Modified ovine whey proteins having iron aggregation prevention and iron ion absorption promotion and preparation and use thereof

Abstract

The application discloses modified sheep whey protein with the functions of preventing iron aggregation and promoting iron ion absorption, and preparation and application thereof, and belongs to the technical field of protein material modification. The modified sheep whey protein is prepared by using sheep whey protein as raw material, and a protein modification structure is formed through one-step acid-heat method to induce protein structure recombination, so that the modified sheep whey protein has good iron ion combination capacity and reduction activity. The prepared modified sheep whey protein has stable fiber structure and active action sites, can effectively adsorb iron ions and promote Fe 3+ Fe 2+ conversion, while inhibiting the aggregation of nano-iron particles and improving the stability of the iron system. The iron complex system based on the modified sheep whey protein has good iron valence state stability and application potential, and can be applied to the field of food processing and nutrition supplement as a new type of iron fortification material. The preparation method is simple, green and safe, and realizes the functional modification and high-value utilization of natural whey protein.

Description

Technical Field

[0001] This invention belongs to the field of protein material modification technology, specifically relating to a modified sheep whey protein that prevents iron aggregation and promotes iron ion absorption, as well as its preparation and application. Background Technology

[0002] Dietary iron deficiency is a key cause of anemia-related disabilities and shortened lifespan. To reduce anemia, the World Health Organization (WHO) recommends using iron salts as food fortification for most foods; however, most iron fortifications have negative sensory impacts due to low bioavailability and poor sensory performance. Meanwhile, the recommended daily iron intake for young women is 18 mg, a high requirement that is typically met solely through diet. Therefore, there is a need to develop innovative technologies that can efficiently introduce highly bioavailable iron into food.

[0003] To date, several compounds have been developed, such as sodium ferric ethylenediaminetetraacetate (NaFeEDTA), which exhibit superior absorption compared to ferrous salts when used in food matrices containing inhibitory compounds. Meanwhile, ferric diglycinate, an amino-chelated iron fortifier, has shown significant efficacy in specific applications (such as milk), but the World Health Organization does not recommend its large-scale food fortification due to its high cost and potential sensory problems in food. In recent years, nano-iron composites have been widely used in iron-fortified foods due to their excellent biocompatibility and toxicological safety. However, their tendency to oxidize easily and rapidly aggregate in solution when used alone results in less than ideal practical application effects. Therefore, finding a method to effectively stabilize and improve iron absorption without affecting the sensory properties of food remains a key technical challenge.

[0004] Proteins can be modified through physical and chemical methods. Globular proteins, under acidic and heated conditions, can undergo protein unfolding and structural recombination to form various structures such as amyloid fibers, fractal aggregates, or amorphous aggregates. These structures can serve as scaffolds for the synthesis of mineral nanoparticles, carriers of bioactive compounds, and building blocks for functional composite materials. However, different preparation methods and raw material sources can lead to significant differences in the structural morphology of the obtained modified proteins, potentially resulting in different functions. Sheep whey protein, an important byproduct of sheep cheese processing, is a typical natural globular protein with good biocompatibility, high nutritional value, excellent interfacial activity, and biodegradability, making it widely used in food processing, functional materials, biomedicine, and environmental remediation. However, no modified sheep whey protein has yet been used to achieve in-situ generation and stable loading of nano-iron. Therefore, there is an urgent need to obtain natural modified sheep whey protein rapidly in a one-step process while reducing costs, and to apply it to the stable adsorption of nano-iron, thereby controlling iron aggregation. This is of great significance for realizing the high-value utilization of whey protein. Summary of the Invention

[0005] To address the problem of the lack of an effective carrier in the existing technology that can prevent iron aggregation and promote iron ion absorption, the present invention aims to provide a modified sheep whey protein that can prevent iron aggregation and promote iron ion absorption, as well as its preparation and application.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a method for preparing modified sheep whey protein that prevents iron aggregation and promotes iron ion absorption, comprising the following steps: The whey protein isolate from sheep was dispersed in an acidic solution to prepare a protein dispersion. Modified sheep whey protein was obtained by high-temperature heating under stirring conditions and acidic conditions with a pH of 1.5–2.5, followed by cooling.

[0007] Preferably, the heat treatment temperature is 80–95°C, and the time is 3–12 h. This ensures that the sheep whey protein can fully expand and repolymerize, while simultaneously enhancing the adhesion and reducing power of these aggregates.

[0008] More preferably, the heat treatment temperature is 90°C and the heat treatment time is 12 h.

[0009] Preferably, the mass concentration of the protein dispersion is 1% to 5%.

[0010] More preferably, the pH value of the acidic solution is 2.0, and the mass concentration of the protein dispersion is 2%.

[0011] The stirring speed is 300 rpm to 500 rpm.

[0012] The present invention provides a modified sheep whey protein obtained by the above preparation method, which has the functions of preventing iron aggregation and promoting iron ion absorption.

[0013] It contains rigid long fibers with an average height of 3-7 nm, and the modified sheep whey protein has iron ion reducing activity and an adhesive surface.

[0014] This invention provides a modified sheep whey protein-nano iron composite material, comprising: The matrix is ​​the modified sheep whey protein described above; and the iron nanoparticles are loaded on the surface of the matrix.

[0015] The nano-iron particles are loaded on the surface of the modified sheep whey protein. The nano-iron particles generated by the reduction of ferric salt have a particle size of 5-20 nm. The mass ratio of the modified sheep whey protein to iron is 1:(3-7).

[0016] More preferably, the mass ratio of the modified sheep whey protein to iron is 1:5.

[0017] This invention provides the application of the modified sheep whey protein-nano iron composite material in the preparation of iron-fortified foods or iron supplements.

[0018] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a method for preparing modified sheep whey protein that prevents iron aggregation and promotes iron ion absorption. The method uses sheep whey protein isolate as raw material and induces it to become fibrous through acid-heat induction. During the fibrosis process, the adhesion and iron ion reduction capacity of sheep whey protein are simultaneously improved, laying a dual functional foundation for its subsequent use as a carrier of nano-iron materials. This solves the technical problem in the prior art of lacking a preparation method that simultaneously improves the adhesion and reduction capacity of sheep whey protein.

[0019] This invention provides a modified sheep whey protein that prevents iron aggregation and promotes iron ion absorption. Using sheep whey protein as a raw material, after a one-step acid-thermal induction process, its structure differs from the globular conformation of natural sheep whey protein. The modified sheep whey protein obtained by this invention possesses specific structural features and functional properties, making it a novel protein modification material. It also exhibits an adhesive active surface and long-term ability to reduce ferric iron. Furthermore, it can be used as an intermediate product for the subsequent construction of iron nanoparticles or other functional materials.

[0020] The modified sheep whey protein-iron nanocomposite material provided by this invention has both adhesive and reducing properties in its modified sheep whey protein matrix. The former ensures that ferric ions can be effectively adsorbed onto the fiber surface, while the latter reduces Fe... 3+ Reduced to Fe 2+ Furthermore, the modified protein fibers achieved stable loading, enabling in-situ generation and uniform distribution of iron nanoparticles and avoiding nanoparticle aggregation. More importantly, the rigid long fiber structure (average height 3–7 nm) and active surface of the modified protein fibers can form a physical barrier and chemical protective layer around the nanoparticles, effectively blocking contact with dissolved oxygen and thus significantly delaying the degradation of Fe. 2+ The composite material exhibited stable ferrous ion concentration during a 21-day storage period, demonstrating the significant advantage of this invention in terms of iron valence stability.

[0021] The application provided by this invention is the application of modified sheep whey protein-nano iron composite material in the field of iron-fortified foods. This composite material has the following advantages: sheep whey protein has lower allergenicity than bovine whey protein, making it more suitable for sensitive individuals; the nano iron particles are small in size, do not aggregate, and are uniformly dispersed, which is beneficial for improving iron bioavailability; especially in dairy products, it is superior to traditional iron fortifiers, producing no unpleasant odors or sensory differences. It solves the application technology bottlenecks of nano iron and traditional iron fortifiers, expands the application scope of high-value utilization of whey protein, and enhances its application value and market potential. Attached Figure Description

[0022] Figure 1 The images show the modified structures of sheep whey protein obtained at different heating temperatures in Example 1 of this invention. A represents TEM images of the four modified structures, and B represents fluorescence changes at the same heating temperature.

[0023] Figure 2 The images show the atomic force microscopy (AFM) morphology and fiber height distribution of sheep whey protein after heating at 90°C for 12 hours in Example 1 of this invention; where A1-D1: AFM morphology images; A2-B2: magnified modified protein morphology images; A3-D3: height changes of protein aggregates at different times. Figure 3 This is a graph showing the viscosity of sheep whey protein in Example 1 of the present invention as a function of shear rate after heating at 90°C for different times. Figure 4 The figure shows the effect of different concentrations and forms of whey protein fiber on the reduction capacity of iron ions. In this figure, A represents the reduction effect of modified protein on iron ions at different concentrations after 12 hours, and B represents the reduction effect of modified protein on iron ions at different heating times. Figure 5 The images show a comparison of transmission electron microscopy (TEM) images of the modified protein-nano iron composite material before and after preparation, where A is a process flow diagram for the preparation of the composite material, B is pure fiber without iron loading, and C is the modified protein-nano iron composite material after loading with nano iron particles. Figure 6 This diagram shows the stability changes of the modified protein-nano iron composite material prepared in this invention at different storage times. In this diagram, A represents the colloidal appearance at different storage times; B represents the Fe content during storage. 2+ Concentration change bar chart; Figure 7 The color of the composite material prepared in this invention and the traditional iron fortifier were evaluated in different food systems. In this paper, A shows photographs of the appearance of six different food matrices after the addition of the iron fortifier, and B is a bar chart comparing the total color difference (ΔE) of the corresponding samples. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the examples, they can be performed according to the techniques or conditions described in the literature or the product instructions.

[0026] Unless otherwise stated, the following terms as used herein have the following meanings: Goat whey protein: refers to the whey protein component isolated and extracted from goat milk, whose main components are β-lactoglobulin and α-lactalbumin, with a protein content of not less than 90% (on a dry basis). The goat whey protein used in this invention can be obtained commercially or prepared by referring to existing literature methods. It can be prepared from goat whey using membrane separation technology (such as ultrafiltration or nanofiltration) combined with spray drying, or purchased directly from food-grade protein suppliers.

[0027] Adhesion: In this invention, adhesion refers to the adhesive properties exhibited by the modified sheep whey protein, determined by rheological methods at shear rates of 0.1–100 s⁻¹. -1 The viscosity values ​​are evaluated within a certain range; the higher the viscosity value, the stronger the adhesion.

[0028] Reducing power: refers to the ability of modified sheep whey protein to reduce ferric ions (Fe3+). 3+ ) is reduced to ferrous ions (Fe) 2+ The ability of modified sheep whey protein to absorb iron was quantitatively evaluated by measuring absorbance at a wavelength of 512 nm using the o-phenanthroline colorimetric method. Nano-iron particles: These refer to nano-sized iron particles loaded on the surface of modified sheep whey protein, with a particle size of 5-20 nm, spherical in shape, and generated by in-situ reduction of ferric salts with sodium ascorbate. Iron-fortified foods: These are foods with iron-supplementing functions prepared by adding modified sheep whey protein-nano-iron composite materials as iron fortifiers to food matrices (including but not limited to infant formula, milk beverages, solid beverages, and cereal products).

[0029] The goat whey protein used in this embodiment of the invention was purchased from Shanghai Kaiyang Biotechnology Co., Ltd., but can also be prepared by referring to existing technical methods: using fresh goat milk as raw material, the protein is degreased by centrifugation, the pH is adjusted to 4.6 to precipitate casein, the whey supernatant is collected, and then concentrated by ultrafiltration (molecular weight cutoff 10 kDa) and spray dried. Sodium ascorbate and 1,10-phenanthroline were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and 1,10-phenanthroline was purchased from Tianjin Benchmark Chemical Reagent Co., Ltd. Traditional iron fortification agents: food-grade ferrous sulfate (FeSO4) and ferric pyrophosphate (FePP) were purchased from Jiangsu Kelunduo Food Ingredients Co., Ltd., and ferrous glycinate (Fe-bisglycinate) was purchased from Hebei Liweisu Technology Co., Ltd.

[0030] Example 1: Preparation and Process Optimization of Modified Sheep Whey Protein 1. Effects of different heating temperatures on the fibrinization of sheep whey protein Goat whey protein (WPI) was dispersed in deionized water to prepare a 2% (w / w) protein dispersion. The dispersion was stirred overnight at 600 rpm using a magnetic stirrer to ensure complete protein hydration. The pH was adjusted to 2.0 with 1 M HCl solution, and the samples were then heated in water baths at 80℃, 85℃, 90℃, and 95℃ for 12 h, with stirring speeds ranging from 300 rpm to 500 rpm during heating. After heating, the samples were immediately cooled to room temperature in an ice bath and stored at 4℃ for later use.

[0031] Transmission electron microscopy (TEM) observation: Fiber solutions prepared under different conditions were diluted with deionized water to 0.01% (w / v). 20 μL of each solution was dropped onto a copper grid supporting a carbon film and allowed to stand for 1 min. 20 μL of a 2% (w / v) phosphotungstic acid solution was used for negative staining, and after standing for 1 min, excess stain was absorbed with filter paper. Imaging and observation were performed using a Hitachi HT7700 Exalens TEM at an accelerating voltage of 100 kV. See [link to TEM description]. Figure 1 Different structural morphologies were observed at four temperatures: 80℃, 85℃, 90℃, and 95℃. At 80℃ and 85℃, some unaggregated globular protein monomers remained besides fibers; at 90℃ and 95℃, only fibers were observed, and these fibers aggregated with increasing temperature. Thiamine T (ThT) fluorescence detection results showed that the β-sheet content did not increase significantly above 90℃, indicating that the sheep whey protein had been fully unfolded, thus determining the optimal modification temperature to be 90℃.

[0032] 2. Effect of different heating times on the morphology of modified sheep whey protein Goat whey protein isolate was dispersed in deionized water to prepare a 2% (w / w) protein dispersion. The dispersion was stirred overnight at 600 rpm using a magnetic stirrer. The pH was adjusted to 2.0 with 1 M HCl. The samples were then heated in a 90°C water bath for 0, 3, 6, 8, and 12 h, respectively. The resulting samples were labeled WPI (unheated control), WPIF-3, WPIF-6, WPIF-8, and WPIF-12, respectively. After heating, the samples were cooled in an ice bath and stored at 4°C.

[0033] (1) Morphological characteristics of modified sheep whey protein during heating Atomic force microscopy (AFM) observation: The fiber solution was diluted to 0.01 g / L with an acidic solution at pH 2.0. 10 μL of the solution was dropped onto freshly peeled mica sheets and allowed to air dry at room temperature. Imaging was performed using a Bruker Dimension Icon atomic force microscope in tapping mode, with a scanning range of 0.5–5 μm. Statistical analysis of the inner contour length and height of the structure was conducted using Nanoscope Analysis 1.5 and FiberApp software.

[0034] See Figure 2 When heated for 3 h and 6 h, the formed protein aggregates were relatively short, with a height of 1.8–3.5 nm and a wide pitch range. The overall fibrous structure exhibited a rod-like shape and was mainly composed of flexible short fibers. When the heating time exceeded 8 h, protein aggregation was further enhanced, and the length and height of the aggregates increased significantly. As shown in Figures C2 and D2, when the heating time reached 12 h, the average height of the formed protein aggregates was 5 nm.

[0035] (2) Adhesion characteristics of sheep whey protein during fibrinization The viscosity of the samples at different time points was measured using a Haake MARS rheometer, with shear rates ranging from 0.1 to 100 s⁻¹. -1 See also Figure 3 As heating time increased, the viscosity of the samples showed an increasing trend. The viscosity difference between samples formed at 12 h and 8 h was not significant, indicating that the more mature the aggregates formed by protein unfolding, the better the adhesion properties of sheep whey protein, making it more suitable as an adsorbent material.

[0036] (3) The reducing effect of modified sheep whey protein on iron ions Fe was determined by the o-phenanthroline colorimetric method. 2+Content. 10 μL of samples modified at 90℃ for 12 h at different concentrations (2 mg / mL, 10 mg / mL, and 20 mg / mL) were mixed with 30 μL of 0.1 M FeCl3·6H2O solution, and 1.8 mL of 5 mM 1,10-phenanthroline colorimetric reagent was added. The absorbance was measured at 512 nm every half hour for 16 h. FeCl3 solution and pure sample solution were used as blank controls. The effect of different time points (0, 3, 6, 8, and 12 h) on iron reducing power was detected using the same method. 2+ The content was quantified using the FeSO4·7H2O standard curve (0–10 μM).

[0037] See Figure 4 As the sample concentration increased, the absorbance at 512 nm increased, indicating that the higher the fiber concentration, the better the iron ion reduction effect. With prolonged heating time, the Fe content measured at 512 nm... 2+ A higher content indicates more mature protein aggregation and a better reduction effect on iron ions. Therefore, considering the adhesion characteristics and iron ion reduction effect of the modified protein, heating at 90℃ for 12 hours was determined to be the optimal preparation condition for fully modifying sheep whey protein.

[0038] Example 2: Preparation of modified sheep whey protein-nano-iron composite material This embodiment is based on embodiment 1, with reference to... Figure 5 The preparation flow chart for protein A is shown below. Based on a solution obtained from sheep whey protein modified at 90℃ for 12 h, it was mixed with FeCl3·6H2O solution (0.1 M) at a mass ratio of 1:5. Sodium ascorbate (SA) was prepared as a 3 M solution. The SA solution was added dropwise to the mixed dispersion at a molar ratio of iron to sodium ascorbate of 1:2, and the mixture was gently stirred. The final protein concentration in the mixed dispersion was 1.16% (mass-volume ratio), the FeCl3 concentration was 40 mM, and the SA concentration was 81 mM. Pure modified protein without added iron salts served as a control.

[0039] After appropriate dilution of the modified protein solution or the modified protein-nano iron complex dispersion, 20 μL was dropped onto the copper grid of the carbon support membrane, allowed to stand for 1 min, rinsed with ultrapure water, and dried under an infrared lamp. Imaging and observation were performed using a Hitachi HT7700 Exalens transmission electron microscope at 100 kV.

[0040] See Figure 5 Compared to modified protein without iron adsorption, the fiber surface after loading with iron nanoparticles is covered with a large number of small spherical particles with a particle size of 5–20 nm, which are uniformly distributed. During the 21-day storage period, see [reference needed]. Figure 6 Fe in the composite system2+ The concentration remained relatively stable, indicating that the modified sheep whey protein has good iron ion adsorption capacity and long-term iron reduction stability.

[0041] Example 3: Application of modified sheep whey protein-nano-iron composite material in iron-fortified foods The modified sheep whey protein-nano iron composite material obtained in Example 2 was freeze-dried under vacuum (-60℃, 100Pa, 48 h) to obtain a powder. This powder, calculated based on iron content, was added to infant formula, milk beverage, or solid beverage base in an appropriate proportion, and thoroughly mixed using a mixer to obtain iron-fortified food. See also Figure 7 The color of food systems with four iron fortifiers added was measured using a colorimeter (Shenzhen Sanenchi Technology Co., Ltd., NS800). A blank group without iron fortifiers was used as a control, and the total color difference value (ΔE) was calculated. A larger color difference value indicates a more significant color change compared to the blank group; a smaller ΔE indicates a lower degree of interference with the original color of the food. The results showed that the total color difference value ΔE of the composite material (WPIFs-SA-Fe) added to various food matrices (infant formula, milk beverages, solid beverages, etc.) remained at a low level. The color of the food system before and after addition remained basically consistent, with little noticeable change to the naked eye, indicating that the composite material had minimal interference with the original color of the food. In stark contrast, the three traditional iron fortifier groups all showed varying degrees of color deterioration. Ferrous sulfate (FeSO4) group: The ΔE value was significantly higher than that of the present invention group in all test matrices, especially in milk beverages and solid beverages, where obvious browning or darkening was observed, and the color change was clearly distinguishable to the naked eye; Ferrous pyrophosphate (FePP) group: The ΔE value was the second highest, mainly showing a decrease in whiteness and the formation of a pale yellow precipitate, which seriously affected the appearance and acceptability of the product; Ferrous glycinate (Fe-bisglycinate) group: Although the ΔE value was lower than the previous two, it was still significantly higher than that of the present invention group, and the resulting color deepening was also easily identifiable to the naked eye.

[0042] The above comparative results clearly demonstrate that the modified sheep whey protein-nano iron composite material of this invention exhibits superior color compatibility in food matrices compared to commercially available traditional iron fortifiers. This is because the nano-iron particles are uniformly coated and stably loaded onto the surface by the modified protein fibers, effectively preventing direct contact and oxidative complexation reactions between free iron ions and components such as phenols and lipids in the food matrix, thus maximizing the protection of the original color quality of the food. This advantage is particularly valuable for high-end food systems with stringent color requirements, such as infant formula and transparent milk beverages.

[0043] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing modified sheep whey protein that prevents iron aggregation and promotes iron ion absorption, characterized in that, Includes the following steps: The whey protein isolate from sheep was dispersed in an acidic solution to prepare a protein dispersion. Modified sheep whey protein was obtained by high-temperature heating under stirring conditions and acidic conditions with a pH of 1.5–2.5, followed by cooling.

2. The method for preparing modified sheep whey protein with the properties of preventing iron aggregation and promoting iron ion absorption according to claim 1, characterized in that, The heat treatment temperature is 80–95℃, and the time is 3–12 h.

3. The method for preparing modified sheep whey protein with the properties of preventing iron aggregation and promoting iron ion absorption according to claim 2, characterized in that, The heat treatment temperature was 90℃, and the heat treatment time was 12 h.

4. The method for preparing modified sheep whey protein with the properties of preventing iron aggregation and promoting iron ion absorption according to claim 1, characterized in that, The mass concentration of the protein dispersion is 1% to 5%.

5. The method for preparing modified sheep whey protein with the properties of preventing iron aggregation and promoting iron ion absorption according to claim 1, characterized in that, The stirring speed is 300-500 rpm.

6. The modified sheep whey protein obtained by the preparation method according to any one of claims 1 to 5, which has the functions of preventing iron aggregation and promoting iron ion absorption.

7. The modified sheep whey protein according to claim 6, which prevents iron aggregation and promotes iron ion absorption, is characterized in that, It contains rigid long fibers with an average height of 3-7 nm, and the modified sheep whey protein has iron ion reducing active sites and an adhesive surface.

8. A modified sheep whey protein-nano iron composite material, characterized in that, include: The matrix is ​​the modified sheep whey protein as described in claim 6 or 7; And nano-iron particles, loaded on the surface of the substrate.

9. The modified sheep whey protein-nano iron composite material according to claim 8, characterized in that, The nano-iron particles are loaded on the surface of the modified sheep whey protein. The nano-iron particles generated by the reduction of ferric salt have a particle size of 5-20 nm. The mass ratio of the modified sheep whey protein to iron is 1:(3-7).

10. The application of the modified sheep whey protein-nano iron composite material according to claim 8 or 9 in the preparation of iron-fortified foods or iron supplements.

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