Monascus red pigment nanoscale stabilized carrier based on graded self-assembly technology and preparation method and application of monascus red pigment nanoscale stabilized carrier

Abstract

The invention discloses a monascus red pigment nano stabilized carrier constructed based on a hierarchical self-assembly strategy as well as a preparation method and application thereof, and belongs to the technical field of nano carriers. According to the invention, a beta-cyclodextrin-enzymolysis sodium caseinate-Arabic gum ternary supramolecular composite system is constructed, and efficient encapsulation, bitter taste shielding and stability improvement of pigment molecules are realized by virtue of a multi-stage self-assembly mechanism. According to the carrier, a hydrophobic core domain is constructed by virtue of an enzymolysis peptide fragment, bitter components are captured by virtue of an inclusion effect of beta-cyclodextrin, and the interface stability is enhanced by virtue of Arabic gum, so that a nano-scale delivery system with a clear structure and excellent performance is finally formed, and the photo-thermal stability and sensory quality of the pigment are remarkably improved; and the powder obtained by spray drying is good in flowability, easy to re-disperse and high in product applicability. The carrier is wide in raw material source, the preparation process is green and mild, large-scale amplification is easy, and the carrier has good application potential in the high-value-added fields such as high-end functional food, clarified beverages and pharmaceutical preparations.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanocarriers, and particularly relates to a nanoscale stabilized carrier of monascus red pigment constructed by a hierarchical self-assembly strategy, and a preparation method and application thereof. BACKGROUND

[0002] Monascus red pigment is a natural water-soluble pigment produced by Monascus spp. fermentation, and its main coloring components are Monascorubramine and Monascin. The pigment is widely used in coloring of meat products, condiments, beverages, candies and other foods due to its natural color (color tone from red to purple), high safety and certain physiological activity (such as lowering blood lipids), and is one of the ideal choices to replace synthetic red pigments (such as carmine). However, there are two main technical bottlenecks in the practical application of monascus red pigment: poor stability: the chromophore in the molecular structure of monascus red pigment is highly sensitive to light, heat, oxygen and other environmental factors, and is prone to degradation and discoloration during processing and storage, resulting in a shortened shelf life of the product and severely limiting its application in foods requiring heat processing or transparent packaging. Inherent bitter taste: Monascus fermentation will also produce Monacolin K and other secondary metabolites with strong bitter taste. These bitter substances are difficult to separate from the pigment, making ordinary monascus red products have obvious bitter and peculiar taste, affecting the taste of the final product, and making it difficult to be widely used in flavor-sensitive foods such as beverages and candies. At present, the industry often uses microencapsulation, liposome embedding, cyclodextrin inclusion and other technologies to improve the stability or flavor of the pigment. However, the existing monascus red pigment stabilization technology still has certain limitations.

[0003] As described in patent CN102702780A, dissolving monascus red pigment in a specific amino acid solution can effectively inhibit photobleaching, but the cost of amino acids is relatively high, and the metabolism of some amino acids may produce harmful substances (such as mycosin), which limits its wide application.

[0004] Patent CN104212839A adds tea polyphenols and jujube polysaccharides during monascus fermentation to effectively improve the light resistance of the pigment. This method has the advantages of low cost and high safety, but its color protection efficiency still needs to be improved, and high-dose antioxidants may cause aggregation of pigment molecules. In addition, the introduction of tea polyphenols will introduce additional bitter flavor, which may have an adverse effect on the sensory quality of the product.

[0005] Patent CN113480870A uses monascus orange pigment to react with amino acids to derive, which significantly improves the water solubility and thermal stability, especially in acidic environment, but the process is complex, involves the use of organic solvents, and the large-scale production and food compliance still need to be further verified.

[0006] Therefore, developing more efficient, economical, safe, and easily industrialized stabilization technologies for red yeast rice pigment remains an important research direction in this field. However, existing technologies often have limitations: for example, single cyclodextrin encapsulation has limited masking effect on bitterness and insufficient improvement in stability; while nanoliposomes offer good encapsulation effects, they are costly, complex in process, and difficult to industrialize; protein-polysaccharide complexes have stringent requirements for process conditions such as pH and ionic strength, and the raw materials (such as whey protein) are expensive. Therefore, developing an encapsulation technology that can simultaneously solve the problems of stability and bitterness, is low-cost, simple in process, and suitable for industrialization has become a key requirement for the high-value application of red yeast rice pigment. Summary of the Invention

[0007] This invention constructs a ternary composite nanomicelle carrier through a hierarchical self-assembly strategy, effectively solving the stability and flavor problems of red yeast rice pigment. This carrier possesses a multi-layered protective structure, achieving efficient encapsulation of the pigment and masking of bitterness through the synergistic effect of enzymatically hydrolyzed peptides, β-cyclodextrin, and gum arabic.

[0008] This invention is based on the principle of hierarchical self-assembly and constructs a ternary nanocarrier through a three-step synergistic mechanism: First, the hydrophobic cavity of β-cyclodextrin is used to encapsulate red yeast rice pigment and bitter molecules at the molecular level, achieving primary stability and bitterness shielding; then, amphiphilic peptides generated by enzymatic hydrolysis of sodium caseinate self-assemble to form a micelle hydrophobic core, effectively anchoring the encapsulated complex; finally, a spatial stability barrier is formed by interface modification with gum arabic, and a three-dimensional protective layer is constructed with its hydrophilic segments, ultimately achieving a synergistic enhancement of the photothermal stability of the pigment and the optimization of sensory quality.

[0009] To achieve the technical objective of this invention, the technical solution of this invention includes the following steps: (1) Construct a red yeast rice-β-cyclodextrin inclusion complex to achieve primary stabilization at the molecular level; (2) Prepare sodium caseinate colloidal solution as an enzymatic hydrolysis precursor; (3) Papain-directed enzymatic hydrolysis of sodium caseinate colloid was performed to achieve specific peptide chain cleavage and functionalization modification. (4) The enzyme reaction is terminated by high-temperature inactivation of the colloid after enzymatic hydrolysis; (5) The pretreated gum arabic after vacuum degassing is introduced into the enzyme-inactivated colloid in a gradient manner and subjected to high-intensity shear homogenization to induce intermolecular interweaving and adsorption, forming a thermodynamically stable composite colloidal matrix. (6) The inclusion compound obtained in step (1) is mixed with the colloid obtained in step (5) and a composite nanomicelle is formed by self-assembly, which is the nanoscale stabilized carrier of red yeast rice pigment of the present invention.

[0010] Preferably, the concentration of red yeast rice pigment in step (1) is 1–4 mg / mL, and the working concentration of β-cyclodextrin is 2–8 mg / mL; after the β-cyclodextrin is completely dissolved in ultrapure water at 40–60℃ under constant stirring, red yeast rice pigment is added quantitatively, and the mixture is continuously and gently stirred for 30 minutes within the same temperature range to complete the inclusion through hydrophobic interaction and size matching effect.

[0011] Preferably, the concentration of sodium caseinate in step (2) is 0.25 mg / mL. It is dispersed in ultrapure water and continuously mechanically stirred for 60 minutes to form a homogeneous and stable colloidal solution as an enzymatic hydrolysis precursor.

[0012] Preferably, the concentration of papain used in step (3) is 0.0125 mg / mL; the enzymatic hydrolysis reaction is carried out under constant temperature water bath shaking conditions at 50±1℃, and the pH of the system is precisely maintained at 6.5–7.0 by pH meter, and the enzymatic hydrolysis time is 20–40 minutes, so as to achieve specific cleavage and functional modification of caseinate sodium peptide chain.

[0013] Preferably, the enzyme inactivation treatment in step (4) is carried out in a water bath at 90±2℃ for 10 minutes to ensure complete inactivation of enzyme activity. After treatment, the enzyme is rapidly cooled to below 40℃ to avoid degradation of heat-sensitive components.

[0014] Preferably, the concentration of gum arabic added in step (5) is 0.05 mg / mL, and it needs to be pretreated by vacuum degassing before addition; after adding the enzyme-inactivated and cooled colloidal system, it is homogenized by high-intensity shearing at 8000 rpm for 2 minutes to induce interweaving and adsorption between gum arabic and enzymatically hydrolyzed colloidal molecules to form a thermodynamically stable composite colloidal matrix.

[0015] Preferably, the self-assembly process in step (6) is carried out at 35±1℃. The inclusion compound obtained in step (1) and the composite colloidal matrix obtained in step (5) are mixed at low speed for 20 minutes to form a composite nanomicelle with uniform structure and stable performance through molecular self-assembly.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Synergistic stability: The hydrophobic micelle core formed by enzymatic hydrolysis of casein provides a physical barrier to isolate the pigment from light and oxygen; β-cyclodextrin and gum arabic further reinforce the structure, forming a synergistic and stable system. Experiments show that the pigment retention rate of this complex under light and heating conditions is significantly improved compared with ordinary red yeast rice water solution.

[0017] (2) High efficiency masking: Through the dual mechanism of β-cyclodextrin inclusion (host-guest interaction) and micellar physical isolation (steric hindrance), bitter molecules such as Monacolin K are effectively masked, the bitterness value of the electronic tongue is significantly reduced, and the structure is controllable and the performance is highly adjustable.

[0018] (3) Through a hierarchical self-assembly strategy, multi-level precise control of the carrier structure is achieved. The stepwise assembly of enzymatically hydrolyzed peptides, β-cyclodextrin and gum arabic not only enhances the stability and masking performance of the system, but also makes key performance parameters such as carrier particle size, encapsulation efficiency and release behavior adjustable. The carrier formulation and process conditions can be flexibly optimized according to different application scenarios (such as acidic beverages and high-temperature processed foods), which has high adaptability and application flexibility.

[0019] (4) Low cost and simple process: The core raw materials sodium caseinate and β-cyclodextrin are inexpensive. The process does not require complex equipment such as high pressure homogenization. It achieves efficient encapsulation through biomolecule self-assembly, with low energy consumption, and is suitable for industrial production.

[0020] (5) Excellent product performance: The powder product obtained by spray drying the dispersion prepared by this patent has good flowability, is not easy to absorb moisture, and has good resolubility. It can be widely used in various food systems, including acidic beverages. The product can be quickly dispersed and restored in the aqueous phase, forming a uniform and stable suspension within seconds, without clumping or precipitation.

[0021] (6) Environmentally friendly and highly safe: All raw materials are food-grade or GRAS-recognized safe ingredients. The preparation process does not require the use of organic solvents or harmful chemical reagents, thus avoiding potential safety risks. It conforms to the development trend of green food additives and has good biocompatibility and degradability. Attached Figure Description

[0022] Figure 1 The pigment retention rate is the pigment sample dispersion after different light exposure times.

[0023] Figure 2 The improvement rate of pigment retention rate after pigment sample dispersions are exposed to different light exposure times.

[0024] Figure 3 The images show the appearance of the pigment sample dispersion after different light exposure times.

[0025] Figure 4 The pigment retention rate is the pigment sample dispersion after different heating treatments.

[0026] Figure 5 The improvement rate of pigment retention after different heating treatments of the pigment sample dispersion.

[0027] Figure 6The initial average particle size of the pigment sample solution is given.

[0028] Figure 7 Appearance diagram for evaluating the flowability and resolubility of pigment powder.

[0029] Figure 8 The transmittance is the light transmittance of the pigment sample dispersion.

[0030] Figure 9 This is a translucent image of the pigment sample dispersion.

[0031] Figure 10 This is an electronic tongue analysis diagram of the pigment sample dispersion.

[0032] Figure 11 This is an electronic nose analysis diagram of the pigment sample dispersion. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0034] All operations not mentioned in this invention are conventional operations in the field, and all materials not specifically mentioned in this invention are conventional materials that can be purchased from the market.

[0035] Example 1: Preparation of Enzymatic Hydrolysis-Inclusion Complex Micellar Dispersion First, β-cyclodextrin (2 mg / mL) was dissolved completely in ultrapure water at 40℃ by constant stirring. Then, red yeast rice pigment (1 mg / mL) was added, and the mixture was stirred continuously at the same temperature for 30 minutes. Inclusion was achieved through hydrophobic interactions, forming a red yeast rice pigment-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2℃ and stored in the dark. Simultaneously, sodium caseinate (0.25 mg / mL) was dispersed in ultrapure water and stirred to form a homogeneous colloidal solution. Papain (0.0125 mg / mL) was added to this solution, and the pH was adjusted to 6.5–7.0. Enzymatic hydrolysis was performed at 50±1℃ for 20–40 minutes. After hydrolysis, the colloidal system was incubated in a 90±2℃ water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled to 30±2℃ in an ice-water bath. Subsequently, gum arabic (0.05 mg / mL) was added to the enzyme-inactivated and cooled colloid, and the mixture was homogenized by high-speed shearing at 8000 rpm for 2 minutes to form a composite colloidal matrix. Finally, this matrix was mixed with the red yeast rice-β-cyclodextrin inclusion complex and assembled with gentle stirring at 35±1℃ for 20 minutes to obtain a liquid red yeast rice composite micelle dispersion. The dispersion was then continuously irradiated under D65 fluorescent lamps for 10 days.

[0036] Example 2: Preparation of Enzymatic Hydrolysis-Inclusion Complex Micellar Dispersion First, β-cyclodextrin (8 mg / mL) was dissolved completely in ultrapure water at 40℃ by constant stirring. Then, red yeast rice pigment (4 mg / mL) was added, and the mixture was stirred continuously at the same temperature for 30 minutes. Inclusion was achieved through hydrophobic interactions, forming a red yeast rice pigment-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2℃ and stored in the dark. Simultaneously, sodium caseinate (0.25 mg / mL) was dispersed in ultrapure water and stirred to form a homogeneous colloidal solution. Papain (0.0125 mg / mL) was added to this solution, and the pH was adjusted to 6.5–7.0. Enzymatic hydrolysis was performed at 50±1℃ for 20–40 minutes. After hydrolysis, the colloidal system was incubated in a 90±2℃ water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled to 30±2℃ in an ice-water bath. Subsequently, gum arabic (0.05 mg / mL) was added to the enzyme-inactivated and cooled colloid, and the mixture was homogenized by high-speed shearing at 8000 rpm for 2 minutes to form a composite colloidal matrix. Finally, this matrix was mixed with the red yeast rice-β-cyclodextrin inclusion complex and assembled with gentle stirring at 35±1℃ for 20 minutes to obtain a liquid red yeast rice composite micelle dispersion. The dispersion was then continuously irradiated under D65 fluorescent lamps for 10 days.

[0037] Example 3: Preparation of Enzymatic Hydrolysis-Inclusion Complex Micellar Dispersion First, β-cyclodextrin (2 mg / mL) was dissolved completely in ultrapure water at 40℃ by constant stirring. Then, red yeast rice pigment (1 mg / mL) was added, and the mixture was stirred continuously at the same temperature for 30 minutes. Inclusion was achieved through hydrophobic interactions, forming a red yeast rice pigment-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2℃ and stored in the dark. Simultaneously, sodium caseinate (0.25 mg / mL) was dispersed in ultrapure water and stirred to form a homogeneous colloidal solution. Papain (0.0125 mg / mL) was added to this solution, and the pH was adjusted to 6.5–7.0. Enzymatic hydrolysis was performed at 50±1℃ for 20–40 minutes. After hydrolysis, the colloidal system was incubated in a 90±2℃ water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled to 30±2℃ in an ice-water bath. Subsequently, gum arabic (0.05 mg / mL) was added to the enzyme-inactivated and cooled colloid, and the mixture was homogenized by high-speed shearing at 8000 rpm for 2 minutes to form a composite colloidal matrix. Finally, this matrix was mixed with the red yeast rice-β-cyclodextrin inclusion complex and assembled with gentle stirring at 35±1℃ for 20 minutes to obtain a liquid red yeast rice composite micelle dispersion. The dispersion was then heated in a water bath at 95℃ for 2 hours.

[0038] Example 4: Preparation of Enzymatic Hydrolysis-Inclusion Complex Micellar Dispersion First, β-cyclodextrin (8 mg / mL) was dissolved completely in ultrapure water at 40℃ by constant stirring. Then, red yeast rice pigment (4 mg / mL) was added, and the mixture was stirred continuously at the same temperature for 30 minutes. Inclusion was achieved through hydrophobic interactions, forming a red yeast rice pigment-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2℃ and stored in the dark. Simultaneously, sodium caseinate (0.25 mg / mL) was dispersed in ultrapure water and stirred to form a homogeneous colloidal solution. Papain (0.0125 mg / mL) was added to this solution, and the pH was adjusted to 6.5–7.0. Enzymatic hydrolysis was performed at 50±1℃ for 20–40 minutes. After hydrolysis, the colloidal system was incubated in a 90±2℃ water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled to 30±2℃ in an ice-water bath. Subsequently, gum arabic (0.05 mg / mL) was added to the enzyme-inactivated and cooled colloid, and the mixture was homogenized by high-speed shearing at 8000 rpm for 2 minutes to form a composite colloidal matrix. Finally, this matrix was mixed with the red yeast rice-β-cyclodextrin inclusion complex and assembled with gentle stirring at 35±1℃ for 20 minutes to obtain a liquid red yeast rice composite micelle dispersion. The dispersion was then heated in a water bath at 95℃ for 2 hours.

[0039] Example 5: Enzymatic hydrolysis-inclusion complex micelle dispersion and spray drying. First, β-cyclodextrin (2 mg / mL) was dissolved completely in ultrapure water at 40℃ by constant stirring. Then, red yeast rice pigment (1 mg / mL) was added, and the mixture was stirred continuously at the same temperature for 30 minutes. Inclusion was achieved through hydrophobic interactions, forming a red yeast rice pigment-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2℃ and stored in the dark. Simultaneously, sodium caseinate (0.25 mg / mL) was dispersed in ultrapure water and stirred to form a homogeneous colloidal solution. Papain (0.0125 mg / mL) was added to this solution, and the pH was adjusted to 6.5–7.0. Enzymatic hydrolysis was performed at 50±1℃ for 20–40 minutes. After hydrolysis, the colloidal system was incubated in a 90±2℃ water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled to 30±2℃ in an ice-water bath. Subsequently, gum arabic (0.05 mg / mL) was added to the enzyme-inactivated and cooled colloid, and the mixture was homogenized by high-speed shearing at 8000 rpm for 2 minutes to form a composite colloidal matrix. Finally, this matrix was mixed with the red yeast rice-β-cyclodextrin inclusion complex and assembled with gentle stirring at 35±1℃ for 20 minutes to obtain a liquid red yeast rice composite micelle dispersion. Maltodextrin was added to the dispersion so that the solid particles accounted for 10% of the dispersion mass. The dispersion was spray-dried, and then the complex powder was reconstituted to obtain a red yeast rice pigment (1 mg / mL) composite micelle dispersion.

[0040] Preparation of red yeast rice solution in Comparative Example 1 A 1 mg / ml red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water, and the solution was continuously irradiated under a D65 fluorescent lamp for 10 days.

[0041] Comparative Example 2: Preparation of Red Yeast Rice Solution A 4 mg / ml red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water. A 1 mg / ml red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water. The solution was then continuously irradiated under a D65 fluorescent lamp for 10 days.

[0042] Comparative Example 3: Preparation of Composite Micellar Dispersion A 1 mg / mL solution of red yeast rice pigment was prepared by dissolving red yeast rice in pure water. This solution was then used to dissolve β-cyclodextrin (final concentration 2.0 mg / mL) in ultrapure water at 40°C. Red yeast rice pigment (final concentration 1 mg / mL) was then added, and the mixture was stirred continuously at a constant temperature for 30 minutes to form a red yeast rice-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2°C. Gum arabic (final concentration 0.05 mg / mL) was then added, and after uniform dispersion, the mixture was homogenized at 8000 rpm for 2 minutes to obtain a composite micelle dispersion. This dispersion was then continuously irradiated under a D65 fluorescent lamp for 10 days.

[0043] Comparative Example 4: Preparation of Composite Micellar Dispersion A 4 mg / mL solution of red yeast rice pigment was prepared by dissolving red yeast rice in pure water. This solution was then used to dissolve β-cyclodextrin (final concentration 8 mg / mL) in ultrapure water at 40°C. Red yeast rice pigment (final concentration 4 mg / mL) was then added, and the mixture was stirred continuously at a constant temperature for 30 minutes to form a red yeast rice-β-cyclodextrin inclusion complex. The mixture was then cooled to 25±2°C. Gum arabic (final concentration 0.05 mg / mL) was then added, and after uniform dispersion, the mixture was homogenized at 8000 rpm for 2 minutes to obtain a composite micelle dispersion. This dispersion was then continuously irradiated under a D65 fluorescent lamp for 10 days.

[0044] Preparation of red yeast rice solution in Comparative Example 5 A 1 mg / ml red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water, and the red yeast rice solution was heated in a water bath at 95°C for 2 hours.

[0045] Preparation of red yeast rice solution in Comparative Example 6 A 4 mg / ml red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water, and the red yeast rice solution was heated in a water bath at 95°C for 2 hours.

[0046] Comparative Example 7: Preparation of Composite Micellar Dispersion A 1 mg / mL solution of red yeast rice pigment was prepared by dissolving red yeast rice in pure water. This solution was then used to dissolve β-cyclodextrin (final concentration 2 mg / mL) in ultrapure water at 40 °C. Red yeast rice pigment (final concentration 1 mg / mL) was then added, and the mixture was stirred continuously at a constant temperature for 30 minutes to form a red yeast rice-β-cyclodextrin inclusion complex. The mixture was then cooled to 25 ± 2 °C. Next, gum arabic (final concentration 0.05 mg / mL) was added, and after uniform dispersion, the mixture was homogenized at 8000 rpm for 2 minutes to obtain a composite micelle dispersion. The dispersion was then heated in a 95 °C water bath for 2 hours.

[0047] Comparative Example 8: Preparation of Composite Micellar Dispersion A 4 mg / mL solution of red yeast rice pigment was prepared by dissolving red yeast rice in pure water. This solution was then used to dissolve β-cyclodextrin (final concentration 8 mg / mL) in ultrapure water at 40 °C. Red yeast rice pigment (final concentration 4 mg / mL) was then added, and the mixture was stirred continuously at a constant temperature for 30 minutes to form a red yeast rice-β-cyclodextrin inclusion complex. The mixture was then cooled to 25 ± 2 °C. Next, gum arabic (final concentration 0.05 mg / mL) was added, and after uniform dispersion, the mixture was homogenized at 8000 rpm for 2 minutes to obtain a composite micelle dispersion. The dispersion was then heated in a 95 °C water bath for 2 hours.

[0048] Comparative Example 9: An enzymatic hydrolysis-inclusion complex micelle dispersion was prepared and spray-dried. A 1 mg / mL red yeast rice pigment solution was prepared by dissolving red yeast rice in pure water. Maltodextrin was added so that the solid particles accounted for 10% of the mass of the dispersion. The dispersion was spray-dried, and then the complex powder was reconstituted to obtain a complex micelle dispersion of red yeast rice pigment (1 mg / mL).

[0049] Experiment Example 1: Light Stability Test The pigment retention rate and pigment retention rate improvement rate of the composite micelle dispersions prepared in Examples 1, 2, and Comparative Examples 1-4 were measured after 5 and 10 days of illumination. The dispersions were diluted, and their absorbance at 488 nm was measured. Calculation method: ； ； R t : Absorbance retention rate (%) at time point t; A 0 Initial absorbance (ABS) of the sample before illumination; A t : Absorbance value of the sample at time point t (ABS); RRtPigment retention rate of the experimental group (i.e., pigment retention rate after using the new method); RRc Pigment retention rate of the control group (pigment retention rate of the blank control group) Figure 1 It can be seen that, under the same light conditions, the pigment retention rate of the red yeast rice-β-cyclodextrin enzymatic hydrolysis composite micelles decreased more slowly compared to red yeast rice aqueous solution and red yeast rice-β-cyclodextrin composite micelles. The pigment retention rate of the sample dispersion with a red yeast rice concentration of 1 mg / ml was generally higher than that of 4 mg / ml. The pigment retention rate of this composite micelle dispersion reached 70% after 10 days of light exposure. Figure 2 This reflects the improvement in pigment retention rate; the composite micelle dispersion showed a maximum improvement in pigment retention rate of 32% after 10 days of light exposure. Figure 3 As shown, the color of the sample dispersion with a red yeast rice concentration of 1 mg / ml became significantly lighter after 10 days of light exposure.

[0050] Experiment Example 2: Thermal Stability Test The pigment retention rate and pigment retention rate enhancement rate of the composite micelle dispersions prepared in Examples 1, 2, and Comparative Examples 1-4 were measured after heating at 95°C for two hours. The dispersions were diluted and their absorbance at 488 nm was measured (calculated using the same method as above).

[0051] Figure 4 It can be seen that the pigment retention rate of the 1 mg / ml sample dispersion is generally higher than that of the 4 mg / ml dispersion. The pigment retention rate is higher at 45℃ for 2 h than at 95℃. The higher pigment retention rate at 45℃ is because the composite micelle system remains intact, providing good protection; while at 95℃, the high temperature destroys the micelle structure and directly leads to pigment molecule degradation. The pigment retention rate of the red yeast rice-β-cyclodextrin enzymatically hydrolyzed composite micelles is ≥80% after heating in a 95℃ water bath for 2 h. Figure 5 As shown, the pigment retention rate improvement rate of 1 mg / ml can reach up to 23% at 95℃.

[0052] Experimental Example 3: Particle Size Determination of Red Yeast Rice Dispersion The average particle size of the dispersions prepared before and after spray drying in Examples 1, 2, 1, 2, 5, and 6 was determined using a Malvern nanoparticle size analyzer (ZS90, Malvern, UK).

[0053] Figure 6It can be seen that the 1 mg / ml red yeast rice-β-cyclodextrin enzymatic hydrolysis composite micelle dispersion has the smallest particle size, with an average particle size of 125 nm. The red yeast rice solution has a larger particle size, with an average particle size greater than 250 nm. The "particle size reduction" of the composite micelles is relative to the aggregated state of the red yeast rice solution, not its single-molecule state. Essentially, the composite material, through encapsulation and nanoengineering, blocks the pigment aggregation pathway, forming smaller and more stable nanoparticles.

[0054] Experimental Example 4: Powder Flowability and Reconstitution Test The storage properties and flowability of curcumin were evaluated by measuring bulk density, bulk density, Karl Fischer index, and Hausner ratio. Each sample powder (mg) was placed into a 10 mL graduated cylinder, and gently tapped to remove powder adhering to the cylinder wall until the graduation mark was reached. The volume was read directly from the graduated cylinder. V B Then, the bulk density is calculated using formula (1-1). For the tapped density ( p T The cylinder was manually tapped approximately 50 times from a height of 10 cm onto the surface of the red yeast rice powder granules until the powder volume no longer changed, and the final volume of the powder was measured. V T The Cartesian exponent can be obtained using formula (1-2). C ) and Hausnerby ( HR The result can be calculated using formulas (1-3) and (1-4).

[0055] (1-1); (1-2); (1-3); (1-4); As shown in Table 1, the significant difference in the Hausner ratio (approximately 3%) between Example 5 (spray-dried complex powder (1.18%)) and Comparative Example 9 (red yeast rice powder before spray drying (1.42%)) indicates that the red yeast rice-β-cyclodextrin enzymatic hydrolysis complex has better flowability, and Example 5 (spray-dried complex powder) is looser, less prone to clumping, and flows easily like fine sand. Figure 7 As shown, the red yeast rice-β-cyclodextrin enzymatic hydrolysis complex has good resolubility, and there is no phenomenon of insolubility after resolubilization (agglomeration, wall adhesion, precipitation, floating, etc.). However, Comparative Example 9 (red yeast rice powder before spray drying) has strong hygroscopicity and requires long-term stirring to dissolve in water.

[0056] Table 1 shows the parameters for evaluating the flowability of pigment powder.

[0057]

[0058] Experimental Example 5: Transmittance Analysis of Red Yeast Rice Dispersion The absorbance of the red yeast rice dispersion was measured at 680 nm using a UV-Vis spectrophotometer with deionized water as a reference. Transmittance was calculated using the following formula:

[0059] in T For light transmittance, A Absorbance at a wavelength of 680 nm Figure 8 The transmittance of red yeast rice red and red yeast rice red-β-cyclodextrin enzymatic hydrolysis complex solutions before and after drying was shown, and the transmittance was found to be greater than 95% in both cases. Figure 9 The results show that the initial solution of red yeast rice and the solution of red yeast rice-β-cyclodextrin enzymatic hydrolysis complex before and after drying have high transmittance under strong light: light can easily and brightly penetrate the solution, and the light spot appears dazzling and bright with a pure color (bright red), indicating that the nanoscale stabilizing carrier of red yeast rice pigment has no significant effect on the transmittance of the solution.

[0060] Experimental Example 6: Electronic tongue analysis and sensory evaluation of red yeast rice dispersion The SA402B Plus-EX electronic tongue was used to detect umami, bitterness, sourness, richness, astringency, aftertaste-A (astringent aftertaste), and aftertaste-B (bitter aftertaste) in a red yeast rice pigment dispersion. When using the SA402B Plus-EX electronic tongue, the sensor must first be calibrated with a standard solution. Then, the red yeast rice pigment dispersion and ultrapure water are placed in the autosampler. The instrument automatically performs a cycle of cleaning, measuring the reference solution, re-cleaning, measuring the sample, and deep cleaning according to a preset sequence. Finally, the pigment sample dispersion is subjected to sensory evaluation, and its score is calculated.

[0061] from Figure 10 It can be seen that the red yeast rice-β-cyclodextrin enzymatic hydrolysis complex micelles (Example 5) can significantly reduce bitterness. Sensory evaluation showed that when the bitterness value of the red yeast rice solution was 7, there was no obvious bitterness (Table 2). This is achieved through the triple synergistic effect of β-cyclodextrin molecules encapsulating bitter substances, enzymatic hydrolysis peptides inhibiting bitterness perception, and the physical barrier of micelles, resulting in highly efficient masking of bitterness. Furthermore, the bitterness masking effect was better after spray drying than before spray drying, possibly because the maltodextrin added during spray drying also has a certain bitterness masking effect.

[0062] Table 2 shows the sensory evaluation of the pigment sample dispersions.

[0063] Experimental Example 7: Analysis of Red Yeast Rice Dispersion with Electronic Nose The AIRSENSE-PEN 3 electronic nose and electronic tongue were used to detect volatile odor molecules in pigment sample dispersions. 5 mL of red yeast rice pigment dispersion was placed in a 40 mL headspace vial, sealed, and allowed to stand at room temperature for 5 min before inserting the electronic nose probe to measure volatile aroma substances. Parameter settings: sampling interval 1 s, rinsing time 150 s, zeroing time 5 s, pre-sampling time 5 s, detection time 120 s, carrier gas flow rate 300 mL / min, and injection flow rate 300 mL / min. Three replicates were set for each sample, and the response values ​​under steady-state conditions were selected for analysis.

[0064] from Figure 11 As can be seen, the vectors of the three key driving sensors (PC1): W1S, W1W, and W2W are the longest, and their direction is almost parallel to the PC1 axis (pointing to the right). This means that the higher the response values ​​of these sensors, the more the sample tends to be on the right side of the score map. W1S is sensitive to methane, while W1W and W2W are sensitive to sulfides, organosulfur compounds, and aromatic components. Therefore, the dried sample of Example 5 is likely to have significantly higher levels of sulfur-containing compounds, methane, or certain aromatic substances than other groups, which is related to the peptides in the sodium caseinate hydrolysate added to the red yeast rice-β-cyclodextrin enzymatic hydrolysis complex micelles. The sample of Example 5 is clearly distinguishable from other groups on the PC2 axis, and its response value is relatively low, which intuitively indicates that the escape of these volatile bitter and off-flavor substances is significantly suppressed after encapsulation with the carrier system of this invention. The results are highly consistent with the bitterness masking conclusions of electronic tongue analysis, further confirming from the perspective of volatile flavor substances that the ternary composite carrier system described in this invention achieves efficient and comprehensive masking of various off-flavor components in red yeast rice pigment through inclusion, isolation and masking effects, which is strong evidence of its excellent flavor improvement performance.

Claims

1. A method for preparing a nanoscale stabilized carrier for red yeast rice pigment, characterized in that, This method is based on a hierarchical self-assembly strategy. By constructing a ternary supramolecular complex system of β-cyclodextrin-enzymatically hydrolyzed sodium caseinate-gum arabic, it achieves efficient encapsulation of pigment molecules, bitterness shielding, and synergistic enhancement of stability through a multi-level self-assembly mechanism.

2. A method for preparing a nanoscale stabilized carrier of red yeast rice pigment based on hierarchical self-assembly technology, characterized in that, Includes the following steps: (1) Construct a red yeast rice-β-cyclodextrin inclusion complex to achieve primary stabilization at the molecular level; (2) Prepare sodium caseinate colloidal solution as an enzymatic hydrolysis precursor; (3) Papain-directed enzymatic hydrolysis of sodium caseinate colloid; Achieve specific peptide chain cleavage and functionalization modification; (4) The enzyme reaction is terminated by high-temperature inactivation of the colloid after enzymatic hydrolysis; (5) The pretreated gum arabic after vacuum degassing is introduced into the enzyme-inactivated colloid in a gradient manner and subjected to high-intensity shear homogenization to induce intermolecular interweaving and adsorption, forming a thermodynamically stable composite colloidal matrix. (6) The inclusion compound obtained in step (1) is mixed with the colloid obtained in step (5) and self-assembled to form a composite nanomicelle, namely a nanoscale stabilized carrier of red yeast rice pigment.

3. The preparation method according to claim 2, characterized in that, The composite nanomicelles contain red yeast rice pigment, β-cyclodextrin, sodium caseinate hydrolysate, and gum arabic.

4. The preparation method according to claim 2, characterized in that, The concentrations of each component in each step are as follows: red yeast rice pigment: 1–4 mg / mL; β-cyclodextrin: 2–8 mg / mL; Sodium caseinate: 0.25 mg / mL; Papain: 0.0125 mg / mL; Gum arabic: 0.05 mg / mL.

5. The preparation method according to claim 2, characterized in that, The specific steps of step (1) include: β-cyclodextrin, which has been precisely measured, is completely hydrated and dissolved in ultrapure water at 40–60°C under constant stirring. Then, under continuous constant temperature and stirring conditions, red yeast rice pigment is quantitatively introduced through a precision feeding system. The mixture is continuously and gently stirred for 30 minutes within the above temperature range, so that the host molecule β-cyclodextrin and the guest molecule red yeast rice pigment can complete molecular recognition and inclusion through hydrophobic interaction and size matching effect. Finally, the mixture is cooled to room temperature by a program and stored under light-proof conditions throughout the process to form a stable inclusion complex initial dispersion system.

6. The preparation method according to claim 2, characterized in that, The specific steps of step (2) include: dispersing sodium caseinate in ultrapure water and stirring for 1 hour to form a homogeneous solution.

7. The preparation method according to claim 2, characterized in that, The specific steps of step (3) include: using a pH meter to precisely stabilize the pH value of the reaction system in the optimal enzyme activity range of 6.5–7.0, and then in a constant temperature water bath oscillation system equipped with a gradient temperature program, a directional enzymatic hydrolysis process is carried out for 20–40 minutes under a strictly controlled temperature environment of 50±1℃ to achieve specific cleavage and functional modification of sodium caseinate molecules.

8. The preparation method according to claim 2, characterized in that, The specific steps of step (4) include: placing the enzymatically hydrolyzed colloid in a water bath at 90±2℃ for 10 minutes to inactivate the enzyme, and then rapidly cooling it to below 40℃.

9. The preparation method according to claim 2, characterized in that, The specific steps of step (5) include: adding the pretreated gum arabic by low-speed stirring and vacuum degassing in a gradient to the enzyme-inactivated and cooled colloidal solution, and then gently stirring and assembling at 35±1℃ for 20 minutes to promote molecular-level interweaving and adsorption between gum arabic and enzymatically hydrolyzed colloid, and finally obtaining a composite colloidal dispersion system with uniform structure and thermodynamic stability.

10. The application of the red yeast rice pigment nanoscale stabilized carrier prepared by the preparation method according to any one of claims 2-9 in the fields of functional foods, clarified beverages, and pharmaceutical preparations.

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