A starch-anthocyanin complex, and a preparation method and application thereof

Abstract

The application discloses a starch-anthocyanin compound and a preparation method and application thereof. The compound is obtained by blending high amylose starch after partial gelatinization with anthocyanin, then retrogradation, dialysis, drying, crushing and sieving, wherein the amylose content of the high amylose starch is more than 50% of the total mass of the starch; the starch / anthocyanin compound is in a granular form, and the granules are in a state of concave and mutual aggregation or adhesion. The high amylose starch is used to replace common starch, and the high amylose starch is partially gelatinized, so that the granular form of the starch is retained, and the hydrophobicity of the starch granules is enhanced, then the starch is compounded with anthocyanin, and the obtained compound has the stability, hydrophobicity and pH response color changing capacity; and the compound can be directly used for freshness indication of food.

Description

Technical Field

[0001] This invention relates to an anthocyanin complex, its preparation method and application, and particularly to a starch-anthocyanin complex, its preparation method and application. Background Technology

[0002] Anthocyanins are water-soluble natural pigments widely found in plant petals, fruits, and stem and leaf tissues. Anthocyanins possess vibrant colors and various biological activities, including antioxidant, anti-inflammatory, lipid-regulating, and vision-protecting properties, making them promising for applications in food coloring, functional foods, pharmaceuticals, and cosmetics. Furthermore, in the field of smart food packaging, anthocyanins, due to their excellent pH-responsive color-changing ability, can be used to indicate food freshness. However, the structure of natural anthocyanin molecules is unstable, making them susceptible to the effects of light, humidity, high temperature, pH, oxidation, and metal ions in the natural environment, causing fading or discoloration and significantly reducing their application value.

[0003] Currently, various technologies, such as physical compounding, co-coloring, metal ion complexation, encapsulation, and structural modification, are used to improve the stability of anthocyanins. Among these, physical compounding involves mixing anthocyanins with polysaccharides or proteins in a solvent, followed by drying the mixture to obtain a complex. This method primarily utilizes non-covalent interactions such as hydrogen bonds and hydrophobic bonds between anthocyanins and polysaccharides or proteins to stabilize the anthocyanins. Compared to other methods, physical compounding offers advantages such as simple processing and environmental friendliness.

[0004] Starch is a widely available, edible, and renewable natural polysaccharide that can interact with anthocyanins through hydrogen bonding to prepare starch / anthocyanin complexes. Existing research mainly focuses on the development of starch / anthocyanin complexes for functional foods such as resistant starch. Currently, the preparation of starch / anthocyanin complexes often employs a hydrothermal method, the basic steps of which include: first, gelatinizing ordinary starch in water at high temperature; then adding anthocyanins to the starch gelatinized solution for compounding; then cooling the compound solution at room temperature or low temperature; and finally, simply washing or directly drying the cooled compound solution to obtain the starch / anthocyanin complex. The above method has the following main drawbacks: first, using ordinary starch as a raw material, after complete gelatinization and compounding with anthocyanins, the prepared complex is porous and spongy, easily absorbing water and exhibiting unstable properties; second, using water as the compounding solvent for starch and anthocyanins makes it difficult for anthocyanins to maintain stability during the complex preparation process; and third, the complex contains free anthocyanins. In summary, starch-anthocyanin complexes prepared using existing hydrothermal methods are not suitable for use in the field of intelligent food packaging. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide a starch-anthocyanin complex that combines stability, hydrophobicity, and pH-responsive color change capability, and can be used for indicating food freshness; the second objective of this invention is to provide a method for preparing the starch-anthocyanin complex; and the third objective of this invention is to provide applications of the starch-anthocyanin complex.

[0006] Technical solution: The starch-anthocyanin complex of the present invention is obtained by partially gelatinizing high amylose and blending it with anthocyanins, then regenerating, dialyzing, drying, pulverizing and sieving to obtain the starch-anthocyanin complex, wherein the amylose content of the high amylose accounts for more than 50% of the total starch mass; the starch / anthocyanin complex is granular, and the granules are in a state of depression and mutual aggregation or adhesion.

[0007] Preferably, the high amylose is high amylose corn starch, high amylose rice starch, high amylose wheat starch, high amylose potato starch, high amylose barley starch, high amylose pea starch, or high amylose cassava starch.

[0008] Preferably, the high amylose undergoes partial gelatinization at a temperature of 100–130°C and a gelatinization time of 30–60 min. Gelatinization temperature and time significantly affect the morphology of the high amylose. When the gelatinization temperature is 100–130°C and the gelatinization time is 30–60 min, the high amylose undergoes partial gelatinization. The starch granules swell but still maintain their granule shape. Simultaneously, some amylose migrates from the interior of the granules and coats the outside. Anthocyanins, during their complexation with starch, enter the interior of the starch granules, and the amylose located on the outside of the starch granules provides hydrophobic protection for the anthocyanins inside. Further increasing the gelatinization temperature and extending the gelatinization time completely destroys the morphology of the starch granules, rendering them unable to provide hydrophobic protection for the anthocyanins.

[0009] Preferably, the mass ratio of anthocyanins to high amylose is 1:5 to 1:20 (w / w). The mass ratio of anthocyanins to high amylose mainly affects the complexation rate. When the mass ratio of anthocyanins to high amylose is too low, the internal space of the starch granules cannot be fully utilized, and the resulting complex is too light in color, which is not conducive to its pH-responsive color change ability. When the mass ratio of anthocyanins to high amylose is too high, exceeding the internal loading capacity of the starch granules, a large amount of anthocyanins cannot enter the starch granules and will be removed during dialysis, resulting in waste of anthocyanins.

[0010] The preparation method of the starch-anthocyanin complex of the present invention includes the following steps:

[0011] (1) Disperse high amylose in a buffer solution and heat it to obtain a gelatinized liquid in which high amylose is partially gelatinized;

[0012] (2) Cool the high amylose gelatinized liquid obtained in step (1), add anthocyanins to it, mix evenly, and obtain a high amylose / anthocyanin composite solution.

[0013] (3) Cool and regenerate the high amylose-anthocyanin complex solution obtained in step (2) to obtain a regenerated high amylose / anthocyanin complex solution;

[0014] (4) Dialyze the regenerated high amylose / anthocyanin complex solution obtained in step (3) to obtain high amylose-anthocyanin complex dialysate;

[0015] (5) The high amylose-anthocyanin complex dialysate obtained in step (4) is freeze-dried, pulverized, and sieved to obtain the high amylose-anthocyanin complex with food freshness indication function.

[0016] Preferably, in step (1), the buffer solution is a buffer solution with a pH of 2 to 5. The pH of the buffer solution mainly affects the structural stability and color development performance of anthocyanins in the complex. Under acidic conditions of pH 2 to 5, the anthocyanin structure is relatively stable, which is beneficial to maintaining its pH-responsive color change characteristics. If the pH of the buffer solution is too high, the anthocyanins are prone to degradation in a neutral or alkaline environment, resulting in fading or even loss of pH-responsive color change characteristics.

[0017] Preferably, in step (1), the mass ratio of the high amylose to the volume ratio of the buffer solution is 1:50 to 1:100 (w / v).

[0018] Preferably, in step (2), the anthocyanins are purple cabbage anthocyanins, purple sweet potato anthocyanins, blueberry anthocyanins, purple rice anthocyanins, or black goji berry anthocyanins.

[0019] Preferably, in step (2), both cooling and stirring are carried out in a water bath at a temperature of 80–90 °C.

[0020] Preferably, the anthocyanin extraction method is as follows: plant tissue containing anthocyanins is soaked in an acidified ethanol aqueous solution, and then crushed, extracted, and filtered to obtain a crude anthocyanin extract; the crude anthocyanin extract is concentrated by vacuum rotary evaporation, purified by resin, and vacuum dried to obtain anthocyanins. Further preferred embodiments include: the volume ratio of hydrochloric acid, ethanol, and water in the acidified ethanol aqueous solution is (0.1%–0.5%):(60%–80%):(20%–40%) (v / v / v); the mass ratio of plant tissue to the volume ratio of the acidified ethanol aqueous solution is 1:10–3:10 (w / v); the plant tissue is crushed using a juicer; the extraction temperature is 2–8 ºC, and the extraction time is 8–24 h; filtration is performed using 4–8 layers of gauze; the conditions for vacuum rotary evaporation concentration are: vacuum degree ≤98 kPa, temperature 30–50 ºC, and rotary evaporation speed 10–300 rpm; the conditions for resin purification are: loading 10–20 mL of anthocyanin concentrate onto an AB-8 macroporous resin column, eluting with 20%–80% (v / v) ethanol aqueous solution, and collecting the eluent; and the conditions for vacuum drying are: vacuum degree <133 Pa, drying temperature 30–50 ºC, and drying time 24–72 h.

[0021] Preferably, in step (3), the cooling and regeneration involves placing the high amylose-anthocyanin composite solution in a low-temperature environment for regeneration. Preferably, the temperature of the low-temperature environment is 4~25℃, and the placement time is 1~7 days.

[0022] Preferably, in step (4), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000–12000 Da and distilled water. More preferably, the dialysis time is 24–72 h, and the water change interval is 4–8 h.

[0023] Preferably, in step (5), the sieve aperture is 100-200 mesh. Preferably, the pulverization is performed using a grinding mill.

[0024] Preferably, in step (5), the drying is freeze drying, with the conditions being a drying temperature of -45 to -50°C and a drying time of 24 to 48 hours.

[0025] The application of the starch-anthocyanin complex described in this invention in food freshness indicators.

[0026] Invention Mechanism:

[0027] Existing starch-anthocyanin complexes have a porous, sponge-like structure, which easily absorbs water, leading to anthocyanin instability. This invention uses high-amylose starch instead of ordinary starch and partially gelatinizes the high-amylose starch, thus preserving the starch granule morphology and enhancing the hydrophobicity of the starch granules.

[0028] Existing technologies use water as the solvent for the composite of starch and anthocyanins, but anthocyanins are not easily stabilized during preparation. This invention uses a buffer solution with a pH of 2-5 as the solvent, leveraging the fact that anthocyanins are more stable in acidic environments to enhance their stability during the composite process.

[0029] Furthermore, starch-anthocyanin complexes prepared by existing technologies contain free anthocyanins, which can easily interfere with the stability of the complex. This invention employs a mild dialysis process, which can effectively remove free anthocyanins that are not bound to starch particles.

[0030] Its specific principle is as follows Figure 1 As shown: After partial gelatinization of high amylose, starch granules swell but still maintain their granular state. Some amylose inside the starch granules migrates to the outside, enhancing the hydrophobicity of the granules. When anthocyanins are added to the starch gelatinization solution, some anthocyanins enter the swollen starch granules and interact with the amylose and amylopectin within them through hydrogen bonds, while the remaining anthocyanins remain on the outside of the starch granules. When the starch cools and retrogrades, the linear portions of amylose and amylopectin recrystallize to form a double helix structure. Simultaneously, the starch granules undergo dehydration and shrinkage, further strengthening the hydrogen bonding between the anthocyanins inside the starch granules and the amylose and amylopectin. When the complex is dialyzed, the anthocyanins located on the outside of the starch granules can be removed, while the anthocyanins located inside the starch granules do not migrate due to hydrogen bonding, thus causing the complex to exhibit pH-responsive color change.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention uses high amylose to replace ordinary starch and partially gelatinizes the high amylose, which not only retains the granular shape of starch, but also enhances the hydrophobicity of starch granules. Then, it is compounded with anthocyanins, and the resulting complex has stability, hydrophobicity and pH-responsive color change ability; (2) The present invention uses buffer solution instead of water as the composite solvent of high amylose and anthocyanins, which effectively improves the stability of anthocyanins in the preparation process of the complex; (3) The present invention uses dialysis to remove free anthocyanins in the complex, which helps to accurately reveal the interaction between starch and anthocyanins; (4) The high amylose-anthocyanin complex prepared by the present invention retains the pH-responsive color change ability of anthocyanins, and compared with anthocyanin powder, the complex is lighter in color and the color change process is easier to be directly observed by the naked eye, which can be directly used for food freshness indication. Attached Figure Description

[0032] Figure 1 This is a mechanism diagram of the present invention;

[0033] Figure 2A is an appearance diagram of Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5 of the present invention; Figure 2 B represents the anthocyanin content and composite rate of Examples 1, 2, 3, Comparative Example 4, and Comparative Example 5 of this invention;

[0034] Figure 3 The images shown are magnified tenfold from the exterior views of Embodiments 1, 2, 3, 2, 3, 4, and 5 of the present invention.

[0035] Figure 4 These are scanning electron microscope images of Embodiments 1, 2, and 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention;

[0036] Figure 5 These are polarizing microscope images of Embodiments 1, 2, 3, Comparative Example 2, and Comparative Example 3 of the present invention;

[0037] Figure 6 A is the infrared spectrum of Examples 1, 2, 3, Comparative Examples 1, 2, and 3 of this invention; Figure 6 B is the deconvolutioned infrared spectrum of Examples 1, 2, 3, Comparative Example 2, and Comparative Example 3 in this invention; Figure 6 C represents the short-range order of Examples 1, 2, 3, Comparative Example 2, and Comparative Example 3 in this invention;

[0038] Figure 7 A is the X-ray diffraction pattern of Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 in this invention; Figure 7 B represents the small-angle X-ray scattering diagrams of Examples 1, 2, 3, Comparative Example 2, and Comparative Example 3 of this invention;

[0039] Figure 8 This refers to the iodine binding capacity of Examples 1, 2, 3, Comparative Example 2, and Comparative Example 3 in this invention;

[0040] Figure 9 A is a diagram showing the dissolution behavior of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 in water in this invention; Figure 9 B represents the cumulative anthocyanin release rate in water for Examples 1, 2, 3, Comparative Example 4, and Comparative Example 5 of this invention; Figure 9 C represents the DPPH removal capacity of the supernatant from Examples 1, 2, 3, Comparative Example 4, and Comparative Example 5 of this invention.

[0041] Figure 10A is a stability diagram of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 in hot water at 80 °C. Figure 10 B represents the anthocyanin retention rates of Examples 1, 2, 3, Comparative Examples 1, 4, and 5. Figure 10 C represents the first-order kinetic fitting curve of the degradation process in Examples 1, 2, 3, Comparative Examples 1, 4, and 5; Figure 10 D is the TGA diagram of Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 in this invention; Figure 10 E is the DTG diagram of Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 in this invention;

[0042] Figure 11 A is a graph showing the color-changing ability of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 in buffer solutions of pH 3-12 in this invention. Figure 11 B is a graph showing the color-changing ability of Examples 1, 2, 3 and Comparative Example 1 in an ammonia atmosphere. Figure 11 C-11F is a color reversibility diagram of Examples 1, 2, 3 and Comparative Example 1 in the present invention under hydrochloric acid and ammonia atmosphere;

[0043] Figure 12 A is a diagram showing the freshness indication effect of shrimp in Examples 1, 2, 3 and Comparative Example 1 of this invention; Figure 12 B is an enlarged view of the color change diagrams of shrimp during storage in Examples 1, 2, 3 and Comparative Example 1 of this invention;

[0044] Figure 13 The relevant physicochemical indicators for the freshness of shrimp in Examples 1, 2, 3, and Comparative Example 1 of this invention are provided; wherein, Figure 13 A represents the pH change of shrimp during storage; Figure 13 B represents the change in total volatile basic nitrogen (TVB-N) value of shrimp during storage; Figure 13 C represents the change in ΔE value during shrimp storage for Examples 1, 2, 3, and Comparative Example 1 in this invention; Figure 13 D represents the change in G / R value during shrimp storage for Examples 1, 2, 3, and Comparative Example 1 in this invention; Figure 13 E represents the correlation between the ΔE value and the TVB-N level of shrimp in Examples 1, 2, 3 and Comparative Example 1 of this invention; Figure 13 F represents the correlation between the G / R values ​​and the TVB-N levels of shrimp in Examples 1, 2, 3, and Comparative Example 1 of this invention. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the embodiments.

[0046] Example 1

[0047] The starch-anthocyanin complex 1 of the present invention is prepared by the following steps:

[0048] (1) Purple cabbage was soaked in acidified ethanol aqueous solution (the volume ratio of hydrochloric acid, ethanol and water was 0.5%:80%:20% (v / v / v)). The mass ratio of purple cabbage to acidified ethanol aqueous solution was 2:10 (w / v). The solution was crushed by a juicer, extracted at 4 ºC for 24 h, and filtered through 4 layers of gauze to obtain crude anthocyanin extract of purple cabbage. The crude anthocyanin extract of purple cabbage was concentrated by rotary evaporation under reduced pressure (vacuum degree 95 kPa, temperature 45 ºC, rotary evaporation speed 100 rpm), purified by AB-8 macroporous resin (15 mL of anthocyanin concentrate was loaded onto AB-8 macroporous resin column, eluted with 80% (v / v) ethanol aqueous solution and the eluted components were collected), and vacuum dried (vacuum degree 105 Pa, drying temperature 50 ºC, drying time 72 h) to obtain purple cabbage anthocyanins.

[0049] (2) High amylose corn starch (70% amylose content) was dispersed in a phosphate buffer solution at pH 3. The mass ratio of high amylose corn starch to the volume ratio of the buffer solution was 1:50 (w / v). The mixture was heated in an oil bath at 120 °C for 30 min with magnetic stirring (600 rpm) to obtain a high amylose corn starch gelatinized solution.

[0050] (3) Cool the high amylose corn starch gelatinized liquid to 90 °C in a water bath; add purple cabbage anthocyanins to it, with a mass ratio of purple cabbage anthocyanins to high amylose corn starch of 1:20 (w / w); stir and mix to obtain a high amylose corn starch / purple cabbage anthocyanin complex solution.

[0051] (4) The high amylose corn starch / purple cabbage anthocyanin complex solution was placed at 4 ℃ for 2 days to obtain the regenerated high amylose corn starch / purple cabbage anthocyanin complex solution;

[0052] (5) The regenerated high amylose corn starch / purple cabbage anthocyanin complex solution was loaded into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 48 h with a water exchange interval of 4 h to obtain the high amylose corn starch / purple cabbage anthocyanin complex dialysate.

[0053] (6) The high amylose corn starch / purple cabbage anthocyanin complex dialysate was freeze-dried (drying temperature -50 ℃, drying time 48 h), pulverized by a mill, and passed through a 100-mesh sieve to obtain the starch / anthocyanin complex 1, which was named HACS / RCA5.

[0054] Example 2

[0055] The starch-anthocyanin complex 2 of the present invention is prepared by the following steps:

[0056] The mass ratio of purple cabbage anthocyanins to high amylose corn starch in step (3) of Example 1 was adjusted to 1:10 (w / w), while the other preparation conditions remained unchanged. It was named HACS / RCA10.

[0057] Example 3

[0058] The high amylose / anthocyanin complex 3 with food freshness indication function described in this invention is prepared by the following steps:

[0059] The mass ratio of purple cabbage anthocyanins to high amylose corn starch in step (3) of Example 1 was adjusted to 1:5 (w / w), while the other preparation conditions remained unchanged. It was named HACS / RCA20.

[0060] Comparative Example 1

[0061] A purple cabbage anthocyanin, the preparation method includes the following steps;

[0062] The purple cabbage anthocyanin obtained in step (1) of Example 1 is named RCA.

[0063] Comparative Example 2

[0064] A high amylose corn starch (amylose content is 70%).

[0065] The high amylose corn starch used in step (2) of Example 1 is named HACS.

[0066] Comparative Example 3

[0067] A gelatinized retrograde high amylose corn starch without added purple cabbage anthocyanins is prepared by the following steps;

[0068] The amount of purple cabbage anthocyanins added in step (3) of Example 1 was adjusted to 0, while the other preparation conditions remained unchanged. It was named HACS / RCA0.

[0069] Comparative Example 4

[0070] A fully gelatinized high amylose-anthocyanin complex 4 is prepared by the following steps:

[0071] The heating temperature of the oil bath in step (2) of Example 3 was adjusted to 140℃, while the other preparation conditions remained unchanged. It was named HACS-140 / RCA20.

[0072] Comparative Example 5

[0073] A fully gelatinized ordinary starch-anthocyanin complex 5 is prepared by the following steps:

[0074] In step (2) of Example 3, the high amylose content was changed to ordinary corn starch (amylose content 25%), the heating temperature of the oil bath was changed to 100℃, and the other preparation conditions remained unchanged. It was named CS / RCA20.

[0075] 1. Appearance and composite rate of the complex

[0076] (1) Observe the appearance of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5. The results are as follows: Figure 2 As shown in Figure A.

[0077] Depend on Figure 2 As shown in Figure A, RCA powder is purplish-black, while HACS / RCA5, HACS / RCA10, and HACS / RCA20 are purple, with the color gradually deepening, but all lighter than RCA. This is because the HACS in the complex is milky white, which dilutes the dark-colored RCA. HACS-140 / RCA20 differs in color from the other samples, being dark red. Compared to HACS / RCA20, CS / RCA20 is lighter in color, indicating that it contains fewer anthocyanins in its complex with ordinary corn starch.

[0078] (2) Examples 1, 2, 3, Comparative Example 4, and 5 were completely dissolved in hydrochloric acid solution. The RCA content in the complex was determined and the composite rate was calculated. The results are as follows: Figure 2 As shown in B.

[0079] Depend on Figure 2As shown in section B, the RCA contents of HACS / RCA5, HACS / RCA10, and HACS / RCA20 are 10.43 mg / g, 14.22 mg / g, and 26.89 mg / g, respectively, consistent with the gradually deepening purple color of the three complexes. The RCA contents of HACS-140 / RCA20 and CS / RCA20 are 30.41 mg / g and 24.11 mg / g, respectively. Calculations show that the composite rates of HACS / RCA5, HACS / RCA10, HACS / RCA20, HACS-140 / RCA20, and CS / RCA20 are 20.88%, 14.22%, 13.44%, 15.04%, and 11.87%, respectively. Comparing the composite rates of HACS / RCA20, HACS-140 / RCA20, and CS / RCA20, it can be seen that the composite rate of high amylose corn starch with anthocyanins is higher than that of ordinary corn starch.

[0080] (3) The appearance of Examples 1, 2, 3, 2, 3, 4, and 5 was observed under a 10x magnifying lens. The results are as follows: Figure 3 As shown.

[0081] Depend on Figure 3 It can be seen that natural HACS presents as fine granules. HACS / RCA0, HACS / RCA5, HACS / RCA10, and HACS / RCA20 exhibit a similar morphology to HACS, and their color gradually deepens with increasing RCA content, indicating that high amylose corn starch is partially gelatinized when heated to 120 ºC, while still maintaining its granular shape. However, HACS-140 / RCA20 and CS / RCA20 both present as large, spongy masses, indicating that high amylose corn starch is completely gelatinized when heated to 140 ºC, while ordinary corn starch is completely gelatinized when heated to 100 ºC.

[0082] 2. Structural characteristics of the complex

[0083] The structures of Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were characterized using a scanning electron microscope, a polarizing microscope, an infrared spectrometer, an X-ray diffractometer, a small-angle X-ray scattering instrument, and a UV-Vis spectrophotometer.

[0084] (1) The microstructure of the sample was observed using a scanning electron microscope, and the results are as follows: Figure 4 As shown.

[0085] Depend on Figure 4As can be seen, RCA exhibits an irregular blocky structure. Natural HACS are spherical, elliptical, or irregular in shape, with smooth surfaces and varying sizes, and the particles remain separated from each other. Unlike natural HACS, HACS / RCA0 exhibits a concave surface shape, which is due to the loss of amylose located in the center of the starch granules after gelatinization and retrogradation. Similar to HACS / RCA0, HACS / RCA5, HACS / RCA10, and HACS / RCA20 also exhibit concave surfaces and a state of mutual aggregation / adhesion. Compared with HACS / RCA0, the particle surfaces of HACS / RCA5, HACS / RCA10, and HACS / RCA20 show more filaments, which may be because RCA acts as a "bridge" to promote the aggregation and adhesion of HACS particles.

[0086] (2) The morphology of the sample was observed using a polarizing microscope under normal light and polarized light. The results are as follows: Figure 5 As shown.

[0087] Depend on Figure 5 It is known that under normal illumination, native HACS particles exhibit various shapes and sizes. Unlike natural HACS particles, HACS / RCA particles tend to aggregate and adhere to each other. Under polarized light, natural HACS particles display typical Maltese cross birefringence, which originates from the ordered radial arrangement of the semi-crystalline structure in natural starch particles. However, no birefringence was observed in HACS / RCA0 particles, indicating that the ordered structure of HACS was disrupted during gelatinization. Similar to HACS / RCA0, the three HACS / RCA complexes also did not exhibit birefringence.

[0088] (3) Record the sample at 400–4000 cm⁻¹ using an infrared spectrometer. -1 The infrared spectrum within the range, the results are as follows Figure 6 As shown in Figure A.

[0089] Depend on Figure 6 As shown in A, RCA exhibits the characteristic infrared absorption bands of anthocyanins, specifically including the band at 3275 cm⁻¹. -1 OH stretching vibration at 2931 cm -1 CH stretching vibration at 1513 cm -1 C=C stretching vibration at 1066 cm -1 The C-OH stretching vibration is located at 3286–3299 cm⁻¹. HACS and the HACS / RCA complex exhibit the typical characteristic bands of HACS, with the OH stretching vibration located between 3286 and 3299 cm⁻¹. -1 In the range of 2921–2931 cm⁻¹, CH stretching vibrations occur. -1 In the interval, the stretching vibration of COC is located at 1148 cm.-1 Compared to HACS / RCA0, the OH stretching vibration peaks of HACS / RCA5, HACS / RCA10, and HACS / RCA20 all show a certain degree of shift. This phenomenon originates from the hydrogen bond interaction formed between HACS and RCA molecules.

[0090] Further analysis of the samples at 800–1200 cm⁻¹ -1 The infrared spectrum within the range is deconvolved. Figure 6 B), and through 1041 cm -1 (The ordered structure of starch) and 1022 cm -1 Peak intensity ratio (I) of the amorphous region of starch 1041 / I 1022 Compare the short-range order of the samples ( Figure 6 C).

[0091] Depend on Figure 6 As shown in C, HACS / RCA0 exhibits higher short-range order compared to HACS, which is related to the recrystallization of gelatinized starch molecules through hydrogen bonds during retrogradation. HACS / RCA5, HACS / RCA10, and HACS / RCA20 also show higher short-range order than HACS / RCA0, and the short-range order of the HACS / RCA complex increases with increasing RCA content. This is because RCA contains abundant hydroxyl groups, which can interact with amylose and amylopectin through hydrogen bonds, facilitating the rearrangement of amylose and amylopectin into a more ordered conformation during retrogradation.

[0092] (5) The crystallinity and long-range order of the sample were determined using an X-ray diffractometer, and the results are as follows: Figure 7 As shown in Figure A.

[0093] Depend on Figure 7As shown in Figure A, RCA exhibits broad amorphous peaks. Natural HACS displays typical type B crystal polymorphism, with a strong diffraction peak at 17.4° and four weak diffraction peaks at 5.8°, 15.1°, 22.5°, and 24.5°. The strong diffraction peak of HACS at 20.2° corresponds to the type V crystal polymorphism of the amylose-lipid complex. The XRD pattern of HACS / RCA0 shows only one type B diffraction peak (17.1°) and one type V diffraction peak (20.1°), and its relative crystallinity is lower than that of natural HACS. The decrease in long-range order of HACS / RCA0 is mainly related to the destruction of the amylopectin structure during gelatinization. The XRD patterns of HACS / RCA5, HACS / RCA10, and HACS / RCA20 are similar to those of HACS / RCA0. However, with the increase of RCA content, the relative crystallinity of the HACS / RCA complex decreases slightly, indicating that RCA hinders the orderly arrangement and assembly of the amylose and amylopectin double helix structures during the retrogradation process of high amylose.

[0094] (6) The layered structure of the sample was determined using a small-angle X-ray scattering instrument, and the results are as follows: Figure 7 As shown in B.

[0095] Depend on Figure 7 As shown in B, the natural HACS exhibits a scattering vector peak at approximately 0.67 nm⁻¹, indicating that the HACS possesses a layered structure with an average repeatability distance of 9.38 nm. No scattering vector peak was observed in HACS / RCA0, suggesting that the ordered layered structure of the HACS was disrupted during the gelatinization process. Similarly, no scattering vector peak was observed in HACS / RCA5, HACS / RCA10, and HACS / RCA20.

[0096] (7) The iodine binding capacity of the sample was determined using a UV-Vis spectrophotometer, and the results are as follows: Figure 8 As shown.

[0097] Depend on Figure 8 The HACS-iodine complex is blue, with a maximum absorption peak at 575 nm. Compared to HACS, the HACS / RCA0-iodine complex exhibits a weakened absorption intensity and a blue shift effect. This is because the amylose in HACS partially forms a double helix during retrogradation, limiting its iodine-binding capacity. The iodine-binding capacity of HACS further decreases upon complexation with RCA, with HACS / RCA20 showing the lowest iodine-binding capacity. This is because RCA promotes the formation of a double helix in amylose during retrogradation, further inhibiting the formation of the amylose-iodine complex.

[0098] 3. Water solubility of the complex

[0099] The water solubility of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 was determined, and the results are as follows: Figure 9 As shown.

[0100] (1) Take photos to record the dissolution of the sample in water. The results are as follows: Figure 9 As shown in Figure A.

[0101] Depend on Figure 9 As shown in Figure A, RCA is completely soluble in water, and the solution is a uniform purple color. Conversely, HACS / RCA5, HACS / RCA10, and HACS / RCA20 are all insoluble in water and settle stably to the bottom of the bottle as particles; over time, only a small amount of RCA is released from the complex, giving the supernatant a pale purple color. However, HACS-140 / RCA20 and CS / RCA20, due to their sponge-like structure, can rapidly absorb water, swell, and release anthocyanins. If applied to a high-humidity packaging environment, they are difficult to maintain the stability of the anthocyanins in the complex. The results indicate that combining with partially gelatinized HACS improves the hydrophobicity of RCA. Applying the obtained HACS / RCA5, HACS / RCA10, and HACS / RCA20 to a high-humidity packaging environment helps maintain the stability of the anthocyanins in the complex.

[0102] (2) The cumulative RCA release rate and DPPH free radical scavenging activity of the sample in water were determined, and the results are as follows: Figure 9 B and Figure 9 As shown in C.

[0103] Depend on Figure 9 B and Figure 9 As shown in Figure C, the cumulative release rates of HACS / RCA5, HACS / RCA10, and HACS / RCA20 decrease sequentially, indicating that HACS / RCA20 is the most stable in water. Nevertheless, HACS / RCA20 releases the largest amount of RCA into water, thus exhibiting the highest DPPH radical scavenging activity in its supernatant. Compared to HACS / RCA20, HACS-140 / RCA20 and CS / RCA20 show higher cumulative RCA release rates in water. This is because HACS-140 / RCA20 and CS / RCA20 are sponge-like, allowing them to rapidly absorb water, swell, and release anthocyanins. Furthermore, the DPPH radical scavenging activity of the supernatants of HACS-140 / RCA20 and CS / RCA20 is significantly higher than that of HACS / RCA20. These results demonstrate that partially gelatinized HACS can effectively control the slow release of anthocyanins.

[0104] 4. Thermal stability of the composite

[0105] (1) The stability of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 in 80 °C hot water was determined. Samples were taken out at predetermined time intervals, and their state was recorded by photograph. The residual RCA content in the samples was determined and linear fitting was performed. The results are as follows: Figure 10 As shown in A-10C.

[0106] Depend on Figure 10 As shown in Figure A, the RCA solution gradually faded from purple to light yellow, indicating degradation in 80 °C hot water, demonstrating the low thermal stability of natural anthocyanins. HACS / RCA5, HACS / RCA10, and HACS / RCA20 are insoluble in water, with only a small amount of RCA slowly released into the supernatant. Compared to HACS / RCA20, HACS-140 / RCA20 and CS / RCA20 are spongy, and due to their higher water solubility, more RCA is released into the supernatant, resulting in a darker supernatant color. Figure 10 As shown in Figure B, the RCA retention rate (C / C0) decreased rapidly within 24 hours. Compared to RCA, the HACS / RCA complex exhibited a higher RCA retention rate. This is because the HACS compound effectively slowed down the release and degradation of RCA, which is beneficial for maintaining the stability of anthocyanins in the complex under high temperature and high humidity packaging conditions. Compared to HACS / RCA20, HACS-140 / RCA20 and CS / RCA20 showed lower RCA retention rates, indicating that HACS-140 / RCA20 and CS / RCA20 have poorer thermal stability; therefore, HACS-140 / RCA20 and CS / RCA20 are unlikely to maintain the stability of anthocyanins in the complex under high temperature and high humidity packaging conditions. Figure 10 According to C, the thermal degradation of RCA, HACS / RCA5, HACS / RCA10, HACS / RCA20, HACS-140 / RCA20, and CS / RCA20 all conform to a first-order kinetic model. The results indicate that combining with partially gelatinized HACS significantly improves the thermal stability of RCA.

[0107] (2) The TGA and DTG curves of Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were measured at 50–650 °C, and the results are as follows: Figure 10 D and Figure 10 As shown in E.

[0108] Depend on Figure 10 D and Figure 10E indicates that RCA undergoes a continuous degradation process, with the fastest weight loss occurring at 209 °C. Both HACS and HACS / RCA exhibit a three-stage degradation characteristic: weight loss between 50 and 270 °C is due to water evaporation and RCA degradation; weight loss between 270 and 360 °C is due to starch chain depolymerization and degradation; and weight loss between 360 and 650 °C is due to the oxidative decomposition of carbonaceous residues. The maximum weight loss rates for both HACS and HACS / RCA occur between 315 and 320 °C. Among them, HACS exhibits the highest weight loss rate, while the weight loss rates of both HACS and HACS / RCA gradually decrease with increasing RCA content. The results indicate that combining HACS with partially gelatinized HACS significantly improves the thermal stability of RCA.

[0109] 5. pH-responsive color change ability of the complex

[0110] The color-changing ability of Examples 1, 2, 3, Comparative Examples 1, 4, and 5 was measured, and the results are as follows: Figure 11 As shown.

[0111] (1) Add a buffer solution with pH 3-12 to the sample and take a picture to record the color change of the sample. The results are as follows: Figure 11 As shown in Figure A.

[0112] Depend on Figure 11 As shown in Figure A, RCA, HACS / RCA5, HACS / RCA10, HACS / RCA20, HACS-140 / RCA20, and CS / RCA20 exhibited a color change trend of red → purplish-red → purple → blue → green in different pH buffer solutions. This indicates that the complexes retained the pH-responsive color-changing ability of anthocyanins. It is noteworthy that RCA, due to its high water solubility, completely dissolved in the buffer solution; furthermore, HACS-140 / RCA20 and CS / RCA20, being sponge-like and highly hygroscopic, also formed solutions. If these three substances are applied to a high-humidity food packaging environment, they are highly susceptible to moisture absorption and liquid formation. If the samples are shaken or tipped over, they will contaminate the food, adversely affecting its sensory properties. Therefore, RCA, HACS-140 / RCA20, and CS / RCA20 are unsuitable for intelligent food packaging. In contrast, HACS / RCA5, HACS / RCA10, and HACS / RCA20, due to their strong hydrophobicity, were not wetted by the buffer solution. In practical applications, they can be packaged in perforated containers, maintaining a solid state in high-humidity packaging environments and avoiding contact with food, thus preventing negative impacts on food quality. The results indicate that HACS / RCA5, HACS / RCA10, and HACS / RCA20 retain the excellent pH-responsive color-changing properties of RCA and are more valuable for application in high-humidity packaging environments.

[0113] (2) Expose the sample to a 0.25 mol / L ammonia atmosphere and take photos to record the color change of the sample. The results are as follows: Figure 11 As shown in B.

[0114] Depend on Figure 11 As shown in Figure B, RCA remained a purplish-black color without any noticeable color change. In contrast, HACS / RCA5, HACS / RCA10, and HACS / RCA20 exhibited relatively consistent and distinct color changes (purplish-red → purple → blue-green) in an ammonia-filled atmosphere. These results indicate that HACS / RCA5, HACS / RCA10, and HACS / RCA20 possess excellent ammonia-responsive color-changing capabilities.

[0115] (3) The sample was alternately exposed to hydrochloric acid atmosphere (6 mol / L) for 10 min and ammonia atmosphere (3 mol / L) for 10 min, repeated 7 times, and the color and a value of the sample were recorded. The results are as follows. Figure 11 As shown in C-11F.

[0116] Depend on Figure 11 As shown in C-11F, RCA powders alternately exposed to hydrochloric acid and ammonia atmospheres did not exhibit any visually perceptible color change, and their color parameter α value remained almost unchanged. In contrast, the colors of HACS / RCA5, HACS / RCA10, and HACS / RCA20 alternately exposed to hydrochloric acid and ammonia atmospheres could change between red and green, and their α values ​​showed periodic fluctuations. These results indicate that HACS / RCA5, HACS / RCA10, and HACS / RCA20 possess good color reversibility.

[0117] 6. The effect of the compound on the freshness indication of shrimp

[0118] Examples 1, 2, 3, and Comparative Example 1 were used to indicate the freshness of shrimp. RCA, HACS / RCA5, HACS / RCA10, and HACS / RCA20 powders were placed in the top caps of centrifuge tubes, which were then placed at the bottom of petri dishes. Fresh shrimp were placed in the petri dishes, the lids were closed, and the dishes were sealed with plastic wrap. The shrimp were stored at 4 °C for 5 days. Color changes in the membrane samples were recorded every 24 hours, and the pH and TVB-N values ​​of the shrimp were measured. The results are as follows: Figure 12-13 As shown.

[0119] Depend on Figure 12It was found that during shrimp storage, RCA powder rapidly absorbed moisture and remained a dark purple color, indicating that it did not possess the ability to indicate shrimp freshness. In contrast, HACS / RCA5, HACS / RCA10, and HACS / RCA20 exhibited good moisture resistance, all showing visible color changes during shrimp storage. On the third day, HACS / RCA5, HACS / RCA10, and HACS / RCA20 all showed a significant color change, shifting from purple to blue.

[0120] Depend on Figure 13 As shown in A and 13B, the pH and TVB-N values ​​of the shrimp continuously increased during storage; by the third day of storage, the TVB-N value of the shrimp had exceeded 20 mg / 100 g. Figure 13 From C and 13D, it can be seen that during shrimp storage, the ΔE and G / R values ​​of HACS / RCA5, HACS / RCA10, and HACS / RCA20 all increased slowly, while the E and G / R values ​​of RCA changed irregularly. From Figure 13 The correlation analysis of E and 13F showed that the changes in ΔE and G / R values ​​of HACS / RCA5, HACS / RCA10, and HACS / RCA20 were highly correlated with the TVB-N value of shrimp. In contrast, the changes in ΔE and G / R values ​​of RCA were not correlated with the TVB-N value of shrimp. These results indicate that HACS / RCA5, HACS / RCA10, and HACS / RCA20 possess excellent moisture resistance and shrimp freshness indication capabilities in high-humidity environments.

Claims

1. A starch-anthocyanin complex, characterized in that, A starch-anthocyanin complex is obtained by partially gelatinizing high amylose and blending it with anthocyanins, followed by retrogradation, dialysis, drying, pulverizing, and sieving. The amylose content of the high amylose accounts for more than 50% of the total starch mass. The starch / anthocyanin complex is granular, and the granules are in a concave and aggregated or adhered state.

2. The starch-anthocyanin complex according to claim 1, characterized in that, The high amylose starch undergoes partial gelatinization at a temperature of 100–130°C for a gelatinization time of 30–60 min.

3. The starch-anthocyanin complex according to claim 1, characterized in that, The mass ratio of anthocyanins to high amylose is 1:5 to 1:20 (w / w).

4. A method for preparing the starch-anthocyanin complex according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Disperse high amylose in a buffer solution and heat it to obtain a gelatinized liquid in which high amylose is partially gelatinized; (2) Cool the high amylose gelatinized liquid obtained in step (1), add anthocyanins to it, mix evenly, and obtain a high amylose / anthocyanin composite solution. (3) Cool and regenerate the high amylose-anthocyanin complex solution obtained in step (2) to obtain a regenerated high amylose / anthocyanin complex solution; (4) Dialyze the regenerated high amylose / anthocyanin complex solution obtained in step (3) to obtain high amylose-anthocyanin complex dialysate; (5) The high amylose-anthocyanin complex dialysate obtained in step (4) is freeze-dried, pulverized, and sieved to obtain the high amylose-anthocyanin complex with food freshness indication function.

5. The method for preparing the starch-anthocyanin complex according to claim 4, characterized in that, In step (1), the buffer solution is a buffer solution with a pH of 2 to 5.

6. The method for preparing the starch-anthocyanin complex according to claim 4, characterized in that, In step (1), the mass ratio of the high amylose to the volume ratio of the buffer solution is 1:50 to 1:100 (w / v).

7. The method for preparing the starch-anthocyanin complex according to claim 4, characterized in that, In step (4), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000 to 12000 Da for distilled water dialysis.

8. The method for preparing the starch-anthocyanin complex according to claim 7, characterized in that, The dialysis time is 24–72 h, and the water exchange interval is 4–8 h.

9. The method for preparing the starch-anthocyanin complex according to claim 4, characterized in that, In step (5), the sieve aperture is 100-200 mesh.

10. The use of the starch-anthocyanin complex according to any one of claims 1 to 3 in a food freshness indicator.

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