myofibrillar protein suspension; Step 2: Mix the myofibrillar protein suspension with protein glutaminase and dextran aqueous solution, and heat and stir in a water bath at 37 ℃ for 8 hours to carry out glycosylation and enzymatic deamidation reactions, to obtain the modified myofibrillar protein suspension; Step 3: Add 25% soybean oil (by volume) to the modified myofibrillar protein suspension, and homogenize at 12,000 rpm for 2 minutes to obtain a modified myofibrillar protein emulsion; The average particle size of the emulsion, measured by a laser particle size analyzer, was 14.15 μm at the initial stage of preparation (day 1), increasing to 20.78 μm after 7 days of storage. The zeta potential was -23.08 mV. The absorbance of the emulsion was measured by spectrophotometry. Based on the formula: emulsifying activity (%) = (2 × 2.303 × absorbance of freshly prepared emulsion × dilution factor) / (protein concentration × (1 - oil phase volume fraction) × 1 × 10000), the emulsifying activity was calculated to be 3.37%. The emulsion stability (%) = (absorbance of emulsion after 30 min of preparation / absorbance of freshly prepared emulsion) × 100, and the emulsion stability was assessed to be 71.22%. Example 2:

[0012] The method described in this embodiment is basically the same as that in Embodiment 1, except that the volume fraction of soybean oil added in step 3 is 33%. The average particle size of the emulsion, measured by a laser particle size analyzer, was 29.77 μm at the initial stage of preparation (day 1), increasing to 34.90 μm after 7 days of storage. The zeta potential was -26.52 mV. The absorbance of the emulsion was measured by spectrophotometry. Based on the formula: emulsifying activity (%) = (2 × 2.303 × absorbance of freshly prepared emulsion × dilution factor) / (protein concentration × (1 - oil phase volume fraction) × 1 × 10000), the emulsifying activity was calculated to be 3.93%. The emulsion stability (%) = (absorbance of emulsion after 30 min of preparation / absorbance of freshly prepared emulsion) × 100, and the emulsion stability was assessed to be 82.26%. Example 3:

[0013] This embodiment is basically the same as the method described in Embodiment 1, except that the volume fraction of soybean oil added in step 3 is 50%. The average particle size of the emulsion, measured by a laser particle size analyzer, was 32.45 μm at the initial stage of preparation (day 1), increasing to 64.35 μm after 7 days of storage. The zeta potential was -25.72 mV. The absorbance of the emulsion was measured by spectrophotometry. Based on the formula: emulsifying activity (%) = (2 × 2.303 × absorbance of freshly prepared emulsion × dilution factor) / (protein concentration × (1 - oil phase volume fraction) × 1 × 10000), the emulsifying activity was calculated to be 2.79%. The emulsion stability (%) = (absorbance of emulsion after 30 min of preparation / absorbance of freshly prepared emulsion) × 100, and the emulsion stability was assessed to be 68.03%. Example 4:

[0014] The method described in this embodiment is basically the same as that in Embodiment 1, except that the volume fraction of soybean oil added in step 3 is 66%. The average particle size of the emulsion, measured by a laser particle size analyzer, was 39.63 μm at the initial stage of preparation (day 1), increasing to 43.43 μm after 7 days of storage. The zeta potential was -33.03 mV. The absorbance of the emulsion was measured by spectrophotometry. Based on the formula: emulsifying activity (%) = (2 × 2.303 × absorbance of freshly prepared emulsion × dilution factor) / (protein concentration × (1 - oil phase volume fraction) × 1 × 10000), the emulsifying activity was calculated to be 4.83%. The emulsion stability (%) = (absorbance of emulsion after 30 min of preparation / absorbance of freshly prepared emulsion) × 100, and the emulsion stability was assessed to be 86.44%. Example 5:

[0015] This embodiment is basically the same as the method described in Embodiment 1, except that the volume fraction of soybean oil added in step 3 is 75%. The average particle size of the emulsion, measured by a laser particle size analyzer, was 41.21 μm at the initial stage of preparation (day 1), increasing to 45.74 μm after 7 days of storage. The zeta potential was -37.12 mV. The absorbance of the emulsion was measured by spectrophotometry. Based on the formula: Emulsifying activity (%) = (2 × 2.303 × absorbance of freshly prepared emulsion × dilution factor) / (protein concentration × (1 - oil phase volume fraction) × 1 × 10000), the emulsifying activity was calculated to be 6.49%. The emulsion stability (%) = (absorbance of emulsion after 30 min of preparation / absorbance of freshly prepared emulsion) × 100, indicating an emulsion stability of 88.09%. from Figure 1 It was found that the centrifugal stability of Pickering emulsions was significantly positively correlated with the oil phase content. As the oil phase volume fraction increased from 25% to 75%, the observed water phase separation height after centrifugation decreased significantly. The sample with an oil phase volume fraction of 75% showed almost no stratification, exhibiting the best centrifugal stability. This phenomenon confirms that a high oil phase ratio effectively enhances the ability of Pickering emulsions to resist centrifugation-induced phase separation, mainly due to the three-dimensional network structure formed by the high internal phase emulsion, the increased coverage of the interfacial particle film, and the enhanced interdroplet interaction forces. from Figure 2It was found that the emulsion droplet size showed a significant positive correlation with the water-to-oil ratio. As the water-to-oil ratio increased from 3:1 to 1:3 (corresponding to an increase in the oil phase volume fraction φ from 25% to 75%), the average droplet diameter showed a significant increasing trend. This change is mainly attributed to the fact that the increased oil phase proportion leads to a relative decrease in the number of available solid stable particles per unit interface area, thereby reducing the coverage density of the interfacial film and prompting droplets to coalesce into larger dispersed phases. Specifically, the smallest droplet diameter (14.15 μm) was observed at a water-to-oil ratio of 3:1 (φ=25%), while the largest droplet diameter (45.75 μm) was obtained at 1:3 (φ=75%). During the 7-day storage stability test, the droplet size changes of emulsions with different water-to-oil ratios showed significant differences. The emulsion with a water-to-oil ratio of 1:1 (φ=50%) exhibited the most significant droplet growth (49.57%), followed by the emulsions with a water-to-oil ratio of 3:1 (φ=25%, 31.90%) and 2:1 (φ=33%, 14.69%). The emulsion with a water-to-oil ratio of 1:3 (φ=75%) showed only a 9.90% droplet size growth throughout the storage period (p<0.05), demonstrating the best storage stability. This is mainly attributed to the effective confinement of droplet movement by the three-dimensional network structure formed under a high oil phase ratio. from Figure 3 It was found that the absolute value of the zeta potential of the emulsion increased significantly with the increase of the oil phase volume fraction (φ). When the water-oil ratio decreased from 3:1 to 1:3 (φ increased from 25% to 75%), the absolute value of the zeta potential increased from 23.08 mV to 37.12 mV (p<0.01). This phenomenon indicates that the increased surface charge density interfered with the tail-tail interaction between myosin molecules through electrostatic repulsion, effectively inhibiting the formation of filamentous polymers, thereby significantly enhancing the stability of myosin at the oil-water interface. It is particularly noteworthy that when φ=75% (water-oil ratio 1:3), the system obtained the highest absolute value of the zeta potential (37.12±1.63 mV), indicating that the surface of the emulsion droplets had the strongest charge density at this time. This indicates that the enhanced electrostatic repulsion significantly reduced the collision frequency between droplets, overcame van der Waals attraction, and enabled the system to resist unstable processes such as flocculation and aggregation for a long time. from Figure 4It was observed that emulsions with a low oil-to-water ratio (3:1) exhibited smaller droplet sizes and more uniform distribution, primarily due to the effective suppression of protein self-assembly by MP molecules when fully covering the interface. As the oil-to-water ratio increased to 1:2, the average droplet diameter significantly increased, and the uniformity of distribution decreased. This was attributed to insufficient interfacial film coverage and reduced homogenization efficiency caused by increased system viscosity. Notably, emulsions with water-to-oil ratios of 3:1 and 1:3 exhibited unique microstructural characteristics: laser confocal microscopy revealed that both ratios formed ordered core-shell structures, with the oil phase completely encapsulated within the droplets. MP molecules formed a dense protein layer with significantly enhanced fluorescence intensity at the interface, while intermediate ratios (2:1, 1:1, and 1:2) showed obvious aggregation. from Figure 5 It can be seen that as the oil phase volume fraction gradually increases from 25% to 75%, the emulsifying activity and emulsifying stability of the myofibrillar protein emulsion show an upward trend. When the oil phase volume fraction reaches 75%, the emulsifying activity and emulsifying stability reach their peak values. This phenomenon indicates that the adsorption of myofibrillar protein molecules at the oil-water interface has reached saturation. Through hydrophobic interactions and intermolecular cross-linking, protein molecules form a dense and irreversible interfacial protein film on the surface of oil droplets. This interfacial film structure with high mechanical strength can effectively prevent oil droplet aggregation, enabling the emulsion system to achieve optimal physical stability. from Figure 6 The rheological analysis of the emulsion revealed that the system exhibited typical shear-thinning behavior, with its apparent viscosity decreasing significantly with increasing shear rate. Notably, the oil phase volume fraction had a decisive influence on the emulsion viscosity: as the water-to-oil ratio decreased from 3:1 to 1:3 (corresponding to an increase in the oil phase volume fraction φ from 25% to 75%), the apparent viscosity showed a monotonically increasing trend, reaching its maximum at φ=75%. This phenomenon can be explained in two ways: firstly, a high oil phase ratio leads to a significant increase in the number of droplets per unit volume; secondly, the closely packed droplets (φ>0.74) form a spatially confined three-dimensional network structure, significantly enhancing the flow resistance of the system through inter-droplet hydrodynamic interactions and interfacial friction effects. Rheological analysis further confirmed that the emulsion exhibited the strongest shear-thinning index at φ=75%, which is highly consistent with its closely packed droplet microstructure characteristics.

Claims

1. This invention uses myofibrillar protein as raw material, and obtains a modified aqueous emulsifier by modifying the myofibrillar protein solution. Soybean oil is used as the oil phase, and emulsions with different oil phase volume fractions are formed by homogenization.

2. According to claim 1, a method for preparing a myofibrillar protein emulsion, characterized by the following steps: S1: Myofibrillar protein is uniformly dispersed in water to obtain a myofibrillar protein suspension; S2: The myofibrillar protein suspension obtained in S1 was mixed with protein glutaminase and dextran aqueous solution, and heated and stirred in a water bath at 37 ℃ to carry out glycosylation and enzymatic deamidation reactions to obtain the modified myofibrillar protein suspension; S3: Add soybean oil to the modified myofibrillar protein suspension and homogenize at 12,000 rpm for 2 minutes to obtain a modified myofibrillar protein emulsion.

3. The method for preparing myofibrillar protein emulsion according to claim 2, characterized in that, The concentration of the myofibrillar protein suspension described in S1 is 80 mg / mL.

4. The method for preparing myofibrillar protein emulsion according to claim 2, characterized in that, The mass of the added protein glutaminase in S2 is 3.2% of the protein mass.

5. The method for preparing a myofibrillar protein emulsion according to claim 2, characterized in that, The concentration of the dextran aqueous solution in S2 is 40 mg / mL, and the molecular weight of the dextran in the dextran aqueous solution is 70 kDa.

6. The method for preparing a myofibrillar protein emulsion according to claim 2, characterized in that the volume ratio of the myofibrillar protein suspension and the dextran aqueous solution in S2 is 2:1, and the heating time is 8 h.

7. The method for preparing a myofibrillar protein emulsion according to claim 2, characterized in that, The volume fractions of soybean oil added in S3 are 25%, 33%, 50%, 66%, and 75%, respectively.

Images