Method for improving color stability of black corn anthocyanin-protein compound

Abstract

A method for improving color stability of a black corn anthocyanin-protein compound relates to the technical field of anthocyanin-protein compound preservation, and comprises the following steps: dissolving a black corn anthocyanin-protein compound in a phosphate buffer solution to prepare a black corn anthocyanin-protein compound solution; dissolving an auxiliary color agent in a phosphate buffer solution to prepare an auxiliary color agent solution, and adding the auxiliary color agent solution into the black corn anthocyanin-protein compound solution; the method is used for solving the technical problems that the anthocyanin-protein compound is low in stability and easy to decompose.

Description

Technical Field

[0001] The present invention relates to the technical field of anthocyanin - protein complex preservation, and specifically relates to a method for improving the color stability of anthocyanin - protein complexes in black corn. Background Art

[0002] Black corn, also known as black waxy corn, is the general term for corn with black or purple kernel color, also called purple corn, and belongs to the genus Zea of the Gramineae family. Black corn not only contains a large amount of nutrients such as carbohydrates and proteins, but also contains a variety of bioactive substances, such as anthocyanins, phenolic acids, and flavonoids. Research shows that black corn is rich in anthocyanins, which have biological activities such as antioxidant, anti - inflammatory, anti - cancer, anti - obesity, and blood - sugar - inhibiting effects. The contents of protein, fat, and selenium in black corn kernels are 1.23 times, 1.3 times, and 3 - 8.5 times that of common corn respectively, and the anthocyanin content is 4.3 times that of blueberries. In recent years, it has attracted many scholars at home and abroad to research and discuss because of its rich anthocyanin substances.

[0003] Anthocyanins are water - soluble flavonoid plant pigments with strong antioxidant activity, and their basic structure is a flavonoid cation. They are also natural pigments that give various colors to fruits and vegetables and are widely used as food - grade colorants. Research has found that anthocyanins exist in some purple, black, or red vegetables, fruits, and grains, as well as in their stems and leaves. For example, grapes, berries, black rice, eggplants, purple corn, purple sweet potatoes, etc. are all contributors of anthocyanins. Some studies have shown that anthocyanins have many biological activities such as antioxidant, anti - inflammatory, anti - cancer, heart - protecting, obesity - inhibiting, immune - system - strengthening, and anti - inflammatory effects. Due to their excellent properties such as natural source, non - toxicity, safety, and biological functions, anthocyanins have now attracted wide attention from researchers and are often used in fields such as medicine, food, and cosmetics.

[0004] As one of the three essential nutrients for the human body, protein has good biocompatibility and biodegradability and is often used in food processing, but its functional properties need to be improved. During the processing, storage, and consumption of agricultural products, the interaction between their active ingredients and proteins is inevitable, and they can form new complexes in the form of covalent or non - covalent bonds. For example, some studies have shown that proteins have good surface properties, self - assembly properties, and gelation characteristics. Without changing the structure of anthocyanins, they can form conjugates through non - covalent and covalent interactions. The anthocyanins are fixed in the protein structure cavity, which can reduce the loss of pyran cations, thus having a positive impact on the stability of anthocyanins.

[0005] The applicant previously applied for a Chinese invention patent with a publication number of CN119326127A and a patent name of "Preparation Method of Black Corn Anthocyanin-Protein Complex", and studied the method of extracting anthocyanin-protein complex from black corn. However, the stability of the anthocyanin-protein complex is relatively general. If the anthocyanin-protein complex decomposes, it is easy to cause the anthocyanin to be affected and degrade and fade.

[0006] In recent years, scientific and technological workers at home and abroad have carried out research and development on anthocyanins, mainly studying the extraction methods of anthocyanins and how to maintain the steady state of anthocyanins, specifically including research on the extraction, purification and storage of anthocyanins, and research on the influence of co-colorants on the color stability of black corn anthocyanins. At present, there is no method to improve the color stability of black corn anthocyanin-protein complex. Summary of the Invention

[0007] The present invention provides a method for improving the color stability of black corn anthocyanin-protein complex to solve the technical problems of low stability and easy decomposition of the anthocyanin-protein complex.

[0008] To achieve the above object, the specific solution adopted by the present invention is: a method for improving the color stability of black corn anthocyanin-protein complex, mixing a co-colorant solution and a black corn anthocyanin-protein complex solution, and the co-colorant in the co-colorant solution is gallic acid, tartaric acid, citric acid or boric acid.

[0009] As a further optimization of the above technical solution, the mass ratio of the co-colorant to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 2-40:1.

[0010] As a further optimization of the above technical solution, when the co-colorant is gallic acid, the mass ratio of gallic acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 10:1;

[0011] When the co-colorant is tartaric acid, the mass ratio of tartaric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 5:1;

[0012] When the co-colorant is citric acid, the mass ratio of citric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 10:1;

[0013] When the co-colorant is boric acid, the mass ratio of boric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 2:1.

[0014] As a further optimization of the above technical solution, after mixing the co-colorant and the black corn anthocyanin-protein complex solution, the mass concentration of the co-colorant in the obtained mixed solution is 0.1-2.0 mg / mL.

[0015] As a further optimization of the above technical solution, dissolve the black corn anthocyanin-protein complex in a phosphate buffer to prepare the black corn anthocyanin-protein complex solution; dissolve the co-colorant in a phosphate buffer to prepare a co-colorant solution, add the co-colorant solution to the black corn anthocyanin-protein complex solution, and adjust the pH to 4.

[0016] As a further optimization of the above technical solution, after adding the co-colorant solution to the black corn anthocyanin-protein complex solution and adjusting the pH to 4, store it in the dark.

[0017] As a further optimization of the above technical solution, the pH of the phosphate buffer is 7.4.

[0018] As a further optimization of the above technical solution, the concentration of the black corn anthocyanin-protein complex solution is 0.1 mg / mL.

[0019] As a further optimization of the above technical solution, the concentration of the co-colorant solution is 0.2-4.0 mg / mL.

[0020] As a further optimization of the above technical solution, the concentration of the black corn anthocyanin-protein complex solution is 0.1 mg / mL, the concentration of the co-colorant solution is 0.2-4.0 mg / mL, the volume ratio of the black corn anthocyanin-protein complex solution to the co-colorant solution is 1:1, and the mass concentration of the co-colorant in the mixed solution obtained by adding the co-colorant solution to the black corn anthocyanin-protein complex solution is 0.1-2.0 mg / mL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] By adding a co-colorant (gallic acid, tartaric acid, citric acid or boric acid) to the anthocyanin-protein complex solution, the present invention enhances the binding force between anthocyanins and proteins and stabilizes the structure of the anthocyanin-protein complex, thereby further improving the color stability of the complex.

[0023] The anthocyanins and proteins in the black corn anthocyanin-protein complex carry different charges. The addition of organic acids will regulate the charge distribution of the system, enhancing the electrostatic interaction between anthocyanins and proteins. In addition, the co-pigment molecules bound to the black corn anthocyanin-protein complex can form a steric hindrance around the complex, preventing other molecules or ions that may cause the dissociation of the complex or the destruction of the anthocyanin structure from approaching, thereby protecting the structural integrity of the complex and enabling anthocyanins to better exert their color-forming function and achieve a co-pigmentation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effect of gallic acid on the Zeta potential of the black corn anthocyanin-protein complex;

[0025] Figure 2 Effect of tartaric acid on the Zeta potential of the black corn anthocyanin-protein complex;

[0026] Figure 3 Effect of citric acid on the Zeta potential of the black corn anthocyanin-protein complex;

[0027] Figure 4 Effect of boric acid on the Zeta potential of the black corn anthocyanin-protein complex;

[0028] Figure 5 Effect of gallic acid on the color stability of the black corn anthocyanin-protein complex;

[0029] Figure 6 Effect of tartaric acid on the color stability of the black corn anthocyanin-protein complex;

[0030] Figure 7 Effect of citric acid on the color stability of the black corn anthocyanin-protein complex;

[0031] Figure 8 Effect of boric acid on the color stability of the black corn anthocyanin-protein complex. DETAILED DESCRIPTION OF THE INVENTION

[0032] The technical solutions of the present invention will be further elaborated in detail below in conjunction with specific embodiments. For parts not detailedly described and disclosed in the following embodiments of the present invention, they should all be understood as the prior art known or should be known to those skilled in the art.

[0033] To avoid repetition, the raw materials used in the following examples and comparative examples are described as follows: The black corn anthocyanin-protein complex used in the following examples and comparative examples was prepared by the method disclosed in "Publication No. CN119326127A, Patent Name: A Preparation Method of a Black Corn Anthocyanin-Protein Complex"; the phosphate buffer solution, gallic acid, tartaric acid, citric acid, and boric acid used are all commercially available products.

[0034] Example 1

[0035] A method for improving the color stability of a black corn anthocyanin-protein complex, using a phosphate buffer solution with a pH of 7.4 to prepare a 0.1 mg / mL black corn anthocyanin-protein complex solution and a 0.2 mg / mL co-colorant solution respectively, wherein the co-colorant solution is a gallic acid solution;

[0036] After taking equal volumes of the black corn anthocyanin-protein complex solution and the gallic acid solution and mixing them evenly, the gallic acid solution in the mixed solution becomes 0.1 mg / mL, and the pH is adjusted to 4, and it is placed in the dark at room temperature.

[0037] Example 2

[0038] A method for improving the color stability of a black corn anthocyanin-protein complex, using a phosphate buffer solution with a pH of 7.4 to prepare a 0.1 mg / mL black corn anthocyanin-protein complex solution and a 0.5 mg / mL co-colorant solution respectively, wherein the co-colorant solution is a gallic acid solution;

[0039] After taking equal volumes of the black corn anthocyanin-protein complex solution and the gallic acid solution and mixing them evenly, the gallic acid solution in the mixed solution becomes 0.25 mg / mL, and the pH is adjusted to 4, and it is placed in the dark at room temperature.

[0040] Example 3

[0041] A method for improving the color stability of a black corn anthocyanin-protein complex, using a phosphate buffer solution with a pH of 7.4 to prepare a 0.1 mg / mL black corn anthocyanin-protein complex solution and a 1.0 mg / mL co-colorant solution respectively, wherein the co-colorant solution is a gallic acid solution;

[0042] After taking equal volumes of the black corn anthocyanin-protein complex solution and the gallic acid solution and mixing them evenly, the gallic acid solution in the mixed solution becomes 0.5 mg / mL, and the pH is adjusted to 4, and it is placed in the dark at room temperature.

[0043] Example 4

[0044] A method for improving the color stability of black corn anthocyanin-protein complex, using a phosphate buffer solution with a pH of 7.4 to prepare a 0.1 mg / mL black corn anthocyanin-protein complex solution and a 2.0 mg / mL co-colorant solution respectively, wherein the co-colorant solution is a gallic acid solution;

[0045] Take equal volumes of the black corn anthocyanin-protein complex solution and the gallic acid solution, mix them evenly, the gallic acid solution in the mixed solution becomes 1.0 mg / mL, adjust the pH to 4, and place it in the dark at room temperature.

[0046] Example 5

[0047] A method for improving the color stability of black corn anthocyanin-protein complex, using a phosphate buffer solution with a pH of 7.4 to prepare a 0.1 mg / mL black corn anthocyanin-protein complex solution and a 4.0 mg / mL co-colorant solution respectively, wherein the co-colorant solution is a gallic acid solution;

[0048] Take equal volumes of the black corn anthocyanin-protein complex solution and the gallic acid solution, mix them evenly, the gallic acid solution in the mixed solution becomes 2.0 mg / mL, adjust the pH to 4, and place it in the dark at room temperature.

[0049] Examples 6 - 10

[0050] The general steps of Examples 6 - 10 are the same as those of Example 1, the difference is that the co-colorant in Examples 6 - 10 is tartaric acid, and the specific concentrations are as shown in Table 1 below:

[0051] Table 1

[0052] Examples 11 - 15

[0053] The general steps of Examples 11 - 15 are the same as those of Example 1, the difference is that the co-colorant in Examples 11 - 15 is citric acid, and the specific concentrations are as shown in Table 2 below:

[0054] Table 2

[0055] Examples 16 - 20

[0056] The general steps of Examples 16 - 20 are the same as those of Example 1, the difference is that the co-colorant in Examples 16 - 20 is boric acid, and the specific concentrations are as shown in Table 3 below:

[0057] Table 3

[0058] Comparative Examples 1 - 20

[0059] Preparation of black corn anthocyanins: Take 20 mL of black corn anthocyanin - protein complex, add 1 g of PVPP to it, vortex, and then add acetone according to a volume ratio of 1:1, leave overnight, centrifuge at 6000 r / min at 4°C for 20 min. The supernatant is the black corn anthocyanins, which are diluted to a concentration of 0.1 mg / mL with the same phosphate buffer as the black corn anthocyanin - protein complex below.

[0060] Prepare 0.1 mg / mL black corn anthocyanin solution and different - concentration co - colorant solutions using phosphate buffer with a pH of 7.4. The co - colorant solutions are gallic acid solution, tartaric acid solution, citric acid solution, or boric acid solution;

[0061] After taking equal volumes of the black corn anthocyanin solution and the co - colorant solution and mixing them evenly, the concentration of the co - colorant solution is equivalent to being diluted by half, and adjust the pH to 4, and place it in the dark at room temperature. The specific concentration of the co - colorant solution after mixing is shown in Table 4 below:

[0062] To detect the effect of the method described in the present invention on the color stability of the black corn anthocyanin - protein complex, the mixed solution is stored in the dark for 1 hour, and samples are taken for analysis:

[0063] It should be noted that the co - colorant mass concentration and the attached Figure 1-8 The concentrations shown are the co - colorant mass concentrations in the mixed solution after the co - colorant solution and the black corn anthocyanin solution or the black corn anthocyanin - protein complex solution are mixed evenly in equal volumes.

[0064] I. Influence of co - colorant types on the co - coloration effect of black corn anthocyanin - protein complex

[0065] Use an ultraviolet spectrophotometer to measure the absorbance at the maximum absorption wavelength of the above samples in the range of 200 - 800 nm, and calculate the hyperchromic effect and the value of the red - shift effect of each co - colorant. The hyperchromic effect and the percentage change in absorbance (ΔΑ λmax ) at the ultraviolet maximum absorption wavelength of the anthocyanin solution, and the red - shift effect is the increase in the ultraviolet maximum absorption wavelength of the anthocyanin - protein complex solution (Δλ max ). The larger the values of the hyperchromic effect and the red - shift effect, the better the co - coloration property of the co - colorant on the black corn anthocyanin - protein complex. Table 5 Hyperchromic effect and red - shift effect of co - colorants on black corn anthocyanin - protein complex Note: The lowercase letters in the same column superscript indicate significant differences (P < 0.05).

[0066] As shown in Table 5, four organic acids were selected as co-colorants. All four co-colorants could improve the hyperchromic effect and red-shift effect of the black corn anthocyanin-protein complex. At pH 4.0, the anthocyanin-protein solution with boric acid had the best hyperchromic and red-shift effects (P < 0.05), increasing by 25.64% and 8 nm respectively. However, the anthocyanin-protein solution with citric acid had a relatively poor hyperchromic effect, with a hyperchromic effect of 3.35% and no red-shift occurred. In addition, the hyperchromic effects of gallic acid (9.36%) and tartaric acid (7.22%) were average. At pH 4.0, the hyperchromic effects of the four co-colorants on the black corn anthocyanin-protein complex from large to small were boric acid (25.64%), gallic acid (9.36%), tartaric acid (7.22%), and citric acid (3.35%). In the present invention, the co-coloring effect of boric acid on the black corn anthocyanin-protein complex was significantly better than that of gallic acid (P < 0.05). In addition, the solution pH value also affected the hyperchromic and red-shift effects of the co-colorant on the black corn anthocyanin-protein complex. It was speculated that at pH 4.0, the intramolecular charge of the anthocyanin in the complex was prone to transfer, and the double bond of the C ring of the flavylium cation was hydrolyzed, resulting in a decrease in the stability of the anthocyanin in the complex.

[0067] Second, use an ultraviolet spectrophotometer to measure the absorbance at the maximum absorption wavelength of the above samples in the range of 200 - 800 nm, and calculate the hyperchromic effect and red-shift effect values of each co-colorant. Determine the optimal concentration of the co-colorant.

[0068] <Effect of gallic acid mass concentration on the co-coloring effect of black corn anthocyanin-protein complex and black corn anthocyanin> Table 6 Hyperchromic effect and red-shift effect of gallic acid on black corn anthocyanin-protein complex Note: The lowercase letters in the same column superscript indicate significant differences (P < 0.05).

[0069] As shown in Table 6, when the mass concentration of gallic acid increased from 0.1 mg / mL to 2.0 mg / mL, the hyperchromic effect of the black corn anthocyanin-protein solution showed a trend of first increasing and then decreasing, increasing from 15.25% to 37.58%, and then decreasing to 9.37%. The maximum value of the red shift effect was 25 and the minimum value was 5. This indicates that the co-coloring effect is related to the mass concentration of gallic acid. With the increase in the mass concentration of gallic acid, the co-coloring effect gradually increases, but when it exceeds 0.5 mg / mL, its co-coloring effect also gradually weakens. Research shows that the phenomenon that the co-coloring effect of gallic acid on the black corn anthocyanin-protein complex first increases and then decreases is mainly related to various factors such as intermolecular interaction, steric hindrance, and environmental factor changes. Gallic acid can form a more stable complex with the black corn anthocyanin-protein complex through π-π stacking and hydrogen bond connection, which helps to maintain the chromophore structure in the complex, thereby enhancing its color stability and achieving the co-coloring effect. When the concentration of gallic acid is too high, a large number of gallic acid molecules will form steric hindrance around the complex, competing with anthocyanins for the binding sites with proteins, disturbing the normal interaction between anthocyanins and proteins, and being unfavorable to the stable formation of the complex, thus weakening the co-coloring effect. The optimal mass concentration of gallic acid for co-coloring the black corn anthocyanin-protein complex is 0.5 mg / mL. Table 7 Hyperchromic effect and red shift effect of gallic acid on black corn anthocyanins Note: The lowercase superscripts in the same column indicate significant differences (P < 0.05)

[0070] Combined with Table 6 and Table 7, with the increase in the concentration of gallic acid, its co-coloring effect on the black corn anthocyanin-protein complex shows a phenomenon of first increasing and then decreasing. When the mass concentration of gallic acid increases, the co-coloring effect gradually increases, but when it exceeds 0.5 mg / mL, its co-coloring effect also gradually weakens. On the contrary, as shown in Table 7, the hyperchromic effect of gallic acid on black corn anthocyanins shows an upward trend, but there are negative values, that is, a hypochromic effect. With the increase in the concentration of gallic acid, the hyperchromic effect changes from negative to positive.

[0071] <Effect of tartaric acid mass concentration on the co-coloring effect of black corn anthocyanin-protein complex and black corn anthocyanins> Table 8 Hyperchromic effect and red shift effect of tartaric acid on black corn anthocyanin-protein complex Note: The lowercase superscripts in the same column indicate significant differences (P < 0.05)

[0072] As shown in Table 8, for the co - color effect of tartaric acid on the black corn anthocyanin - protein complex, the phenomenon of first increasing and then decreasing occurs. When the mass concentration is 0.25 mg / mL, the hyperchromic effect increases by 25.56%, and the corresponding red - shift effect is 14 nm. This indicates that the co - color effect is related to the mass concentration of tartaric acid. As the mass concentration of tartaric acid increases, the co - color effect gradually enhances. However, when it exceeds 0.25 mg / mL, its co - color effect gradually weakens. This phenomenon shows that low - concentration tartaric acid can bind to the complex through interactions such as hydrogen bonds and van der Waals forces. This binding can make the molecular structure of the complex more stable, thereby enhancing color stability. At high concentrations, tartaric acid will ionize a large number of hydrogen ions and tartrate ions in the solution, changing the charge state that was originally conducive to binding, which is not conducive to the formation and stability of the anthocyanin - protein complex, and the co - color effect weakens. The optimal mass concentration of tartaric acid for the co - color of the black corn anthocyanin - protein complex is 0.25 mg / mL. Table 9 Hyperchromic effect and red - shift effect of tartaric acid on black corn anthocyanin Note: The lowercase superscripts in the same column indicate significant differences (P < 0.05)

[0073] Combined with Table 8 and Table 9, as the concentration of tartaric acid increases, its hyperchromic effect and red - shift effect on the black corn anthocyanin - protein complex show a trend of first increasing and then decreasing. As the mass concentration of tartaric acid increases, the co - color effect gradually enhances. However, when it exceeds 0.25 mg / mL, its co - color effect gradually weakens. Conversely, from Table 9, it can be seen that the hyperchromic effect of tartaric acid on black corn anthocyanin shows a downward trend. When the mass concentration of tartaric acid exceeds 0.5 mg / mL, the hyperchromic effect shows a negative value, that is, a hypochromic effect.

[0074] <Effect of citric acid mass concentration on the co - color effect of black corn anthocyanin - protein complex and black corn anthocyanin> Table 10 Hyperchromic effect and red - shift effect of citric acid on black corn anthocyanin - protein complex Note: The lowercase superscripts in the same column indicate significant differences (P < 0.05)

[0075] As shown in Table 10, the co - color enhancement effect of citric acid on the black corn anthocyanin - protein complex first increases and then decreases. When the mass concentration is 0.5 mg / mL, the hyperchromic effect is the highest at 35.36%, and the corresponding red - shift effect is 16 nm. This indicates that the co - color enhancement effect is related to the mass concentration of citric acid. As the mass concentration of citric acid increases, the co - color enhancement effect gradually increases, but when it exceeds 0.5 mg / mL, the co - color enhancement effect gradually decreases. Research shows that low - concentration citric acid interacts with the complex through functional groups such as carboxyl and hydroxyl in the molecule, exposing more binding sites that can combine with anthocyanins in the complex, thus promoting the formation of the black corn anthocyanin - protein complex and enhancing the co - color enhancement effect. Citric acid has a certain antioxidant capacity. At low concentrations, it can scavenge some free radicals in the system, reduce the oxidative damage of free radicals to anthocyanin molecules, and further enhance the co - color enhancement effect. High - concentration citric acid will compete with anthocyanins for the binding sites on the protein and increase the ionic strength of the solution, which is not conducive to the formation of the black corn anthocyanin - protein complex, resulting in a decrease in the co - color enhancement effect. The optimal mass concentration of citric acid for the co - color enhancement of the black corn anthocyanin - protein complex is 0.5 mg / mL. Table 11 Hyperchromic effect and red - shift effect of citric acid on black corn anthocyanin Note: The lowercase letters with subscripts in the same column indicate significant differences (P < 0.05)

[0076] Combined with Table 10 and Table 11, as the concentration of citric acid increases, its hyperchromic effect and red - shift effect on the black corn anthocyanin - protein complex show a trend of first increasing and then decreasing. When the mass concentration of citric acid increases, the co - color enhancement effect gradually increases, but when it exceeds 0.5 mg / mL, the co - color enhancement effect gradually decreases. Similarly, it can be seen from Table 11 that the co - color enhancement effect of citric acid on black corn anthocyanin also shows a phenomenon of first increasing and then decreasing. When the mass concentration of citric acid exceeds 0.5 mg / mL, the hyperchromic effect shows a negative value, that is, a hypochromic effect.

[0077] <Effect of boric acid mass concentration on the co - color enhancement effect of black corn anthocyanin - protein complex and black corn anthocyanin> Table 12 Hyperchromic effect and red - shift effect of boric acid on black corn anthocyanin - protein complex Note: The lowercase letters with subscripts in the same column indicate significant differences (P < 0.05)

[0078] As shown in Table 12, the co-coloring effect of boric acid on the black corn anthocyanin-protein complex shows a downward trend. When the mass concentration is 0.2 mg / mL, the hyperchromic effect is the highest at 40.05%, and the corresponding red shift effect is 25 nm. When the mass concentration increases, both the hyperchromic effect and the red shift effect on the complex decrease. This indicates that the co-coloring effect is related to the mass concentration of boric acid. With the increase of the mass concentration of boric acid (0.2 mg / mL), the co-coloring effect gradually weakens. This phenomenon shows that low-concentration boric acid can form stable borate bonds with the complex and interact with the protein molecules in the complex, thus facilitating the formation of a more stable complex and enhancing the co-coloring effect. At high concentrations, boric acid will form too many borate bonds with the complex. The formation of too many chemical bonds will cause excessive distortion of the spatial structure of the complex, destroying its original conjugated structure that is beneficial to color development. Moreover, the large presence of high-concentration boric acid molecules will compete with anthocyanins for the binding sites on the protein, resulting in a significant weakening of the co-coloring effect. The optimal mass concentration of boric acid for the co-coloring of the black corn anthocyanin-protein complex is 0.1 mg / mL. Table 13 Hyperchromic Effect and Red Shift Effect of Boric Acid on Black Corn Anthocyanins Note: The lowercase superscripts in the same column indicate significant differences (P < 0.05).

[0079] Combined with Table 12 and Table 13, with the increase of the concentration of boric acid, its hyperchromic effect and red shift effect on the black corn anthocyanin-protein complex show a downward trend. And it can be seen from Table 13 that the co-coloring effect of boric acid on black corn anthocyanins shows a phenomenon of first increasing and then decreasing. When the mass concentration of boric acid exceeds 0.5 mg / mL, the hyperchromic effect value decreases and the co-coloring effect weakens.

[0080] In summary, the co-coloring effects of the four organic acids on the black corn anthocyanin-protein complex and black corn anthocyanins are not the same. Organic acids such as gallic acid, tartaric acid, and citric acid show a hypochromic effect on black corn anthocyanins, while gallic acid, tartaric acid, citric acid, and boric acid all show a hyperchromic effect on the black corn anthocyanin-protein complex, which can enhance the co-coloring effect of the complex.

[0081] Different organic acids showed different co - color effects on black corn anthocyanin - protein complexes and black corn anthocyanins. It is speculated that this is due to the differences in the intermolecular interactions, steric hindrance effects, and system stabilities between black corn anthocyanin - protein complexes and black corn anthocyanins. The molecular structure of anthocyanins is relatively simple, and organic acids mainly interact directly with anthocyanin molecules through hydrogen bonding. In the complex, in addition to the interaction between anthocyanins and organic acids, proteins also interact with organic acids. Moreover, anthocyanin molecules are relatively small, and the interaction between organic acids and anthocyanins is less affected by steric hindrance. In the complex, due to the relatively large size and complex spatial structure of protein molecules, steric hindrance will be generated on the binding of organic acids and anthocyanins. In addition, anthocyanins mainly exist in the form of monomers or simple polymers in solution, and the system is relatively simple. The stability is mainly affected by the interaction between gallic acid and anthocyanins. The stability of the complex not only depends on the interaction between anthocyanins and organic acids, but is also closely related to the stability of proteins.

[0082] III. Zeta Potential

[0083] Zeta potential refers to the potential of the shear plane, which reflects the charge property and charge density of the particle surface. The initial potential of the black corn anthocyanin - protein complex is - 5.84 mV, carrying a negative charge, and the initial potential of black corn anthocyanins is - 3.39 mV, carrying a negative charge.

[0084]

[0085] After different concentrations of co - colorants were mixed with black corn anthocyanin - protein complexes and black corn anthocyanins respectively, the pH was adjusted to 4.0, and the Zeta potential of the mixed solution was measured using a Zeta potential analyzer.

[0086] <Zeta Potential of Added Different Concentrations of Gallic Acid>

[0087] As Figure 1As shown, when the pH was adjusted to 4.0 by adding gallic acid at different concentrations to the black corn anthocyanin-protein complex, the absolute value of the Zeta potential showed a trend of first increasing and then decreasing, involving principles such as electrostatic interaction, hydrogen bond interaction, steric hindrance effect, and hydrophobic interaction. The Zeta potential of the black corn anthocyanin-protein complex without adding gallic acid was negative, being negatively charged due to the presence of many acidic groups such as carboxyl groups on the surface. As the concentration of gallic acid increased, the absolute value of the Zeta potential showed a trend of first increasing and then decreasing. Gallic acid is a polyphenolic compound that can dissociate hydrogen ions in the solution, increasing the hydrogen ion concentration in the system. It is speculated that gallic acid binds to the negatively charged black corn anthocyanin-protein complex through electrostatic attraction, increasing the charge density on the surface of the complex, thus increasing the absolute value of the Zeta potential. Moreover, there are multiple hydroxyl groups in the gallic acid molecule, and there are also many groups that can form hydrogen bonds on the black corn anthocyanin and protein molecules, such as hydroxyl groups, amino groups, carboxyl groups, etc. Gallic acid binds to the black corn anthocyanin-protein complex through hydrogen bonds, increasing the effective charge on the surface of the complex. In addition, there are some hydrophobic regions in both the black corn anthocyanin and protein molecules, and gallic acid also has a certain degree of hydrophobicity. Within a certain concentration range, gallic acid binds to the complex through hydrophobic interaction, making the charge distribution on the surface of the complex more uneven, which also leads to an increase in the absolute value of the potential. When the concentration of gallic acid exceeds 0.5 mg / mL, the absolute value of the potential shows a downward trend. This is because when the concentration of gallic acid continues to increase to a certain extent, gallic acid molecules will bind excessively on the surface of the complex. Since it also carries a certain charge itself, it will neutralize some of the negative charges on the surface of the complex, resulting in a decrease in the absolute value of the Zeta potential. And too high a concentration of gallic acid will disrupt the hydrophobic interaction inside the complex, changing the structure of the complex and redistributing the surface charges, thus causing the absolute value of the potential to decrease. After an appropriate amount of gallic acid binds to the black corn anthocyanin-protein complex, a relatively regular molecular layer will form on the surface of the complex, thus increasing the absolute value of the Zeta potential. However, when a large number of gallic acid molecules accumulate on the surface of the complex, the spatial structure of the complex will become disordered, and the repulsive force between particles will weaken, resulting in a decrease in the absolute value of the Zeta potential.

[0088] <Zeta potential with different concentrations of tartaric acid added>

[0089] When the pH was adjusted to 4.0 after adding different concentrations of tartaric acid to the black corn anthocyanin-protein complex, Figure 2The absolute value of the Zeta potential shows a trend of first rising and then falling, which involves the principles of electrostatic interaction and hydrogen bonding. Tartaric acid is a dibasic weak acid that will dissociate in the solution to produce hydrogen ions and corresponding acid radical ions. As the concentration of tartaric acid increases, the dissociated acid radical ions will bind to the negatively charged complex through electrostatic attraction, increasing the negative charge density on the surface of the complex and the absolute value of the Zeta potential rising. In addition, the tartaric acid molecule contains multiple hydroxyl and carboxyl groups, and there are also many groups that can form hydrogen bonds on black corn anthocyanins and protein molecules. As the concentration of tartaric acid increases, the number of tartaric acid and complexes combined through hydrogen bonds increases, which changes the charge distribution on the surface of the complex, increases the effective charge, and increases the absolute value of the Zeta potential. When the tartaric acid concentration exceeds 0.25 mg / mL, the absolute value of the potential shows a downward trend. The decrease in the absolute value of the potential is due to the fact that when the tartaric acid concentration is too high, a large amount of hydrogen ions in the solution will neutralize part of the negative charge on the surface of the complex, resulting in a decrease in the net charge on the surface of the complex. When the concentration of tartaric acid is too high, too much hydrogen bonding will destroy the original structure of the complex, making the charge distribution on the surface of the complex chaotic, and part of the charge being neutralized or shielded, thereby increasing the absolute value of the Zeta potential. High concentrations of tartaric acid will also cause excessive changes in the conformation of the complex, causing the structure of the complex to become loose or disordered, the surface charge distribution to be disrupted, and the charges to be neutralized or shielded.

[0090] <Zeta potential with different concentrations of citric acid>

[0091] When the pH of black corn anthocyanin-protein complex was adjusted to 4.0 after adding different concentrations of citric acid, Figure 3As shown in the figure, the absolute value of Zeta potential shows a trend of first rising and then falling, which involves the principles of electrostatic interaction, hydrogen bonding and steric hindrance. Citric acid is a ternary weak acid, which will gradually dissociate into hydrogen ions and citrate ions in the solution. As the concentration of citric acid increases, the concentration of citrate ions in the solution also increases. These citrate ions will bind to the negatively charged complex through electrostatic attraction, further increasing the negative charge density on the surface of the complex. Citric acid molecules contain multiple hydroxyl and carboxyl groups. As the concentration of citric acid increases, the number of hydrogen bonds between citric acid and black corn anthocyanin-protein complex increases. After an appropriate amount of citric acid binds to the complex, a relatively regular molecular layer will be formed on the surface of the complex, which increases the steric hindrance of the complex, changes the dispersion state of the complex in the solution, and enhances the interaction between particles, thereby increasing the absolute value of Zeta potential. When the concentration of citric acid is further increased to exceed 0.5 mg / mL, excessive hydrogen bonding and complexation will destroy the original structure and charge distribution of the complex, causing the charge on the surface of the complex to be neutralized or shielded. High concentrations of citric acid will cause excessive steric hindrance of the complex, resulting in chaotic interactions between the complexes and an imbalance in the repulsion and attraction between particles, leading to a decrease in the absolute value of the Zeta potential.

[0092] <Zeta potential with different concentrations of boric acid added>

[0093] When the pH of black corn anthocyanin-protein complex was adjusted to 4.0 after adding different concentrations of boric acid, Figure 4 The absolute value of the Zeta potential shows a downward trend, which involves the principles of electrostatic interaction and competitive adsorption. Boric acid is a weak acid that will undergo weak dissociation in aqueous solution to produce a small amount of hydrogen ions and borate ions. As the concentration of boric acid increases, the hydrogen ions produced by its dissociation will neutralize part of the negative charge on the surface of the complex, causing the net negative charge on the surface of the complex to gradually decrease. Boric acid molecules or borate ions in the solution will compete with other charged particles originally adsorbed on the surface of the complex for adsorption. As the concentration of boric acid increases, more boric acid molecules or borate ions are adsorbed on the surface of the complex, replacing some of the particles that originally made the complex negatively charged, thereby reducing the negative charge density on the surface of the complex and the absolute value of the Zeta potential decreases.

[0094] In summary, organic acid co-colorants can further improve the color stability of the complex by enhancing the binding force between anthocyanins and proteins and stabilizing the complex structure. Under certain conditions, black corn anthocyanins and proteins carry different charges. The addition of organic acid co-colorants will regulate the charge distribution in the system, enhancing the electrostatic interaction between anthocyanins and proteins. In addition, the organic acid molecules bound to the black corn anthocyanin-protein complex can form a steric hindrance around the complex, preventing other molecules or ions that may cause the dissociation of the complex or the destruction of the anthocyanin structure from approaching, thus protecting the structural integrity of the complex and enabling anthocyanins to better exert their color-developing effect and achieve the co-coloring effect.

[0095] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the color stability of black corn anthocyanin-protein complex, characterized in that: Mix the co-colorant solution and the black corn anthocyanin-protein complex solution. The co-colorant in the co-colorant solution is gallic acid, tartaric acid, citric acid or boric acid.

2. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 1, characterized in that: The mass ratio of the co-colorant to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 2 - 40:

1.

3. A method for improving the color stability of a black corn anthocyanin-protein complex according to claim 1, characterized in that: When the co-colorant is gallic acid, the mass ratio of gallic acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 5:1; When the co-colorant is tartaric acid, the mass ratio of tartaric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 2.5:1; When the co-colorant is citric acid, the mass ratio of citric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 5:1; When the co-colorant is boric acid, the mass ratio of boric acid to the black corn anthocyanin-protein complex in the black corn anthocyanin-protein complex solution is 2:

1.

4. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 1, characterized in that: After mixing the co-colorant and the black corn anthocyanin-protein complex solution, the mass concentration of the co-colorant in the resulting mixed solution is 0.1 - 2.0 mg / mL.

5. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 1, characterized in that: Dissolve the black corn anthocyanin-protein complex in phosphate buffer to prepare the black corn anthocyanin-protein complex solution; dissolve the co-colorant in phosphate buffer to prepare the co-colorant solution, add the co-colorant solution to the black corn anthocyanin-protein complex solution, and adjust the pH to 4.

6. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 5, characterized in that: Add the co-colorant solution to the black corn anthocyanin-protein complex solution, and after adjusting the pH to 4, store it in the dark.

7. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 5, characterized in that: The pH of the phosphate buffer is 7.

4.

8. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 5, characterized in that: The concentration of the black corn anthocyanin-protein complex solution is 0.1 mg / mL.

9. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 5, characterized in that: The concentration of the co-colorant solution is 0.2 - 4.0 mg / mL.

10. A method for improving the color stability of black corn anthocyanin-protein complex according to claim 5, characterized in that: The concentration of the black corn anthocyanin-protein complex solution is 0.1 mg / mL, the concentration of the co-colorant solution is 0.2 - 4.0 mg / mL, the volume ratio of the black corn anthocyanin-protein complex solution to the co-colorant solution is 1:1, and the mass concentration of the co-colorant in the mixed solution obtained by adding the co-colorant solution to the black corn anthocyanin-protein complex solution is 0.1 - 2.0 mg / mL.

Images