Petunidin-based anthocyanins and blue pigments, and methods of making and using the same

Abstract

The present application belongs to the technical field of pigment preparation, and provides petunidin anthocyanins and blue pigments, and a preparation method and application thereof. The present application extracts anthocyanins from Lycium ruthenicum Murr by alcohol extraction and concentration; removes sugars, proteins and other impurities in the crude extract of Lycium ruthenicum Murr anthocyanins by using a macroporous resin; and then purifies the anthocyanin freeze-dried powder by using a preparative liquid chromatography system, and collects petunidin anthocyanins mainly composed of petunidin-3-O-(trans-p-coumaroyl) rutinoside-5-O-glucoside. The petunidin anthocyanins obtained by the present application are complexed with metal ions, and the blue pigments obtained have good color and stability. Compared with the prior art technical solution of adding polysaccharides to improve the stability of natural blue pigments, the petunidin anthocyanins of the present application can also ensure the stability of the blue pigments without adding polysaccharides, and at the same time, will not reduce the color value of the blue pigments.

Description

Technical Field

[0001] This invention relates to the field of natural pigment preparation technology, and in particular to a petunia anthocyanin and its extraction method, a blue pigment and its preparation method and application. Background Technology

[0002] As people pay increasing attention to food safety and health, the demand for natural food colorings is also growing. Blue, as one of the three primary colors, plays a crucial role in coloring food. However, compared to red and yellow pigments, natural blue pigment resources are relatively scarce. Currently reported and applied natural blue pigments mainly include phycocyanin, gardenia blue, and indigo, which are insufficient to meet the color-matching needs and wide range of applications in the food industry. Therefore, developing novel natural blue pigments is of great significance for promoting the development of natural food colorings and the food industry.

[0003] Black goji berries (Lycium ruthenicum Murr.) are the fruit of a plant belonging to the genus Lycium in the Solanaceae family. Black goji berries contain a large amount of petunia-like anthocyanins, which give the fruit its purplish-black color. Anthocyanins are common natural pigments, and some anthocyanin-rich substances have been approved as food additives in China, the United States, and the European Union. In my country, anthocyanin-rich natural pigments such as purple sweet potato pigment and bayberry red are also used as colorants in candies, beverages, and pastries. Although anthocyanin-based natural pigments are more readily accepted by consumers, these anthocyanins have poor stability, are sensitive to light, heat, and pH, and are prone to fading, which limits their application prospects in the food industry.

[0004] Previous studies have shown that anthocyanins and other polyphenolic compounds can form complexes with metal ions through coordination bonds. In this process, some anthocyanins transform into a blue, negatively charged ionic quinone base structure, giving the anthocyanin complex its blue color. Furthermore, anthocyanin complexes exhibit greater stability in weakly acidic and neutral environments compared to free anthocyanins. Therefore, anthocyanin complexes have the potential to be used as natural blue pigments in food.

[0005] Based on the unique color-producing properties of anthocyanins, some studies in recent years have attempted to develop food-grade blue pigments using anthocyanins as raw materials. Chinese patent CN111685261A discloses a method for preparing iron-supplemented anthocyanin blue pigments. This method first extracts delphinidin anthocyanins from eggplant peel, black wheat bran, or black goji berry residue; then, it complexes the delphinidin anthocyanins with ferrous ions to obtain ferrous delphinidin anthocyanin compounds; finally, using polysaccharides (xanthan gum, sodium alginate, pectin) and ferrous delphinidin anthocyanin compounds as raw materials, it prepares an iron-rich functional blue pigment product. Because natural anthocyanins vary in the type of parent nucleus, the type and amount of glycosylation substitution, whether they are acylated, the acylation sites and numbers, and the type of acyl groups, different types of anthocyanins exhibit different color-producing properties and stability after binding with metal ions. Delphinidin anthocyanins have three hydroxyl groups on their B ring, resulting in relatively lower stability. To improve stability, patent CN111685261A added polysaccharides such as xanthan gum. Test Example 1 shows that a 1:10 mass ratio of delphinidin anthocyanins to xanthan gum is required to achieve good stability. The addition of a large amount of xanthan gum reduces the color value (coloring power per unit mass of pigment) of the functional blue pigment. Furthermore, purification using macroporous resin alone is insufficient to remove a large number of non-coloring impurities from eggplant peel, black wheat bran, or black goji berry residue, which also affects the coloring effect of the functional blue pigment formed from delphinidin anthocyanins. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide petunia anthocyanins, their extraction method, blue pigment, their preparation method, and applications. The petunia anthocyanins provided by this invention exhibit good color development and stability after complexing with metal ions.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for extracting petunia anthocyanins, comprising the following steps:

[0009] Using black goji berries as raw material, crude extract of black goji berry anthocyanins was obtained through alcohol extraction and concentration.

[0010] The crude extract of anthocyanins from black goji berries was injected into a macroporous resin, and after elution, concentration and freeze-drying, anthocyanin freeze-dried powder was obtained.

[0011] The anthocyanin lyophilized powder was dissolved and injected into a preparative liquid chromatography system for preparative-grade liquid chromatography separation and purification. The target component was detected by an ultraviolet detector and collected. The target component was then subjected to vacuum evaporation and lyophilization to obtain the petunia anthocyanins.

[0012] The parameters for preparative-grade liquid chromatography separation and purification include:

[0013] Mobile phase: Phase A is pure acetonitrile, and Phase B is an aqueous solution of formic acid with a volume percentage concentration of 1% to 2%.

[0014] The gradient elution procedure is as follows:

[0015] 0-6min: The volume fraction of phase A changes uniformly from 10% to 15%;

[0016] 6-21 min: The volume fraction of phase A changes from 15% to 21% at a constant rate;

[0017] 21-24 min: The volume fraction of phase A changes uniformly from 21% to 60%;

[0018] 24-27 min: The volume fraction of phase A changes uniformly from 60% to 10%;

[0019] 27-30 min: The volume fraction of phase A is maintained at 10%.

[0020] Preferably, the alcohol extraction and concentration includes: mixing black goji berries with an acidic ethanol solution and pulping, followed by ultrasonic-assisted extraction and filtration, and then concentrating the filtrate under reduced pressure to remove the ethanol, thereby obtaining the crude extract of black goji berry anthocyanins.

[0021] In the acidic ethanol solution, the volume concentration of ethanol is 50% to 80%, and the volume concentration of acid is 0.1% to 1%, wherein the acid includes at least one of hydrochloric acid, formic acid, and acetic acid;

[0022] The ratio of black goji berries to acidic ethanol solution is 1g: 5-20mL;

[0023] The ultrasound-assisted extraction time is 40–120 min;

[0024] The temperature for vacuum concentration is 40–50°C.

[0025] Preferably, the macroporous resin includes AB-8, D101, XAD-7, HPD-100, or DM-130; the specific surface area of ​​the macroporous resin is 450–550 m². 2 / g, with an average pore size of 10–50 nm and a particle size range of 0.3–1.25 mm;

[0026] The elution process includes: sequentially eluting the macroporous resin with four column volumes of each of the first elution reagent, the second elution reagent, the third elution reagent solution, and the fourth elution reagent, and collecting the eluent of the fourth elution reagent.

[0027] The first elution reagent is an acidic ethanol solution with a volume concentration of 0%.

[0028] The second elution reagent is an acidic ethanol solution with a volume concentration of 5%.

[0029] The third elution reagent is an acidic ethanol solution with a volume concentration of 20%.

[0030] The fourth elution reagent is an acidic ethanol solution with a volume concentration of 40%.

[0031] In the first elution reagent, the second elution reagent, the third elution reagent, and the fourth elution reagent, the volume concentration of the acid is independently 0.1% to 1%, and the acid independently includes at least one of hydrochloric acid, formic acid, and acetic acid.

[0032] Preferably, the parameters for the preparative liquid chromatography separation and purification further include: the flow rate of the mobile phase is 10-12 mL / min, the column temperature is 30℃, and the detection wavelength of the ultraviolet detector is 280 nm or 520 nm.

[0033] Preferably, the target component is a component with a retention time of 19.0 to 21.0 min.

[0034] The present invention also provides petunia anthocyanins obtained by the extraction method described above, wherein the main component of the petunia anthocyanins is petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside, and the mass content of petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside in the petunia anthocyanins is 5-100%.

[0035] The present invention also provides a blue pigment, which is an anthocyanin complex formed by petunia-type anthocyanins and metal ions as described in the above technical solution, wherein the metal ions include at least one of calcium ions, zinc ions, ferrous ions and magnesium ions.

[0036] The present invention also provides a method for preparing the blue pigment described in the above technical solution, comprising the following steps:

[0037] The anthocyanin solution of petunia and the metal ion solution were mixed, the pH of the resulting mixture was adjusted to 6-8, and then allowed to stand and freeze-dried to obtain the blue pigment.

[0038] Based on petunidin-3-O-(trans-p-coumaryl(rutin-5-O-glucoside), the molar ratio of metal ions to petunidin anthocyanins in the mixture is 1:0.2 to 1:6.

[0039] Preferably, the reagents for the petunia anthocyanin solution and the metal ion solution are water, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, or sodium carbonate-sodium bicarbonate buffer.

[0040] The concentration of the petunia anthocyanin solution is 0.05–20 g / L;

[0041] The concentration of the metal ion solution is 0.05–20 g / L.

[0042] The present invention also provides the application of the blue pigment described in the above technical solution or the blue pigment prepared by the above technical solution in food.

[0043] This invention provides a method for extracting petunia-like anthocyanins, comprising the following steps: using black goji berries as raw material, extracting and concentrating the extract with alcohol to obtain a crude extract of black goji berry anthocyanins; injecting the crude extract of black goji berry anthocyanins into a macroporous resin, eluting, concentrating, and lyophilizing to obtain anthocyanin lyophilized powder; dissolving the anthocyanin lyophilized powder, injecting it into a preparative liquid chromatography system for preparative-grade liquid chromatography separation and purification, detecting it with a UV detector, collecting the target component, and subjecting the target component to vacuum evaporation and lyophilization to obtain the petunia-like anthocyanins; the preparative-grade liquid chromatography separation and purification... The parameters include: mobile phase: phase A is pure acetonitrile, and phase B is an aqueous solution of formic acid with a volume percentage concentration of 1% to 2%; the gradient elution program is as follows: 0-6 min: the volume fraction of phase A changes from 10% to 15% at a constant rate; 6-21 min: the volume fraction of phase A changes from 15% to 21% at a constant rate; 21-24 min: the volume fraction of phase A changes from 21% to 60% at a constant rate; 24-27 min: the volume fraction of phase A changes from 60% to 10% at a constant rate; 27-30 min: the volume fraction of phase A is maintained at 10%.

[0044] In the extraction method provided by this invention, alcohol extraction and concentration can extract anthocyanins from black goji berries. The crude anthocyanin extract is purified using macroporous resin to remove impurities such as sugars and proteins. Then, the lyophilized anthocyanin powder is purified using a preparative liquid chromatography system to collect petunia-based anthocyanins, with petunidin-3-O-(trans-p-coumaryl)rutin-5-O-glucoside as the main component. The petunia-based anthocyanins obtained by this invention, after complexing with metal ions, produce a blue pigment with good color rendering and stability. Compared to existing technologies that add polysaccharides to improve the stability of natural blue pigments, the petunia-based anthocyanins of this application can maintain the stability of the blue pigment without adding polysaccharides; at the same time, it does not reduce the color value of the blue pigment. Attached Figure Description

[0045] Figure 1 The high-performance liquid chromatogram of the crude extract of black wolfberry anthocyanins obtained in Example 1;

[0046] Figure 2 The high-performance liquid chromatogram of the anthocyanin lyophilized powder obtained in Example 1 is shown below.

[0047] Figure 3 This is the liquid chromatogram of petunia anthocyanins obtained in Example 1;

[0048] Figure 4 Infrared spectra of ferrous sulfate, petunia anthocyanins, and blue pigment;

[0049] Figure 5 The circular dichroism spectrum of blue pigment;

[0050] Figure 6 This is a characterization diagram of the color stability of blue pigment in phosphate buffer (pH 7);

[0051] Figure 7 This is an image showing the appearance of the blue pigment in cream in Example 2.

[0052] Figure 8 This is an image showing the appearance of the blue pigment used in the gummies in Example 3.

[0053] Figure 9 The image shows the color stability of the blue pigment prepared using anthocyanins other than petunia anthocyanins in Comparative Example 1 in phosphate buffer (pH 7). Detailed Implementation

[0054] This invention provides a method for extracting petunia anthocyanins, comprising the following steps:

[0055] Using black goji berries as raw material, crude extract of black goji berry anthocyanins was obtained through alcohol extraction and concentration.

[0056] The crude extract of anthocyanins from black goji berries was injected into a macroporous resin, and after elution, concentration and freeze-drying, anthocyanin freeze-dried powder was obtained.

[0057] The anthocyanin lyophilized powder was dissolved and injected into a preparative liquid chromatography system for preparative-grade liquid chromatography separation and purification. The target component was detected by an ultraviolet detector and collected. The target component was then subjected to vacuum evaporation and lyophilization to obtain the petunia anthocyanins.

[0058] The parameters for preparative-grade liquid chromatography separation and purification include:

[0059] Mobile phase: Phase A is pure acetonitrile, and Phase B is an aqueous solution of formic acid with a volume percentage concentration of 1% to 2%.

[0060] The gradient elution procedure is as follows:

[0061] 0-6min: The volume fraction of phase A changes uniformly from 10% to 15%;

[0062] 6-21 min: The volume fraction of phase A changes from 15% to 21% at a constant rate;

[0063] 21-24 min: The volume fraction of phase A changes uniformly from 21% to 60%;

[0064] 24-27 min: The volume fraction of phase A changes uniformly from 60% to 10%;

[0065] 27-30 min: The volume fraction of phase A is maintained at 10%.

[0066] Unless otherwise specified, all raw materials used in this invention are preferably commercially available products.

[0067] This invention uses black goji berries as raw material, and obtains a crude extract of black goji berry anthocyanins through alcohol extraction and concentration.

[0068] In this invention, the alcohol extraction and concentration preferably includes: mixing black goji berries with an acidic ethanol solution and pulping them, then extracting and filtering them with ultrasonic assistance, and concentrating the resulting filtrate under reduced pressure to remove the ethanol, thereby obtaining the crude extract of black goji berry anthocyanins.

[0069] In this invention, the black goji berries preferably include fresh black goji berries and / or dried black goji berries; the black goji berries are preferably washed before alcohol extraction and concentration; this invention does not specifically limit the washing operation.

[0070] In this invention, the alcohol extraction and concentration is preferably performed once.

[0071] In this invention, the volume concentration of ethanol in the acidic ethanol solution is preferably 50% to 80%, more preferably 60% to 70%; the volume concentration of acid is preferably 0.1% to 1%, more preferably 0.3% to 0.7%; the acid preferably includes at least one of hydrochloric acid, formic acid and acetic acid.

[0072] In this invention, the preferred material-to-liquid ratio of black goji berries to acidic ethanol solution is 1g:5-20mL, and more preferably 1g:10-15mL.

[0073] In this invention, the temperature of the ultrasound-assisted extraction is preferably room temperature, and the time is preferably 40-120 min, more preferably 60-80 min.

[0074] In this invention, the temperature for vacuum concentration is preferably 40–50°C.

[0075] After obtaining the crude extract of black goji berry anthocyanins, the present invention injects the crude extract of black goji berry anthocyanins into a macroporous resin, and after elution, concentration and freeze-drying, obtains anthocyanin freeze-dried powder.

[0076] In this invention, the macroporous resin preferably includes AB-8, D101, XAD-7, HPD-100, or DM-130, and more preferably AB-8. In this invention, the specific surface area of ​​the macroporous resin is preferably 450–550 m². 2 The average pore size is preferably 10–50 nm, and the particle size range is preferably 0.3–1.25 mm. In this invention, the macroporous resin is preferably packed into a chromatography column; the chromatography column packed with macroporous resin is preferably cleaned before use, and the cleaning reagents are preferably ethanol, 0.5 mol / L hydrochloric acid solution, 0.5 mol / L sodium hydroxide solution, and water in that order.

[0077] In this invention, the preferred injection flow rate of the crude extract of black goji berry anthocyanins is 0.2 times column volume / h.

[0078] In this invention, the elution preferably includes: eluting the macroporous resin sequentially with four column volumes of each of a first elution reagent, a second elution reagent, a third elution reagent, and a fourth elution reagent, and collecting the eluent of the fourth elution reagent. In this invention, the first elution reagent is preferably an acidic ethanol solution with a volume concentration of 0%; the second elution reagent is preferably an acidic ethanol solution with a volume concentration of 5%; the third elution reagent is preferably an acidic ethanol solution with a volume concentration of 20%; and the fourth elution reagent is preferably an acidic ethanol solution with a volume concentration of 40%. In this invention, the volume concentration of the acid in the first, second, third, and fourth elution reagents is independently preferably 0.1% to 1%, more preferably 0.4% to 0.6%, and more preferably 0.5%; the acid independently preferably includes at least one of hydrochloric acid, formic acid, and acetic acid, and more preferably hydrochloric acid.

[0079] In this invention, the concentration temperature is preferably 40-50°C, and more preferably 45°C.

[0080] The present invention does not specifically limit the freeze-drying operation; any operation known to those skilled in the art can be used.

[0081] After obtaining the anthocyanin lyophilized powder, the present invention dissolves the anthocyanin lyophilized powder and injects it into a preparative liquid chromatography system for preparative-grade liquid chromatography separation and purification. The target component is detected by an ultraviolet detector and collected. The target component is then subjected to vacuum evaporation and lyophilization to obtain the petunia anthocyanins.

[0082] In this invention, the reagent used to dissolve the anthocyanin lyophilized powder is preferably one or more of the following: an acidic aqueous solution with a formic acid volume percentage of 1% to 2%, an acidic methanol solution with a methanol volume percentage of 1% to 20%, or an acidic acetonitrile solution with a acetonitrile volume percentage of 1% to 10%. The concentration of the anthocyanin lyophilized powder solution obtained by dissolving the anthocyanin lyophilized powder is preferably 5 to 40 mg / mL, more preferably 10 to 30 mg / mL, and even more preferably 20 mg / mL.

[0083] In this invention, the liquid chromatography column of the preparative liquid chromatography system is preferably a C18 column; the single injection volume is preferably 5-50 mg based on anthocyanin lyophilized powder.

[0084] In this invention, the parameters for preparative liquid chromatography separation and purification include:

[0085] Mobile phase: Phase A is pure acetonitrile, and Phase B is an aqueous solution of formic acid with a volume percentage concentration of 1% to 2%, preferably an aqueous solution of formic acid with a volume percentage concentration of 1.5%.

[0086] The gradient elution procedure is as follows:

[0087] 0-6min: The volume fraction of phase A changes uniformly from 10% to 15%;

[0088] 6-21 min: The volume fraction of phase A changes from 15% to 21% at a constant rate;

[0089] 21-24 min: The volume fraction of phase A changes uniformly from 21% to 60%;

[0090] 24-27 min: The volume fraction of phase A changes uniformly from 60% to 10%;

[0091] 27-30 min: The volume fraction of phase A is maintained at 10%.

[0092] In this invention, the parameters for preparative liquid chromatography separation and purification also include: a flow rate preferably of 10-12 mL / min and a column temperature preferably of 30°C.

[0093] In this invention, the detection wavelength of the ultraviolet detector is preferably 280nm or 520nm, and more preferably 280nm.

[0094] In this invention, the target component is preferably a component with a retention time of 19.0 to 21.0 min.

[0095] The present invention does not specifically limit the operation and parameters of the vacuum evaporation and freeze-drying of the target component; any operation known to those skilled in the art can be used.

[0096] This invention also provides petunia-based anthocyanins obtained by the extraction method described in the above technical solution. In this invention, the main component of the petunia-based anthocyanins is petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside, and the mass content of petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside in the petunia-based anthocyanins is 5-100%.

[0097] In this invention, the petunidin-3-O-(trans-p-coumaryl)rutin-5-O-glucoside has the structure shown in Formula 1:

[0098]

[0099] The present invention also provides a blue pigment, which is an anthocyanin complex formed by petunia-type anthocyanins and metal ions as described in the above technical solution.

[0100] In this invention, the metal ions include at least one of calcium ions, zinc ions, ferrous ions, and magnesium ions.

[0101] The present invention also provides a method for preparing the blue pigment described in the above technical solution, comprising the following steps:

[0102] The anthocyanin solution of petunia and the metal ion solution were mixed, the pH of the resulting mixture was adjusted to 6-8, and then allowed to stand and freeze-dried to obtain the blue pigment.

[0103] In this invention, the reagents for the petunia anthocyanin solution and the metal ion solution are preferably water, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, or sodium carbonate-sodium bicarbonate buffer. In this invention, the pH value of the reagents for the petunia anthocyanin solution and the metal ion solution is preferably 6.5–7.5, more preferably 7–7.2.

[0104] In this invention, the concentration of the petunia anthocyanin solution is preferably 0.05–20 g / L, more preferably 1–10 g / L, and even more preferably 5 g / L.

[0105] In this invention, the concentration of the metal ion solution is preferably 0.05–20 g / L, more preferably 1–10 g / L, and even more preferably 5 g / L. In this invention, the metal ions in the metal ion solution preferably include at least one of calcium ions, zinc ions, ferrous ions, and magnesium ions, further including ferrous ions. In this invention, the calcium ions are preferably used in the form of calcium salts, which preferably include one or more of calcium dihydrogen phosphate, dicalcium hydrogen phosphate, calcium sulfate, calcium chloride, and calcium lactate; the zinc ions are preferably used in the form of zinc salts, which preferably include zinc gluconate and / or zinc sulfate; the ferrous ions are preferably used in the form of ferrous salts, which preferably include one or more of ferrous sulfate, ferrous gluconate, and ferrous lactate; and the magnesium ions are preferably used in the form of magnesium salts, which preferably include magnesium sulfate and / or magnesium chloride.

[0106] In this invention, the molar ratio of metal ions to petunia anthocyanins in the mixture is preferably 1:0.2 to 1:6, more preferably 1:2 to 1:5, and even more preferably 1:3 to 1:4, based on petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside.

[0107] In this invention, the settling time is preferably 1 hour. This invention does not specifically limit the freeze-drying operation; any operation well-known to those skilled in the art can be used.

[0108] This invention also provides the application of the blue pigment described in the above-described technical solution in food. In this invention, the food is preferably a low-moisture active food, which preferably includes gummies, sugar coatings, or cream.

[0109] The following detailed description, in conjunction with embodiments, illustrates the anthocyanins of petunia genus and their extraction methods, as well as the blue pigments, their preparation methods, and applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0110] Example 1

[0111] Wash the dried black goji berries. Add 1 kg of black goji berries to a 70% acidic ethanol solution containing 0.5% (v / v) hydrochloric acid at a ratio of 1 g:10 mL. Mix thoroughly using a stirrer, and extract with ultrasonic assistance for 60 min. Filter sequentially through gauze and a Buchner funnel. Evaporate the filtrate under reduced pressure at 45℃ to remove ethanol and concentrate to obtain a crude extract of black goji berry anthocyanins. The high-performance liquid chromatogram of the obtained crude extract of black goji berry anthocyanins is shown below. Figure 1 As shown, from Figure 1 It can be seen that the crude extract of black goji berry anthocyanins contains a variety of polyphenols; in addition, the crude extract also contains impurities such as sugars, proteins and lipids.

[0112] An AB-8 macroporous resin was packed into a chromatography column, which was then washed sequentially with ethanol, 0.5 mol / L hydrochloric acid solution, 0.5 mol / L sodium hydroxide solution, and water. The crude extract of black goji berry anthocyanins was then injected into the column at a flow rate of 0.2 column volumes per hour. After loading, the column was eluted sequentially with 4 column volumes each of acidic ethanol solutions of 0% (containing 0.5% hydrochloric acid, v / v), 5% (containing 0.5% hydrochloric acid, v / v), 20% (containing 0.5% hydrochloric acid, v / v), and 40% (containing 0.5% hydrochloric acid, v / v). The eluent containing 40% (containing 0.5% hydrochloric acid, v / v) acidic ethanol solution was collected, and the ethanol was removed by vacuum evaporation at 45°C. The eluent was then lyophilized to obtain anthocyanin lyophilized powder and stored at -20°C for later use. The high-performance liquid chromatogram of the obtained anthocyanin lyophilized powder is shown below. Figure 2 As shown, from Figure 2 It can be seen that: after purification with macroporous resin, the content of polyphenolic impurities in the black wolfberry anthocyanin extract is reduced, and the purity of the target anthocyanin petunidin-3-O-(trans-p-coumaryl)rutin-5-O-glucoside is improved.

[0113] Liquid chromatography was performed using an Ultimate XB-C18 column (7 μm, 21.2 × 250 mm). The mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid aqueous solution (phase B). The gradient elution program was as follows: 0–6 min: 10%–15% phase A, 6–21 min: 15%–21% phase A, 21–24 min: 21%–60% phase A, 24–27 min: 60%–10% phase A, 27–30 min: 10% phase A; the mobile phase flow rate was 12 mL / min, the column temperature was 30 °C, and the detection wavelength was 280 nm.

[0114] The above-mentioned anthocyanin lyophilized powder was dissolved in a 1.5% (v / v) acidic aqueous solution to prepare an anthocyanin lyophilized powder solution with a concentration of 20 mg / mL. The injection volume was 2 mL. Based on the high-performance liquid chromatography (HPLC) chromatogram, components with retention times of 19.0–21.0 min were collected. The solution was then evaporated under reduced pressure and freeze-dried to finally obtain 10 g of petunia anthocyanins. The HPLC chromatogram of the obtained petunia anthocyanins is shown below. Figure 3 As shown. From Figure 3 It can be seen that the mass content of petunia-3-O-(trans-p-coumaroyl)rutin-5-O-glucoside in petunia anthocyanins is 95%.

[0115] Using a 10 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate solution (pH = 7.2) as a solvent, prepare 5 g / L petunia anthocyanin solutions and 5 g / L ferrous sulfate solutions respectively. Mix the ferrous sulfate solution and petunia anthocyanin solution at a molar ratio of 1:3, adjust the pH of the resulting mixture to 7.2, let it stand for 1 hour, and then freeze-dry to obtain the blue pigment.

[0116] The blue pigment obtained in Example 1 was subjected to Fourier transform infrared spectroscopy and circular dichroism spectroscopy characterization tests, and its color stability was characterized under neutral conditions (pH=7).

[0117] 1) Fourier transform infrared spectroscopy analysis: Potassium bromide was pre-dried at 110℃ for 1 hour, and the sample was pre-lyophilized to remove moisture and stored in a vacuum desiccator for later use. 0.5 g of the dried potassium bromide sample was weighed, ground for 10 min, then compressed into a pellet and placed in the sample cell to scan the background spectrum. Approximately 0.005 g of the test sample and 0.5 g of potassium bromide were weighed, mixed, ground for 10 min, compressed into a pellet, and the sample spectrum was scanned. The scanning parameters were: wavenumber range 4000-400 cm⁻¹. -1 (Mid-infrared region), resolution 4cm -1 The infrared spectral data were processed and analyzed using OMNIC 8.2 software.

[0118] 2) Circular dichroism spectroscopy analysis: The blue pigment sample was dissolved in 10 mmol / L PBS (pH=7) to prepare a solution of approximately 0.05 mmol / L. The solution was thoroughly mixed, and 2 mL was added to a 10 mm cuvette. The circular dichroism spectral signal in the 200-800 nm range was scanned using a circular dichroism spectrometer. To improve the signal-to-noise ratio, each sample was scanned three times, and the average millisecond value (mdeg) was calculated. An equal volume of 10 mmol / L PBS was used as a blank control.

[0119] 3) Characterization of color stability: A buffer solution with pH=7 was prepared using sodium dihydrogen phosphate and disodium hydrogen phosphate. The blue pigment was dissolved in the buffer solution to prepare a solution of approximately 0.3 g / L, which was then placed in a screw-top bottle. Photos were taken at regular intervals for observation. 200 μL of the solution was added to a 96-well microplate, and the visible absorption spectrum (380-780 nm) was detected using a microplate reader. The maximum absorption wavelength λ was recorded. m Maximum absorbance A m Based on this information, calculate the color retention rate using the following formula:

[0120]

[0121] Figure 4 Infrared spectra of ferrous sulfate, petunia anthocyanins, and blue pigments, from... Figure 4 It can be seen that the blue pigment prepared in this embodiment has a wavelength of 548 cm⁻¹ in the infrared spectrum.-1 The absorption peak is generated by the Fe-O coordination bond.

[0122] Figure 5 The circular dichroism spectrum of blue pigment, from Figure 5 It can be seen that the blue pigment prepared in this embodiment exhibits a significant positive Cotton effect in the region around 280 nm (one of the characteristic absorption wavelengths of anthocyanins), indicating that there is a supramolecular chiral conformation in the blue pigment.

[0123] Figure 6 This is a characterization diagram of the color stability of blue pigment in phosphate buffer (pH 7), from... Figure 6 It can be seen that the blue pigment exhibits relatively stable color development in neutral phosphate buffer. Calculations show that the color retention rate of this blue pigment is 74.5% after 7 days.

[0124] Example 2

[0125] This embodiment is an example of the application of the blue pigment obtained in Example 1 in cream.

[0126] Mix 100mL of chilled butter, 10g of granulated sugar, and 120mg of blue food coloring (or blue food coloring and lemon yellow food coloring). Whip the mixture thoroughly with a butter whipper, then pipe it into a piping bag for decoration. Use a common food additive, brilliant blue food coloring, as a control. Figure 7 As shown. From Figure 7 It can be seen that the food additive brilliant blue pigment exhibits a "cyan" color, while the blue pigment obtained in Example 1 exhibits an "indigo" color; the combination of brilliant blue pigment and lemon yellow pigment produces a "bright green" color, while the combination of the blue pigment obtained in Example 1 and lemon yellow pigment produces a "green" matcha color. In summary, the blue pigment provided by this invention can enrich the hues of food colorings.

[0127] Example 3

[0128] This embodiment is an example of the application of the blue pigment obtained in Example 1 in gummy candies.

[0129] Weigh out 200g of purified water, 20g of gelatin sheets, and 40g of granulated sugar. Dissolve them in a 70℃ water bath. After cooling to 35℃, add 12mg of blue food coloring (or a mixture of blue and lemon yellow food coloring), mix thoroughly, pour into a mold, and let it solidify to obtain blue gummy candies. Use a commonly used food additive, brilliant blue, as a control. Figure 8 As shown. From Figure 8 It can be seen that when the blue pigment prepared in Example 1 is applied to gummies, no obvious fading phenomenon occurs after 30 days of storage, indicating that the blue pigment provided by the present invention has strong stability under suitable application scenarios.

[0130] Comparative Example 1

[0131] The preparation process is the same as in Example 1, except that during the liquid chromatography purification process, other anthocyanins besides petunia anthocyanins are collected for the preparation of blue pigment.

[0132] The color stability characterization diagram of the obtained blue pigment in phosphate buffer (pH 7) is shown in the figure below. Figure 9 As shown, from Figure 9 It can be seen that the blue pigments prepared from anthocyanins other than petunia anthocyanins faded significantly under neutral conditions, indicating that the blue pigments prepared from other anthocyanins in black goji berries have low stability.

[0133] Comparative Example 2

[0134] The preparation process was the same as in Example 1, except that in the preparation of the blue pigment, the reagent used to dissolve the petunia anthocyanins and ferrous ions was a disodium hydrogen phosphate-sodium dihydrogen phosphate solution with a pH of 5, and the pH of the mixed solution was not adjusted to 6.0–8.0. The resulting blue pigment did not completely self-assemble to form a complex, had a low color value, and faded faster.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A blue pigment, characterized in that, It is an anthocyanin complex formed by petunia-type anthocyanins and metal ions, wherein the metal ion is ferrous ion; the main component of the petunia-type anthocyanins is petunia-3-O-(trans-p-coumaryl)rutin-5-O-glucoside. The petunia-type anthocyanins contain 95-100% petunia-3-O-(trans-p-coumaroyl)rutin-5-O-glucoside by mass. The extraction method of petunia anthocyanins includes the following steps: Using black goji berries as raw material, crude extract of black goji berry anthocyanins was obtained through alcohol extraction and concentration. The crude extract of anthocyanins from black goji berries was injected into a macroporous resin, and after elution, concentration and freeze-drying, anthocyanin freeze-dried powder was obtained. The anthocyanin lyophilized powder was dissolved and injected into a preparative liquid chromatography system for preparative-grade liquid chromatography separation and purification. The target component was detected by an ultraviolet detector and collected. The target component was then subjected to vacuum evaporation and lyophilization to obtain the petunia anthocyanins. The parameters for preparative-grade liquid chromatography separation and purification include: Mobile phase: Phase A is pure acetonitrile, and Phase B is an aqueous solution of formic acid with a volume percentage concentration of 1%~2%; The gradient elution procedure is as follows: 0-6 min: The volume fraction of phase A changes uniformly from 10% to 15%; 6-21 min: The volume fraction of phase A changes uniformly from 15% to 21%; 21-24 min: The volume fraction of phase A changes uniformly from 21% to 60%; 24-27 min: The volume fraction of phase A changes uniformly from 60% to 10%; 27-30 min: The volume fraction of phase A is maintained at 10%; The method for preparing the blue pigment includes the following steps: The anthocyanin solution of petunia and the metal ion solution were mixed, the pH of the resulting mixture was adjusted to 6-8, and then allowed to stand and freeze-dried to obtain the blue pigment. Based on petunidin-3-O-(trans-p-coumaroyl)rutin-5-O-glucoside, the molar ratio of metal ions to petunidin anthocyanins in the mixture is 1:0.2 to 1:

6.

2. The blue pigment according to claim 1, characterized in that, The alcohol extraction and concentration process includes: mixing black goji berries with an acidic ethanol solution and pulping them, then extracting and filtering them with ultrasonic assistance. The resulting filtrate is concentrated under reduced pressure to remove the ethanol, thus obtaining the crude extract of black goji berry anthocyanins. In the acidic ethanol solution, the volume concentration of ethanol is 50% to 80%, and the volume concentration of acid is 0.1% to 1%, wherein the acid is selected from at least one of hydrochloric acid, formic acid, and acetic acid; The ratio of black goji berries to acidic ethanol solution is 1g: 5~20mL; The ultrasound-assisted extraction time is 40-120 min; The temperature for vacuum concentration is 40~50℃.

3. The blue pigment according to claim 1, characterized in that, The macroporous resin is selected from AB-8, D101, XAD-7, HPD-100, or DM-130; the specific surface area of ​​the macroporous resin is 450~550 m². 2 / g, with an average pore size of 10~50nm and a particle size range of 0.3~1.25mm; Elution after injection of macroporous resin includes: eluting the macroporous resin sequentially with 4 column volumes of each of the first, second, third, and fourth elution reagents, and collecting the eluent of the fourth elution reagent; The first elution reagent is an acidic ethanol solution with a volume concentration of 0%. The second elution reagent is an acidic ethanol solution with a volume concentration of 5%. The third elution reagent is an acidic ethanol solution with a volume concentration of 20%. The fourth elution reagent is an acidic ethanol solution with a volume concentration of 40%. In the first elution reagent, the second elution reagent, the third elution reagent, and the fourth elution reagent, the volume concentration of the acid is independently 0.1% to 1%, and the acid is independently selected from at least one of hydrochloric acid, formic acid, and acetic acid.

4. The blue pigment according to claim 1, characterized in that, The parameters for the preparative liquid chromatography separation and purification also include: the flow rate of the mobile phase is 10~12 mL / min, the column temperature is 30℃, and the detection wavelength of the ultraviolet detector is 280nm or 520nm.

5. The blue pigment according to claim 4, characterized in that, The target component is the component with a retention time of 19.0~21.0 min.

6. The method for preparing the blue pigment according to any one of claims 1 to 5, characterized in that, Includes the following steps: The anthocyanin solution of petunia and the metal ion solution were mixed, the pH of the resulting mixture was adjusted to 6-8, and then allowed to stand and freeze-dried to obtain the blue pigment. Based on petunidin-3-O-(trans-p-coumaroyl)rutin-5-O-glucoside, the molar ratio of metal ions to petunidin anthocyanins in the mixture is 1:0.2 to 1:

6.

7. The preparation method according to claim 6, characterized in that, The reagents for the petunia anthocyanin solution and the metal ion solution are water, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, or sodium carbonate-sodium bicarbonate buffer. The concentration of the petunia anthocyanin solution is 0.05~20g / L; The concentration of the metal ion solution is 0.05~20 g / L.

8. The use of the blue pigment according to any one of claims 1 to 5 or the blue pigment prepared by the preparation method according to claim 6 or 7 in food.

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