Method for extracting anthocyanin in hybrid prunus armeniaca tissue

Abstract

The invention provides a method for extracting anthocyanin in hybrid prunus armeniaca tissue, and belongs to the technical field of anthocyanin extraction. The extraction method of the anthocyanin in the hybrid prunus armeniaca tissue comprises the following steps: performing vacuum freeze drying on the hybrid prunus armeniaca tissue, and crushing to obtain powder; mixing the powder, a compound enzyme and the extracting solution, extracting, centrifuging, taking supernate, filtering, taking filtrate, mixing with a 60% methanol aqueous solution, adsorbing with macroporous adsorption resin, eluting with an acidified ethanol solution, collecting eluent, and removing ethanol in the eluent to obtain the anthocyanin. According to the method for extracting the anthocyanin in the hybrid prunus armeniaca tissue, the anthocyanin in the hybrid prunus armeniaca tissue can be efficiently extracted, separated and purified, the reaction is mild, the extraction efficiency is high, the purity is high, and the anthocyanin with the purity being 95% or above can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of anthocyanin extraction, and particularly relates to a method for extracting anthocyanin from hybrid apricot plum tissue. Background Art

[0002] With the improvement of living standards, more and more people tend to pursue natural organic substances from natural plant sources, which are non-toxic and harmless, and are beneficial to protecting health, preventing and curing diseases. At the same time, since artificial anthocyanidins have great toxic side effects on the human body and have been banned by the World Health Organization, highly efficient and non-toxic natural plant anthocyanidins are becoming more and more popular.

[0003] Hybrid apricot plum (Prunus salicina x armeniaca) is a special economic forest tree species bred by hybridization between plum and apricot. Its fruits generally have the characteristics of good taste, rich nutrition and high yield. In addition to the characteristics of large fruit, high yield and excellent flavor, the peel and flesh of hybrid apricot plum are rich in anthocyanins. The overall anthocyanin content is equivalent to that of blueberry fruit, and it has higher health value than green-skinned and yellow-fleshed apricot plum varieties. Anthocyanins have the effects of preventing brain nerve aging, strengthening the heart, anti-cancer, softening blood vessels, and enhancing immunity. They have broad application prospects in the fields of food, medicine, cosmetics, etc. Therefore, how to efficiently extract natural anthocyanins has great practical significance for protecting people's health and preventing and treating diseases. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for extracting anthocyanins from hybrid apricot plum tissue, which can efficiently extract anthocyanins with a purity of more than 95%.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for extracting anthocyanins from hybrid apricot plum tissue, comprising the following steps: vacuum freeze-drying the hybrid apricot plum tissue and crushing it to obtain powder; mixing the powder, a composite enzyme and an extracting solution for extraction, centrifuging to obtain a supernatant, filtering and mixing the filtrate with a 60% methanol aqueous solution for macroporous adsorption resin adsorption, eluting with an acidified ethanol solution, collecting the eluate, and removing ethanol from the eluate to obtain anthocyanins;

[0007] The complex enzyme is composed of cellulase and pectinase, the weight ratio of the complex enzyme to powder is 5-10:100, the extracting solution is 0.1% HCl methanol solution, the extraction temperature is 25-60° C., and the extraction time is 20-50 minutes.

[0008] Preferably, the weight-to-volume ratio of the powder to the extract is 1 g:15-40 mL.

[0009] Preferably, the mass ratio of cellulase to pectinase in the complex enzyme is (1-2): (1-2).

[0010] Preferably, the tissue comprises peel and / or pulp.

[0011] Preferably, the powder is sieved after crushing, and the mesh size of the sieve is 20 meshes.

[0012] Preferably, the pH of the extract is 3, and the pH of the 60% methanol aqueous solution is 3.

[0013] Preferably, the filtration is 0.45 μm filter membrane filtration.

[0014] Preferably, the acidified ethanol solution is a 70% by volume ethanol solution, and the acidified ethanol solution contains 1% by volume HCl.

[0015] Preferably, the volume ratio of the filtrate to the 60% methanol aqueous solution is 1:1.

[0016] Preferably, the adsorption time of the macroporous adsorption resin is 10 hours.

[0017] Beneficial effects of the present invention:

[0018] The method for extracting anthocyanins from hybrid apricot plum tissue provided by the present invention can efficiently extract, separate and purify anthocyanins from hybrid apricot plum tissue, has a mild reaction, high extraction efficiency and high purity, and can obtain anthocyanins with a purity of more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the peak time diagram of high performance liquid chromatography;

[0020] Figure 2 This is the standard curve used to calculate the anthocyanin content in anthocyanin powder. DETAILED DESCRIPTION

[0021] The invention provides a method for extracting anthocyanins from hybrid apricot plum tissue, comprising the following steps: vacuum freeze-drying the hybrid apricot plum tissue and crushing it to obtain powder; mixing the powder, a composite enzyme and an extracting solution for extraction, centrifuging to obtain a supernatant, filtering and mixing the filtrate with a 60% methanol aqueous solution for macroporous adsorption resin adsorption, eluting with an acidified ethanol solution, collecting the eluate, and removing ethanol from the eluate to obtain anthocyanins;

[0022] The complex enzyme is composed of cellulase and pectinase, the weight ratio of the complex enzyme to powder is 5-10:100, the extracting solution is 0.1% HCl methanol solution, the extraction temperature is 25-60° C., and the extraction time is 20-50 minutes.

[0023] The present invention does not specifically limit the specific source of hybrid apricot plums. In the present invention, it is preferred that the hybrid apricot plums are first washed, frozen overnight at -80°C to avoid browning of the hybrid apricot plum tissue, and then vacuum freeze-dried. In the present invention, the tissue preferably includes peel and / or pulp; the vacuum freeze-drying is preferably performed by precooling the machine for 1 hour to -80°C, preheating the machine vacuum pump for 30 minutes, placing the sample in a freeze dryer, main drying for 24 hours, and final drying for 6 hours. After vacuum freeze-drying, the sample is crushed. The present invention does not specifically limit the specific method of crushing. After crushing, the sample is sieved, and the mesh number of the sieve is preferably 20 meshes.

[0024] In the present invention, the weight ratio of the complex enzyme to the powder is preferably 5 to 10:100, more preferably 6 to 9:100, and the weight-volume ratio of the powder to the extract is preferably 1 g:15 to 40 mL, more preferably 1 g:20 to 30 mL. In the present invention, the mass ratio of cellulase to pectinase in the complex enzyme is preferably (1 to 2): (1 to 2), more preferably 1:2 or 2:1. The present invention does not specifically limit the specific sources of cellulase and pectinase. The cellulase is preferably cellulase 9012-54-8 source leaf S10042-10g, with an enzyme activity of 400 U / mg, and the pectinase is preferably pectinase 9032-75-1 source leaf S10007-5g, with an enzyme activity of 50,000 U / g. In the present invention, the 0.1% HCl methanol solution refers to an extract containing 0.1% HCl by volume, and the pH of the extract is preferably 3; the extraction temperature is preferably 25-60°C, more preferably 30-50°C, and the extraction time is preferably 20-50 min, more preferably 30-40 min.

[0025] In the present invention, the centrifugal condition is preferably 12000r / min centrifugation for 5-10min, the filtration is preferably filtered with a 0.45μm filter membrane, and after filtration, the filtrate is mixed with a 60% methanol aqueous solution for macroporous adsorption resin adsorption, the macroporous adsorption resin is preferably an AB-8 medium polar macroporous resin, the adsorption time is preferably 10h, the adsorption temperature is preferably 20-30°C, and after the adsorption is completed, it preferably flows out at 3BV / h (volume flow rate), and the BV / h refers to the volume of the liquid flowing through the adsorption column relative to the volume of the filler per unit time. Then it is preferably eluted with an acidified ethanol solution, the acidified ethanol solution is preferably a 70% ethanol solution by volume, and the acidified ethanol solution contains 1% HCl by volume. In the present invention, the 60% methanol aqueous solution refers to a methanol volume fraction of 60%, the pH value of the 60% methanol aqueous solution is preferably 3, and the volume ratio of the filtrate to the 60% methanol aqueous solution is preferably 1:1. In the present invention, the pretreatment method of the AB-8 medium-polar macroporous resin is preferably: soaking the resin in distilled water until it is fully expanded, washing the resin with a 50%-70% ethanol solution to remove impurities and residues, and finally rinsing the resin with deionized water until it is neutral. In the present invention, the method of removing ethanol from the eluent is preferably evaporation under reduced pressure, and the reduced pressure is preferably -0.08MPa to -0.09MPa.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] A method for extracting anthocyanins from hybrid apricot and plum tissues comprises the following steps:

[0031] (1) The hybrid apricot plum 'Flavor Rose' was washed with purified water, the skin and flesh were separated, and the peel was placed in a -80°C refrigerator overnight. The frozen apricot plum peel was taken out and vacuum freeze-dried (the vacuum freeze-drying was as follows: the machine was pre-cooled for 1 hour to -80°C, the machine vacuum pump was preheated for 30 minutes, the sample was placed in a freeze dryer, the main drying was 24 hours, and the final drying was 6 hours). The vacuum freeze-dried sample was placed in a grinder for grinding and passed through a 20-mesh sieve to obtain a powder.

[0032] (2) Mix the powder, compound enzyme and extract, and extract at 35° C. for 35 min; the weight ratio of the compound enzyme to the powder is 10:100, the compound enzyme is composed of cellulase (the cellulase is cellulase 9012-54-8 source leaf S10042-10 g, with an enzyme activity of 400 U / mg) and pectinase (the pectinase is pectinase 9032-75-1 source leaf S10007-5 g, with an enzyme activity of 50,000 U / g), the mass ratio of cellulase to pectinase is 1:1.5, the extract is a 0.1% HCl methanol solution with a pH value of 3, and the weight-to-volume ratio of the powder to the extract is 1 g:25 mL.

[0033] (3) Centrifuge at 12000 r / min for 5 min, take the supernatant and filter it through a 0.45 μm filter membrane to remove fine particles and insoluble impurities, take the filtrate and mix it with a 60% methanol aqueous solution in a volume ratio of 1:1, and perform adsorption on an AB-8 medium-polarity macroporous resin column for 10 h (the pretreatment method of the AB-8 medium-polarity macroporous resin is: soak the resin in distilled water until it is fully expanded, wash the resin with a 70% ethanol solution to remove impurities and residues, and finally rinse the resin with deionized water until it is neutral). During the adsorption process, the temperature is maintained at no more than 30° C., and then it flows out at a volume flow rate of 3 BV / h. Then, the resin is first rinsed with deionized water to remove weakly bound impurity components, and then desorbed with an acidified ethanol solution (the acidified ethanol solution is composed of 1% HCl and 70% ethanol). The desorption flow rate is 1.5 BV / h, the desorption temperature is no more than 30° C., and the desorption solvent volume is 4 times the resin volume. The eluate was collected and evaporated to remove ethanol under reduced pressure of -0.08 MPa to -0.09 MPa to obtain anthocyanidins; the anthocyanidins were freeze-dried at -80°C for 1 hour using a vacuum freeze dryer to obtain anthocyanidin powder. The purity of anthocyanidins in the obtained anthocyanidin powder was measured by a spectrophotometer to be 98%.

[0034] Example 2

[0035] A method for extracting anthocyanins from hybrid apricot and plum tissues comprises the following steps:

[0036] (1) The hybrid apricot plum 'Flavor Rose' was washed with purified water, the skin and flesh were separated, and the flesh was placed in a -80°C refrigerator overnight. The frozen apricot plum flesh was taken out and vacuum freeze-dried (the vacuum freeze-drying was as follows: the machine was pre-cooled for 1 hour to -80°C, the machine vacuum pump was preheated for 30 minutes, the sample was placed in a freeze dryer, the main drying was 24 hours, and the final drying was 6 hours). The vacuum freeze-dried sample was placed in a grinder for grinding, and the powder was obtained by passing through a 20-mesh sieve.

[0037] (2) Mix the powder, the composite enzyme and the extract, and extract at 40° C. for 40 min; the weight ratio of the composite enzyme to the powder is 5:100, the composite enzyme is composed of cellulase (the cellulase is cellulase 9012-54-8 source leaf S10042-10 g, with an enzyme activity of 400 U / mg) and pectinase (the pectinase is pectinase 9032-75-1 source leaf S10007-5 g, with an enzyme activity of 50,000 U / g), the mass ratio of cellulase to pectinase is 1.5:1, the extract is a 0.1% HCl methanol solution with a pH value of 3, and the weight-to-volume ratio of the powder to the extract is 1 g:30 mL.

[0038] (3) Centrifuge at 12000 r / min for 10 min, take the supernatant and filter it through a 0.45 μm filter membrane to remove fine particles and insoluble impurities, take the filtrate and mix it with 60% methanol aqueous solution in a volume ratio of 1:1, and perform adsorption on an AB-8 medium-polarity macroporous resin column for 10 h (the pretreatment method of the AB-8 medium-polarity macroporous resin is: soak the resin in distilled water until it is fully expanded, wash the resin with 70% ethanol solution to remove impurities and residues, and finally rinse the resin with deionized water until it is neutral). During the adsorption process, the temperature is maintained at no more than 30° C., and then it flows out at 3 BV / h (volume flow rate). Then, the resin is first rinsed with deionized water to remove weakly bound impurity components, and then desorbed with an acidified ethanol solution (the acidified ethanol solution consists of 1% HCl and 70% ethanol). The desorption flow rate is 1.5 BV / h, the desorption temperature is no more than 30° C., and the desorption solvent volume is 4 times the resin volume. The eluate was collected, and the ethanol was removed by evaporation under reduced pressure of -0.08 MPa to -0.09 MPa to obtain anthocyanidin; the anthocyanidin was freeze-dried at -80°C for 1 hour using a vacuum freeze dryer to obtain anthocyanidin powder. The purity of anthocyanidin in the obtained anthocyanidin powder was measured by a spectrophotometer and was 95%.

[0039] Example 3

[0040] Single factor test of material-liquid ratio

[0041] The difference from Example 1 is that the weight-to-volume ratio of the powder and the extract in step (2) is set to 1g:10mL, 1g:15mL, 1g:20mL, 1g:30mL and 1g:40mL respectively, and the rest is the same as Example 1.

[0042] The content of anthocyanins in anthocyanin powder was determined by high performance liquid chromatography. The specific method is as follows:

[0043] All reagents used were analytical grade, acetonitrile and methanol were chromatographic grade. The chromatographic column was a C18 column, and the column temperature was 40°C. Mobile phase A was 1.5% phosphoric acid aqueous solution; mobile phase B was 1.5% phosphoric acid, 20% acetic acid and 25% acetonitrile aqueous solution; a diode array detector was used, and the anthocyanin detection wavelength was set to 520nm. The sample injection volume was 10μL. The mobile phase gradient changes are shown in Table 1 below, and the peak time diagram is shown in Figure 1 As shown, the peak area of ​​the first peak was collected and measured.

[0044] Table 1 Mobile phase gradient changes

[0045] Time (min) Flow rate (mL / min) ％A ％B 0 0.75 85.0 15.0 30 0.75 45.0 55.0 45 0.75 0.0 100.0 50 0.75 85.0 15.0 60 0.75 85.0 15.0

[0046] The standard substance used for quantitative analysis is cyanidin-3-O-glucoside, with a purity of more than 97%. The obtained standard substance curve is as follows: Figure 2 Substitute the peak area value obtained by HPLC into Figure 2 The content of anthocyanin in anthocyanin powder was calculated from the standard curve shown in Table 2.

[0047] Table 2 Anthocyanin content obtained with different material-liquid ratios

[0048]

[0049]

[0050] Example 4

[0051] Extraction temperature single factor test

[0052] The difference from Example 1 is that the extraction temperatures in step (2) are 25°C, 30°C, 40°C, 50°C and 60°C respectively, and the rest are the same as Example 1. The content of anthocyanin in anthocyanin powder was determined by HPLC, and the specific method was the same as Example 3. The results are shown in Table 3.

[0053] Table 3 Anthocyanin content obtained at different extraction temperatures

[0054]

[0055] Example 5

[0056] Extraction time single factor test

[0057] The difference from Example 1 is that the extraction time in step (2) is 10 min, 20 min, 30 min, 40 min and 50 min respectively, and the rest is the same as Example 1. The content of anthocyanin in anthocyanin powder was determined by HPLC, and the specific method was the same as Example 3. The results are shown in Table 4.

[0058] Table 4 Anthocyanin content obtained at different extraction times

[0059] Extraction time (min) Anthocyanin content (mg / g) 10 4.55 20 5.79 30 6.21 40 6.86 50 6.62

[0060] Example 6

[0061] Single factor test on the ratio of cellulase and pectinase

[0062] The difference from Example 1 is that in step (2), the mass ratios of cellulase and pectinase are 1:0, 2:1, 1:1, 1:2 and 0:1 respectively, and the rest are the same as Example 1.

[0063] The difference from Example 2 is that in step (2), the mass ratios of cellulase and pectinase are 1:0, 2:1, 1:1, 1:2 and 0:1 respectively, and the rest are the same as Example 2.

[0064] The content of anthocyanidin in anthocyanidin powder was determined by high performance liquid chromatography, and the specific method was the same as that in Example 3. The results are shown in Table 5.

[0065] Table 5 Anthocyanin content obtained by different ratios of cellulase and pectinase

[0066]

[0067] Example 7

[0068] The five-level central composite design CCD (centra composite design) combined with the response surface method was used to test the solid-liquid ratio, extraction temperature, and the ratio of cellulase and pectinase in the anthocyanin extraction method described in Example 1 and Example 2. The other conditions and methods were the same as those in Example 1 and Example 2. The content of anthocyanin in anthocyanin powder was determined by high performance liquid chromatography, and the specific method was the same as in Example 3. The results are shown in Table 6.

[0069] Table 6 CCD test results

[0070]

[0071]

[0072] It can be seen from Table 6 that the highest anthocyanin content in the peel of hybrid apricot plum is 316.84 mg / 100 g, and the highest anthocyanin content in the pulp is 147.82 mg / 100 g. The optimal extraction conditions in the two tissues are the same, with an extraction temperature of 54 °C, a solid-liquid ratio of 1 g:34 mL, and a ratio of cellulase to pectinase of 1:2.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting anthocyanins from hybrid apricot and plum tissues, characterized in that: The method comprises the following steps: vacuum freeze-drying hybrid apricot plum tissue and crushing it to obtain powder; mixing the powder, complex enzyme and extracting solution for extraction, centrifuging to obtain supernatant, filtering and mixing the filtrate with 60% methanol aqueous solution for macroporous adsorption resin adsorption, eluting with acidified ethanol solution, collecting the eluate, removing ethanol in the eluate, and obtaining anthocyanin; The complex enzyme is composed of cellulase and pectinase, the weight ratio of the complex enzyme to powder is 5-10:100, the extracting solution is 0.1% HCl methanol solution, the extraction temperature is 25-60° C., and the extraction time is 20-50 minutes.

2. The extraction method according to claim 1, characterized in that The weight-to-volume ratio of the powder and the extract is 1 g:15-40 mL.

3. The extraction method according to claim 1, characterized in that The mass ratio of cellulase to pectinase in the complex enzyme is (1-2): (1-2).

4. The extraction method according to claim 1, characterized in that The tissue includes peel and / or pulp.

5. The extraction method according to claim 1, characterized in that After crushing, the product is sieved, and the mesh number of the sieve is 20 meshes.

6. The extraction method according to claim 1, characterized in that The pH of the extract is 3, and the pH of the 60% methanol aqueous solution is 3.

7. The extraction method according to claim 1, characterized in that The filtration is carried out by 0.45 μm filter membrane filtration.

8. The extraction method according to claim 1, characterized in that The acidified ethanol solution is an ethanol solution with a volume fraction of 70%, and the acidified ethanol solution contains HCl with a volume fraction of 1%.

9. The extraction method according to claim 1, characterized in that The volume ratio of the filtrate to the 60% methanol aqueous solution is 1:

1.

10. The extraction method according to claim 1, characterized in that The adsorption time of the macroporous adsorption resin is 10h.

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