A complex DHA bovine milk protein peptide, its preparation method and application
By using nanoscale DHA emulsion encapsulation and compound bio-enzymatic hydrolysis technology, the problems of easy oxidation and low bioavailability of DHA in milk have been solved, and DHA milk protein peptides with high stability and easy absorption have been prepared. These peptides can be applied to liquid milk, fermented milk, milk powder and high-protein nutrition bars, thereby improving the nutritional value and flavor of the products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YUNNAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-30
AI Technical Summary
Traditional milk lacks DHA, which is easily oxidized and its content decreases in liquid milk. The bioavailability of milk protein is low, and high-temperature treatment destroys protein activity. The challenge is to combine the stabilization and protection of DHA with the targeted enzymatic hydrolysis of milk protein under mild conditions to prepare a milk base material that has both brain-boosting functions and easy absorption properties.
A nanoscale DHA emulsion encapsulation technology was combined with complex biological enzymatic hydrolysis. DHA emulsion was prepared by high-pressure homogenization, and after pH adjustment, trypsin and flavor protease were added for enzymatic hydrolysis. After enzyme inactivation and drying, complex DHA milk protein peptides were obtained, and after secondary homogenization, they were sterilized to prepare liquid milk.
It significantly improves the stability and bioavailability of DHA, avoids oxidation and bitterness, enhances nutrient absorption and product flavor, and achieves stable protection and efficient enzymatic hydrolysis of DHA in dairy products.
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Figure CN122296483A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of dairy processing technology, and in particular to a complex DHA bovine milk protein peptide, its preparation method, and its application. Background Technology
[0002] Docosahexaenoic acid (DHA) is an essential Omega-3 polyunsaturated fatty acid, commonly known as "brain gold," and is crucial for brain development and nerve health. Milk is hailed as "white blood," a vital source of high-quality protein and calcium. However, traditional milk contains almost no DHA, and adding DHA directly to liquid milk faces two major technical bottlenecks: first, DHA is highly susceptible to oxidation, leading to a fishy smell and increased peroxide value in the milk; second, DHA content significantly decreases during sterilization and long-term storage. Furthermore, the bioavailability of milk protein directly affects the body's absorption efficiency.
[0003] Traditional dairy processing often employs high-temperature or acid / alkali treatments, which can easily destroy the natural activity of proteins. In recent years, targeted enzymatic hydrolysis technology has been used to extract active proteins from fresh milk. However, how to combine the stabilization and protection of DHA with the enzymatic modification of milk proteins to prepare milk base materials that possess both brain-boosting functions and easy-to-absorb properties remains a challenge in the industry.
[0004] Therefore, there is an urgent need for a method to stabilize DHA, which can achieve stable protection of DHA under mild processing conditions without affecting the efficiency of targeted enzymatic hydrolysis of milk proteins and the activity of the products, so as to promote the upgrading and development of functional dairy products towards a more efficient, more natural, and more synergistic nutritional value. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art. On one hand, this application provides a method for preparing a complex DHA milk protein peptide, characterized by comprising the following steps: S1, DHA algal oil is mixed with emulsifier and prepared into nano-sized DHA emulsion by high pressure homogenization; S2, mix the DHA emulsion obtained in step S1 with skim milk, adjust the pH, add compound biological enzyme and enzymatically hydrolyze for 2-4 hours to obtain enzymatic hydrolysate; The complex bioenzyme consists of 0.1% trypsin by volume of DHA emulsion and 0.05% flavor protease by volume of DHA emulsion. S3, after inactivating the enzyme in the enzymatic hydrolysate and drying it, yields a complex DHA milk protein peptide.
[0006] Furthermore, in step S1, the pressure of high-pressure homogenization is 30-50 MPa, and the number of homogenization cycles is 2-3.
[0007] Furthermore, the enzyme inactivation treatment in step S3 is performed by heating at 85-95°C for 10-15 minutes.
[0008] Furthermore, the enzymatic hydrolysis temperature in step S3 is 45-55℃.
[0009] Furthermore, in step S3, the drying process involves drying the material through a spray drying tower; the inlet air temperature of the spray drying tower is 160°C, and the outlet air temperature is 80°C.
[0010] On the other hand, this application also provides a complex DHA milk protein peptide obtained according to the above preparation method.
[0011] On the other hand, this application also provides a method for preparing DHA-rich liquid milk, the preparation method comprising: subjecting a complex DHA milk protein peptide to secondary homogenization followed by sterilization to obtain liquid milk; wherein the complex DHA milk protein peptide is prepared by the above-described preparation method.
[0012] Furthermore, the pressure of the secondary homogenizer is 20 MPa / 5 MPa.
[0013] Furthermore, the ultra-high temperature sterilization temperature is 137°C, and the time is 4 seconds.
[0014] On the other hand, this application also provides the application of the complex DHA milk protein peptide obtained by the above preparation method in the preparation of liquid milk, fermented milk, milk powder or high-protein nutrition bars.
[0015] Beneficial effects 1. This application provides a compound DHA milk protein peptide with an in vitro digestibility of 94.8% (Example 3), significantly improving the bioavailability of the protein. Compared to ordinary milk powder without enzymatic hydrolysis (Comparative Example 1, digestibility 78.3%), the protein peptide of this application, due to the use of a targeted enzymatic hydrolysis process, produces mainly small molecule peptides (with a high proportion of molecular weight <1000Da), making it easier for the human body to absorb. Furthermore, compared to Comparative Example 2, which only involves enzymatic hydrolysis without encapsulation, this application, through encapsulation with nano-sized DHA emulsion, effectively avoids the bitterness produced during enzymatic hydrolysis while maintaining a high digestibility (94.8% vs. 96.1%), resulting in a better sensory flavor (no obvious fishy or bitter taste), achieving a synergistic improvement in nutrient absorption and product flavor.
[0016] 2. This application provides a method for preparing a complex DHA milk protein peptide, optimizing the composition of the complex bioenzyme and the enzymatic hydrolysis conditions. By selecting trypsin and flavor protease for compounding (Example 1), on the one hand, trypsin specifically cleaves the basic amino acid sites in milk protein, improving hydrolysis efficiency; on the other hand, flavor protease removes terminal hydrophobic amino acids, effectively controlling the formation of bitter peptides. Combining the effect of enzymatic hydrolysis time on product performance in Comparative Example 3 (as shown in Table 2), the enzymatic hydrolysis time was determined to be 3 hours. At this time, the degree of hydrolysis is moderate (18%), the bitterness value is low, and the DHA retention rate is high (96%), solving the technical problem of insufficient degree of hydrolysis or excessive bitterness caused by single enzymatic hydrolysis.
[0017] 3. This application also provides a method for preparing DHA-rich liquid milk. The liquid milk obtained by this method has a DHA retention rate as high as 92% (Example 2), which is significantly improved compared to Comparative Example 1 (conventional addition, retention rate 45%). Meanwhile, after 6 months of storage at room temperature, the peroxide value of this liquid milk is only 0.8 meq / kg (Example 2), far lower than the 3.5 meq / kg of Comparative Example 1, indicating that this application significantly enhances the oxidative stability of DHA through the synergistic effect of nano-encapsulation and enzymatic hydrolysis technologies. Furthermore, thanks to the small molecule characteristics of protein peptides, the resulting liquid milk not only retains the pure milk aroma, but also has nutrients that are more easily absorbed, improving the overall nutritional value of the product.
[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description
[0019] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.
[0020] Figure 1 This describes the effect of enzymatic hydrolysis time on the degree of protein hydrolysis and DHA retention.
[0021] Figure 2 This relates to the effect of homogenization pressure on the particle size and stability of DHA emulsions.
[0022] Figure 3 This study investigates the effect of pH on the degree of hydrolysis and sensory evaluation. Detailed Implementation
[0023] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.
[0024] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.
[0025] Example 1 Take 1000 kg of fresh milk and sterilize it by microfiltration at 50°C using a ceramic membrane microfiltration device to obtain skim milk for later use.
[0026] DHA emulsion preparation: Weigh 5 kg of DHA algal oil (DHA content ≥ 40%), mix it with 1.5 kg of sodium caseinate and 0.5 kg of glyceryl monostearate, add an appropriate amount of softened water, and homogenize it 3 times under a pressure of 45 MPa to obtain a DHA emulsion with an average particle size of 200 nm.
[0027] Mixed enzymatic hydrolysis: Mix DHA emulsion and skim milk evenly in an enzymatic hydrolysis tank, adjust the pH to 6.8, raise the temperature to 50°C, add 0.1% trypsin and 0.05% flavor protease by volume of DHA emulsion, and enzymatically hydrolyze for 3 hours.
[0028] Post-processing: The temperature was raised to 90℃ and held for 10 minutes to inactivate the enzyme. The material was then dried in a spray drying tower with an inlet air temperature of 160℃ and an outlet air temperature of 80℃, yielding a composite DHA milk protein peptide. In this product, DHA is uniformly distributed in a nano-encapsulated form within a small molecule milk protein peptide matrix, forming a composite system in which DHA and active peptides work synergistically.
[0029] Table 1. Comparison of physicochemical properties between the compound DHA milk protein peptide and ordinary protein peptides in this application. Example 2 After preparing DHA bovine milk protein peptides according to the method in Example 1, the peptides were subjected to secondary homogenization at 20 MPa / 5 MPa pressure, and finally sterilized by ultra-high temperature sterilization (137°C, 4 seconds) and aseptically filled to obtain liquid milk rich in DHA.
[0030] Comparative Example 1: Effect of enzymatic hydrolysis time on protein hydrolysis degree and DHA retention rate The enzymatic hydrolysis time in Example 1 was adjusted to 0, 1, 2, 3, 4, and 5 hours, with a fixed hydrolysis temperature of 50°C, pH 6.8, and the amount of the compound enzyme (trypsin:flavor protease volume ratio = 2:1) added was 0.15% of the DHA emulsion volume. The degree of hydrolysis and DHA retention rate were measured at different time points. The remaining steps were the same as in Example 1. The results are as follows Figure 1 As shown, the optimal enzymatic hydrolysis time is determined to ensure a high degree of hydrolysis (which is beneficial for absorption) while avoiding excessive loss of DHA or the production of bitterness. Figure 1The results showed that as the enzymatic hydrolysis time increased from 0 hours to 5 hours, the degree of protein hydrolysis continued to rise. The degree of hydrolysis increased rapidly from 0 to 3 hours, and the rate of increase slowed down after 3 hours. The DHA retention rate remained above 95% in the early stage of enzymatic hydrolysis, but began to decline significantly after 3 hours, dropping to about 88% by 5 hours. Based on the trends of the two curves, 3 hours was the optimal enzymatic hydrolysis time, at which the degree of hydrolysis reached about 18%, the product was mainly small molecule peptides, and the DHA retention rate was still as high as 96%, achieving a synergistic balance between efficient protein hydrolysis and stable DHA protection.
[0031] Comparative Example 2: Effect of Homogenization Pressure on Particle Size and Stability of DHA Emulsion In Example 1, the homogenization pressure was adjusted to 10, 20, 30, 40, 50, and 60 MPa. The DHA algal oil content was fixed at 5%, and the compound emulsifier content was fixed at 2% (sodium caseinate: monoglyceride = 3:1). The homogenization was performed 3 times. The average particle size under different pressures and the particle size growth rate (stability index) after being placed at room temperature for 30 days were measured. The remaining steps were the same as in Example 1.
[0032] The results are as follows Figure 2 As shown, the optimal homogenization pressure was determined to prepare a DHA emulsion with fine and uniform particle size distribution, thereby improving its antioxidant capacity. Figure 2 This study demonstrates the effect of homogenization pressure on the particle size and stability of DHA emulsions, providing crucial experimental evidence for determining the process parameters for preparing nanoscale DHA emulsions. The curves show that as the homogenization pressure increases from 10 MPa to 60 MPa, the initial average particle size of the DHA emulsion exhibits a trend of first rapidly decreasing and then leveling off: in the 10-30 MPa range, the particle size rapidly decreases from approximately 400 nm to around 220 nm; in the 30-50 MPa range, the particle size further decreases slowly to approximately 180 nm; when the pressure exceeds 50 MPa, the decrease in particle size is minimal, remaining essentially at 170-180 nm. Meanwhile, the particle size growth rate after 30 days at room temperature was used as a stability evaluation index. The results showed that the particle size growth rate was the lowest (approximately 5%-8%) in the 30-50 MPa range, indicating the best emulsion stability. When the pressure was below 30 MPa, the initial particle size was larger and unevenly distributed, and the particle size growth rate after 30 days was as high as 15% or more, making the emulsion prone to stratification and aggregation. When the pressure exceeded 50 MPa, although the initial particle size was smaller, the excessive shear force may destroy the emulsifier structure, resulting in a slight increase in the particle size growth rate (approximately 10%). Considering both the particle size refinement effect and stability performance, 30-50 MPa is the optimal homogenization pressure range. Under this condition, DHA emulsions with small particle size (100-300 nm), uniform distribution, and excellent long-term stability can be prepared, providing a good stabilization basis for subsequent enzymatic hydrolysis with skim milk.
[0033] Comparative Example 3: Effect of enzymatic hydrolysis pH on degree of hydrolysis and sensory score In Example 1, the pH was adjusted to 5.5, 6.0, 6.5, 7.0, and 7.5 MPa. The enzymatic hydrolysis temperature was fixed at 50°C, the time was 3 hours, and the compound enzyme concentration was 0.15%. The degree of hydrolysis was measured at different pH values, and the final product was sensory evaluated (blind test by a 10-person panel, with a maximum score of 10 points, including bitterness, fishy smell, and overall acceptability). The remaining steps were the same as in Example 1. The results are as follows: Figure 3 As shown, determine the optimal pH for enzymatic hydrolysis to maximize enzyme activity and ensure good product flavor (avoiding excessive acidity or alkalinity that can lead to off-flavors). Figure 3 This study demonstrates the impact of pH on the degree of protein hydrolysis and the sensory score of the product, providing important experimental evidence for optimizing enzymatic hydrolysis process conditions. The curves show that as the pH increases from 5.5 to 7.5, the degree of protein hydrolysis exhibits a peak-shaped change: in the pH range of 5.5-6.5, the degree of hydrolysis rapidly increases from approximately 10% to 18%; it reaches its peak at pH 6.8 (approximately 18.5%); subsequently, in the pH range of 7.0-7.5, the degree of hydrolysis gradually decreases to approximately 15%. The sensory score trend is largely consistent with the degree of hydrolysis. The highest sensory score (9.2-9.5 points) is achieved in the pH range of 6.5-7.0, with the product exhibiting a pure milky aroma and no obvious bitterness or fishy smell. When the pH is below 6.0, the sensory score drops significantly to below 7.0 points, mainly manifested as a pronounced sour taste and unbalanced flavor. When the pH is above 7.2, the sensory score also decreases (approximately 8.0 points), with the product exhibiting a slight alkaline taste and bitterness. Comprehensive analysis shows that the optimal pH range for enzymatic hydrolysis is 6.5-7.0. Under this condition, the activity of the complex enzymes (trypsin and flavor protease) can be kept at its best, the degree of hydrolysis can reach more than 18%, and good product flavor can be obtained, thus achieving synergistic optimization of enzymatic hydrolysis efficiency and sensory quality.
[0034] Comparative Example 4: Preparation of Conventional Liquid Milk Following standard procedures, an equal amount of DHA algal oil is directly added to ordinary fresh milk without emulsification, encapsulation, or enzymatic hydrolysis, and then the milk is filled and sterilized under the same conditions.
[0035] Comparative Example 5: Enzymatic digestion only, without encapsulation 1000 kg of fresh milk was subjected to microfiltration sterilization and defatting treatment according to the method in Example 1 to obtain defatted milk for later use. The difference from Example 1 is that the DHA emulsion preparation step was omitted. Instead, 5 kg of DHA algal oil (DHA content ≥40%) was directly mixed with defatted milk in an enzymatic hydrolysis tank without adding any emulsifier or undergoing high-pressure homogenization pretreatment. Only mechanical stirring was used to initially disperse the DHA algal oil in the milk. Subsequently, the pH was adjusted to 6.8, the temperature was raised to 50°C, and 0.1% trypsin and 0.05% flavor protease were added. Enzymatic hydrolysis was carried out for 3 hours. After enzymatic hydrolysis, the temperature was raised to 90°C and maintained for 10 minutes to inactivate the enzymes. The material was then dried in a spray drying tower with an inlet air temperature of 160°C and an outlet air temperature of 80°C, and the product was collected. This comparative example was used to verify the protective effect of nano-encapsulation pretreatment on DHA during enzymatic hydrolysis and drying.
[0036] Example 3 Effect Experiment The liquid milk prepared in Example 2 and the sample of Comparative Example 1 were stored at room temperature for 6 months to detect the retention rate of DHA and sensory flavor.
[0037] The results in Tables 2 and 3 show that Example 2 exhibited a DHA retention rate as high as 92% and no oxidative fishy odor; while Comparative Example 1 showed a DHA retention rate of only 45% and exhibited a distinct fishy and oxidized fat odor. This indicates that the present invention significantly improves the stability of DHA in dairy products through the synergistic effect of emulsification encapsulation and targeted enzymatic hydrolysis technologies.
[0038] The protein peptide powder prepared in Example 1 was subjected to an in vitro simulated digestion experiment. The results showed that its digestibility and absorption rate was significantly higher than that of ordinary milk powder that had not been enzymatically hydrolyzed, confirming that the enzymatic hydrolysis process improved the bioavailability of the protein.
[0039] Table 2: Comparison of Effects / Performance between Examples and Comparative Examples Table 3: Effects of different enzymatic hydrolysis conditions on product performance The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A method for preparing a complex DHA bovine milk protein peptide, characterized in that, Includes the following steps: S1, DHA algal oil is mixed with emulsifier and prepared into nano-sized DHA emulsion by high pressure homogenization; S2, mix the DHA emulsion obtained in step S1 with skim milk, adjust the pH, add compound biological enzyme and enzymatically hydrolyze for 2-4 hours to obtain enzymatic hydrolysate; The complex bioenzyme consists of 0.1% trypsin by volume of DHA emulsion and 0.05% flavor protease by volume of DHA emulsion. S3, after inactivating the enzyme in the enzymatic hydrolysate and drying it, yields a complex DHA milk protein peptide.
2. The preparation method according to claim 1, characterized in that, In step S1, the pressure of high-pressure homogenization is 30-50 MPa, and the number of homogenization cycles is 2-3.
3. The preparation method according to claim 1, characterized in that, The enzyme inactivation treatment in step S3 is to heat at 85-95°C for 10-15 minutes.
4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature in step S3 is 45-55℃.
5. The preparation method according to claim 1, characterized in that, In step S3, drying involves passing the material through a spray drying tower; the inlet temperature of the spray drying tower is 160°C, and the outlet temperature is 80°C.
6. A complex DHA bovine milk protein peptide obtained by the preparation method according to any one of claims 1-5.
7. A method for preparing DHA-rich liquid milk, characterized in that, The preparation method includes: subjecting the complex DHA milk protein peptide to secondary homogenization and then sterilizing to obtain liquid milk; the complex DHA milk protein peptide is prepared by the preparation method according to any one of claims 1-5.
8. The method according to claim 7, characterized in that, The pressure of the secondary homogenizer is 20 MPa / 5 MPa.
9. The method according to claim 7, characterized in that, The ultra-high temperature sterilization was performed at 137°C for 4 seconds.
10. The application of the complex DHA bovine milk protein peptide obtained by the preparation method according to any one of claims 1-5 in the preparation of liquid milk, fermented milk, milk powder or high-protein nutrition bars.