Method for improving stability of monascus pigment by preparing polysaccharide-protein-monascus pigment compound and obtained product

Abstract

The invention discloses a method for improving the stability of monascus pigment by preparing a polysaccharide-protein-monascus pigment compound and an obtained product, and relates to the technical field of food additives and natural pigment stabilization. Beta-cyclodextrin and ovotransferrin are used as stabilizers and react with the monascorubin in a certain proportion, and a ternary composite system is constructed through interaction between ovotransferrin and the monascorubin and interaction between ovotransferrin and beta-cyclodextrin so as to improve the stability of the monascorubin. And finally, the beta-cyclodextrin-ovotransferrin-monascus pigment compound is obtained. A thermal stability test and a light stability test prove that the beta-cyclodextrin and the ovotransferrin have a synergistic effect, and the thermal stability and the illumination stability of the monascus pigment can be effectively improved.

Description

Technical Field

[0001] This invention relates to the fields of food additives and natural pigment stabilization technology, and in particular to a method for improving the stability of red yeast rice pigment by preparing a polysaccharide-protein-red yeast rice pigment complex and the resulting product. Background Technology

[0002] Monascus pigment (MP) is a secondary metabolite produced by the filamentous fungus Monascus purpureus. As a natural food coloring, it has a long history of widespread application in the food industry. The molecular structure of Monascus pigment is characterized by the presence of a carbonyl group, a γ-lactone ring, and a unique conjugated system based on conjugated double bonds (-C=CC=CC-). (The structural formula of Monascus pigment is shown in Figure 1). Figure 1 (As shown). Red yeast rice pigment possesses excellent coloring properties and reliable safety. Besides its coloring characteristics, red yeast rice pigment also exhibits various beneficial properties, including antioxidant, antitumor, and antibacterial activities. However, red yeast rice pigment is sensitive to temperature and light, exhibits poor stability and varying solubility under different pH conditions, limiting its widespread application. To address these challenges, researchers have conducted extensive studies on the stability of red yeast rice pigment from different perspectives. For example, adding antioxidants and adjusting the pH of the usage environment have been explored, but these methods often suffer from drawbacks such as high cost, complex operation, and limited effectiveness in improving stability. While adding antioxidants and other chemicals can delay fading caused by oxidation to some extent, it may introduce new chemical residue risks, which does not align with the trend of green and natural consumption, and its improvement on stability changes caused by other factors such as temperature and pH is not ideal. Adjusting the pH of the usage environment has significant limitations; practical application scenarios are complex and varied, making it difficult to precisely control the environmental pH to maintain a suitable range to ensure the stability of red yeast rice pigment. Therefore, it is essential to develop a method that can effectively improve the safety, efficiency, and widespread applicability of red yeast rice pigment. Summary of the Invention

[0003] The purpose of this invention is to provide a method for improving the stability of red yeast rice pigment by preparing a polysaccharide-protein-red yeast rice pigment complex and the resulting product, so as to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] One of the technical solutions of the present invention is a method for improving the stability of red yeast rice pigment by preparing a polysaccharide-protein-red yeast rice pigment complex. β-cyclodextrin and ovotransferrin are used as stabilizers and mixed with red yeast rice pigment to prepare the polysaccharide-protein-red yeast rice pigment complex, thereby improving the stability of red yeast rice pigment.

[0006] Ovotransferrin (OVT) is an iron-binding glycoprotein derived from eggs, possessing two high-affinity iron-binding sites. The main reasons for choosing OVT as a stabilizer to improve the stability of red yeast rice pigment in this invention are as follows:

[0007] (1) Ovotransferrin can form a stable complex with pigments through intermolecular interactions.

[0008] Ovotransferrin, a glycoprotein with a specific three-dimensional structure, can interact with pigments through hydrophobic interactions, hydrogen bonds, or electrostatic forces to form stable complexes. This binding "encapsulates" pigment molecules within its structural cavity or on its surface, reducing the contact between pigment-sensitive groups (such as conjugated double bonds or functional groups susceptible to thermal / photodegradation) and the external environment. This encapsulation effect restricts the thermal motion of pigment molecules or their sensitivity to photochemical reactions, thereby improving pigment stability.

[0009] (2) Barrier effect against external pressure

[0010] The macromolecular structure of ovotransferrin may act as a physical barrier. Regarding thermal stability, it can reduce the direct impact of heat on pigment molecules, slowing down heat-induced molecular rearrangement, decomposition, or oxidation of the pigment. Regarding photostability, it can absorb or scatter some ultraviolet (UV) or visible light, reducing the pigment's absorption of harmful light (which usually triggers photochemical reactions such as photolysis or photooxidation).

[0011] (3) Chelating metal ions

[0012] ovotransferrin's resistance to metal ions (such as Fe) 3+ Cu 2+ Zn 2+ Ovotransferrin possesses high affinity and can chelate free metal ions in the system. Many metal ions (especially transition metal ions) play a catalytic role in pigment degradation reactions—they promote the oxidative degradation of pigments under thermal or light conditions (e.g., through the Fenton reaction to generate reactive oxygen species). By chelating these metal ions, ovotransferrin can inhibit their catalytic activity, thereby protecting pigments from degradation.

[0013] (4) Antioxidant activity

[0014] Ovotransferrin and its hydrolysates possess certain antioxidant properties. They can scavenge reactive oxygen species (ROS, such as superoxide anions and hydroxyl radicals) generated under heat or light stress, and ROS are key factors leading to pigment oxidation and degradation. This antioxidant capacity helps reduce ROS-mediated pigment damage, indirectly enhancing its stability.

[0015] While ovotransferrin possesses the aforementioned advantages, the inventors discovered during their research that its solubility decreases near its isoelectric point, resulting in low efficiency in stabilizing red yeast rice pigments. Using ovotransferrin alone to stabilize red yeast rice pigments also presents application limitations. To improve the stabilization effect, this invention further introduces the synergistic effect of β-cyclodextrin and ovotransferrin. β-cyclodextrin (β-CD) has a truncated conical cavity structure composed of seven units of pyranose, which can enhance the stability of small molecules (pigments) in an aqueous environment. Moreover, the hydrophobic groups of ovotransferrin can penetrate the hydrophobic cavity of β-CD, forming a more stable β-cyclodextrin-ovotransferrin complex. This β-cyclodextrin-ovotransferrin complex further binds to red yeast rice pigments (the β-cyclodextrin-ovotransferrin complex can be considered a carrier of red yeast rice pigments), yielding a stable polysaccharide-protein-red yeast rice pigment ternary complex. Therefore, this invention effectively enhances pigment stability by forming a ternary complex of β-cyclodextrin, ovotransferrin, and red yeast rice pigments.

[0016] The second technical solution of the present invention: a highly stable polysaccharide-protein-red yeast pigment complex, which is obtained by combining red yeast pigment, β-cyclodextrin and ovotransferrin.

[0017] The third technical solution of the present invention: a method for preparing the above-mentioned highly stable polysaccharide-protein-red yeast rice pigment complex, comprising the following steps:

[0018] The highly stable polysaccharide-protein-red yeast rice pigment complex was obtained by stirring and reacting red yeast rice pigment solution, β-cyclodextrin solution and ovotransferrin solution at 20-60℃.

[0019] Furthermore, the step of stirring and reacting the red yeast rice pigment solution, β-cyclodextrin solution, and ovotransferrin solution at 20-60°C as raw materials includes: first mixing the β-cyclodextrin solution and the ovotransferrin solution, stirring and reacting at 20-60°C for 30-60 minutes, then adding the red yeast rice pigment solution, and continuing to stir and react at 20-60°C for 30-60 minutes.

[0020] Furthermore, the concentration of the red yeast rice pigment solution is 1-5 mM, and the solvent is ethanol.

[0021] Furthermore, the concentration of the β-cyclodextrin solution is 10-50 mg / mL, and the solvent is water.

[0022] Furthermore, the concentration of the ovotransferrin solution is 0.01-0.05 mM, and the solvent is a Tris-HCl buffer solution containing 0.01 mol / L NaCl.

[0023] Furthermore, the volume ratio of the red yeast rice pigment solution, β-cyclodextrin solution, and ovotransferrin solution is 1:1:1.

[0024] Furthermore, the stirring speed is 200-400 r / min.

[0025] Furthermore, after the stirring reaction is completed, the process also includes steps of impurity removal and vacuum freeze-drying.

[0026] Furthermore, the impurity removal method includes ultrafiltration or dialysis.

[0027] The present invention discloses the following technical effects:

[0028] (1) The present invention can effectively overcome the problem of poor stability of red yeast pigment in the prior art by preparing a polysaccharide-protein-red yeast pigment ternary complex, and broaden the application range of red yeast pigment.

[0029] (2) This invention uses β-cyclodextrin and ovotransferrin as stabilizers, reacting them with red yeast rice pigment in a certain ratio. A ternary complex system is constructed through the interaction between ovotransferrin and red yeast rice pigment, and between ovotransferrin and β-cyclodextrin, to improve the stability of red yeast rice pigment, ultimately obtaining a β-cyclodextrin-ovotransferrin-red yeast rice pigment complex. This invention measures the thermal stability of the β-cyclodextrin-ovotransferrin-red yeast rice pigment complex at different temperatures (55, 75, and 95°C) and the photostability under constant temperature (27°C) light conditions. The results are compared with free red yeast rice pigment and the ovotransferrin-red yeast rice pigment binary complex, demonstrating that the synergistic effect of β-cyclodextrin and ovotransferrin can effectively improve the thermal and photostability of red yeast rice pigment.

[0030] (3) The method of the present invention is simple, efficient and low cost. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is the structural formula of Monascus purpureus.

[0033] Figure 2 A photograph of the β-CD-OVT-MP complex prepared in Example 1.

[0034] Figure 3The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 55℃.

[0035] Figure 4 The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 75℃.

[0036] Figure 5 The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 95℃.

[0037] Figure 6 The results show the photostability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex.

[0038] Figure 7 The results show the pH stability of the MP, OVT-MP complex, and β-CD-OVT-MP complex at pH 5.5.

[0039] Figure 8 The results show the pH stability of the MP, OVT-MP complex, and β-CD-OVT-MP complex at pH 7.5.

[0040] Figure 9 The results show the pH stability of the MP, OVT-MP complex, and β-CD-OVT-MP complex at pH 9.5.

[0041] Figure 10 The UV-Vis absorption spectra of OVT, OVT-MP complex and β-CD-OVT-MP complex are shown. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0048] In specific embodiments of the present invention, room temperature refers to 25±5℃.

[0049] Unless otherwise specified, all raw materials used in the specific embodiments of this invention are commercially available products, and the ethanol is anhydrous ethanol.

[0050] The preparation method of ovotransferrin used in the following examples and comparative examples is as follows: Egg whites from fresh eggs were separated and an equal volume of distilled water was added, followed by thorough mixing. The pH of the diluted egg whites was adjusted to 6.0, and the mixture was centrifuged at 10,000 × g for 30 minutes to remove ovomucoid. The supernatant was collected, and 8% (w / v) ammonium sulfate and 3.5% (w / v) citric acid were added. The mixture was allowed to stand at 4°C for 12 hours, and then centrifuged at 10,000 × g for 30 minutes. 4% (w / v) ammonium sulfate and 1.5% (w / v) citric acid were added to the supernatant again, and the mixture was allowed to stand at 4°C for 12 hours, followed by another centrifugation. The precipitates from both centrifugation processes were collected, dissolved in distilled water, and the pH was adjusted to 9.0. The mixture was desalted using a dialysis membrane with a molecular weight cutoff of 30 kDa, and then freeze-dried to obtain ovotransferrin.

[0051] The above-described preparation method for ovotransferrin is inexpensive and simple to prepare.

[0052] Example 1

[0053] A highly stable polysaccharide-protein-red yeast rice pigment complex is prepared by the following steps:

[0054] (1) Preparation of β-cyclodextrin (β-CD) solution, ovotransferrin (OVT) solution and red yeast rice pigment (MP) solution Preparation of β-CD solution: Accurately weigh 50 mg of β-cyclodextrin and dissolve it in 5 mL of distilled water to obtain a β-CD solution with a concentration of 10 mg / mL. Store in a water bath at 40 °C.

[0055] Preparation of OVT solution: Prepare a 0.01 mM OVT solution using Tris-HCl buffer (pH = 7.4) containing 0.01 mol / L NaCl, and store at 4°C.

[0056] Preparation of MP solution: Prepare a 1 mM MP solution with ethanol and store it away from light.

[0057] (2) Preparation of β-cyclodextrin-ovoferrin-monas pigment complex (abbreviated as β-CD-OVT-MP complex)

[0058] The OVT and β-CD solutions were mixed at a 1:1 volume ratio and mechanically stirred at 200 rpm for 30 min at 40 °C to ensure thorough mixing and dissolution, yielding an OVT / β-CD mixed solution. MP solution (the volume of MP solution was the same as the volume of OVT solution used in preparing the OVT / β-CD mixed solution, i.e., the volume ratio of OVT, β-CD, and MP solutions was 1:1:1) was added to the OVT / β-CD mixed solution, and the mixture was stirred at 200 rpm for 30 min at 40 °C to ensure thorough mixing and dissolution. Unbound small molecule impurities were then removed by ultrafiltration to obtain a β-CD-OVT-MP complex solution.

[0059] The β-CD-OVT-MP complex solution was dried by vacuum freeze drying (vacuum freeze drying conditions: -40℃, 200Pa) to obtain a powdered β-CD-OVT-MP complex product, which was stored in a cool, dry, and light-protected place for later use.

[0060] Figure 2 The photograph shows the β-CD-OVT-MP complex prepared in this embodiment. It can be seen that the β-CD-OVT-MP complex is in powder form, fine and uniform, and the powder color is light brown. Some areas shimmer under light.

[0061] Comparative Example 1

[0062] The preparation steps of the ovotransferrin-monas pigment complex (OVT-MP complex) are as follows:

[0063] The OVT solution (same as the OVT solution in Example 1) and Tris-HCl buffer solution (pH = 7.4) containing 0.01 mol / L NaCl were mixed at a 1:1 volume ratio. The mixture was mechanically stirred at 200 rpm for 30 min at 40 °C to ensure thorough mixing. Then, MP solution (the volume of MP solution was the same as the original OVT solution) was added, and the mixture was stirred at 200 rpm for 30 min at 40 °C to ensure thorough mixing and dissolution. Unbound small molecule impurities were then removed by ultrafiltration to obtain the OVT-MP complex solution.

[0064] The OVT-MP complex solution was dried by vacuum freeze drying (at -40°C and 200Pa) to obtain a powdered OVT-MP complex product, which was then stored in a cool, dry, and dark place for later use.

[0065] Comparative Example 2

[0066] Similar to Comparative Example 1, the only difference is that the ovalbumin solution was replaced with an ovalbumin solution (the concentration, preparation method, and amount of ovalbumin solution used in preparing the complex were the same as in Comparative Example 1, and the ovalbumin was a commercially available ovalbumin). The complex solution obtained in this comparative example is called the ovalbumin-MP complex solution, and the powdered product obtained after vacuum freeze-drying is called the ovalbumin-MP complex.

[0067] Comparative Example 3

[0068] Same as Example 1, except that the OVT solution was replaced with an ovalbumin solution (the concentration, preparation method and amount of ovalbumin solution used in preparing the complex were the same as in Example 1, and the ovalbumin was a commercially available ovalbumin). The complex solution obtained in this comparative example is called the β-CD-ovalbumin-MP complex solution, and the powdered product obtained after vacuum freeze-drying is called the β-CD-ovalbumin-MP complex.

[0069] Comparative Example 4

[0070] Same as Example 1, except that the β-CD solution is replaced with gum arabic solution (the concentration, preparation method and amount of gum arabic solution used in preparing the complex are the same as in Example 1). The complex solution obtained in this comparative example is called gum arabic-OVT-MP complex solution, and the powdered product obtained after vacuum freeze-drying is called gum arabic-OVT-MP complex.

[0071] Test Example 1

[0072] Stability testing of the complex

[0073] (1) Plotting the standard curve

[0074] Red yeast rice pigment standard was prepared into standard solutions of different concentrations (0.2, 0.4, 0.8, and 1 mM) using ethanol. The absorbance (OD value) of each standard solution was measured at the maximum absorption wavelength of red yeast rice pigment (470 nm). A concentration-absorbance standard curve was plotted, yielding the linear regression equation y = 0.1922x + 0.0076, R0. 2 =0.9996.

[0075] (2) Changes in the thermal stability of red yeast rice pigment in the complex at different temperatures

[0076] Take 5 mL of MP solution (0.33 mM, prepared with ethanol), OVT-MP complex solution (prepared in Comparative Example 1), β-CD-OVT-MP complex solution (prepared in Example 1), ovalbumin-MP complex solution (prepared in Comparative Example 2), β-CD-ovalbumin-MP complex solution (prepared in Comparative Example 3), and gum arabic-OVT-MP complex solution (prepared in Comparative Example 4), respectively, and heat them in constant temperature water baths at 55, 75, and 95 °C for 0, 1, 2, 3, and 4 h. Measure the absorbance at 470 nm at different temperatures and heating times. Substitute the absorbance into the standard curve to obtain the red yeast rice pigment concentration of the six solutions (all six solutions had the same initial MP concentration) after different temperatures and heating times. Then calculate the pigment retention rate using formula (1). The results are as follows: Figures 3-5 As shown.

[0077] Pigment retention rate (%) = N 加热 / N 原始 ×100% (1)

[0078] Where, N 加热 The MP concentration and N concentration obtained from the OD value after heating the solution for a certain period of time represent the concentrations of N and N. 原始 This represents the original MP concentration of the solution before heating (i.e., 0.33 mM).

[0079] Figure 3 The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 55℃. Figure 4 The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 75℃. Figure 5 The results show the thermal stability of MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex at 95℃.

[0080] Depend on Figures 3-5It can be seen that under heating conditions, the MP in free MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex are all unstable. With increasing temperature and treatment time, the pigment retention rate of MP in free MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex all decrease to varying degrees. Moreover, the higher the temperature and the longer the treatment time, the greater the decrease in pigment retention rate of MP. However, at the same temperature and heating time, the pigment retention rate of MP among the six substances was significantly higher for the β-CD-OVT-MP complex than for the other complexes and free MP (the pigment retention rates of the β-CD-OVT-MP complex after heating at 55, 75, and 95℃ for 4 hours were 81.0%, 70.2%, and 50.6%, respectively, while the corresponding pigment retention rates for free MP were 65.4%, 60.3%, and 20.0%). This indicates that the degradation rate of MP is slowed down by the co-coloring effect of proteins and polysaccharides, especially at high temperatures above 75℃, where the co-coloring effect of β-CD and OVT provides good protection for the thermal stability of MP.

[0081] (3) Changes in the photostability of the composite

[0082] Take 5 mL of each of the following solutions: MP solution (0.33 mM, prepared with ethanol), OVT-MP complex solution (prepared in Comparative Example 1), β-CD-OVT-MP complex solution (prepared in Example 1), ovalbumin-MP complex solution (prepared in Comparative Example 2), β-CD-ovalbumin-MP complex solution (prepared in Comparative Example 3), and gum arabic-OVT-MP complex solution (prepared in Comparative Example 4). Place them under constant temperature (27℃) and light conditions for 0, 3, 6, 9, and 12 days. Measure the absorbance at 470 nm. Substitute the absorbance into the standard curve to obtain the red yeast rice pigment concentration of the six solutions after different light exposure times. Then calculate the pigment retention rate using formula (2). The results are as follows: Figure 6 As shown.

[0083] Pigment retention rate (%) = N 光照 / N 原始 ×100% (2)

[0084] Where, N 光照 The MP concentration, N, is obtained from the OD value after the solution has been exposed to light for a certain period of time. 原始 This represents the initial MP concentration of the solution before illumination (i.e., 0.33 mM).

[0085] Depend on Figure 6It was found that the pigment retention rate of MP in free MP, OVT-MP complex, β-CD-OVT-MP complex, ovalbumin-MP complex, β-CD-ovalbumin-MP complex, and gum arabic-OVT-MP complex all decreased with increasing light exposure time. However, after day 3, the pigment retention rate of free MP gradually became significantly lower than that of MP in the complexes. Furthermore, with prolonged exposure, the pigment retention rate of MP in the β-CD-OVT-MP complex showed a significant advantage (the pigment retention rate of the β-CD-OVT-MP complex was 95.1% after 12 days of storage under light conditions, while the corresponding pigment retention rate of free MP was 74.8%). This indicates that during this period, the pigment retention rate of MP in the β-CD-OVT-MP complex was significantly more stable than that of other complexes and free MP. This suggests that under constant temperature and light conditions at 27℃, β-CD and OVT work synergistically to provide good protection for the light stability of MP.

[0086] (4) Changes in pH stability of the complex

[0087] 0.1 mg of solid MP, OVT-MP complex (prepared in Comparative Example 1), and β-CD-OVT-MP complex (prepared in Example 1) were dissolved in 1 mL of Tris-HCl buffer (pH = 7.4) containing 0.01 mol / L NaCl. Then, 4 mL of phosphate buffer with different pH values ​​(5.5, 7.5, 9.5) were added respectively. The solutions were incubated at room temperature for 2 h. The absorbance was measured at 470 nm before and after incubation at room temperature. The absorbance of the three solutions after different light exposure times was obtained by substituting the values ​​into the standard curve. The pigment retention rate was then calculated using formula (3). The results are as follows: Figure 7-9 As shown.

[0088] Pigment retention rate (%) = N 孵育 / N 原始 ×100% (3)

[0089] Where, N 孵育 The MP concentration and N concentration were obtained based on OD values ​​after adding phosphate buffer at different pH values ​​(5.5, 7.5, 9.5) and incubating for 2 hours. 原始 This represents the MP concentration immediately after adding phosphate buffer at different pH values ​​(5.5, 7.5, 9.5) (i.e., before incubation or at 0h).

[0090] Figure 7 The results show the pH stability of MP, OVT-MP complex, and β-CD-OVT-MP at pH 5.5. Figure 8 The results show the pH stability of MP, OVT-MP complex, and β-CD-OVT-MP at pH 7.5. Figure 9 The results show the pH stability of MP, OVT-MP complex and β-CD-OVT-MP at pH=9.5.

[0091] Depend on Figures 7-9 It was found that MP in the free MP, OVT-MP complex, and β-CD-OVT-MP complex were unstable at different pH levels. The free MP exhibited the worst stability under acidic conditions (pH = 5.5), with stability increasing with increasing pH. Under acidic conditions, the formation of the complex significantly improved MP stability, increasing the stability of MP in the OVT-MP complex and β-CD-OVT-MP complex by 31% and 48%, respectively (the pigment retention rates of MP, OVT-MP complex, and β-CD-OVT-MP complex were 44.9%, 64.8%, and 87.1%, respectively). This indicates that the co-coloring effect of proteins and polysaccharides slows down the degradation rate of MP, especially under acidic conditions, where the co-coloring effect of β-CD and OVT provides good protection for the pH stability of MP.

[0092] Test Example 2

[0093] Structural changes of the complex

[0094] (1) Ultraviolet-Vis (UV-Vis) absorption spectroscopy analysis

[0095] The absorption spectra of OVT solution (0.0033 mM, prepared with Tris-HCl buffer (pH = 7.4) containing 0.01 mol / L NaCl), OVT-MP complex solution (prepared in Comparative Example 1), and β-CD-OVT-MP complex solution (prepared in Example 1) were measured at 298 K.

[0096] Figure 10 The UV-Vis absorption spectra of OVT, the OVT-MP complex, and the β-CD-OVT-MP complex are shown. All spectra exhibit distinct absorption peaks of aromatic amino acids at approximately 280 nm, primarily due to π-π* electronic transitions of chromophores such as tryptophan (Trp) and tyrosine (Tyr) residues. Typically, ligand-protein interactions are accompanied by hyperchromic or hypochromic effects in UV-Vis spectroscopy. Figure 10 As shown, the absorbance of OVT at 280 nm decreased with the addition of MP and β-CD, indicating the formation of a complex. The environment of the Trp residues was altered due to contact with MP. Furthermore, the decrease in peak intensity at 280 nm also indicates a conformational change in the OVT framework after the binding of β-CD, OVT, and MP. Therefore, these findings demonstrate that MP can bind to β-CD and OVT, inducing a conformational change in OVT. This also confirms the successful preparation of the complex.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the stability of red yeast rice pigment by preparing a polysaccharide-protein-red yeast rice pigment complex, characterized in that, A polysaccharide-protein-red yeast pigment complex was prepared by mixing β-cyclodextrin and ovotransferrin with red yeast pigment to improve the stability of red yeast pigment.

2. A highly stable polysaccharide-protein-red yeast rice pigment complex, characterized in that, It is obtained by combining red yeast rice pigment, β-cyclodextrin and ovotransferrin.

3. A method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 2, characterized in that, The following steps are involved: The highly stable polysaccharide-protein-red yeast rice pigment complex was obtained by stirring and reacting red yeast rice pigment solution, β-cyclodextrin solution and ovotransferrin solution at 20-60℃.

4. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 3, characterized in that, The process of using red yeast rice pigment solution, β-cyclodextrin solution, and ovotransferrin solution as raw materials and stirring at 20-60°C includes: first mixing the β-cyclodextrin solution and the ovotransferrin solution, stirring at 20-60°C for 30-60 minutes, then adding the red yeast rice pigment solution, and continuing to stir at 20-60°C for another 30-60 minutes.

5. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, The concentration of the red yeast rice pigment solution is 1-5 mM, and the solvent is ethanol.

6. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, The concentration of the β-cyclodextrin solution is 10-50 mg / mL, and the solvent is water.

7. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, The concentration of the ovotransferrin solution is 0.01-0.05 mM, and the solvent is Tris-HCl buffer containing 0.01 mol / L NaCl.

8. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, The volume ratio of the red yeast rice pigment solution, β-cyclodextrin solution, and ovotransferrin solution is 1:1:

1.

9. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, The stirring speed is 200-400 r / min.

10. The method for preparing the highly stable polysaccharide-protein-red yeast rice pigment complex as described in claim 4, characterized in that, After the stirring reaction is completed, the process also includes impurity removal and vacuum freeze-drying.

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