Anthocyanin monomer-plant oil body compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of functional plant-based beverages, in particular to an anthocyanin monomer-plant oil body compound as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing peanuts with water, pulping, centrifuging, adjusting the pH value to 9.5, taking an upper-layer enriched product, and repeatedly centrifuging for three times to obtain a peanut oil body. And diluting the pH value of the peanut oil body to 3 by using 1M HCl to prepare the peanut oil body emulsion. Dissolving anthocyanin in water, mixing with the peanut oil body emulsion in proportion, homogenizing, and centrifuging to obtain an upper-layer enriched product, namely the anthocyanin-peanut oil body compound. The obtained compound is mainly used for preparing a raw material matrix of a plant-based beverage. Spectroscopy and molecular docking find that the compound is relatively high in combination degree and relatively good in stability, and shows excellent oxidation resistance and color generation performance.

Description

Technical Field

[0001] The present invention relates to the technical field of functional plant-based beverages, and particularly relates to an anthocyanin monomer-plant oil body complex, a preparation method thereof, and an application thereof. Background Art

[0002] With the upgrading of the global health consumption concept, the market demand for plant-based beverages with both nutritional functionality and visual attractiveness has been continuously increasing. However, there are two major technical bottlenecks in the existing technologies: First, the lack of functional ingredients and the abuse of artificial synthetic pigments are significant. According to statistics, about 78% of commercially available products rely on synthetic pigments for color adjustment, and their potential allergenicity and metabolic toxicity have attracted widespread attention. Second, the stability of the natural pigment application system has defects, especially the easy degradation characteristics of water-soluble anthocyanin substances have severely restricted their industrial applications.

[0003] As a typical representative of natural pigments and functional ingredients, cyanidin-3-O-glucoside (C3G) has six biological activities such as antioxidant and hypoglycemic effects. However, during processing and storage, due to its sensitivity to light and heat, the fading rate is as high as 60-80%, and the loss rate of biological activity exceeds 40%.

[0004] The existing methods have three significant defects: relying on a single type of interaction, the composite system is easily dissociated in a complex food matrix; most of the existing carriers use modified proteins, and there is a risk of chemical reagent residues; the composite process requires multiple steps of purification, and the industrial production cost is high.

[0005] In the application of natural emulsion systems, the existing technologies mainly focus on the combination of fat-soluble pigments (such as β-carotene) and oil bodies. The curcumin-oil body composite technology based on phase transition temperature regulation, but this method requires an accurate gradient heating program (±0.5 °C control accuracy), and can only present a single orange color system.

[0006] Currently, there is no technical report on the interfacial binding of water-soluble anthocyanins and plant oil bodies (POB), especially this special system with a natural interfacial protein layer of peanut oil bodies. The unique amino acid composition of the outer membrane protein of peanut oil bodies (containing 22.6% hydrophobic amino acids and 14.3% charged amino acids) can theoretically form multiple binding sites, but the existing research has not yet clarified its binding mechanism and synergistic effect law with C3G.

[0007] In summary, the existing technologies have the following key problems that need to be solved urgently: First, there is a lack of a technology for the directional compounding of water-soluble natural pigments and plant oil bodies; second, the action mechanism of the existing protein-pigment composite system is single, and it is impossible to achieve color stability and functional synergy; finally, the traditional emulsion coloring technology relies on physical embedding, resulting in a low pigment loading rate (<35%) and a single color. In view of the above technical gaps, the present invention innovatively constructs an anthocyanin - peanut oil body composite system, and realizes multi-mechanism synergistic stability through interface engineering, providing a new technical path for the development of a new generation of intelligent coloring functional beverages. Summary of the Invention

[0008] The present invention aims to provide a preparation method of an anthocyanin monomer - plant oil body complex. Using plant oil bodies as substrates, anthocyanins are combined through non-covalent interactions, enabling the anthocyanin - plant oil body emulsion to maintain stability and its color-forming and biological activity capabilities, thus broadening the technical path of anthocyanins in the new generation of intelligent coloring functional beverages.

[0009] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0010] A preparation method of an anthocyanin - plant oil body complex, the preparation method comprising the following steps:

[0011] S1. Preparation of peanut slurry

[0012] Peanuts are soaked, peeled, washed, mixed with deionized water, ground, filtered to obtain a filtrate, and then the pH of the filtrate is adjusted to 9 - 11 to obtain peanut slurry;

[0013] S2. Preparation of peanut oil body emulsion

[0014] The treated peanut slurry is centrifuged, and the upper enriched material is taken, and dispersed and centrifuged at least three times repeatedly to obtain pure peanut oil bodies; the obtained peanut oil bodies are uniformly dispersed in deionized water to obtain a peanut oil body emulsion with a mass concentration of 1 - 3%;

[0015] S3. Preparation of anthocyanin solution

[0016] Anthocyanins are dissolved in deionized water, and the pH value is adjusted to 3 - 4 with an acidic regulator to obtain an anthocyanin solution;

[0017] S4. Preparation of anthocyanin - plant oil body complex

[0018] The peanut oil body emulsion and the anthocyanin solution are mixed and homogenized to obtain an anthocyanin - plant oil body emulsion, which is centrifuged, and the upper enriched material obtained is the anthocyanin - plant oil body complex; in the obtained anthocyanin - plant oil body emulsion, the anthocyanin content is 2.5 - 10.0 mg / 100 mL.

[0019] Preferably, in step S1, the specific conditions for soaking are: soaking temperature 4°C - 5°C, soaking time 18 - 24h. In the above technical solution, soaking peanuts at 4°C can delay the oxidation of oil and improve the quality of peanut oil bodies.

[0020] In the above technical solution, the post-treatment of the filtrate of the peanut slurry is crucial. Using a pH regulator to adjust the pH of the filtrate of the peanut slurry is further preferably 9 - 10, and most preferably 9.5. When the pH of the peanut slurry is too small, the obtained peanut oil body particles are larger and there are many foreign proteins at the interface. When the pH of the peanut slurry is too large, it will cause the demulsification of high-oleic acid peanut oil bodies, forming liquid oil and unable to maintain the interface structure.

[0021] Preferably, in step S1, the pH regulator used to adjust the pH is alkaline KOH or NaOH.

[0022] Preferably, in step S2, the specific conditions for centrifugation are: centrifugation speed 6000 - 12000 rpm, centrifugation time 10 - 25 min.

[0023] Preferably, in step S3, the anthocyanin monomer is cyanidin-3-O-glucoside. The selected anthocyanin monomer is cyanidin-3-O-glucoside, which is the most widely distributed in nature.

[0024] Preferably, in step S3, the acid regulator is 1M HCl solution, and the pH value is adjusted to 3.

[0025] Preferably, in step S4, in the obtained anthocyanin - peanut oil body emulsion, the anthocyanin content is 5.0 - 10.0 mg / 100 mL, and the optimal value is 7.5 mg / 100 mL.

[0026] Preferably, in step S4, the specific conditions for centrifugation are: centrifugation speed 8000 - 10000 rpm, centrifugation time 15 - 20 min.

[0027] Preferably, in S2, a peanut oil body emulsion with a mass concentration of 2% is prepared.

[0028] An anthocyanin - peanut oil body complex prepared by the preparation method of the present invention.

[0029] The beneficial effects of the present invention are:

[0030] 1. The anthocyanin - peanut oil body complex prepared by the present invention can be directly used as a plant - based beverage matrix, realizing two major characteristics of natural coloration and functionality.

[0031] 2. The preparation method of the anthocyanin - peanut oil body complex of the present invention is simple, and peanut oil bodies can be used as effective stabilizers for sensitive anthocyanins.

[0032] 3. The anthocyanin-plant oil body complex prepared by the present invention is natural in source and has high stability. Through molecular docking analysis, it is found that the two are combined through non-covalent interactions and the binding effect is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The physicochemical properties of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Examples 1-4 are shown. a shows the appearance of POB emulsions with different concentrations of C3G (from left to right: Comparative Example 1, Example 1, Example 2, Example 3, Example 4), b shows the effect of different concentrations of C3G on the ζ potential of peanut oil body emulsions, c shows the chromaticity change (from left to right: Comparative Example 1, Example 1, Example 2, Example 3, Example 4), d shows the antioxidant property (from left to right: Comparative Example 1, Example 1, Example 2, Example 3, Example 4), and e shows the particle size and PDI (from left to right: Comparative Example 1, Example 1, Example 2, Example 3, Example 4);

[0034] Figure 2 The fluorescence (a) and ultraviolet spectrum (b) diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3;

[0035] Figure 3 The infrared (a) and surface-enhanced Raman spectrum (b) diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3;

[0036] Figure 4 The crystal structure diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3;

[0037] Figure 5 The microscopic structure diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3. a shows the microscopic structures of POB, C3G, and POB-C3G, and b shows the morphology of POB-C3G observed under magnification;

[0038] Figure 6 The SDS interface diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3;

[0039] Figure 7 The molecular docking of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3. a shows the prediction of the docking binding site of POB-C3G, b shows the 3D docking binding site, c shows the electrostatic binding, d shows the binding energy, and e shows the visualization diagram of the docking site;

[0040] Figure 8 The binding mechanism diagrams of the anthocyanin-plant oil body complexes prepared in Comparative Example 1 and Example 3; Detailed implementation manners

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0042] It should be noted that the terms used herein are only for describing the specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not constitute a limitation to the present invention. The test samples and test procedures used in the following examples include the following content (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels).

[0044] In the following examples, the peanuts used are the high-oleic acid peanut "Weihua No. 25".

[0045] Comparative Example 1 (POB)

[0046] A preparation method and application of a vegetable oil body complex. The specific steps of the preparation method are as follows:

[0047] S1. Preparation of peanut slurry

[0048] The peanuts are selected to remove mildewed and deteriorated peanuts; soaked in water at 4°C for 20 h; the peanut skins are removed; 8 times the mass of water is added, and the slurry is ground at 18,000 r / min for 3 min. A 1 mol / L KOH solution is added to the peanut slurry to adjust the pH of the filtrate to 9.5 to obtain the peanut slurry;

[0049] S2. Preparation of peanut oil body emulsion

[0050] The treated peanut slurry is centrifuged at 10,000 rpm for 20 min, and the upper white enriched material is taken. The dispersion and centrifugation are repeated three times to obtain the peanut oil bodies; the obtained peanut oil bodies are uniformly dispersed in deionized water to obtain a peanut oil body emulsion with a mass concentration of 2%.

[0051] S3. Preparation of anthocyanin solution

[0052] Dissolve 0 mg of anthocyanin in 50 mL of deionized water, and adjust the pH value to 3 using 1 M HCl solution to obtain a solution without anthocyanin (for control).

[0053] S4. Preparation of anthocyanin - phytosome complex

[0054] Mix the peanut phytosome emulsion with the above - mentioned anthocyanin solution in a ratio of 1:1 (v / v), homogenize to obtain an anthocyanin - phytosome emulsion, and centrifuge it. The upper - layer enrichment is the anthocyanin - phytosome complex, and at this time, the anthocyanin content is 0 mg / 100 mL.

[0055] Example 1 (POB - C3G2.5)

[0056] S1. Preparation of peanut slurry

[0057] Select peanuts, remove moldy and deteriorated peanuts; soak them in water at 4 °C for 20 h; remove the peanut skins; add 8 times the mass of water, grind the slurry at 18000 r / min for 3 min, add 1 mol / L KOH solution to the peanut slurry, and adjust the pH of the filtrate to 9.5 to obtain the peanut slurry.

[0058] S2. Preparation of peanut phytosome emulsion

[0059] Centrifuge the treated peanut slurry at 10000 rpm for 20 min, take the upper - layer white enrichment, and repeat the dispersion and centrifugation three times to obtain peanut phytosomes; uniformly disperse the obtained peanut phytosomes into deionized water to obtain a 2% peanut phytosome emulsion.

[0060] S3. Preparation of anthocyanin solution

[0061] Dissolve 2.5 mg of anthocyanin in 50 mL of deionized water, and adjust the pH value to 3 using 1 M HCl solution to obtain the anthocyanin solution.

[0062] S4. Preparation of anthocyanin - phytosome complex

[0063] Mix the peanut phytosome emulsion of S2 with the anthocyanin solution of S3 in a ratio of 1:1 (v / v), homogenize to obtain an anthocyanin - phytosome emulsion, and centrifuge it. The upper - layer enrichment is the anthocyanin - phytosome complex, and at this time, the anthocyanin content is 2.5 mg / 100 mL.

[0064] Example 2 (POB - C3G5.0)

[0065] A preparation method and application of an anthocyanin - phytosome complex. The specific steps of the preparation method are as follows:

[0066] S1. Preparation of peanut slurry

[0067] The peanuts are selected to remove the moldy and deteriorated peanuts; soaked in water at 4°C for 20 h; the peanut skins are removed; 8 times the mass of water is added, and the mixture is ground at 18,000 r / min for 3 min. Then, 1 mol / L KOH solution is added to the peanut slurry to adjust the pH of the filtrate to 9.5, obtaining the peanut slurry.

[0068] S2. Preparation of peanut oil body emulsion

[0069] The processed peanut slurry is centrifuged at 10,000 rpm for 20 min, and the upper white enrichment is taken. The dispersion and centrifugation are repeated three times to obtain peanut oil bodies. The obtained peanut oil bodies are uniformly dispersed in deionized water to obtain a peanut oil body emulsion with a concentration of 2%.

[0070] S3. Preparation of anthocyanin solution

[0071] 5.0 mg of anthocyanin is dissolved in 50 mL of deionized water, and the pH value is adjusted to 3 using 1 M HCl solution to obtain the anthocyanin solution.

[0072] S4. Preparation of anthocyanin - peanut oil body complex

[0073] The peanut oil body emulsion and the above - mentioned anthocyanin solution are mixed in a ratio of 1:1 (v / v), homogenized to obtain an anthocyanin - peanut oil body emulsion. After centrifugation, the upper enrichment is the anthocyanin - peanut oil body complex, and the anthocyanin content is 5.0 mg / 100 mL at this time.

[0074] Example 3 (POB - C3G7.5)

[0075] A preparation method and application of an anthocyanin - peanut oil body complex. The specific steps of the preparation method are as follows:

[0076] S1. Preparation of peanut slurry

[0077] The peanuts are selected to remove the moldy and deteriorated peanuts; soaked in water at 4°C for 20 h; the peanut skins are removed; 8 times the mass of water is added, and the mixture is ground at 18,000 r / min for 3 min. Then, 1 mol / L KOH solution is added to the peanut slurry to adjust the pH of the filtrate to 9.5, obtaining the peanut slurry.

[0078] S2. Preparation of peanut oil body emulsion

[0079] The processed peanut slurry is centrifuged at 10,000 rpm for 20 min, and the upper white enrichment is taken. The dispersion and centrifugation are repeated three times to obtain peanut oil bodies. The obtained peanut oil bodies are uniformly dispersed in deionized water to obtain a peanut oil body emulsion with a concentration of 2%.

[0080] S3. Preparation of anthocyanin solution

[0081] Dissolve 7.5 mg of anthocyanin in 50 mL of deionized water, and adjust the pH value to 3 using 1 M HCl solution to obtain an anthocyanin solution.

[0082] S4. Preparation of anthocyanin - plant oil body complex

[0083] Mix the peanut oil body emulsion with the above - mentioned anthocyanin solution in a ratio of 1:1 (v / v), homogenize to obtain an anthocyanin - plant oil body emulsion, and centrifuge it. The upper enrichment is the anthocyanin - plant oil body complex, and the anthocyanin content is 7.5 mg / 100 mL at this time.

[0084] Example 4 (POB - C3G10.0)

[0085] S1. Preparation of peanut slurry

[0086] Select peanuts, remove moldy and deteriorated peanuts; soak them in water at 4 °C for 20 h; remove the peanut red skin; add 8 times the mass of water, grind the slurry at 18000 r / min for 3 min, add 1 mol / L KOH solution to the peanut slurry, and adjust the pH of the filtrate to 9.5 to obtain the peanut slurry.

[0087] S2. Preparation of peanut oil body emulsion

[0088] Centrifuge the treated peanut slurry at 10000 rpm for 20 min, take the upper white enrichment, and repeat the dispersion - centrifugation three times to obtain the peanut oil body; uniformly disperse the obtained peanut oil body into deionized water to obtain a 2% peanut oil body emulsion.

[0089] S3. Preparation of anthocyanin solution

[0090] Dissolve 10.0 mg of anthocyanin in 50 mL of deionized water, and adjust the pH value to 3 using 1 M HCl solution to obtain an anthocyanin solution.

[0091] S4. Preparation of anthocyanin - plant oil body complex

[0092] Mix the peanut oil body emulsion with the above - mentioned anthocyanin solution in a ratio of 1:1 (v / v), homogenize to obtain an anthocyanin - plant oil body emulsion, and centrifuge it. The upper enrichment is the anthocyanin - plant oil body complex, and the anthocyanin content is 10.0 mg / 100 mL at this time.

[0093] Analyze the physicochemical properties, binding state, morphological characteristics, and molecular structure of the anthocyanin - plant oil body complexes prepared in Examples 1 - 4 and Comparative Example 1. The specific test methods are as follows:

[0094] 1. Analysis of physicochemical properties:

[0095] The color characteristics of the samples were evaluated using a precision colorimeter (Threenh, NR10QC, China). CIELab parameters were collected: L* (brightness), a* (red - green), and b* (yellow - blue).

[0096] The size parameters of POB and POB - C3G solutions (3 mL) were evaluated using a Zetasizer from Malvern Instruments (UK). The average particle size obtained from the particle size distribution was reported in nanometers (nm). Additionally, the ζ - potential of each sample (200 μL per sample) was measured using the same Zetasizer at a controlled temperature of 25 ± 1 °C.

[0097] According to the protocol provided by the kit, the free - radical scavenging activity of the samples was evaluated using a DPPH free - radical scavenging ability assay kit (Nanjing Jiancheng Bioengineering Institute, China). After mixing the samples with the working solution, the mixture was incubated in the dark at 25 °C for 30 minutes, followed by centrifugation at 4000 rpm for 5 minutes. A Trolox solution was used as a standard. The free - radical scavenging ability was quantified at 517 nm using a UV - Vis spectrophotometer (U2800, Hitachi, Japan). The calculation is as shown in the equation, where A1 represents the absorbance of the sample and A0 represents the absorbance of the blank:

[0098]

[0099] The ability to scavenge ABTS free radicals was evaluated using a previously established method. A mixture containing 7 mM ABTS and 2.5 mM potassium persulfate was prepared at 25 °C for 16 hours. After that, the treated solution was diluted with ultrapure water and the absorbance was recorded at a wavelength of 734 nm. Next, 3 mL of the treated solution was mixed with 0.4 mL of the sample solution and incubated in the dark at room temperature for 30 minutes. Then the absorbance rate of the sample was measured at 734 nm using an ultraviolet spectrophotometer (UV - 1000, produced by Co., Ltd). The specific calculation is as the equation, where A1 represents the absorbance of the sample and A0 represents the absorbance of the blank:

[0100]

[0101] Shown by Figure 1 a shows the appearance of POB emulsions with different concentrations of C3G. Obviously, as the C3G content increases, the color of the POB - C3G composite emulsion becomes darker. As Figure 1 shown in c, the L* value gradually decreases, while the a* value gradually increases, and the b* value remains relatively stable. At a pH value of 3.0, C3G mainly appears red. As the C3G concentration increases, the number of color - forming factors in the emulsion increases, resulting in a decrease in the brightness value and an increase in red color. As Figure 1As shown in Figure e, the addition of C3G to POB results in an increase in the average particle size of the emulsion. Specifically, the average particle size of POB increases from 1064.14 ± 119.75 nm to 1540.05 ± 57.40 nm for POB-C3G10.0. This increase can be attributed to the molecular interaction between POB and C3G, which leads to the fusion of some droplets, thus producing larger droplet sizes. In addition, PDI is a parameter that quantifies the uniformity of the particle size distribution. A smaller PDI value indicates a more uniform particle size distribution, while a larger PDI value indicates a greater degree of non-uniformity in the particle size distribution. Therefore, PDI is a key indicator for evaluating the uniformity of the particle size distribution. As Figure 1 shown in Figure e, after the addition of C3G, the PDI of POB-C3G remains below 0.3, indicating a relatively uniform particle size distribution on the surface. It is worth noting that POB-C3G7.5 exhibits the smallest PDI, indicating its highest stability.

[0102] Zeta potential is used to evaluate the potential stability of a colloidal system because it reflects the degree of interaction between colloidal particles. The positive charge of the sample can be attributed to the presence of C3G. Figure 1 As shown in Figure b, C3G significantly affects the change in ζ potential within the system. POB is negatively charged during extraction. However, to ensure the consistency of the entire experiment, the ζ potential of POB was measured at a pH value of 3. As the amount of C3G added increases, the ζ potential of the sample continuously rises, thereby enhancing the stability of the system

[0103] C3G scavenges free radicals by reacting with the hydroxyl groups on the ring, thereby generating stable semiquinone free radicals. Using DPPH and ABTS + The study of antioxidant properties by free radical scavenging assays shows that Figure 1 POB in Figure d exhibits certain antioxidant properties, which may be due to the presence of antioxidant compounds such as tocopherols and phytosterols in its composition. However, compared with POB, the free radical scavenging ability of the POB-C3G complex is enhanced. As the concentration of C3G increases, the DPPH and ABTS + free radical scavenging rates of POB-C3G gradually increase, showing excellent antioxidant activity. In summary, POB-C3G7.5 was selected as the sample in this paper for subsequent studies on the binding mechanism between POB and C3G.

[0104] 2. Fluorescence and UV spectroscopy test methods:

[0105] Using the method described above, the fluorescence spectrum of the complex was obtained using a fluorescence spectrophotometer (Cary Eclipse, Agilent, USA). The excitation wavelength was set at 280 nm, while the emission wavelength range was between 250 - 450 nm.

[0106] Each sample was equilibrated using 3 mL for 10 minutes and then scanned with a UV-visible spectrophotometer (UV-2550, Shimadzu, Japan) in the wavelength range of 250 to 400 nm, where the slit width was configured to 0.5 nm to capture spectral changes.

[0107] Fluorescence spectroscopy is often used to study the interaction mechanism between proteins and small molecules. Figure 2 a shows the trend observed in the fluorescence spectrum of the complex. In the fluorescence spectrum range of 340 to 350 nm, the fluorescence intensity associated with the interaction between POB and C3G is lower than that of POB alone, indicating that the interaction with C3G reduces the intrinsic fluorescence of POB. As the concentration of C3G increases, the fluorescence intensity of POB continues to decrease, and this phenomenon is usually attributed to the quenching of aromatic amino acids in proteins by other substances. This phenomenon is called endogenous fluorescence quenching, indicating a specific interaction between C3G and POB. This observation suggests that the microenvironment of tryptophan residues becomes more polar and less hydrophobic, which may be due to the structural change of POB after the interaction.

[0108] UV-visible absorption spectroscopy is often used to analyze the interaction between small molecule substances and biological macromolecules. By comparing the similarities and differences in the UV-visible absorption spectra of POB with different concentrations of C3G, the interaction between C3G and POB can be preliminarily evaluated. As Figure 2 shown in b, the UV-visible spectral curve of POB is smooth, and its absorption value increases with the increase in the concentration of C3G. Notably, the maximum absorption peak of POB and C3G significantly redshifts from 265 nm to 275 nm, indicating that the interaction with C3G changes the spatial structure of the oil body. This structural change is similar to that observed when walnut protein isolate interacts with C3G, where the peak uptake gradually increases with the increase in the concentration of C3G, indicating the interaction between C3G and the protein. These findings suggest that the interaction between C3G and POB may lead to the extension of the peptide chain and the generation of new conformations. In addition, UV-visible spectroscopy can clarify the change in the hydrophobicity of the POB-C3G complex. The peak in the range of 270 - 280 nm indicates the presence of tryptophan, phenylalanine, and tyrosine residues in POB. As the content of C3G increases, the peak at approximately 275 nm becomes more and more obvious, resulting in a "color enhancement effect", which means that the microenvironment of hydrophobic amino acid residues inside the POB side chain has changed.

[0109] 3. Infrared spectroscopy test method:

[0110] The solution sample was freeze-dried and then stored at -20 °C. Then, using an agate mortar and pestle, it was uniformly mixed and finely pulverized in combination with spectroscopic-grade potassium bromide at a ratio of 1:100. After drying and pressing the obtained sample, an FTIR spectrum was obtained using an FTIR spectrometer (Nicolet 5700, Thermo, USA). The wavelength range was adjusted from 4000 to 400 cm -1 , with a resolution of 4 cm -1 , and a total of 32 scans were performed.

[0111] The structural differences between the POB-C3G complex and its monomers can be characterized by FTIR, and the formation of new chemical bonds can be evaluated by analyzing different peaks. Compared with C3G, new peaks were observed in POB and POB-C3G at 1700 - 1760 cm -1 and 2700 - 3000 cm -1 . These peaks may be due to the presence of grease in POB, which enhances the stretching vibration of CH. In addition, the resonance of C=O and C=C contributes to the appearance of the characteristic peaks related to ketones. The formation of hydrogen bonds usually causes the stretching vibration absorption peak to shift to a lower wavenumber (red shift). This phenomenon occurs because the establishment of hydrogen bonds reduces the bond force constant between the hydrogen atom and the atom it is connected to, resulting in a decrease in the vibration frequency. As Figure 3 shown in a, the broad peak observed in the range of 3200 - 3600 cm -1 corresponds to hydrogen bonds. It is worth noting that compared with POB and C3G, the peak maximum of POB-C3G has a red shift, indicating the generation of new hydrogen bonds. The peaks observed at 1384 and 1359 cm -1 indicate the persistence of the hydrocarbon structure. Compared with POB alone, the glycosidic bond peak of C3G-POB is significantly enhanced after complexation.

[0112] 4. Surface-enhanced Raman spectroscopy test method:

[0113] The Raman spectrum of the sample was measured in situ using a confocal Raman microscope (Via Qontor from Renishaw, UK). The scanning method involves directly focusing the light onto the sample. To capture the white-light microscope image of the sample, a 50x objective lens was used. The Raman spectrum in the range of 400 to 4000 cm -1 was collected using a laser with a power of 100 mW, a scanning wavelength of 532 nm, and a collection duration of 20 s.

[0114] Surface-enhanced Raman spectroscopy studies were carried out to analyze the structural characteristics of POB-C3G and to study the interaction between POB and C3G, which helps in the non-destructive detection of these complexes. Figure 3b shows the surface-enhanced Raman spectra of POB, C3G, and the POB-C3G complex, highlighting the significant differences in peak height. 543 cm -1 、631 cm -1 and 731 cm -1 The peaks at correspond to the in-plane bending vibration of the C-C single bond (δ(CC)). The peak at 879 cm -1 is related to the out-of-plane bending vibration of the C-H bond (γ(CH)). The chemical structures represented by these peaks exhibit relative stability. The peak at 1139 cm -1 is related to the bending vibration of the hydroxyl group (δ(OH)), while the peaks at 1204 cm -1 and 1240 cm -1 are related to the stretching vibration of the C-OH bond (ν(CO)). The peak at 1331 cm -1 corresponds to the stretching vibration of the inter-ring bond (ν(CC), i) and the in-plane bending vibration of the ring (δ(CH)) in the anthocyanin carbon structure. As shown in Figure 3 d, the peaks at 1471 cm -1 and 1597 cm -1 are attributed to the C-C stretching vibration of the benzene ring. The peak at 1487 cm -1 corresponds to the bending vibration of CH2, representing the strongest peak observed in the Raman spectrum. After adding C3G, the peak intensity of POB-C3G decreases, which may be due to the change in molecular structure. These changes may include inductive effect, conjugation effect, steric effect, and hydrogen bond interaction.

[0115] 5. Crystal structure testing method:

[0116] XRD analysis was performed using a Smart Lab instrument from Rigaku, Japan, to evaluate the long-range order of the freeze-dried samples. The instrument operates at 30 mA and 40 kV, using Cu Kα radiation as the X-ray source. The diffraction angle (2θ) was scanned at a rate of 2° / min in the range of 5° to 80°.

[0117] To further emphasize the change in the physical state after mixing C3G and POB, the XRD patterns of POB, C3G, and BOB-C3G were analyzed. It is worth noting that the diffraction pattern of POB-C3G shows significant differences compared to POB and C3G. As shown in Figure 4As shown, the crystal structure of POB shows multiple characteristic peaks, while the C3G monomer shows a typical amorphous pattern, lacking an obvious crystal structure. In the POB-C3G complex, the combined interactions lead to changes in the intensity and position of the characteristic peaks. These XRD results confirm the onset of the amorphous phase after mixing C3G and POB, indicating an interaction between the two components. Specifically, during the binding of POB to C3G, the highest peak at 2θ shifts from 19.55° to 19.72°, indicating that the interaction is non-covalent and mediated by electrostatic forces and other factors. Although the combination on the left and right is successful, the peak area decreases, indicating that the addition of C3G disrupts the original interactions at the POB interface and promotes spatial reconstruction. In addition, the formation of hydrogen bonds between POB and C3G enhances the crystallinity of the POB-C3G complex and reduces the amorphous state of C3G. This finding is highly consistent with the results of Fourier transform infrared spectroscopy analysis.

[0118] 6. Microstructure and interfacial composition analysis:

[0119] Transfer 20 μL of the sample onto a glass microscope slide and place a coverslip on it. Perform morphological observations using an optical microscope (BX22F-3M830F, AOSVI, China) at magnifications of 500 times and 1000 times and at room temperature (25 °C).

[0120] Mix the loading buffer with POB-C3G and then heat it to 100 °C for 5 minutes to denature the protein. Subsequently, centrifuge the mixture at 8000×g for 15 minutes to separate the protein sample. Prepare a 5% stacking gel and a 15% separating gel, and load 10 μL of each sample to initiate electrophoresis. Apply an initial voltage of 80 mV in the stacking gel, which is later increased to 120 mV as the protein transitions to the separating gel. Subsequently, stain the gel with Coomassie Brilliant Blue R-250 for 2 hours and then decolorize it with glacial acetic acid until the protein bands are clearly visible. Visualize the protein bands using a gel imager and analyze them using Image Lab software (Bio-Rad, USA).

[0121] Use a laser confocal microscope to directly observe the microscopic changes in the sample. Since C3G itself is a chromogenic substance, no staining is required. As Figure 5As shown, the POB is spherical and evenly distributed. After adding different proportions of C3G, the composite material maintains its spherical appearance; however, the particle size slightly increases, which may be due to the formation of the POB-C3G composite material. The interaction between particles may lead to the fusion of droplets. In addition, when magnifying and examining the POB-C3G composite material, it is obvious that the surface of the POB is decorated with many granular small molecule substances. Therefore, adding C3G will cause obvious changes in the apparent morphology of the composite. SDS-PAGE was used to show the interfacial proteins of POB-C3G. As Figure 6 shown, the analysis shows that the protein bands are mainly composed of endogenous proteins. By using ImageJ to evaluate the light intensity distribution, the SDS-PAGE results of POB-C3G were further examined, and the results show that oleosin accounts for 90.21% of the protein bands. According to the vegetable oil body model, oleosin is the main endogenous protein on the oil body interface and adopts a hairpin structure to effectively encapsulate other components. Therefore, oleosin was selected as the receptor for docking simulation with small molecule C3G molecules.

[0122] 7. Molecular docking test method and binding mechanism analysis:

[0123] Use ChemDraw to create the molecular structure of cyanide-3-O-glucoside. Subsequently, open the ChemDraw 3D software and import the small molecule to generate its three-dimensional structure. The three-dimensional structure of oleosin can be obtained from the Protein Data Bank (PDB). Use the PyMOL software to eliminate water molecules and receptor proteins. Finally, the molecular docking of the target and active ingredient structures will be performed using Vina in the PyRx software.

[0124] Molecular docking is used to predict molecular conformations and interactions. This technique allows the calculation of the interaction energy between molecules, where a higher negative binding energy indicates a tighter and more stable ligand-receptor interaction. To further verify the interaction between C3G and POB, we visualized all the spatial conformations. The results showed that Oleosin could form a suitable conformation with C3G. Therefore, we selected C3G to dock with Oleosin to determine the optimal binding site. The molecular docking simulation experiment showed that C3G bound to the active site of Oleosin, obtaining the lowest binding energy arrangement and the best 3D molecular docking structure. The lowest binding energy of Oleosin protein with C3G was -5.1 kcal / mol. The binding pocket of Oleosin protein and the small molecule receptor (C3G) showed a high degree of compatibility, both having good binding activity and being able to bind under natural conditions. In addition, C3G formed a hydrogen bond with the amino acid residue ARG-148 in the protein. At the same time, the protein made non-bonded contacts with the small molecule, forming forces represented by electrostatic potential energy and van der Waals forces. In summary, C3G has the potential to form more interactions with oleosin, and these forces can enable the two to form a tight and stable complex.

[0125] C3G is a natural anthocyanin compound characterized by an aromatic anthocyanin core linked to glucose molecules by glycosidic bonds, which imparts polarity to the compound and has significant antioxidant activity and a positive charge when dissolved in water. When POB is dispersed in water, its interface is mainly composed of negatively charged amino acid residues, providing the possibility of interaction with C3G. The results showed that at pH 3.0, negatively charged POB and positively charged C3G could interact through electrostatic interactions. In addition, Oleosin, the main component of the POB interfacial layer, has active groups in its amino acid residues and can form hydrogen bonds with the hydroxyl groups of the C3G ring structure. This interaction indicates that POB enhances the binding of anthocyanins under acidic conditions, thereby stabilizing the color and maintaining the stability of glycosides within the system. In addition, the POB-C3G system is affected by van der Waals forces and hydrophobic interactions; van der Waals forces promote intermolecular attraction and facilitate molecular aggregation. However, steric hindrance and hydrophobic interactions on the POB interface prevent these weak forces from causing excessive fusion and flocculation of droplets, thus maintaining the stability of the system.

[0126] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and reference can be made to the method part for the relevant parts.

[0127] The above has introduced in detail a peanut oil body provided by the present invention and its preparation method. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing an anthocyanin-plant oil body complex, characterized in that: The preparation method includes the following steps: S1. Preparation of peanut slurry The peanuts are soaked, peeled, washed, mixed with deionized water, ground, filtered to obtain a filtrate, and then the pH of the filtrate is adjusted to 9-11 to obtain peanut slurry. S2. Preparation of peanut oil body emulsion The treated peanut slurry is centrifuged, and the upper enriched material is taken and dispersed and centrifuged at least three times to obtain pure peanut oil bodies. The obtained peanut oil bodies are evenly dispersed in deionized water to obtain a peanut oil body emulsion with a mass concentration of 1-3%. S3. Preparation of anthocyanin solution The anthocyanin is dissolved in deionized water, and the pH value is adjusted to 3-4 with an acidic regulator to obtain an anthocyanin solution. S4. Preparation of anthocyanin-plant oil body complex The peanut oil body emulsion is mixed with the anthocyanin solution, homogenized to obtain an anthocyanin-plant oil body emulsion, and then centrifuged. The upper enriched material obtained is the anthocyanin-plant oil body complex. In the obtained anthocyanin-plant oil body emulsion, the anthocyanin content is 2.5-10.0 mg / 100 mL.

2. The preparation method according to claim 1, wherein: In step S1, the specific conditions for soaking are: soaking temperature 4°C - 5°C, soaking time 18-24 h.

3. The preparation method according to claim 1, characterized in that: In step S1, the pH regulator used to adjust the pH is alkaline KOH or NaOH.

4. The preparation method according to claim 1, characterized in that: In step S2, the specific conditions for centrifugation are: centrifugation speed 6000-12000 rpm, centrifugation time 10-25 min.

5. The preparation method according to claim 1, characterized in that: In step S3, the anthocyanin monomer is cyanidin-3-O-glucoside.

6. The preparation method according to claim 1, characterized in that: In step S3, the acidic regulator is 1M HCl solution, and the pH value is adjusted to 3.

7. The preparation method according to claim 1, wherein: In step S4, in the obtained anthocyanin-plant oil body emulsion, the anthocyanin content is 5.0-10.0 mg / 100 mL.

8. The preparation method according to claim 1, wherein: In step S4, the specific conditions for centrifugation are: centrifugation speed 8000-10000 rpm, centrifugation time 15-20 min.

9. An anthocyanin-plant oil body complex prepared by the preparation method as described in claim 1.

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