Mixed protein base material and application thereof

Abstract

The invention provides a mixed protein base material and application thereof, and the mixed protein base material comprises the following components in percentage by mass: 25%-80% of whey protein, 15%-70% of collagen peptide, 2%-4% of moderately hydrolyzed whey protein, 1%-2% of highly hydrolyzed whey protein and 2%-4% of whey protein rich in milk phospholipid membrane. According to the mixed protein base material provided by the invention, an oil-water interface of an emulsion is jointly stabilized through proteins with different molecular weights and compositions, the emulsion and microcapsules can be effectively stabilized, and fat-soluble functional factors are further delivered by utilizing the obtained emulsion and microcapsules, so that the stability of the fat-soluble functional factors can be improved; and the bioavailability can be improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a mixed protein base and its application. Background Technology

[0002] The bioavailability of lipid-soluble functional factors is closely related to their digestive behavior in delivery carriers (especially oil-containing carriers). Generally, after being ingested orally, the carrier loaded with functional factors undergoes a complex physiological process in the gastrointestinal tract. Ultimately, the oil-containing carrier is digested and hydrolyzed in the small intestine, while the functional factors are released and absorbed into the bloodstream to improve health.

[0003] Lipid-soluble functional factors are typically dissolved in the oil phase of an oil-containing carrier. After ingestion, the carrier undergoes a series of complex reactions, including physicochemical processes and enzymatic activity, in the mouth, stomach, and small intestine. First, the oil-containing carrier mixes with saliva and interacts with salivary amylase, mucin, and other substances. Some carriers may flocculate due to consumption or bridging effects. Upon reaching the stomach, the highly acidic environment and peristalsis may further alter the carrier's properties, leading to hydrolysis by lipases and affecting the stability of the lipid-soluble functional factors. Therefore, enhancing the stability of the oil-containing carrier in the gastric environment will improve the bioavailability of the loaded lipid-soluble functional factors. Finally, the oil-containing carrier enters the intestine, mixes with bile, pancreatic juice, and other digestive fluids, and is completely hydrolyzed and digested into mixed micelles or vesicles under the synergistic action of pancreatic lipases and various cofactors (helper lipases, bile salts, and calcium). During the disintegration of the carrier structure, lipid-soluble functional factors are simultaneously released, and some of these released factors are further "dissolved" by the aforementioned mixed micelle load. This loading method facilitates the compatibility of lipid-soluble functional factors with the human body environment, enabling them to effectively enter the small intestinal epithelial mucosa, cross the mucosal barrier, and be absorbed by the body. Therefore, the changes in the oil-containing carrier throughout the digestive process are highly complex, and the evolution of the interfacial properties and micelle structure of the oil-containing carrier during digestion affects the absorption efficiency of functional factors.

[0004] Protein emulsions and microcapsules, as important delivery carriers, have a positive impact on promoting the absorption of lipid-soluble functional factors, and are therefore widely used in the development of functional foods. During gastrointestinal digestion, proteins can not only stabilize oil-containing carriers and prevent the degradation of functional factors by increasing the interaction between lipophilic and water-soluble components, but also participate in driving the formation of mixed micelles, thus promoting the digestion and absorption of functional factors.

[0005] Therefore, providing a stable and efficient delivery system for lipid-soluble functional factors is of great significance. Summary of the Invention

[0006] In view of this, the present invention provides a mixed protein base and its application, which can effectively stabilize emulsions and microcapsules, and further utilize the resulting emulsions and microcapsules to deliver lipid-soluble functional factors, which can not only improve the stability of lipid-soluble functional factors, but also improve their bioavailability.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a mixed protein base comprising, by weight percentage: 25% to 80% whey protein, 15% to 70% collagen peptides, 2% to 4% moderately hydrolyzed whey protein, 1% to 2% highly hydrolyzed whey protein, and 2% to 4% whey protein rich in milk phospholipid membrane.

[0008] Preferably, by weight percentage, the mixed protein base comprises the following components: 30%~50% whey protein, 40%~60% collagen peptides, 3%~4% moderately hydrolyzed whey protein, 1%~2% highly hydrolyzed whey protein, and 3%~4% whey protein rich in milk phospholipid membrane.

[0009] Preferably, the molecular weight of the collagen peptide is 1000 Da to 5000 Da; and / or, The proportion of moderately hydrolyzed whey proteins with a molecular weight of 5000 Da to 20000 Da is greater than 80%; and / or, The proportion of highly hydrolyzed proteins with a molecular weight ≤1000 Da is greater than 90%; and / or, The whey protein rich in milk phospholipid membrane contains 5% to 10% milk phospholipid membrane.

[0010] Secondly, the present invention also provides a method for preparing an emulsion, comprising the following steps: S1. Mix the aforementioned mixed protein base with water to obtain an aqueous phase; S2. Mix the aqueous phase and the oil phase, emulsify, and obtain an emulsion.

[0011] Preferably, in step S2, the method for preparing the oil phase includes: dissolving the fat-soluble functional factor in edible oil to obtain the oil phase; wherein: The fat-soluble functional factor includes at least one of curcumin and carotene; and / or, The edible oil includes at least one of soybean oil, coconut oil, medium-chain triglycerides, and rapeseed oil; and / or, The mass fraction of the lipid-soluble functional factor in the oil phase is 0.1% to 0.5%.

[0012] Preferably, in step S1, the mass fraction of the mixed protein matrix in the aqueous phase is 3% to 10%.

[0013] Preferably, in step S2, the oil phase in the emulsion has a mass fraction of 1% to 8%; and / or, In step S2, the emulsification includes pre-emulsification and secondary emulsification.

[0014] Thirdly, the present invention also provides a method for preparing microcapsules, comprising the following steps: The emulsion prepared by the method described above is spray-dried to obtain microcapsules.

[0015] Fourthly, the present invention also provides a liquid protein beverage, comprising, by mass percentage: 3% to 8% of the protein mixture base, 0% to 1% of medium-chain fatty acid glycerides, 1% to 2% magnesium gluconate, 0.5% to 1.5% beetroot powder, 0.1% to 0.2% peppermint flavoring, 0.05% to 0.15% citric acid, 0.05% to 0.1% zinc citrate, 0.01% to 0.05% vitamins, with the balance being water.

[0016] Fifthly, the present invention also provides a liquid protein beverage, comprising an emulsion prepared by the method described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Based on the in vivo digestion and absorption process of lipid-soluble functional factors, the mixed protein base provided by the present invention can stabilize the oil-water interface of the emulsion by proteins of different molecular weights and compositions, effectively stabilize the emulsion and microcapsules, and further utilize the obtained emulsion and microcapsules to deliver lipid-soluble functional factors, thereby protecting the lipid-soluble functional factors and improving their stability.

[0018] (2) Emulsions and microcapsules modified with various protein interfaces can form hollow vesicle structures in digestive juices after digestion, which increases the solubility of intestinal digestive juices and can improve the bioavailability of lipid-soluble functional factors. Attached Figure Description

[0019] Figure 1 The image shows the sample state and microstructure of the emulsion provided by this invention. Figure 2 The diagram shows the particle size, potential, and particle size variation during storage of the emulsion provided by this invention. Figure 3 The graph shows the curcumin encapsulation efficiency and stability of the emulsion provided by this invention. Figure 4 The graph shows the free fatty acid release rate and curcumin bioavailability of the emulsion provided by this invention. Figure 5 The images show the sample states and microstructures of the emulsion provided by this invention at various stages of digestion. Figure 6 A diagram illustrating the solubility of the emulsion in the small intestine digestive fluid provided by this invention. Figure 7 The sample state and microstructure diagram of the microcapsules provided by this invention; Figure 8 The graph shows the release rate of free fatty acids and the bioavailability of carotene from the microcapsules provided by this invention. Figure 9 Microscopic structural diagrams of the microcapsules provided by this invention at various stages of digestion; Figure 10 A model diagram illustrating the digestion mechanism of the emulsion provided by this invention; Figure 11 This is a graph showing the change in the degree of protein hydrolysis during the gastrointestinal digestion process of the mixed protein base provided by the present invention. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0021] Example 1: Preparation of Mixed Protein Base Material According to Table 1, different masses of whey protein, collagen peptides, partially hydrolyzed whey protein, highly hydrolyzed whey protein, and whey protein rich in milk phospholipid membranes were weighed, mixed, and stirred evenly to obtain a mixed protein base. The raw materials were sourced as follows: whey protein (Hilmar 9010), partially hydrolyzed whey protein (Hilmar 8350), highly hydrolyzed whey protein (Hilmar 8390), and whey protein rich in milk phospholipid membranes (Hilmar 7500) were all purchased from Hilmar Company, USA, and collagen peptides were provided by Shanghai Maixiang Shengli Health Technology Co., Ltd.

[0022] Table 1. Raw materials and their proportions for comparative examples and formulas 1-7

[0023] Example 2: Preparation of Protein-Stabilized Emulsion The protein base was mixed with water to obtain an aqueous phase (the mass fraction of the protein base in the aqueous phase was 5%). The oil phase (coconut oil) was then mixed with the aqueous phase (the mass ratio of the two was 5:95) and pre-emulsified (12000 rpm, 5 min). Then, a secondary emulsification was performed using a high-pressure homogenizer (12000 psi, 5 cycles) to obtain an emulsion.

[0024] Emulsions were prepared using the comparative example and the mixed protein bases of formulations 1-7, respectively. The sample states of each emulsion are shown in Table 2, and the sample states and microstructure diagrams are shown in [the table below]. Figure 1 A.

[0025] Table 2. Sample states of the comparative examples and emulsions obtained from formulations 1-7.

[0026] from Figure 1 As can be seen from Figure A, the emulsion obtained from Formula 7 exhibited significant stratification, while the emulsions obtained from the comparative example and Formulas 1-6 all showed a homogeneous and stable state. This may be because the peptide content in the emulsion obtained from Formula 7 was too high, and due to the hydrophilicity of collagen peptides, they preferentially distributed in the aqueous phase, resulting in only a small portion of peptides migrating to the oil-water interface. This made the structure of the oil-water interface loose, unable to form a tight network structure to stabilize the oil droplets. Under a microscope, the microstructure of the emulsion obtained from Formula 7 showed obvious oil droplet aggregation, while the microstructure of the emulsions obtained from the comparative example and Formulas 1-6 was relatively stable and uniformly distributed.

[0027] Fat-soluble functional factors (including curcumin and carotene) were dissolved in edible oil (coconut oil) to obtain an oil phase (the mass fraction of fat-soluble functional factors in the oil phase was 0.3%). The protein matrix was mixed with water to obtain an aqueous phase (the mass fraction of protein matrix in the aqueous phase was 5%). The oil phase and the aqueous phase were then mixed (mass ratio of 5:95) and pre-emulsified (12000 rpm, 5 min). Then, a secondary emulsification was performed using a high-pressure homogenizer (12000 psi, 5 cycles) to obtain an emulsion loaded with fat-soluble functional factors.

[0028] Curcumin-loaded emulsions were prepared using the mixed protein base of comparative examples and formulations 1-6, respectively, with the fat-soluble functional factor (curcumin). Carotene-loaded emulsions were prepared using the mixed protein base of comparative examples and formulations 1-6, respectively, with the fat-soluble functional factor (carotene). The sample states of each curcumin-loaded emulsion and each carotene-loaded emulsion are shown in the figure. Figure 1 B.

[0029] from Figure 1 As can be seen from B, the curcumin-loaded emulsions obtained from the comparative examples and formulas 1-6, as well as the carotene-loaded emulsions obtained from the comparative examples and formulas 1-6, all exhibit a homogeneous and stable state.

[0030] Particle size potential characterization of the sample in Example 3 Particle size: The average particle size of the emulsion was determined using a laser particle size analyzer. A wet sample injector was used to add the sample. The refractive indices of the dispersed and continuous phases were set to 1.448 and 1.333, respectively. During measurement, the sample was dispersed in clean distilled water, and the addition was stopped when the shading rate stabilized between 4% and 5%. The average particle size was measured using the surface area average particle size (…). d 32 Characterizing the average particle size of the emulsion:

[0031] In the formula, ni represents the number of sample droplets with a particle size of di. Zeta potential: The zeta potential of the emulsion was measured using a nanoparticle size analyzer. The measurement temperature was 25 °C. Before measurement, the sample was diluted 100 times with PBS solution to eliminate the influence of multiple light scattering on the measured value.

[0032] The particle size and zeta potential of the curcumin-loaded emulsions obtained from the comparative example provided in Example 2 and formulations 1-6 were measured respectively, and the results are shown in [Figure 1]. Figure 2 A; The curcumin-loaded emulsions obtained from the comparative examples and formulations 1-6 provided in Example 2 were stored at 25 °C for 9 days. During storage, the sample state of the emulsions was observed, and the particle size change was measured. The particle size changes of each emulsion during storage are shown in [Figure 1]. Figure 2 B.

[0033] from Figure 2 As can be seen from Figure A, although the particle size of the emulsions obtained from formulations 1-6 increased compared to the comparative example, the particle size remained relatively stable. Compared to the comparative example, the absolute value of the zeta potential of the emulsions obtained from formulations 1-6 gradually decreased from 38 mV to 28 mV. This may be due to the reduction in proteins adsorbed at the emulsion interface, thereby weakening the electrostatic repulsion between droplets and leading to a decrease in the absolute value of the zeta potential.

[0034] During storage, the emulsions obtained from formulations 1-6 did not exhibit obvious phase separation or precipitation, indicating that their macroscopic stability remained good. Figure 2 As can be seen from B, the particle size distribution of each emulsion remained stable in the early stage of storage (0~3 days). When the storage time was extended to the 9th day, the particle size of all emulsions except those obtained from formula 4 and formula 5 showed a significant increasing trend.

[0035] Example 4: Encapsulation efficiency and stability test of curcumin Curcumin Standard Curve: A precise amount of curcumin powder was dissolved in anhydrous ethanol, and then serially diluted to obtain a series of curcumin standard solutions of different concentrations. The absorbance values ​​of curcumin at different mass concentrations were measured, and the standard curve was plotted with mass concentration on the x-axis and absorbance on the y-axis as y=0.1531x. 0.0089, R2 =0.9998.

[0036] Curcumin encapsulation efficiency: The curcumin content in the emulsion was calculated based on the standard curve. C 1) The theoretical content is based on the concentration of curcumin added during emulsion preparation. C 0), the encapsulation efficiency of curcumin can be calculated using the following formula:

[0037] Thermal stability: The samples were heated in a 90 ℃ water bath, with portions removed at regular intervals. Undegraded curcumin was then extracted with anhydrous ethanol, and the absorbance of the extract was measured using a UV-Vis spectrophotometer. The curcumin content was determined by referring to a standard curve. Simultaneously, a curcumin-ethanol solution (free curcumin) with a concentration of 50 μg / mL was prepared as a control for comparison with the experimental group. UV stability: The sample was placed at 40 cm from a UV light source with an illumination power of 30 W and a wavelength of 254 nm. Samples were taken out at regular intervals. Undegraded curcumin was then extracted with anhydrous ethanol, and the absorbance was measured using a UV-Vis spectrophotometer. The curcumin content was determined by referring to a standard curve. A control group consisting of a 50 μg / mL curcumin-ethanol solution (free curcumin) was used for comparison with the experimental group.

[0038] The curcumin encapsulation efficiency of the curcumin emulsions obtained from the comparative example provided in Example 2 and formulations 1-6 was determined respectively, and the results are shown in the figure. Figure 3 A; The thermal stability of the comparative examples provided in Example 2 and the curcumin-loaded emulsions and free curcumin obtained from formulations 1-6 were determined respectively. The results are shown in […]. Figure 3 B; The UV stability of the emulsions and free curcumin loaded with the lipid-soluble functional factor (curcumin) obtained from the comparative examples provided in Example 2 and formulations 1-6 was determined, and the results are shown in [Figure 1]. Figure 3 C.

[0039] from Figure 3 As shown in Figure A, the encapsulation efficiency of curcumin in each emulsion reached over 89%. In formulations 5 and 6, the content of collagen peptides increased significantly, while the encapsulation efficiency of curcumin showed a slight decreasing trend. Curcumin is unstable in the environment; therefore, ultraviolet light irradiation and water bath heating treatments were used to evaluate the protective effect of different emulsions on curcumin. Figure 3 B and Figure 3As can be seen from Figure C, compared to free curcumin, each emulsion exhibits a significant ability to inhibit curcumin degradation. After 3 hours of UV irradiation, more than 70% of the curcumin in the emulsions obtained from formulations 1-6 remained undecomposed, while only 31% of the free curcumin remained. Similar results were also observed in the water bath heating treatment. This indicates that the mixed protein matrix provided by this invention can effectively improve the photothermal stability of curcumin in emulsions.

[0040] Example 5: Extracellular digestion of emulsion To study the digestive behavior of emulsions and the absorption efficiency of curcumin, an in vitro simulated digestive model was used to simulate the digestive behavior of emulsions in the mouth, stomach, and small intestine. Oral digestion simulation: Take 5 mL of freshly prepared emulsion sample and mix it with 4 mL of simulated saliva containing 0.015 g of mucin. Then add 25 μL of 0.3 M CaCl2 and 0.975 mL of distilled water, and stir at 37 ℃ for 2 min at a stirring speed of 100 rpm.

[0041] Gastric digestion simulation: 10 mL of oral digestive fluid collected from the oral simulated digestion experiment was mixed with 8 mL of gastric simulated digestive fluid, and then 5 μL of 0.3 M CaCl2 was added. The pH of the mixed digestive fluid was adjusted to 3.00, and then pepsin with a concentration of 0.080 g / mL was added. The mixture was placed in a water bath at 37 ℃ and stirred for 1 h at a stirring speed of 100 rpm.

[0042] Simulated small intestinal digestion: Take 20 mL of the above sample digested with simulated gastric juice, place it at 37 ℃ and stir at 100 rpm, add simulated small intestinal digestion fluid, 40 μL of 0.3 M CaCl2 and 3 mL of bile salt extract (200 mg / mL) solution, and adjust the pH to 7.0 with 3 M NaOH. Add 4 mL of freshly prepared lipase suspension to NaOH (0.25 mol / L) dropwise using an automated potentiometric titrator to maintain the pH of the system at (7.00±0.02), react at 37 ℃ and 100 rpm for 2 h, and record the volume of NaOH consumed.

[0043] Since the complete digestion of one triglyceride molecule produces two molecules of free fatty acid, the free fatty acid release rate (FFA) is expressed by the following formula:

[0044] In the formula, FFA is the free fatty acid release rate (%). V NaOH (t) It is the volume (L) of NaOH consumed at time t. M NaOHThis is the molar concentration of NaOH (mol / L). M lipid It is the molar mass of the oil (650 g / mol for coconut oil). W lipid It is the mass (g) of edible oil in the pre-digested emulsion in the small intestine.

[0045] The free fatty acid release rates of the curcumin-loaded emulsions obtained from the comparative example provided in Example 2 and formulations 1-6 were measured respectively, and the results are shown in the figure. Figure 4 .

[0046] from Figure 4 It can be seen that after undergoing in vitro digestion, the release rate of free fatty acids in each emulsion is between 70% and 80%.

[0047] Example 6: Determination of the bioavailability of functional factors in emulsions Immediately after digestion, the small intestinal digestive fluid was centrifuged (12000 rpm, 30 min, 25 ℃) to separate the oil layer, micelle layer, and precipitate layer. The clear, transparent liquid in the middle layer is the micelle layer, which can be carefully removed using a syringe. The small intestinal digestive fluid or micelle solution was mixed with anhydrous ethanol in an equal proportion and vortexed for 30 s to extract curcumin. Then, it was centrifuged again (10000 rpm, 10 min, 4 ℃), and the supernatant was collected. The absorbance of curcumin was measured at 425 nm. The absorbance value was converted to the concentration of curcumin using a standard curve. Finally, the bioavailability of curcumin was calculated using the following formula:

[0048] In the formula, C digesta This represents the concentration of curcumin in the digestive fluids of the small intestine (mg / mL). C micelle The concentration of curcumin in the micelles is (mg / mL).

[0049] The bioavailability of curcumin in the comparative examples provided in Example 2 and the curcumin-loaded emulsions obtained from formulations 1-6 was determined respectively, and the results are shown in [Figure 1]. Figure 4 .

[0050] from Figure 4It can be seen that, compared with the comparative example, the emulsions obtained from formulations 1-6 can improve the bioavailability of curcumin. Among them, the emulsion obtained from formulation 4 has the highest bioavailability of curcumin, reaching 94%, followed by the emulsions obtained from formulations 5 and 6, with bioavailability of curcumin of 91.5% and 90.3%, respectively. Although the bioavailability of curcumin in the emulsions obtained from formulations 1-3 is not significantly different from that of the comparative example, it is still higher than that of the comparative example (79%). This indicates that the mixed protein matrix provided by the present invention can improve the bioavailability of curcumin.

[0051] Example 7: Microstructure characterization after emulsion digestion The emulsion structure at each digestion stage was meticulously observed using laser confocal microscopy. Before observation, 10 μL of Nile Red (0.01 wt%, dissolved in anhydrous ethanol, used for labeling the oil phase) and 10 μL of Fast Green (FCF, 1 wt%, dissolved in anhydrous ethanol, used for labeling proteins) were added to 0.5 mL of the emulsion and the digestion solution at each stage, respectively, ensuring thorough mixing. Subsequently, the microstructure of each digestion stage was observed and recorded under a 60× objective lens in fluorescence mode to facilitate in-depth analysis of the structural evolution of the emulsion during digestion.

[0052] The microstructure of the comparative examples provided in Example 2 and the curcumin-loaded emulsions obtained from formulations 1-6 were characterized at each digestion stage. The sample states and microstructure diagrams at each digestion stage are shown below. Figure 5 The yellow arrows indicate hollow vesicle structures.

[0053] from Figure 5 It can be seen that the appearance color of each emulsion lightens after oral digestion due to the dilution effect of simulated saliva. From a microscopic perspective, the internal structure of the emulsions shows aggregation, possibly due to electrostatic shielding caused by mineral ions in the simulated oral fluid and bridging or flocculation caused by mucins. During simulated gastric digestion, pepsin hydrolysis leads to changes in the protein emulsion interface structure, resulting in significant aggregation. Upon entering the small intestine, the large protein particles observed in the gastric digestion stage disappear. Compared to the control group, the emulsions obtained from formulations 1-6, after digestion, exhibited some vesicle structures in the small intestinal digestive fluid, and the number of these vesicle structures increased with the increase in collagen peptide content and complexation with other proteins. This indicates that the emulsions obtained from formulations 1-6 affect the digestive properties of the emulsion, possibly by forming special protective vesicle structures to alter digestive and absorptive behavior.

[0054] Example 8: Determination of Solubilizing Capacity of Small Intestinal Digestive Fluids First, the digested small intestine digestive fluid was centrifuged at 3500 rpm for 15 min to separate the soluble fraction. During the experiment, the temperature was maintained at 37 ℃. 5 μL of oleic acid was added to 10 mL of the digestive fluid sample, mixed for 5 min, and the turbidity change was measured at 600 nm using a UV-Vis spectrophotometer. The above experiment was repeated with gradual addition of oleic acid. A small intestine simulated solution (specific composition shown in Table 3) + bile salt system (SIF+BS) was used as a control, i.e., 8 mL SIF + CaCl2 (40 μL SIF, 0.3 M) + bile salt solution (0.08 g bile salt extract + 3 mL SIF) + pancreatic enzyme solution (0.02 g pancreatic enzyme + 3 mL SIF) + lipase solution (0.16 g lipase + 2 mL SIF) + 3 mL H2O. All experiments were repeated three times.

[0055] Table 3 Composition of Small Intestinal Imitation Fluid (SIF)

[0056] The solubilizing ability of the curcumin-loaded emulsion, small intestinal simulated fluid, and bile salt mixture (SIF+BS) obtained from the comparative example provided in Example 2 and formulations 1-6 was determined. The results are shown in [Figure 1]. Figure 6 .

[0057] The intestinal absorption of lipid-soluble functional factors typically depends on their solubilization within the small intestinal micelle system. Figure 6 It can be seen that the gradual addition of oleic acid to a mixture of small intestinal simulated fluid and bile salts (SIF+BS) resulted in an increase in the OD of the solution. 600 The value increased rapidly, while the OD of the small intestinal digestive fluid after digestion of the emulsions obtained in proportions and formulas 1-6 increased rapidly. 600 The increasing trend of the value is more gradual. Specifically, the OD value of the small intestinal digestive fluid after emulsion digestion obtained in the comparative example... 600 The value change can be divided into two stages. When the amount of oleic acid added is less than 60 uL, the measured OD value is... 600 The value changes relatively smoothly; when the amount of oleic acid added exceeds 60 μL, the measured OD value... 600 The value began to rise significantly. The OD values ​​of the small intestinal digestive fluid after digestion of the emulsions obtained from formulas 1-6 and the small intestinal digestive fluid after digestion of the emulsion obtained in the comparative example were compared. 600 The values ​​showed similar trends, divided into two phases: a gradual increase and a rapid rise. However, with the increase in collagen peptide content, the OD of the small intestinal digestive fluid after emulsion digestion decreased. 600 The period of plateauing in the value was prolonged. This indicates that after in vitro digestion of the emulsion, collagen peptides may participate in micelle formation, thereby increasing the solubility of oleic acid in the micelles of the digestive fluid.

[0058] Example 9: Preparation of Protein-Stabilized Microcapsules The protein base was mixed with water to obtain an aqueous phase (protein mass fraction of 5%). The oil phase (soybean oil) was then mixed with the aqueous phase (mass ratio of 5:95) and pre-emulsified (12000 rpm, 5 min). Then, a second emulsification was performed using a high-pressure homogenizer (12000 psi, 5 cycles) to obtain an emulsion. The resulting emulsion was further spray-dried using a 0.5 mm diameter atomizing nozzle, with an inlet air temperature of 180 ± 5 ℃, an outlet air temperature of 90 ± 5 ℃, and a sample flow rate of 5 mL / min to obtain blank microcapsules.

[0059] Blank microcapsules were prepared using the comparative example and the mixed protein bases of formulations 4-6, respectively. The sample state and microstructure of each blank microcapsule are shown in the figure. Figure 7 .

[0060] Fat-soluble functional factor (carotene) was dissolved in edible oil (soybean oil) to obtain an oil phase (carotene mass fraction in the oil phase was 0.3%). Protein matrix was mixed with water to obtain an aqueous phase (protein matrix mass fraction in the aqueous phase was 5%). The oil phase and aqueous phase were then mixed (mass ratio of 5:95) and pre-emulsified (12000 rpm, 5 min). Then, a secondary emulsification was performed using a high-pressure homogenizer (12000 psi, 5 cycles) to obtain an emulsion. The resulting emulsion was spray-dried using a 0.5 mm diameter atomizing nozzle, with an inlet air temperature of 180±5 ℃, an outlet air temperature of 90±5 ℃, and a sample flow rate of 5 mL / min to obtain carotene-loaded microcapsules.

[0061] Carotene-loaded microcapsules were prepared using the comparative example and the mixed protein bases of formulations 4-6, respectively. The sample states and microstructures of each carotene-loaded microcapsule are shown in the figures. Figure 7 .

[0062] from Figure 7 It can be seen that the blank microcapsule powder is uniform and dispersible. Microscopic results show that the blank microcapsule particles are irregular in shape. Loading with carotene does not affect the formation of microcapsules, and microscopic results show that the microcapsule particles loaded with carotene can better present a spherical structure.

[0063] Example 10: In vitro digestion of microcapsules, determination of bioavailability of functional factors, and characterization of post-digestion microstructure. Using the methods of Examples 5 and 6, the free fatty acid release rate and bioavailability of carotene-loaded microcapsules obtained from the comparative example provided in Example 9 and formulations 4-6 were determined, respectively. The results are shown in [Figure 1]. Figure 8Using the method of Example 7, the microstructure of the carotene-loaded microcapsules obtained from the comparative example provided in Example 9 and formulations 4-6 was characterized at each stage of digestion. The results are shown in [Figure 7]. Figure 9 The yellow arrows indicate hollow vesicle structures.

[0064] from Figure 8 It can be seen that after in vitro digestion, the release rate of free fatty acids in each microcapsule exceeded 100%, indicating that the fat digestion was complete. However, the bioavailability of carotene varied significantly among different microcapsules. The bioavailability of carotene in the comparative example and the microcapsules obtained from formulations 4-6 were 72.97%, 77.50%, 74.53%, and 70.69%, respectively. Among them, the microcapsules obtained from formulation 4 had the highest bioavailability of carotene, while the microcapsules obtained from formulation 6 had the lowest bioavailability. This indicates that the dosage of each protein in the mixed protein base affects the bioavailability of carotene; for example, excessively high collagen peptide content may affect the stability of the emulsion. Figure 9 The presence of phosphatidylcholine in whey protein rich in milk phospholipid membranes is beneficial for enhancing the bioavailability of fat-soluble functional factors.

[0065] from Figure 9 It can be seen that after digestion in the stomach, each microcapsule exhibited obvious oil droplets and protein aggregates. The action of pepsin destabilized the droplet structure, and the acidic conditions neutralized the charge in the protein layer surrounding the oil droplets, leading to flocculation. Compared to the microcapsules obtained in the comparative examples and formulations 5-6, the microcapsules obtained in formulation 4 showed less aggregation and fewer particle aggregates during gastric digestion, which may affect the bioavailability of carotene. Based on the above results, the digestion mechanism model diagram of the emulsions obtained in the comparative examples and the embodiments is shown below. Figure 10 .

[0066] Example 11 Gastrointestinal hydrolysis behavior of mixed protein base The in vitro simulated digestion model described in Example 5 was used to simulate the digestion behavior of the protein-based material in the oral cavity, stomach, and small intestine. Samples were removed at different digestion times, and the degree of protein hydrolysis was determined using the o-phenylenediamine (OPA) method. The degree of hydrolysis of the comparative example provided in Example 1 and the mixed protein-based materials of formulations 1-6 were measured at different digestion times, and the results are shown below. Figure 11 .

[0067] from Figure 11It can be seen that the degree of protein hydrolysis increases during gastric digestion, indicating that the protein is hydrolyzed by pepsin. Compared with the control group, the degree of hydrolysis of the mixed protein bases of formulas 1-6 is increased. After small intestinal digestion, the degree of protein hydrolysis increases significantly, indicating that the hydrolytic ability of trypsin is stronger. After 2 hours of small intestinal hydrolysis, compared with the control group (degree of hydrolysis of 70%), the degrees of hydrolysis of the mixed protein bases of formulas 1, 2, and 3 are 75%, 76%, and 84%, respectively. The degree of hydrolysis of the mixed protein bases of formulas 5 and 6 reaches 100% after 15 minutes of small intestinal hydrolysis, and the degree of hydrolysis of the mixed protein base of formula 4 reaches 100% after 2 hours of small intestinal hydrolysis. This indicates that the mixed protein bases provided by the present invention have a higher degree of gastrointestinal hydrolysis, which is beneficial to protein absorption.

[0068] Conclusion: The mixed protein matrix provided by this invention co-stabilizes the oil-water interface of emulsions, effectively stabilizing both the emulsion and microcapsules. Furthermore, by utilizing the resulting emulsion and microcapsules to deliver lipid-soluble functional factors, it protects these factors and improves their stability. The mixed protein matrix provided by this invention can be used as a stabilizer in the preparation of emulsions and microcapsules, and for loading lipid-soluble functional factors. Gastrointestinal digestion evaluation showed that the mixed protein matrix provided by this invention enhances the solubilizing capacity of intestinal digestive fluids and improves the bioavailability of lipid-soluble functional factors. In addition, the mixed protein matrix provided by this invention exhibits a higher degree of hydrolysis.

[0069] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0070] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mixed protein base, characterized in that, By weight percentage, it comprises the following components: 25%–80% whey protein, 15%–70% collagen peptides, 2%–4% partially hydrolyzed whey protein, 1%–2% highly hydrolyzed whey protein, and 2%–4% whey protein rich in milk phospholipid membrane.

2. The mixed protein base according to claim 1, characterized in that, By weight percentage, the mixed protein base comprises the following components: 30%~50% whey protein, 40%~60% collagen peptides, 3%~4% moderately hydrolyzed whey protein, 1%~2% highly hydrolyzed whey protein, and 3%~4% whey protein rich in milk phospholipid membrane.

3. The mixed protein base according to claim 1, characterized in that, The molecular weight of the collagen peptide is 1000 Da to 5000 Da; and / or, The proportion of moderately hydrolyzed whey proteins with a molecular weight of 5000 Da to 20000 Da is greater than 80%; and / or, The proportion of highly hydrolyzed proteins with a molecular weight ≤1000 Da is greater than 90%; and / or, The whey protein rich in milk phospholipid membrane contains 5% to 10% milk phospholipid membrane.

4. A method for preparing an emulsion, characterized in that, Includes the following steps: S1. Mix the mixed protein base according to any one of claims 1 to 3 with water to obtain an aqueous phase; S2. Mix the aqueous phase and the oil phase, emulsify, and obtain an emulsion.

5. The method for preparing the emulsion according to claim 4, characterized in that, In step S2, the method for preparing the oil phase includes: dissolving the fat-soluble functional factor in edible oil to obtain the oil phase; wherein: The fat-soluble functional factor includes at least one of curcumin and carotene; and / or, The edible oil includes at least one of soybean oil, coconut oil, medium-chain triglycerides, and rapeseed oil; and / or, The mass fraction of the lipid-soluble functional factor in the oil phase is 0.1% to 0.5%.

6. The method for preparing the emulsion according to claim 4, characterized in that, In step S1, the mass fraction of the mixed protein matrix in the aqueous phase is 3% to 10%.

7. The method for preparing the emulsion according to claim 4, characterized in that, In step S2, the oil phase in the emulsion has a mass fraction of 1% to 8%; and / or, In step S2, the emulsification includes pre-emulsification and secondary emulsification.

8. A method for preparing microcapsules, characterized in that, Includes the following steps: The emulsion prepared by the method of any one of claims 4 to 7 is spray-dried to obtain microcapsules.

9. A liquid protein beverage, characterized in that, The product comprises, by mass percentage, the following components: 3% to 8% of the protein mixture base as described in any one of claims 1 to 3, 0% to 1% medium-chain fatty acid glycerides, 1% to 2% magnesium gluconate, 0.5% to 1.5% beetroot powder, 0.1% to 0.2% peppermint flavoring, 0.05% to 0.15% citric acid, 0.05% to 0.1% zinc citrate, 0.01% to 0.05% vitamins, with the balance being water.

10. A liquid protein beverage, characterized in that, Emulsions prepared by the method of any one of claims 4 to 7.

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