Method for improving color and stability of anthocyanin by combining with punicalagin

Abstract

The invention discloses a method for improving color and stability of anthocyanin through combination with punicalagin. The method comprises the step that punicalagin and anthocyanin are subjected to an intermolecular co-pigmentation reaction. According to the invention, punicalagin is adopted as a co-pigment molecule and is subjected to a co-pigmentation reaction with anthocyanin to form a punicalagin and anthocyanin compound, so that the color attribute of the anthocyanin is remarkably enhanced, and an anthocyanin solution presents brighter red color; the punicalagin and anthocyanin compound is prepared from punicalagin and anthocyanin, the heat stability, the light stability, the pH stability and the storage stability of anthocyanin are improved, the heat stability, the light stability and the storage stability of anthocyanin are further improved when the punicalagin and anthocyanin compound is prepared into microcapsules, and the punicalagin and anthocyanin compound has the advantages of being simple in process and low in cost. According to the invention, the utilization value of the anthocyanin is obviously improved, and a new thought is provided for the application of the anthocyanin in the food industry.

Description

Technical Field

[0001] The invention relates to the technical field of food chemistry, and in particular to a method for improving the color and stability of anthocyanins by combining with punicalagin. Background Art

[0002] Anthocyanins are a class of flavonoid compounds that exist naturally in flowers, fruits, vegetables and other plants, giving plants a variety of colors such as red, blue, and purple. As a natural colorant, anthocyanins can not only replace synthetic pigments, but also have a variety of biological functional activities, such as antioxidant, anti-inflammatory and anti-tumor. These characteristics have made anthocyanins receive more and more attention in the fields of food, cosmetics and health products. In addition, as people's health awareness continues to improve, products rich in anthocyanins are gradually favored by consumers, and their market potential and application prospects are becoming more and more broad.

[0003] However, anthocyanins are very unstable and are easily affected by external factors such as pH, light, temperature, enzymes, oxygen and metal ions, causing structural changes or degradation, resulting in fading of anthocyanins and loss of biological functional activity, which greatly limits the application of anthocyanins and reduces the quality of products. Therefore, it is crucial to improve the stability of anthocyanins. Studies have shown that co-pigmentation is one of the technologies to improve the stability of anthocyanins, that is, anthocyanins interact with co-pigment molecules (such as phenols, amino acids and alkaloids) in the plant environment, enhancing their stability while making the color of the solution appear brighter red. However, co-pigment molecules with different structures have significant differences in the effect of enhancing the color and stability of anthocyanins. Therefore, choosing the right co-pigment molecule is the key to enhancing the stability and color performance of anthocyanins. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a method for improving the color and stability of anthocyanins by combining with punicalagin, which uses punicalagin as a co-pigmentation molecule to undergo a co-pigmentation reaction with anthocyanins to form a punicalagin and anthocyanin complex, thereby significantly enhancing the color properties of anthocyanins, making the anthocyanin solution appear more vivid red, and at the same time improving the stability of anthocyanins, thereby avoiding the problem of anthocyanins being unstable during processing and storage and being prone to degradation and fading.

[0006] To achieve these objects and other advantages according to the present invention, a method for improving the color and stability of anthocyanins by combining with punicalagins is provided, which is achieved by an intermolecular co-pigmentation reaction between punicalagins and anthocyanins.

[0007] Preferably, the molar ratio of punicalagin to anthocyanin is 1:10-80.

[0008] Preferably, the method specifically comprises the following steps: Step 1, extraction of punicalagin: using pomegranate peel as raw material, and subjecting it to crushing, sieving, ultrasonic extraction, macroporous resin purification, fractional extraction, and liquid chromatography purification to obtain punicalagin; Step 2, dissolving the punicalagin and anthocyanin obtained in step 1 in an acidic buffer solution; Step 3: Mix the punicalagin solution and anthocyanin solution obtained in step 2, and carry out intermolecular co-pigmentation reaction at 4-25° C. to obtain a mixed solution, and the reaction time is 40-120 min.

[0009] Preferably, the method further comprises the following steps: step 4, adding the mixed solution obtained in step 3 to 80 mg / mL chondroitin sulfate or sodium alginate aqueous solution at a volume ratio of 1:1, and homogenizing at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; adding the primary complex solution dropwise to 8 mg / mL carrageenan solution, adjusting the pH to 3.5 at 30°C with 0.1 M HCl solution, and freezing at 0°C for 50 min to obtain punicalagin-anthocyanin microcapsules.

[0010] Preferably, step one specifically includes: S11, placing pomegranate peel in a constant temperature box to dry, crushing the dried pomegranate peel, and passing through a 200-mesh sieve to obtain pomegranate peel powder; S12, take 100-200 g of pomegranate peel powder, add 2 L of 20-80% methanol aqueous solution by volume, perform ultrasonic extraction for 40-120 min, and centrifuge at a centrifugal force of 10,000 g for 10-15 min to obtain a crude extract of punicalagin; S13, removing methanol from the crude extract of punicalagin by rotary evaporation, and concentrating the aqueous phase to obtain a punicalagin concentrate; S14, purifying the punicalagin concentrate with a macroporous resin, performing graded extraction with petroleum ether and ethyl acetate, and rotary evaporating the ethyl acetate extraction component to dryness to obtain an ethyl acetate extraction product; S15, re-dissolving the product extracted with ethyl acetate in methanol, separating and purifying it by liquid chromatography, and freeze-drying it to obtain punicalagin.

[0011] Preferably, step 2 specifically comprises: dissolving punicalagin and anthocyanin in 0.1 M citric acid phosphate buffer solution, wherein the pH range of the buffer solution is 3-6.

[0012] Preferably, step S14 specifically comprises: using AB-8 macroporous resin, a chromatography column with an inner diameter of 35 mm and a height of 50 cm for purification, wherein the loading and elution speeds are both 1 mL / min, the eluent is water, and 0.8 L of methanol aqueous solution with a volume mass fraction of 20%, 40%, 60% and 80% is used for gradient elution, respectively, collecting 40% and 60% elution components, removing methanol by rotary evaporation and concentrating the aqueous phase to 20-50 mL, then adding 2 volumes of petroleum ether for degreasing, repeating 2 times, discarding petroleum ether, adding 2 volumes of ethyl acetate to the remaining aqueous phase for extraction, repeating 4-6 times, retaining the ethyl acetate extract, and rotary evaporating the ethyl acetate extract to obtain the ethyl acetate extract product; in step S15, the conditions for separation and purification by liquid chromatography are: the chromatographic column is ZORBAX-SB C18 (9.4×250 mm, 5 μm), the mobile phase was 0.1% formic acid in water and methanol, and the elution gradient was 0-5 min, 5%-10% methanol, 5-15 min, 10%-30% methanol, 15-20 min, 30%-80% methanol, 20-25 min, 100% methanol, 25-30 min, 100%-5% methanol, 30-35 min, 5% methanol.

[0013] Preferably, the molar ratio of anthocyanin to punicalagin is 1:80.

[0014] Preferably, the anthocyanin is cyanidin-3-O-glucoside monomer.

[0015] The object of the present invention can be further achieved by preparing a punicalagin-anthocyanin complex by combining with punicalagin to improve the color and stability of anthocyanins. The object of the present invention can be further achieved by applying the method for improving the color and stability of anthocyanins by combining with punicalagin in fruit juice processing.

[0016] The present invention has at least the following beneficial effects: First, the present invention uses punicalagin as a co-pigment molecule to undergo a co-pigmentation reaction with anthocyanins to form a complex of punicalagin and anthocyanins, which significantly enhances the color properties of anthocyanins, making the anthocyanin solution appear more vivid red, and improving the depth and saturation of its hue, thereby providing a natural color enhancer for industries such as food, beverages and cosmetics.

[0017] Second, the punicalagin and anthocyanin complex formed by the present invention improves the stability of anthocyanins, including thermal stability, light stability, storage stability, and pH stability, thereby improving the application of anthocyanins in the food industry.

[0018] Third, the co-pigment molecule punicalagin used in the present invention is the main phenolic substance in pomegranate peel, and its extraction process is simple and efficient, and has the advantages of being natural, safe and low-cost.

[0019] Fourthly, the punicalagin used in the present invention is an important phenolic substance in pomegranate peel, which has a variety of biological activities beneficial to human health. It can be applied to food as a co-pigment molecule to improve the functional properties of the food.

[0020] Fifth, the present invention is simple to operate, has low equipment requirements, has significant practicality and economy, and is in line with the current industry's pursuit of green and sustainable development concepts.

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a liquid chromatogram of the high-purity punicalagin obtained in Example 1 of the present invention; Figure 2 It is the physical picture corresponding to Table 1 of the present invention; Figure 3 The UV-visible spectra (a), high color shift graph (b) and dark color shift graph (c) of anthocyanin (C3G) and punicalagin (PUN) in different molar ratios after co-pigmentation reaction in the embodiments of the present invention are shown; Figure 4 This is a graph showing the results of thermodynamic parameter determination in the present invention; Figure 5 This is a graph showing the pH stability of anthocyanins enhanced by punicalagin in an embodiment of the present invention; Figure 6 This is a graph showing the results of punicalagin enhancing the thermal stability of anthocyanins in an embodiment of the present invention; Figure 7 This is a graph showing the results of punicalagin enhancing the storage stability of anthocyanins in an embodiment of the present invention; Figure 8 This is a graph showing the results of punicalagin enhancing the photostability of anthocyanins in an example of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0027] Example 1 A method for improving the color and stability of anthocyanins by combining with punicalagin, comprising the following steps: Step 1: Extraction of Punicalagin (PUN): S11, placing pomegranate peel in a constant temperature box to dry, crushing the dried pomegranate peel, and passing through a 200-mesh sieve to obtain pomegranate peel powder; S12, take 100 g of pomegranate peel powder, add 2 L of 50% methanol aqueous solution (v / v), extract by ultrasonication for 90 min, then centrifuge at 10000 g for 12 min, and take the supernatant to obtain the crude extract of punicalagin; S13, removing methanol from the crude extract of punicalagin by rotary evaporation at 40° C. and 120 rpm, and concentrating the aqueous phase to 40 mL to obtain punicalagin concentrate; S14. The punicalagin concentrate was purified by using a chromatographic column with an AB-8 macroporous resin, an inner diameter of 35 mm and a height of 50 cm, wherein the loading and elution speeds were both 1 mL / min, the eluent was water, and 0.8 L of methanol aqueous solution with a volume mass fraction of 20%, 40%, 60% and 80% was used for gradient elution, respectively, and the 40% and 60% elution components were collected, and the methanol was removed by rotary evaporation and the aqueous phase was concentrated to 30 mL, followed by adding 2 volumes of petroleum ether for degreasing, and the process was repeated 2 times. The petroleum ether was discarded, and 2 volumes of ethyl acetate were added to the remaining aqueous phase for extraction, and the process was repeated 4 to 6 times. The ethyl acetate extract was retained, and the ethyl acetate extract was rotary evaporated to dryness to obtain the ethyl acetate extract product; S15. The ethyl acetate extraction product was redissolved in methanol at a concentration of 10 mg / mL, separated and purified by liquid chromatography, and freeze-dried to obtain high-purity (95.47%) punicalagin; wherein the liquid chromatography conditions were: the chromatographic column was ZORBAX-SB C18 (9.4×250mm, 5 μm), the mobile phase was 0.1% (v / v) formic acid water and methanol, and the elution gradient was 0-5 min, 5%-10% (v / v) methanol, 5-15 min, 10%-30% (v / v) methanol, 15-20 min, 30%-80% (v / v) methanol, 20-25 min, 100% methanol, 25-30 min, 100%-5% (v / v) methanol, 30-35 min, 5% (v / v) methanol; The purity of the obtained punicalagin was determined by high performance liquid chromatography, and the determination method was as follows: the purified punicalagin obtained in step S15 was dissolved in methanol to a concentration of 0.5 mg / mL, and the liquid chromatography conditions were as follows: the chromatographic column model was ZORBAX-SB C18 (4.6×250mm, 5 μm), the mobile phase was 0.1% formic acid aqueous solution and methanol, the column temperature was 25°C, the detection wavelength was 280 nm, and the elution gradient was 0-5 min, 5% methanol, 5-15 min, 5%-30% methanol, 15-20 min, 30%-100% methanol.

[0028] HPLC chromatogram Figure 1 Among them, peaks 11.99 and 13.04 are both punicalagin, which are isomers of each other. The two are converted into each other, and the mass percentage concentration of purified punicalagin is 95.47%.

[0029] Step 2: dissolving the high-purity punicalagin and anthocyanin (cyanidin-3-O-glucoside monomer, C3G) obtained in step S15 in a citric acid phosphate buffer solution (pH=3.5, 0.1 M), wherein the concentration of anthocyanin is 0.2 mM and the concentration of punicalagin is 16 mM; Step 3: Mix equal volumes of anthocyanin solution and punicalagin solution, i.e., the molar ratio of anthocyanin to punicalagin in the solution is 1:80. Vortex for 2 min and place at 25°C for intermolecular co-pigmentation reaction. The reaction time is 120 min.

[0030] Example 2 The difference between the embodiment 1 and the embodiment 1 is that in step 2, the concentration of punicalagin is 2 mM, that is, in step 3, the molar ratio of anthocyanin to punicalagin in the solution is 1:10.

[0031] Example 3 The difference between the embodiment 1 and the embodiment 1 is that in step 2, the concentration of punicalagin is 4 mM, that is, in step 3, the molar ratio of anthocyanins to punicalagin in the solution is 1:20.

[0032] Example 4 The difference between the embodiment 1 and the embodiment 1 is that in step 2, the concentration of punicalagin is 8 mM, that is, in step 3, the molar ratio of anthocyanin to punicalagin in the solution is 1:40.

[0033] Example 5 The difference between the embodiment 4 and the embodiment 4 is that it further comprises the steps of: Step 4: Add the mixed solution obtained after the intermolecular co-pigmentation reaction in step 3 to an 80 mg / mL chondroitin sulfate (CS) aqueous solution at a volume ratio of 1:1, and homogenize at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; add the primary complex solution dropwise to an 8 mg / mL carrageenan (KC) colloidal solution (formed by dissolving carrageenan in 70°C hot water), adjust the pH to 3.5 at 30°C using 0.1 M HCl solution, and freeze at 0°C for 50 min to obtain punicalagin-anthocyanin microcapsules, recorded as CS-PUN-C3G-KC.

[0034] Example 6 The difference from Example 5 is that the 80 mg / mL chondroitin sulfate solution is replaced with an 80 mg / mL sodium alginate aqueous solution to prepare punicalagin-anthocyanin microcapsules, which are recorded as SA-PUN-C3G-KC.

[0035] Comparative Example 1 The difference between it and Example 1 is that: step 1 is not included, and step 2: dissolving anthocyanins in a citric acid phosphate buffer solution (pH = 3.5, 0.1 M), wherein the concentration of anthocyanins is 0.1 mM; step 3 is not included, i.e., the molar ratio of anthocyanins to punicalagin is 1:0, and this comparative example 1 is a control group without adding punicalagin.

[0036] Comparative Example 2 The difference between the embodiment 1 and the embodiment 1 is that in step 2, the concentration of punicalagin is 0.2 mM, that is, in step 3, the molar ratio of anthocyanin to punicalagin in the solution is 1:1.

[0037] Comparative Example 3 The difference between the embodiment 1 and the embodiment 1 is that in step 2, the concentration of punicalagin is 20 mM, that is, in step 3, the molar ratio of anthocyanin to punicalagin in the solution is 1:100.

[0038] Performance Experiment 1. Punicalagin enhances the color properties of anthocyanins 1) Using a colorimeter to measure the L of the reaction solution after the reaction in step 3 in Examples 1 to 4 and Comparative Examples 1 to 3 * , a * , b * The C* value, H* value and △E value are then calculated according to the following formula.

[0039] (1) (2) (3) Among them, C* value represents saturation, H* value represents hue angle, △E value represents color difference, L * Indicates the brightness value of the corresponding sample solution, a * Indicates the red value of the corresponding sample solution, b * Indicates the yellow value of the corresponding sample solution, , , It represents the corresponding color parameter values ​​of the control group (without adding punicalagin, i.e. the control group).

[0040] 2) Use a UV-visible spectrophotometer to scan the entire spectrum of the reaction solution from 400 to 700 nm.

[0041] The results of the test are as follows Figure 2 As shown in Table 1, Figure 2 It can be seen that the ratio of anthocyanin to co-pigment molecules is an important factor affecting the co-pigmentation reaction. With the increase of the molar ratio of anthocyanin to punicalagin, the maximum absorbance and maximum absorption wavelength of anthocyanin also increased significantly, showing high color shift and dark color shift, respectively. This indicates that an effective co-pigmentation reaction occurred between anthocyanin and punicalagin. When the molar ratio of anthocyanin to punicalagin in the solution increased from 1:1 to 1:100, the maximum absorbance of anthocyanin increased from 2.27% to 71.56%, and the maximum absorption wavelength increased from 0.26% to 3.77%. Therefore, the co-pigmentation reaction can significantly enhance the color properties of anthocyanin solution. As shown in Table 1, with the increase of the molar ratio, the solution brightness value L* decreased from 55.61 to 34.65, the red value a* increased from 40.43 to 50.82, and the yellow value b* increased from 17.41 to 40.64, indicating that the color of the solution gradually showed a brighter red. Corresponding to the changes in L*, a*, and b* values, the C* value increased from 44.02 to 65.07, the H* value decreased from 2.18 to 0.97, and the △E increased from 0 to 32.98. These changes also indicate that the addition of punicalagin improves the depth and saturation of the hue of anthocyanins and enhances the color performance of anthocyanins. The obvious reddening of the solution color can also be observed from the actual picture, which further illustrates the suitability of punicalagin as a co-pigment molecule. However, when the molar ratio of anthocyanins to punicalagin is lower than 1:10, the enhancement effect of punicalagin on the color of anthocyanins is small, and when the molar ratio is 1:100, there is no significant difference in the color enhancement effect of anthocyanins compared to the molar ratio of 1:80. Therefore, the preferred molar ratio of anthocyanins to punicalagin in the solution is in the range of 1:10 to 80.

[0042] Table 1 Color L*, a*, b*, C*, H* and △E determination of co-pigmentation reaction of anthocyanin and punicalagin at different molar ratios 2. Determination of thermodynamic parameters of co-pigmentation of punicalagin and anthocyanin Anthocyanin (Cyanidin-3-O-glucoside monomer, C3G) and punicalagin were dissolved in a citric acid phosphate buffer solution (pH = 3.5, 0.1 M), the concentration of anthocyanin was 0.2 mM, the concentration of punicalagin was 8 mM, the anthocyanin solution and the punicalagin solution were mixed in equal volumes, that is, the molar ratio of anthocyanin C3G to punicalagin (PUN) in the mixed solution was 1:40, and after vortexing for 2 min, the solution was placed at 25, 35, 45, 55, and 65 ° C for 120 min. The moderate molar ratio of 1:40 was used for this and subsequent performance tests in order to save costs while meeting the reaction conditions.

[0043] Experimental method: The reaction system was scanned at 400-700 nm at different temperatures. The reaction equilibrium constant K, stoichiometric ratio n, △G 0 , △H 0 and △S 0 Calculated by the following formula: (4) (5) (6) (7) In formula (4), A and A0 represent the maximum absorbance of anthocyanins when different concentrations of punicalagins are added and when no punicalagins are added, respectively (see Figure 3 In a, (Cp)0 represents the concentration of punicalagin added to the anthocyanin solution (0-20 mM), K is the reaction equilibrium constant, and n is the stoichiometric ratio; in formula (5) and formula (6), R is the gas constant (8.314 J / moL / K), T0 represents the Kelvin temperature of 25°C; in formula (7), T represents the Kelvin temperature of the above-mentioned temperatures of 25, 35, 45, 55, and 65°C, and A and A0 represent the maximum absorbance of punicalagin (the molar ratio of anthocyanin C3G to punicalagin in the solution is 1:40) added at different temperatures of 25, 35, 45, 55, and 65°C, respectively. Figure 4 The maximum absorbance of anthocyanins in a) and without adding punicalagin, △G 0 , △H 0 and △S 0 Respectively represent the Gibbs free energy, enthalpy change and entropy change of the reaction between anthocyanin and punicalagin at 25°C.

[0044] Thermodynamic parameters were used to further explore the mechanism of co-pigmentation between anthocyanins and punicalagins in order to determine the probability or spontaneity of the reaction. Figure 4As shown in Figure a, with the increase of temperature, the intensity of the co-pigmentation reaction of anthocyanins and punicalagins gradually decreases, indicating that low temperature will promote the reaction. Figure 3 As shown in a, the UV-visible spectra of anthocyanins at different concentrations of punicalagin are plotted with ln(Cp)0 as the horizontal axis and ln (A-A0) / A as the vertical axis. Figure 3 In b, K and n values ​​were calculated by formula (4). K is the reaction equilibrium constant, and its value indicates the binding strength between the reactants. The K value of the co-pigmentation reaction of anthocyanins and punicalagins is 15.09 mol / L, indicating that the structure of the complex formed by anthocyanins and punicalagins is tight. Substituting K value, n and T0 into formula (5), △G0 is calculated to be -6.73 KJ / mol, indicating that the co-pigmentation between anthocyanins and punicalagins is spontaneous in nature. Figure 4 As shown in a, the UV-visible spectra of punicalagin to anthocyanins at different temperatures are plotted with 1 / T as the horizontal axis and ln (A-A0) / A as the vertical axis. Figure 4 In b, according to formula (7), △H0 is -10.73 KJ / mol when T is equal to T0, which means that the reaction is exothermic, which also shows that anthocyanins and punicalagins are more likely to react at lower temperatures. According to formula (6), △S0 is -13.42 KJ / mol, which reflects that the complex formed by the combination of anthocyanins and punicalagins has a more ordered and stable structure. This is due to the molecular interactions between anthocyanins and punicalagins, such as hydrogen bonds, electrostatic interactions or hydrophobic interactions, which increase the driving force for the formation of the complex.

[0045] 3. Punicalagin enhances the stability of anthocyanin (C3G): (1) pH stability Anthocyanin (cyanidin-3-O-glucoside monomer, C3G) and punicalagin were both dissolved in a citric acid phosphate buffer solution (pH = 1-6, 0.1 M), the concentration of anthocyanin was 0.2 mM, and the concentration of punicalagin was 8 mM. The anthocyanin solution and the punicalagin solution were mixed in equal volumes, that is, the molar ratio of anthocyanin C3G to punicalagin (PUN) in the mixed solution was 1:40. After vortexing for 2 min, the solutions were placed at 25°C for reaction for 120 min.

[0046] Test method: Use UV-visible spectrophotometer to scan the reaction solution at 400-700 nm. The test results are as follows: Figure 5 shown.

[0047] pH is one of the important factors that limit the industrial application of anthocyanins. This is because the structure and color of anthocyanins will change with the change of pH. When the pH range is less than 2, anthocyanins mainly exist in the form of red yellow cations. When the pH range is 3-6, anthocyanins gradually change into colorless methanol pseudobases and chalcone forms, and the red color gradually fades. When the pH range is 6-7, anthocyanins exist in the form of neutral quinone bases, which are purple or light purple; when the pH range is 8-10, anthocyanins exist in the form of resonance-stabilized quinone bases, which are purple or blue. The change in the structure of anthocyanins is manifested as a decrease in the maximum absorption peak of 520nm. For example Figure 5 As shown in the figure, as the pH increases, the absorbance at 520 nm of the anthocyanin solution without punicalagin (where the concentration of anthocyanin is the same as that of anthocyanin added with punicalagin) gradually decreases, while the anthocyanin solution with punicalagin shows two different phenomena with the change of pH. When the solution pH is 1 and 2, the addition of punicalagin reduces the absorbance of anthocyanin at 520 nm, while when the solution pH ranges from 3 to 6, the addition of punicalagin increases the absorbance of anthocyanin at 520 nm. The reason for this phenomenon is related to the structure of anthocyanin. Methanol pseudoalkali and chalcone are the best configurations for anthocyanin to co-pigment with co-pigment molecules, rather than the form of yellow cation. Therefore, under appropriate pH conditions, punicalagin can improve the structural stability of anthocyanin, and most food matrices are acidic, which expands the application range of anthocyanin to a certain extent.

[0048] (2) Thermal stability Test method for non-microcapsules: anthocyanin (C3G) and punicalagin are dissolved in citric acid phosphate buffer solution (pH=3.5, 0.1 M), the concentration of anthocyanin is 0.2 mM, the concentration of punicalagin is 8 mM, the anthocyanin solution and the punicalagin solution are mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution is 1:40, vortex for 2 min, place at 25°C for reaction for 120 min, place at 50°C, 70°C and 90°C for 4 h, respectively, and take samples to determine the anthocyanin content.

[0049] The test method for preparing microcapsules is as follows: anthocyanin (C3G) and punicalagin are dissolved in a citric acid phosphate buffer solution (pH=3.5, 0.1 M), the concentration of anthocyanin is 0.2 mM, the concentration of punicalagin is 8 mM, the anthocyanin solution and the punicalagin solution are mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution is 1:40, vortexed for 2 min, and then reacted at 25°C for 120 min to obtain a mixed solution, the mixed solution is added to an 80 mg / mL chondroitin sulfate (CS) aqueous solution or a sodium alginate (SA) aqueous solution at a volume ratio of 1:1, and homogenized at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; the primary complex solution is added dropwise to an 8 mg / mL carrageenan (KC) colloidal solution (formed by dissolving carrageenan in 70°C hot water), the pH is adjusted to 3.5 at 30°C using 0.1 M HCl solution, and frozen at 0°C for 50 min. The punicalagin-anthocyanin microcapsules, denoted as CS-PUN-C3G-KC or SA-PUN-C3G-KC, were placed at 50°C, 70°C and 90°C for 4 h, respectively, and samples were taken to determine the anthocyanin content.

[0050] Determination test method: The content of anthocyanins in the solution was determined by high performance liquid chromatography. The liquid chromatography conditions were as follows: the chromatographic column was ZORBAX SB-C18 (4.6 × 250 mm, 5μm), the running medium was 5% formic acid water and methanol in a ratio of 7:3, the column temperature was 30°C, and the elution time was 10 min.

[0051] High temperature will accelerate the degradation of anthocyanins, so it is crucial to improve the retention rate of anthocyanins during processing and storage. Figure 6 As shown in the results, the increase of heating temperature and heating time will lead to the decrease of anthocyanin content. However, the addition of punicalagin can improve the retention rate of anthocyanin during heating. h, the retention rates of anthocyanins in the solution without punicalagin were 87.01%, 62.70% and 24.32%, respectively; the retention rates of anthocyanins in the solution with punicalagin were increased to 93.66%, 73.96% and 39.39%, respectively; after the anthocyanins with punicalagin were made into microcapsules CS-PUN-C3G-KC, the retention rates of anthocyanins were increased to 97.89%, 86.37% and 65.74%, respectively; after the anthocyanins with punicalagin were made into microcapsules SA-PUN-C3G-KC, the retention rates of anthocyanins were increased to 94.68%, 80.46% and 58.78%, respectively; the results showed that punicalagin could reduce the effect of temperature on high anthocyanins, and the effect on anthocyanins could be further reduced by forming microcapsules after intermolecular co-pigmentation reaction between punicalagin and anthocyanins, and the best effect was achieved by using chondroitin sulfate and carrageenan as wall materials to form microcapsules.

[0052] (3) Storage stability Test method for non-microcapsules: anthocyanin (C3G) and punicalagin are dissolved in citric acid phosphate buffer solution (pH=3.5, 0.1 M), the concentration of anthocyanin is 0.2 mM, the concentration of punicalagin is 8 mM, the anthocyanin solution and the punicalagin solution are mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution is 1:40, vortex for 2 min, place at 25°C for reaction for 120 min, store at 25°C and 4°C in the dark for 28 d, respectively, and take samples to determine the C3G content.

[0053] The test method for preparing microcapsules is as follows: anthocyanin (C3G) and punicalagin are dissolved in a citric acid phosphate buffer solution (pH=3.5, 0.1 M), the concentration of anthocyanin is 0.2 mM, the concentration of punicalagin is 8 mM, the anthocyanin solution and the punicalagin solution are mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution is 1:40, vortexed for 2 min, and then reacted at 25°C for 120 min to obtain a mixed solution, the mixed solution is added to an 80 mg / mL chondroitin sulfate (CS) aqueous solution or a sodium alginate (SA) aqueous solution at a volume ratio of 1:1, and homogenized at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; the primary complex solution is added dropwise to an 8 mg / mL carrageenan (KC) colloidal solution (formed by dissolving carrageenan in 70°C hot water), the pH is adjusted to 3.5 at 30°C using 0.1 M HCl solution, and frozen at 0°C for 50 min. The mindean punicalagin-anthocyanin microcapsules, denoted as CS-PUN-C3G-KC or SA-PUN-C3G-KC, were stored in the dark at 25°C and 4°C, respectively, for 28 days, and samples were taken to determine the C3G content.

[0054] The test method for determination is the same as the test method for determination of thermal stability.

[0055] The results of the test are as follows Figure 7As shown in Figure 2, with the extension of storage time and the increase of storage temperature, the anthocyanin content in all components gradually decreased. d. The retention rates of anthocyanins in the solution without punicalagin were 55.65% and 71.00%, and those in the solution with punicalagin were increased to 66.89% and 92.63%. After the anthocyanin with punicalagin was made into microcapsules CS-PUN-C3G-KC, the retention rates of anthocyanins were increased to 79.36% and 96.82%, respectively. After the anthocyanin with punicalagin was made into microcapsules SA-PUN-C3G-KC, the retention rates of anthocyanins were increased to 72.67% and 94.79%, respectively. This indicates that the addition of punicalagin can prolong the storage period of anthocyanins, and the microcapsules formed by the intermolecular co-pigmentation reaction of punicalagin and anthocyanins can further prolong the storage period of anthocyanins. The combination of chondroitin sulfate and carrageenan as wall materials to form microcapsules has the best effect.

[0056] (4) Light stability Test method for non-microcapsules: anthocyanin (C3G) and punicalagin were dissolved in citric acid phosphate buffer solution (pH = 3.5, 0.1 M), the concentration of anthocyanin was 0.2 mM, the concentration of punicalagin was 8 mM, the anthocyanin solution and the punicalagin solution were mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution was 1:40, vortexed for 2 min, placed at 25°C for reaction for 120 min, stored under incandescent lamp (5000 lux) and ultraviolet lamp (8 W) for 72 h, respectively, and samples were taken to determine the C3G content.

[0057] The test method for preparing microcapsules is as follows: anthocyanin (C3G) and punicalagin are dissolved in a citric acid phosphate buffer solution (pH=3.5, 0.1 M), the concentration of anthocyanin is 0.2 mM, the concentration of punicalagin is 8 mM, the anthocyanin solution and the punicalagin solution are mixed in equal volumes, that is, the molar ratio of anthocyanin to punicalagin in the solution is 1:40, vortexed for 2 min, and then reacted at 25°C for 120 min to obtain a mixed solution, the mixed solution is added to an 80 mg / mL chondroitin sulfate (CS) aqueous solution or a sodium alginate (SA) aqueous solution at a volume ratio of 1:1, and homogenized at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; the primary complex solution is added dropwise to an 8 mg / mL carrageenan (KC) colloidal solution (formed by dissolving carrageenan in 70°C hot water), the pH is adjusted to 3.5 at 30°C using 0.1 M HCl solution, and frozen at 0°C for 50 min. The mindean punicalagin-anthocyanin microcapsules, denoted as CS-PUN-C3G-KC or SA-PUN-C3G-KC, were stored under incandescent light (5000 lux) and ultraviolet light (8 W) for 72 h, and samples were taken to determine the C3G content.

[0058] The test method for determination is the same as the test method for determination of thermal stability.

[0059] The results of the test are as follows Figure 8 As shown in Figure 2, light can also cause the degradation of anthocyanins. h, the anthocyanin content in all components gradually decreased, the retention rates of anthocyanin in the solution without punicalagin were 88.76% and 42.90%, the effect of ultraviolet light on anthocyanin was greater than that of incandescent light, the retention rates of anthocyanin in the solution with punicalagin increased to 94.31% and 84.67%, the retention rates of anthocyanin in the microcapsules CS-PUN-C3G-KC with punicalagin added increased to 98.67% and 92.56%, respectively, and the retention rates of anthocyanin in the microcapsules SA-PUN-C3G-KC with punicalagin added increased to 96.49% and 89.68%, respectively; this shows that the addition of punicalagin can reduce the effect of light on anthocyanin, and the microcapsules formed by the intermolecular co-pigmentation reaction of punicalagin and anthocyanin can further reduce the effect of light on anthocyanin, and the combination of chondroitin sulfate and carrageenan as wall materials to form microcapsules has the best effect.

[0060] 4. Punicalagin improves the stability of anthocyanins in juice Punicalagin was added to pomegranate juice so that the concentration of punicalagin in the juice was 1 mg / mL, 2 mg / mL and 3 mg / mL, respectively. The juice was then sterilized by ultrahigh pressure (600 Mpa / 5 min), stored at 4°C for 28 days, and samples were taken at 7-day intervals to determine the anthocyanin content.

[0061] Test method: The total anthocyanin content was determined by the pH differential method. 0.6 mL of juice was mixed with 5.4 mL of potassium chloride buffer (0.025 M, pH = 1.0) and sodium acetate buffer (0.4 M, pH = 4.5), respectively. The mixture was incubated in a dark room for 30 minutes. The absorbance of the supernatant at 510 nm and 700 nm was measured using a UV-visible spectrophotometer with a 1 cm path length cuvette. All samples were measured at room temperature, with distilled water as the blank. The results were calculated according to the following formula.

[0062] TAC=A×MW×DF×100 / (ε×L)(8) In the formula, A=(A 510 -A 700 ) pH1.0 - (A 510 -A 700 ) pH4.5 , A 510 , A 700Respectively represent the absorbance of the sample at 510 nm and 700 nm at different pH values. The MW value is 449.2 g / mol, which is the molecular weight of cyanidin-3-glucoside. DF represents the dilution factor. ε is the extinction coefficient of cyanidin-3-glucoside (Cy-3-glu), which is 26,900 L / mol·cm-1. L is the thickness of the quartz cuvette (1 cm).

[0063] As shown in Table 2, after 28 days of storage, the addition of punicalagin significantly increased the retention rate of anthocyanins in the juice. The anthocyanin retention rate of the juice without punicalagin was 63.89%, while the retention rates of the juice with added anthocyanins were 70.62%, 78.12% and 82.87%, respectively. Moreover, the retention rates increased with the increase of punicalagin concentration. The experimental results further proved the effective role of punicalagin in improving the stability of anthocyanins.

[0064] Table 2 Retention rate of anthocyanins in juice In the present invention, punicalagin is used as a co-pigment molecule to undergo a co-pigmentation reaction with anthocyanins to form a complex of punicalagin and anthocyanins, wherein punicalagin and anthocyanins are bound by non-covalent interactions, and hydrogen bonds and van der Waals forces are the main driving forces, and this combination has important molecular recognition and stabilization effects. Hydrogen bonds form strong attraction between molecules through charge interactions between donors and acceptors, especially between functional groups such as hydroxyl, carbonyl, and amino groups, and these specific chemical groups are ubiquitous in the molecular structures of punicalagin and anthocyanins. Van der Waals forces are a weaker interaction that helps molecules maintain a bound state within a shorter distance through the action between instantaneous dipoles and induced dipoles. In addition, this binding process is usually affected by the pairing of the molecular spatial structure, and there is a good spatial conformational match between punicalagin and anthocyanin, thereby improving the binding stability of the two. The formed punicalagin and anthocyanin complex is further made into microcapsules, which have better light, temperature, and storage stability than the punicalagin and anthocyanin complex. The wall material of the microcapsule has a certain influence on the stability. The combination of chondroitin sulfate and carrageenan as the wall material to form microcapsules has the best effect.

[0065] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A method for improving the color and stability of anthocyanins by combining with punicalagin, characterized in that: include: This is achieved through the intermolecular co-pigmentation reaction between punicalagin and anthocyanin.

2. The method according to claim 1, characterized in that The molar ratio of punicalagin to anthocyanin is 1:10-80.

3. The method according to claim 2, characterized in that The specific steps include: Step 1, extraction of punicalagin: using pomegranate peel as raw material, and subjecting it to crushing, sieving, ultrasonic extraction, macroporous resin purification, fractional extraction, and liquid chromatography purification to obtain punicalagin; Step 2, dissolving the punicalagin and anthocyanin obtained in step 1 in an acidic buffer solution; Step 3: Mix the punicalagin solution and anthocyanin solution obtained in step 2, and carry out intermolecular co-pigmentation reaction at 4-25° C. to obtain a mixed solution, and the reaction time is 40-120 min.

4. The method according to claim 3, characterized in that Also includes the steps: Step 4: Add the mixed solution obtained in step 3 to 80 mg / mL chondroitin sulfate or sodium alginate aqueous solution at a volume ratio of 1:1, and homogenize at a speed of 10000-12000 rpm for 2 min to obtain a primary complex solution; add the primary complex solution dropwise to 8 mg / mL carrageenan solution, adjust the pH to 3.5 at 30°C with 0.1 M HCl solution, and freeze at 0°C for 50 min to obtain punicalagin-anthocyanin microcapsules.

5. The method according to claim 3, characterized in that Step 1 specifically includes: S11, placing pomegranate peel in a constant temperature box to dry, crushing the dried pomegranate peel, and passing through a 200-mesh sieve to obtain pomegranate peel powder; S12, take 100-200 g of pomegranate peel powder, add 2 L of 20-80% methanol aqueous solution by volume, perform ultrasonic extraction for 40-120 min, and centrifuge at a centrifugal force of 10,000 g for 10-15 min to obtain a crude extract of punicalagin; S13, removing methanol from the crude extract of punicalagin by rotary evaporation, and concentrating the aqueous phase to obtain a punicalagin concentrate; S14, purifying the punicalagin concentrate with a macroporous resin, performing graded extraction with petroleum ether and ethyl acetate, and rotary evaporating the ethyl acetate extraction component to dryness to obtain an ethyl acetate extraction product; S15, re-dissolving the product extracted with ethyl acetate in methanol, separating and purifying it by liquid chromatography, and freeze-drying it to obtain punicalagin.

6. The method according to claim 3, characterized in that Step 2 specifically includes: dissolving punicalagin and anthocyanin in 0.1 M citric acid phosphate buffer solution, wherein the pH range of the buffer solution is 3-6.

7. The method according to claim 3, characterized in that Step S14 specifically includes: using AB-8 macroporous resin, a chromatography column with an inner diameter of 35 mm and a height of 50 cm for purification, wherein the loading and elution speeds are both 1 mL / min, the eluent is water, and 0.8 L of methanol aqueous solution with a volume mass fraction of 20%, 40%, 60% and 80% is used for gradient elution, respectively, collecting 40% and 60% elution components, removing methanol by rotary evaporation and concentrating the aqueous phase to 20-50 mL, then adding 2 volumes of petroleum ether for degreasing, repeating 2 times, discarding the petroleum ether, adding 2 volumes of ethyl acetate to the remaining aqueous phase for extraction, repeating 4-6 times, retaining the ethyl acetate extract, and rotary evaporating the ethyl acetate extract to obtain the ethyl acetate extract product; in step S15, the conditions for separation and purification by liquid chromatography are: the chromatographic column is ZORBAX-SB C18 (9.4×250 mm, 5 μm), the mobile phase was 0.1% formic acid in water and methanol, and the elution gradient was 0-5 min, 5%-10% methanol, 5-15 min, 10%-30% methanol, 15-20 min, 30%-80% methanol, 20-25 min, 100% methanol, 25-30 min, 100%-5% methanol, 30-35 min, 5% methanol.

8. The method according to claim 2, characterized in that The molar ratio of anthocyanin to punicalagin is 1:80, and anthocyanin is a monomer of cyanidin-3-O-glucoside.

9. Punicalagin-anthocyanin complex prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 in fruit juice processing.

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