Method for improving stability of purple corn anthocyanin

Abstract

The invention discloses a method for improving stability of purple corn anthocyanin, which comprises the following steps: mixing malic acid serving as a modifier with purple corn anthocyanin, dissolving in an ethanol aqueous solution, and modifying the purple corn anthocyanin by using pyridine as a catalyst to obtain acylated purple corn anthocyanin; according to the acylated purple corn anthocyanidin, the stability of the purple corn anthocyanidin is improved, the deterioration speed of the purple corn anthocyanidin in the storage process is slowed down, meanwhile, the original pH developing performance and antioxidant activity of the anthocyanidin are reserved, the adopted experiment steps are simple, the cost is low, and more possibilities are brought to application of the purple corn anthocyanidin.

Description

Technical Field

[0001] The invention relates to the technical field of natural pigments, and in particular to a method for improving the stability of purple corn anthocyanins. Background Art

[0002] Anthocyanidins are a class of water-soluble natural pigments found widely in plants. They are the colored aglycones derived from the hydrolysis of anthocyanins and are the primary colorants in fruits, vegetables, and flowers. There are over 20 known anthocyanidins, six of which are important in food: pelargonium pigment, cyanidin, delphinium pigment, peony pigment, morning glory pigment, and malva pigment. They have a wide range of uses, including food coloring, dyes, pharmaceuticals, and cosmetics.

[0003] Anthocyanins are used in many fields due to their environmental friendliness, rich colors, and wide availability. Firstly, they are often used as dyes in the food coloring industry, as well as in everyday dyes such as cotton fabric and hair dyeing. Secondly, they also have great potential in medicine. Anthocyanins have a strong ability to scavenge free radicals, thereby protecting cells from oxidative damage. Anthocyanins can also inhibit the proliferation of tumor cells, showing potential anti-tumor effects. In the cosmetics industry, anthocyanins have attracted widespread attention due to their ability to resist ultraviolet radiation.

[0004] However, due to the poor stability of anthocyanins, they are easily affected by environmental factors such as temperature, light, and pH, which makes them prone to denaturation during production, storage, and application, thus limiting their scope of application. Therefore, it is crucial to improve the stability of anthocyanins. Summary of the Invention

[0005] The present invention provides a method for improving the stability of purple corn anthocyanidins. Malic acid is used as an acyl donor and pyridine is used as a catalyst to improve the stability of purple corn anthocyanidins. Finally, the thermal stability, light stability, and acid-base stability of the acylated purple corn anthocyanidins are tested. This method provides a new idea for the comprehensive development and utilization of anthocyanidin resources and the long-term preservation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for improving the stability of purple corn anthocyanins, the process is as follows:

[0008] (1) Purple corn anthocyanin (PCA) and acyl donor malic acid were dissolved in an ethanol aqueous solution;

[0009] (2) After pyridine is added to react, the mixed solution is dialyzed and freeze-dried to obtain acylated purple corn anthocyanin powder.

[0010] The mass fraction of the ethanol aqueous solution is 50-60%.

[0011] The mass ratio of the purple corn anthocyanidin to malic acid is 1:100 to 1:600.

[0012] The added amount of the pyridine is not less than 5% of the molar number of the purple corn anthocyanin.

[0013] The reaction is carried out at 60° C. for 30 to 120 minutes.

[0014] The dialysis was performed in a 200 Da dialysis bag for 12 to 24 hours.

[0015] The modified purple corn anthocyanin of the present invention has improved temperature stability, light stability and pH stability to a certain extent, has a simple operation process and low cost, and provides good basic conditions for the storage, transportation and subsequent application of the anthocyanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The infrared spectra of the acylation effect of PCA at different acyl donor ratios;

[0017] Figure 2 The infrared spectra of the acylation effect of PCA at different reaction temperatures;

[0018] Figure 3 The infrared spectra of the acylation effect of PCA at different reaction times;

[0019] Figure 4 The retention rates of PCA and MPCA under different temperature conditions;

[0020] Figure 5 is the retention rate of PCA and MPCA under natural light conditions;

[0021] Figure 6 is the retention rate of PCA and MPCA under light-shielding conditions;

[0022] Figure 7 is the retention rate of PCA and MPCA under different pH environments;

[0023] Figure 8 is the DPPH free radical scavenging ability of PCA and MPCA;

[0024] Figure 9 This is the pH color effect of PCA and MPCA. DETAILED DESCRIPTION

[0025] The present invention is further described below with reference to specific embodiments.

[0026] The purple corn anthocyanidin used in the present invention is obtained by extracting, concentrating and purifying anthocyanidin from purple corn husk according to a conventional method; the malic acid used is commercially available DL-malic acid with an AR content of 99.5%.

[0027] Example 1

[0028] 1 g purple corn anthocyanin (PCA) was mixed with 100 g DL-malic acid and dissolved in 40 mL of 60% ethanol aqueous solution. 1 mL of pyridine was added as a catalyst. The mixed solution was kept in a constant temperature water bath at 60°C for 30 min to obtain an MPCA alcohol solution. After 12 h of dialysis with a 200 Da dialysis bag to remove excess malic acid and pyridine, an MPCA solution was obtained. After freeze-drying, the acylated purple corn anthocyanin powder MPCA was obtained.

[0029] Examples 2-4

[0030] The amount of DL-malic acid added in Example 1 was adjusted to 200 g, 400 g, and 600 g, and the other steps were the same as in Example 1 to obtain different products, which were then detected by FT-IR.

[0031] The functional groups of purple corn anthocyanins were analyzed by Fourier transform infrared spectroscopy (FTIR), and the acylation modification effect was analyzed by comparing samples treated with different proportions of modifier (DL-malic acid). Figure 1 The infrared spectra of the acylation effect of PCA under different acyl donor ratios are shown in the figure. It can be seen from the figure that the untreated purple corn anthocyanin has a wavelength of 1650 cm -1 There is a characteristic peak of benzene ring skeleton vibration near it, reflecting the existence of its aromatic ring structure, at 3400cm -1 The absorption peaks appearing near the phenolic hydroxyl group (-OH) are the stretching vibrations of the anthocyanins treated with different proportions of DL-malic acid at 1750 cm -1 There are absorption peaks near 1750cm, which is the stretching vibration of ester C=O bond, indicating that the carboxyl group of DL-malic acid and the hydroxyl group of anthocyanin have undergone esterification reaction. Further comparison of the treatment effects of different ratios of modifiers found that when the mass ratio of anthocyanin to modifier was 1:100, the absorption peak at 1750cm -1 The ester bond characteristic peak appeared at 1750cm -1 There are characteristic peaks of ester bonds near 3400 cm -1The width of the hydroxyl peak at the position increases, indicating that DL-malic acid is excessive. When the ratio increases to 1:600, the characteristic peak of the benzene ring skeleton vibration is significantly weakened. It may be that excessive DL-malic acid may cause the destruction of the PCA molecular structure or the occurrence of side reactions, thereby affecting its chemical properties. This shows that when the ratio of purple corn anthocyanin to acylating agent is 1:100, the acylation effect is best and does not affect the properties of anthocyanin.

[0032] Examples 5-7

[0033] The water bath temperature of the mixed solution in Example 1 was adjusted to 40° C., 50° C., and 70° C. The other steps were the same as those in Example 1, and different products were obtained. The products were detected by infrared spectroscopy (FT-IR).

[0034] Figure 2 The infrared spectra of the acylation effect of PCA at different reaction temperatures are shown in Figure 2. By comparing the Fourier transform infrared spectroscopy (FT-IR) characteristics of purple corn anthocyanin at different reaction temperatures, the functional group changes are deeply analyzed. It can be seen from the figure that the 1650 cm -1 The absorption peaks on the left and right are the skeleton vibration peaks of the benzene ring in anthocyanin, reflecting the characteristics of the aromatic structure in the anthocyanin molecule. -1 The left and right sides are the stretching vibrations of -OH on the phenolic hydroxyl group; compared with the infrared peaks of the untreated purple corn anthocyanin, the anthocyanin modified by DL-malic acid at 60 ° C has a peak at 1740 cm -1 A new absorption peak appeared near the 1740cm-1, which was the stretching vibration of the ester C=O bond, indicating that the carboxyl group of DL-malic acid and the hydroxyl group of anthocyanin had an esterification reaction, and the acylation modification of purple corn anthocyanin was successfully achieved. When the temperature was controlled at 40℃, 50℃ and 70℃, the absorption peak at 1740cm-1 -1 No obvious ester characteristic peak appeared nearby, indicating that at these temperatures, DL-malic acid failed to be effectively grafted onto the anthocyanin molecule and no acylation reaction of purple corn anthocyanin occurred. Moreover, at 70°C, the absorption peaks of purple corn anthocyanin weakened to varying degrees, which may be due to partial degradation of anthocyanin or increased side reactions caused by high temperature. Based on the above analysis, the most suitable temperature for the acylation of purple corn anthocyanin is 60°C.

[0035] Examples 8-10

[0036] The water bath time of the mixed solution in Example 1 was adjusted to 1 h, 1.5 h, and 2 h. The other steps were the same as those in Example 1, and the experiment was carried out to obtain different products, which were then detected by infrared spectroscopy (FT-IR).

[0037] By comparing the Fourier transform infrared spectra (FT-IR) of purple corn anthocyanins after different reaction times, the changes in their functional groups were analyzed. Figure 3 The infrared spectra of the acylation effect of PCA at different reaction times are shown in the figure. It can be seen from the figure that 1650cm -1 The absorption peaks on the left and right are the C=C stretching vibrations of the benzene ring skeleton in anthocyanin, reflecting the characteristics of the aromatic structure in the anthocyanin molecule. -1 The broad peaks on the left and right are the stretching vibrations of -OH on the phenolic hydroxyl group, indicating that there are abundant active hydroxyl sites in the molecule. After modification with DL-malic acid, all reaction time groups have a peak at 1740 cm -1 A new absorption peak appeared near the ester C=O bond, which indicated that the carboxyl group of DL-malic acid and the hydroxyl group of anthocyanidin had an esterification reaction, and the acylation modification of purple corn anthocyanidin was successfully achieved. However, further comparison of the infrared peaks of the acylated purple corn anthocyanidin in four groups with different treatment times showed that with the increase of treatment time, the benzene ring skeleton vibration peak (1650cm -1 ) and hydroxyl peak (3400cm -1 ) intensity showed a trend of gradual attenuation, which may be due to the partial destruction of the aromatic ring structure in the PCA molecule or excessive consumption of hydroxyl groups caused by long-term reaction. This phenomenon is consistent with the ester bond peak (1740cm -1 ) is contradictory to the stability of the esterification reaction. It is speculated that this may be due to the increase in the proportion of side reactions (such as hydrolysis or oxidation) after the esterification reaction reaches equilibrium; or the thermal degradation of PCA molecules under high temperature conditions. Therefore, considering the comprehensive reaction efficiency and structural stability, 0.5h was confirmed to be the optimal reaction time for acylation modification.

[0038] Example 11

[0039] The method for determining the stability of acylated anthocyanidins is as follows:

[0040] PCA and MPCA (prepared in Example 1) were prepared into two solutions of the same concentration. The two solutions were placed in a water bath at 20°C, 35°C, 50°C, 65°C and 80°C for 3 h under light-shielding conditions, and the anthocyanin retention rate was calculated.

[0041] The retention rate of anthocyanins was detected and calculated by UV-Vis spectrophotometer. The results showed that after anthocyanins were treated at different temperatures for 3 h, the retention rate of anthocyanins decreased with the increase of temperature. Figure 4 Figure 2 shows the retention rates of PCA and MPCA under different temperature conditions. By comparing the retention rates of anthocyanidins (prepared in Example 1) before and after modification, it can be seen that the anthocyanidins modified with DL-malic acid have a higher retention rate than the unmodified anthocyanidins at the same temperature, indicating that the temperature stability of acylated anthocyanidins is significantly improved.

[0042] Example 12

[0043] The method for determining the stability of acylated anthocyanidins is as follows:

[0044] PCA and MPCA (prepared in Example 1) were prepared into two solutions with the same concentration. The two solutions were placed at room temperature under natural light for 10 days. The absorbance was measured every two days to calculate the anthocyanin retention rate.

[0045] The retention rate of anthocyanins was detected and calculated by UV-Vis spectrophotometer. The results showed that under natural light, the retention rate of anthocyanins would gradually decrease over time. Figure 5 is the retention rate of PCA and MPCA under natural light conditions. By comparing the retention rates of anthocyanins before and after modification (prepared in Example 1), it can be seen that under the same time, the retention rate of modified anthocyanins is higher than that of unmodified anthocyanins, indicating that the light stability of acylated anthocyanins is significantly improved.

[0046] Example 13

[0047] The method for determining the stability of acylated anthocyanidins is as follows:

[0048] PCA and MPCA (prepared in Example 1) were prepared into two solutions with the same concentration. The two solutions were placed at room temperature in the dark for 10 days. The absorbance was measured every two days to calculate the anthocyanin retention rate.

[0049] The retention rate of anthocyanins was detected and calculated by UV-Vis spectrophotometer. Figure 6 The retention rates of PCA and MPCA under shading conditions are shown. By comparing the retention rates of anthocyanins (prepared in Example 1) before and after modification, it can be seen that the retention rate of anthocyanins gradually decreases over time. At the same time, the retention rate of modified anthocyanins is significantly higher than that of unmodified anthocyanins, and anthocyanins are almost not degraded in the first six days, indicating that the stability of acylated anthocyanins is significantly improved.

[0050] Example 14

[0051] The method for determining the stability of acylated anthocyanidins is as follows:

[0052] PCA and MPCA (prepared in Example 1) were prepared into two solutions of the same concentration. The two solutions were placed in an environment of pH = 2, 4, 6, 8, 10, and 12, respectively. After standing for 3 hours in the dark, the absorbance was measured and the anthocyanin retention rate was calculated.

[0053] The retention rate of anthocyanins was detected and calculated by UV-Vis spectrophotometer. Figure 7Figure 3 is the concentration change of PCA and MPCA under different pH environments. It can be seen that the retention rate of the modified anthocyanin (prepared in Example 1) at different pH is slightly higher than that of the unmodified anthocyanin, indicating that the stability of the modified anthocyanin in different pH environments is slightly higher than that of the unmodified anthocyanin.

[0054] At the same time, anthocyanidins and acylated anthocyanidins (prepared in Example 1) were compared under pH = 2, 4, 6, 8, 10, and 12. Figure 9 This is the pH color development effect of PCA and MPCA. It can be seen that the modified anthocyanins still maintain good color development ability.

[0055] Example 15

[0056] The method for determining the activity of acylated anthocyanidins is as follows:

[0057] PCA was mixed with MPCA (prepared in Example 1) and 0.1 mmol / L DPPH alcohol solution. 2 mL of PCA and MPCA solutions of different concentrations (0.1, 0.2, 0.4, 0.6, and 0.8 mg / mL) were taken respectively, and 4 mL of DPPH solution was added. The mixture was mixed evenly, and allowed to stand at room temperature for 60 min. The mixture was centrifuged at 3000 rpm for 20 min. The supernatant was taken and the absorbance at 517 nm was measured to calculate the DPPH scavenging rate. VC was used as the control group to verify the antioxidant activity of MPCA.

[0058] The free radical scavenging rates of VC, PCA and MPCA were detected and calculated by UV-Vis spectrophotometer. It was found that VC was used as the control group. Figure 8 is the DPPH free radical scavenging ability of PCA and MPCA (prepared in Example 1). By comparing the scavenging ability of DPPH free radicals, it can be seen that with the increase of anthocyanidin concentration, the DPPH scavenging ability is gradually enhanced. When the concentration of unmodified anthocyanidin is 0.8 mg / mL, the antioxidant activity is greater than that of VC, while when the concentration of modified anthocyanidin is 0.2 mg / mL, the antioxidant activity is greater than that of VC, indicating that the acylated anthocyanidin still maintains good antioxidant activity.

Claims

1. A method for improving the stability of purple corn anthocyanins, characterized in that: The specific steps are as follows: Purple corn anthocyanidin and malic acid are dissolved in an ethanol aqueous solution, pyridine is added for reaction, the mixed solution is dialyzed, and acylated purple corn anthocyanidin powder is obtained after freeze-drying.

2. The method for improving the stability of purple corn anthocyanins according to claim 1, wherein: The mass fraction of the ethanol aqueous solution is 50-60%.

3. The method for improving the stability of purple corn anthocyanidins according to claim 1, wherein: The mass ratio of the purple corn anthocyanidin to malic acid is 1:100 to 1:

600.

4. The method for improving the stability of anthocyanins in purple corn according to claim 1, wherein: The added amount of the pyridine is not less than 5% of the molar number of the purple corn anthocyanin.

5. The method for improving the stability of anthocyanins in purple corn according to claim 1, wherein: The reaction is carried out at 60° C. for 30 to 120 minutes.

6. The method for improving the stability of anthocyanins in purple corn according to claim 1, characterized in that: The dialysis was performed in a 200 Da dialysis bag for 12 to 24 hours.

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