Whey protein-tannic acid-ferrous ion compound and preparation method thereof

Abstract

The invention relates to the technical field of food fortification, in particular to a whey protein-tannic acid-ferrous ion compound and a preparation method thereof. According to the compound, whey protein serves as a carrier, the whey protein is modified through tannic acid, ferrous ions are introduced on the basis, and the stable iron supplement is formed. According to the compound, the stability and bioavailability of ferrous iron can be remarkably improved, the defects of an existing iron supplement in the aspects of cost, stability and side effects are overcome, and oxidative conversion of iron in gastrointestinal tracts is effectively prevented. The preparation method is simple, mild in process condition and easy for industrial production, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of food fortification, specifically to a whey protein-tannic acid-ferrous ion complex and its preparation method. Background Technology

[0002] Iron is an essential trace element for the human body, participating in several important physiological processes such as hemoglobin synthesis, oxygen transport, energy metabolism, and immune function. Iron deficiency anemia (IDA) is the most common nutritional disease worldwide, especially affecting high-risk groups such as pregnant women, infants, children, and the elderly. Insufficient iron intake or malabsorption easily leads to iron deficiency; therefore, iron supplementation through diet or fortification is of great significance in improving iron deficiency anemia.

[0003] Currently, commonly used iron supplements include novel iron supplements such as inorganic iron salts, organic chelated iron, and nanocarriers. While inorganic iron salts (such as ferrous sulfate) are inexpensive and widely used, ferrous ions are prone to oxidation during processing, storage, and gastrointestinal digestion, leading to reduced iron bioavailability and potential side effects such as gastrointestinal discomfort. Although chelated iron can improve iron stability and absorption to some extent, it is more expensive, and its stability in complex digestive environments remains limited.

[0004] In recent years, technologies such as nanocarriers and microcapsules have been used to improve the stability and sensory properties of iron. However, these technologies are often accompanied by complex processes, high production costs, and the need for further evaluation of long-term safety, thus limiting their application in the food industry. Therefore, developing an iron delivery system based on food-grade raw materials, with simple processes, and capable of effectively improving the stability and bioavailability of ferrous iron remains a pressing technical challenge in the field of food fortification.

[0005] In recent years, food proteins have become potential iron ion delivery carriers due to their good biocompatibility and processability. Whey protein (WPI), as a widely available, low-cost protein with good emulsifying and digestible properties, is considered an ideal metal ion carrier. The molecular structure of whey protein contains abundant functional groups, such as carboxyl, amino, and thiol groups, which can form stable complexes with iron ions, enhancing iron stability. However, existing studies have shown that whey protein-iron ion complexes are easily oxidized and degraded in the gastrointestinal tract, affecting iron absorption efficiency.

[0006] To address this issue, this invention introduces tannic acid (TA). As a natural polyphenol, tannic acid not only possesses antioxidant properties but can also form stable complexes with iron ions through multiple coordination interactions, thereby effectively preventing iron oxidation. More importantly, tannic acid can non-covalently modify whey protein, enhancing its antioxidant capacity, protecting iron ions from oxidation, and maintaining its ferrous (Fe2+) iron content during in vitro digestion. 2+ This mechanism improves the absorption efficiency of iron in the digestive tract and significantly enhances the bioavailability of iron. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a whey protein-tannic acid-ferrous ion complex. This complex can enhance the stability and bioavailability of ferrous iron, overcoming the shortcomings of existing iron supplements in terms of stability and absorption efficiency. In particular, the complex of this invention can effectively prevent the oxidation of iron ions in the digestive tract environment, maintain the activity of ferrous iron, thereby significantly improving the bioavailability of iron and ensuring efficient iron supplementation.

[0008] The compound of the present invention is not only applicable to conventional iron supplements, but can also be widely used in food and beverages. It can significantly increase iron content while maintaining good taste and stability, meeting the iron supplementation needs of specific groups (such as pregnant women, infants, the elderly, etc.).

[0009] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for preparing a whey protein-tannic acid-ferrous ion complex, comprising: dissolving whey protein in deionized water and stirring until the whey protein dissolves, and hydrating overnight to obtain a whey protein solution; adding tannic acid to the whey protein solution, adjusting the pH to 7.0, and stirring to form a whey protein-tannic acid complex solution; adding a ferrous iron source to the whey protein-tannic acid complex solution and stirring to coordinate ferrous ions with the functional groups in the whey protein-tannic acid complex to obtain the whey protein-tannic acid-ferrous ion complex.

[0010] The preferred technical solution includes the following steps:

[0011] Step 1: Dissolve whey protein in deionized water at a concentration of 1%-5% (w / v) and stir at room temperature for 1-3 hours. Centrifuge at 4-8 ℃ and 7000-10000 r / min to remove insoluble impurities and obtain a clear whey protein solution. Then hydrate at 3-5 ℃ for 10-12 hours.

[0012] Step 2: Add tannic acid to the whey protein solution at a mass ratio of whey protein to tannic acid of 15-25:1, and adjust the pH to 7.0; stir at room temperature in the dark for 1-3 hours to form a whey protein-tannic acid complex solution.

[0013] Step 3: Add ferrous ions to the whey protein-tannic acid complex solution according to the mass ratio of tannic acid to ferrous ions of 1:0.5-4 to obtain the whey protein-tannic acid-ferrous ion complex.

[0014] To achieve the above and other related objectives, the technical solution provided by the present invention is: the whey protein-tannic acid-ferrous ion complex obtained by the preparation method of the whey protein-tannic acid-ferrous ion complex.

[0015] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0016] 1. The compound of the present invention can be widely used in food and beverages to significantly increase iron content, enhance iron stability, and maintain good sensory quality, thereby meeting the iron supplementation needs of specific populations.

[0017] 2. This invention successfully constructs a stable whey protein-tannic acid-ferrous ion complex by modifying whey protein with tannic acid. Tannic acid binds to functional groups such as amino and carboxyl groups in whey protein in a non-covalent manner, enhancing the antioxidant properties and stability of whey protein. The modified whey protein can effectively prevent the oxidation of ferrous ions in the gastrointestinal tract and maintain its ferrous (Fe2+) activity. The complex of this invention can significantly inhibit the oxidative conversion of iron to ferric (Fe3+). The synergistic effect of whey protein and tannic acid not only improves the absorption efficiency of iron but also ensures a gentler release of iron in the gastrointestinal tract, reducing side effects caused by excessive oxidation or instability of iron.

[0018] 3. This invention has high safety. The whey protein and tannic acid used are food-grade raw materials with good biocompatibility and safety, and avoid the gastrointestinal irritation or oxidative side effects that may be caused by some traditional iron supplements.

[0019] 4. The preparation method of the complex described in this invention has a clear processing flow, mild operating conditions, and is easy to implement and promote. Attached Figure Description

[0020] Figure 1 The images are transmission electron microscope images of Examples 1-4, where (a) is whey protein (WPI), (b) is whey protein-tannic acid complex (WT), and (c)-(f) are whey protein-tannic acid-ferrous ion complex (WTF) at different ferrous concentrations.

[0021] Figure 2 This is a comparison of the ferrous release behavior of whey protein-tannic acid-ferrous ion complex (WTF) and whey protein-ferrous ion complex (WF) at the corresponding ferrous concentration under in vitro digestion conditions in Examples 1-4.

[0022] Figure 3 This is a comparison chart of the antioxidant properties of whey protein-tannic acid-ferrous ion complex (WTF) and whey protein-ferrous ion complex (WF) at the corresponding ferrous concentration in Examples 1-4 under in vitro digestion conditions.

[0023] Figure 4 The figures show a comparison of the biological properties of different iron complexes in Examples 1-4, where (a) represents the cytotoxicity results and (b) represents the ferritin formation rate results.

[0024] Figure 5 The images show a comparison of the appearance of rice milk without added iron and rice milk with three different iron-addition methods during storage, as shown in Test Example 1.

[0025] Figure 6 The graph shows the change in the ferrous / total iron ratio of rice milk with three different iron addition methods during storage, as shown in Test Example 1. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0027] Please see Figure 1-6 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0028] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0029] Example 1: A whey protein-tannic acid-ferrous ion complex and its preparation method

[0030] Step 1: Preparation of whey protein solution

[0031] Whey protein (WPI) was dissolved in deionized water at a concentration of 2% (w / v) and stirred at 500 r / min for 2 h at room temperature to ensure complete dissolution. The solution was then centrifuged at 4-8 ℃ and 7000-10000 r / min to remove insoluble impurities, yielding a clear whey protein solution, which was then hydrated at 4 ℃ for 12 h.

[0032] Step 2: Preparation of whey protein-tannic acid complex

[0033] Tannic acid was added to the whey protein solution at a mass ratio of whey protein to tannic acid of 20:1 (w / w), and the pH was adjusted to 7.0. The solution was stirred in the dark at room temperature for 2 h to form a whey protein-tannic acid complex (WPI-TA), denoted as WT.

[0034] Step 3: Preparation of whey protein-tannic acid-ferrous ion complex

[0035] A certain amount of ferrous sulfate was added to the whey protein-tannic acid solution to make the mass ratio of tannic acid to ferrous ions 2:1. The mixture was stirred rapidly for 60 s to obtain a whey protein-tannic acid-ferrous ion complex, denoted as WTF1.

[0036] Example 2: A whey protein-tannic acid-ferrous ion complex and its preparation method

[0037] Unlike Example 1, ferrous sulfate was added to the whey protein-tannic acid solution, and the mass ratio of tannic acid to ferrous ions was 1:1, resulting in a whey protein-tannic acid-ferrous ion complex, denoted as WTF2.

[0038] Example 3: A whey protein-tannic acid-ferrous ion complex and its preparation method

[0039] Unlike Example 1, ferrous sulfate was added to the whey protein-tannic acid solution, and the mass ratio of tannic acid to ferrous ions was 1:2, resulting in a whey protein-tannic acid-ferrous ion complex, denoted as WTF3.

[0040] Example 4: A whey protein-tannic acid-ferrous ion complex and its preparation method

[0041] Unlike Example 1, ferrous sulfate was added to the whey protein-tannic acid solution, and the mass ratio of tannic acid to ferrous ions was 1:4, resulting in a whey protein-tannic acid-ferrous ion complex, denoted as WTF4.

[0042] Comparative Example 1

[0043] A whey protein solution was prepared according to the method in Example 1. Without the addition of tannic acid, an equal amount of ferrous iron source as WTF1 was added to the whey protein solution, and the mixture was stirred rapidly in the dark for 60 s to obtain a whey protein-ferrous complex (WF), denoted as WF1.

[0044] Comparative Example 2

[0045] Unlike Comparative Example 1, an equal amount of ferrous iron source as WTF2 was added to prepare a whey protein-ferrous complex, denoted as WF2.

[0046] Comparative Example 3

[0047] Unlike Comparative Example 1, an equal amount of ferrous iron source as WTF3 was added to prepare a whey protein-ferrous complex, denoted as WF3.

[0048] Comparative Example 4

[0049] Unlike Comparative Example 1, an equal amount of ferrous iron source as WTF4 was added to prepare a whey protein-ferrous complex, denoted as WF4.

[0050] Comparative Example 5

[0051] Ferrous sulfate was directly dissolved in deionized water without introducing any protein or polyphenol carriers, serving as a control system for traditional iron supplementation methods, denoted as FeSO4.

[0052] The WPI, WT, and WTF1-WTF4 samples prepared in Examples 1-4 were observed by transmission electron microscopy, and the results are shown in the figure. Figure 1 The samples were observed using a transmission electron microscope (model: Tecnai G2 Spirit Twin, FEI, Hillsboro, USA). 5 μL of the diluted sample dispersion was dropped onto a carbon film copper grid, dried in air at room temperature, and then imaged using a transmission electron microscope with an accelerating voltage of 80 kV.

[0053] Depend on Figure 1It is known that pure whey protein (WPI) samples exhibit relatively uniformly distributed, nearly spherical particles with small particle sizes, indicating that whey protein has a relatively stable dispersion in the aqueous phase. After the introduction of tannic acid, the particle size of the resulting whey protein-tannic acid complex (WT) significantly increased, indicating that tannic acid can effectively interact with whey protein, thereby altering its microstructure. Further introduction of ferrous ions resulted in a significant change in the micromorphology of the whey protein-tannic acid-ferrous ion complex (WTF) with the amount of ferrous ion added. Under lower ferrous ion addition conditions, the composite system showed a trend of transforming from dispersed particles to a lamellar or network structure, indicating that a relatively stable assembly structure had been formed within the system. With further increases in ferrous ion addition, the composite system gradually formed a denser aggregate structure, showing a higher degree of structural cross-linking.

[0054] The WTF1-WTF4 samples prepared in Examples 1-4 were used to detect the release behavior of ferrous and total iron under simulated in vitro digestion conditions. The concentrations of ferrous and total iron were determined using the o-phenanthroline colorimetric method and compared with those of whey protein-ferrous complexes (WF1-WF4) at the same ferrous concentration in Comparative Examples 1-4. The results are shown in [Figure 1]. Figure 2 .

[0055] The results showed that, under simulated in vitro digestion conditions, whey protein-tannic acid-ferrous ion complex (WTF) was superior to whey protein-ferrous ion complex (WPI-Fe). 2+ This study demonstrated a more moderate and controllable ferrous iron release behavior, effectively reducing the rapid loss of ferrous iron under gastric conditions and maintaining a high proportion of ferrous iron release during the intestinal phase. This indicates that the introduction of tannic acid helps improve the stability of ferrous iron during digestion, thereby facilitating the effective delivery and utilization of iron.

[0056] The antioxidant capacity of WTF1-WTF4 samples prepared in Examples 1-4 under simulated digestion conditions was determined using the DPPH and ABTS methods, and compared with that of whey protein-ferrous complexes (WF1-WF4) at the same ferrous concentration in Comparative Examples 1-4. The results are shown in [Figure 1]. Figure 3 .

[0057] like Figure 3As shown, the WTF sample exhibited strong antioxidant activity during simulated digestion. WTF demonstrated stronger free radical scavenging ability than WF, especially in the DPPH test, where WTF's scavenging activity was significantly higher than WF. This is attributed to the phenolic hydroxyl groups of tannic acid, which effectively neutralize free radicals and reduce iron oxidation. With increasing ferrous content, the antioxidant activity of WTF further increased, and at higher ferrous contents, WTF maintained high levels of both DPPH and ABTS activities. The ABTS test also showed that WTF exhibited more stable and sustained antioxidant activity in the gastrointestinal digestive environment, especially in the small intestine, where WTF showed a steady increase in activity, while WF reached stability in a shorter time. This indicates that tannic acid maintains the divalent form of iron ions, delays iron oxidation, and enhances antioxidant performance, effectively improving iron bioavailability and antioxidant efficacy.

[0058] The WTF1-WTF4 samples prepared in Examples 1-4 were compared with those in Comparative Examples 1-5 to evaluate their cytotoxicity and ferritin formation ability. The results are shown in [Figure 1]. Figure 4 .

[0059] The results showed that, within the tested concentration range, the whey protein-tannic acid-ferrous ion complex did not have a significant adverse effect on cell viability, and cell survival rates remained at a high level. This indicates that the whey protein-tannic acid-ferrous ion complex constructed in this invention has good biocompatibility and safety, and is suitable for the food and nutritional fortification fields. Compared with the direct addition of ferrous salts or whey protein-ferrous complex, the whey protein-tannic acid-ferrous ion complex exhibited a good level of intracellular ferritin formation, indicating that this delivery system is beneficial for promoting cellular iron absorption and utilization.

[0060] Test Example 1: Application of whey protein-tannic acid-ferrous ion complex in rice milk

[0061] FeSO4, whey protein-ferrous ion complex, and whey protein-tannic acid-ferrous ion complex with the same ferrous concentration were added to rice milk for comparison:

[0062] Rice milk is prepared following the process of "raw material selection - soaking - pulping - gelatinization - enzymatic hydrolysis - enzyme inactivation - filtration - homogenization - blending - secondary homogenization - sterilization". Red brown rice free of mold and sprouts is selected as the raw material. 200 g of red brown rice is accurately weighed, washed with clean water, and impurities are filtered out. It is then soaked for 12 hours at a mass ratio of 1:3. The soaked red brown rice is then added to an appropriate amount of purified water and ground using a high-speed blender to produce a smooth, fluid rice milk. This rice milk is then gelatinized in a 70°C water bath for 30 minutes, with continuous stirring during gelatinization to prevent clumping. Rice milk was sequentially treated with α-amylase (enzymatic hydrolysis temperature 50℃, pH 6.0, enzyme dosage 0.1% (w / v), hydrolysis time 60 min), saccharifying enzyme (enzymatic hydrolysis temperature 55℃, pH 4.5, enzyme dosage 0.15%, hydrolysis time 60 min), and cellulase (enzymatic hydrolysis temperature 50℃, pH 5.0, enzyme dosage 0.1% (w / v), hydrolysis time 100 min). The pH was adjusted to 6.5, and then the enzymes were inactivated in a water bath at 95℃ for 20 min. The rice milk was then filtered through a 100-mesh filter cloth to remove coarse residue, and the filtered rice milk was treated under a homogenization pressure of 20 MPa. Add 6% (w / v) maltose syrup, 0.08% (w / v) sodium carboxymethyl cellulose, 0.06% (w / v) xanthan gum, and 30 mg / L of iron supplements (FeSO4, WF2, WTF2). Homogenize the rice milk again under 20 MPa pressure. Sterilize the homogenized rice milk at 72°C for 15 s. After sterilization, cool and store at low temperature.

[0063] Figure 5 The appearance of rice milk without added iron and rice milk with three different iron-added methods were compared at different time points. As can be seen from the figures, all samples (including the control group, ferrous sulfate (FeSO4), whey protein-ferrous complex (WF), and whey protein-tannic acid-ferrous ion complex (WTF)) maintained good homogeneity throughout the storage process, without any stratification. The color of each sample remained stable at different time points, indicating that the complex of the present invention has good stability and can maintain uniform distribution under different storage conditions.

[0064] Figure 6 The changes in the ratio of ferrous to total iron in rice milk (without added iron) and rice milk with three different iron-fortifying methods were measured at different time points during storage. Under the same storage conditions, the rice milk beverage fortified with the complex (WTF2) of this invention maintained a high ratio of ferrous to total iron during storage from 0 to 14 days, with a relatively small decrease. In contrast, the group with direct addition of ferrous salt (FeSO4) showed a significant decrease in the ratio of ferrous to total iron during storage, while the WPI-Fe... 2+Although the group showed some improvement compared to the FeSO4 group, there was still a significant decrease.

[0065] Example 5: A whey protein-tannic acid-ferrous ion complex and its preparation method

[0066] A method for preparing a whey protein-tannic acid-ferrous ion complex, the specific steps of which are as follows:

[0067] (1) Preparation of whey protein solution

[0068] Whey protein was dissolved in deionized water at a concentration of 2% (w / v) and stirred at room temperature for 2 hours to ensure complete dissolution. Insoluble impurities were removed by centrifugation to obtain a clear whey protein solution, which was then hydrated overnight.

[0069] (2) Preparation of whey protein-tannic acid complex

[0070] Tannic acid was added to the whey protein solution at a mass ratio of whey protein to tannic acid of 20:1 (w / w), and the pH was adjusted to 7.0. The solution was stirred at room temperature in the dark for 2 h to form a whey protein-tannic acid complex (WPI-TA).

[0071] (3) Preparation of whey protein-tannic acid-ferrous ion complex

[0072] Add an appropriate amount of ferrous iron (such as ferrous sulfate) to the whey protein-tannic acid complex solution, and stir rapidly in the dark for 60 s to ensure that the ferrous ions fully coordinate with the functional groups (such as carboxyl and amino groups) in the complex to form a whey protein-tannic acid-ferrous ion complex (WPI-TA-Fe). 2+ (complex).

[0073] The further defined technical solution is as follows:

[0074] In step (1), the stirring conditions are: 400 r / min.

[0075] In step (1), the centrifugation conditions are: temperature 4℃ and rotation speed 7000r / min.

[0076] In step (1), the overnight hydration time is 10 hours.

[0077] In step (2), the stirring conditions are: 400 r / min.

[0078] In step (3), the mass ratio of tannic acid to ferrous ions is 1:1.

[0079] In step (3), the stirring conditions are: 400 r / min.

[0080] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A method for preparing a whey protein-tannic acid-ferrous ion complex, characterized in that: Whey protein was dissolved in deionized water and stirred until dissolved, then hydrated overnight to obtain a whey protein solution. Tannic acid was added to the whey protein solution, the pH was adjusted to 7.0, and the mixture was stirred to form a whey protein-tannic acid complex solution. A ferrous iron source was added to the whey protein-tannic acid complex solution and stirred to coordinate ferrous ions with the functional groups in the whey protein-tannic acid complex, thus obtaining a whey protein-tannic acid-ferrous ion complex.

2. The method for preparing the whey protein-tannic acid-ferrous ion complex according to claim 1, characterized in that, Specifically, the following steps are included: Step 1: Dissolve whey protein in deionized water at a concentration of 1 w / v %-5 w / v % and stir at room temperature for 1-3 hours. Centrifuge at 4-8 ℃ and 7000-10000 r / min to remove insoluble impurities and obtain a clear whey protein solution. Then hydrate at 3-5 ℃ for 10-12 hours. Step 2: Add tannic acid to the whey protein solution at a mass ratio of whey protein to tannic acid of 15-25:1, and adjust the pH to 7.0; stir in the dark at room temperature for 1-3 hours to form a whey protein-tannic acid complex solution. Step 3: Add ferrous ions to the whey protein-tannic acid complex solution according to the mass ratio of tannic acid to ferrous ions of 1:0.5-4 to obtain the whey protein-tannic acid-ferrous ion complex.

3. The whey protein-tannic acid-ferrous ion complex obtained by the preparation method of the whey protein-tannic acid-ferrous ion complex according to any one of claims 1 or 2.

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