Method for extracting and purifying anthocyanin in vaccinium myrtillus

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting and purifying anthocyanin in vaccinium myrtillus. According to the method, extraction, enrichment and purification are completed in one step through dispersive micro solid-phase extraction, and the method is high in sensitivity, easy to operate, short in extraction time, high in extraction efficiency and low in adsorbent and organic reagent consumption and conforms to the principle of'green chemistry '. Compared with the anthocyanin content only reaching about 30% in the current market, the anthocyanin content obtained through the extraction and purification method exceeds 60%, the yield can reach 90%, the purity of the anthocyanin is high, and the yield is remarkably improved. The method for extracting and purifying the anthocyanin is simple to operate, low in cost and beneficial to popularization and application in production practice.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting and purifying anthocyanins in bilberry. Background Art

[0002] Anthocyanins, also known as flower pigments, are flavonoids in the phenolic compounds and are water-soluble natural pigments. Anthocyanins are widely present in the tissues of plant petals and fruits. Anthocyanins are the main substances that cause various fruits and flowers to present bright red and blue colors. Their color changes with different pH values, thus giving plants different colors. Studies have shown that anthocyanins are the most effective antioxidants discovered by humans today. With the development of science and technology, people pay more and more attention to the safety of food additives, and the development of natural additives has become the general trend of the development and use of additives. Anthocyanins are natural pigments, non-toxic and edible, and very safe. Anthocyanins can be used not only as nutritional enhancers in food, but also as food preservatives and food colorants in ordinary beverages and foods. Anthocyanin extraction resources are rich and have certain medicinal value. Its strong antioxidant capacity can eliminate free radicals, a byproduct of human metabolism, protect the health of cells and activate senescent cells, resist human aging, and also have significant effects on preventing and resisting cancer. Therefore, anthocyanins have great application prospects in food, cosmetics and medicine.

[0003] Currently, the main sources of anthocyanin extraction are purple sweet potatoes, grapes, blood oranges, red cabbage, blueberries, eggplants, cherries, cranberries, strawberries, mulberries, hawthorns, morning glories and other plants. However, such a large number of plants still cannot meet the growing demand, and it is necessary to find new sources of extraction to supply anthocyanins.

[0004] The extraction, separation and purification of anthocyanins is an active research area in recent years. Common extraction methods include solvent extraction, ultrasonic-assisted extraction, microwave-assisted extraction, ultrahigh pressure-assisted extraction, etc. The main separation and purification methods include high-efficiency countercurrent chromatography, membrane separation technology, gel chromatography, and macroporous adsorption resin method. However, these separation and purification methods generally have the following problems: long time consumption, low extraction efficiency, high production cost, residual solvent, large consumption of organic solvent, low anthocyanin content, and poor yield. Therefore, a new method for the extraction, separation and purification of anthocyanins is urgently needed. Summary of the invention

[0005] The invention aims to overcome the defects of the prior art in that the extraction of anthocyanins is time-consuming, the extraction efficiency is low, the anthocyanins obtained by separation and purification have low content and low yield, and provides a method for extracting and purifying anthocyanins from bilberry.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A method for extracting and purifying anthocyanidins from bilberry comprises the following steps: (S.1) crushing fresh bilberry fruit into a homogenate, adding water, and ultrasonicating to obtain a mixed solution, adjusting the pH, and extracting at a temperature to obtain an extract; (S.2) Add an extractant to the extract obtained in step (S.1), vortex and oscillate, filter with a microfiltration membrane after the reaction is completed, take the filter residue and elute the filter residue with an ethanol aqueous solution, take the eluate and dry it to obtain anthocyanin dry powder.

[0007] The present invention uses dispersed micro-solid phase extraction to extract, enrich and purify in one step. The method has high sensitivity, simple operation, short extraction time, high extraction efficiency, and small amounts of adsorbents and organic reagents, which complies with the principle of "green chemistry". Compared with the anthocyanin content of only about 30% on the current market, the anthocyanin content obtained by the extraction and purification method of the present invention exceeds 60%, and the yield can reach 90%. The anthocyanin purity is high, and its yield is significantly improved. The extraction and purification method of anthocyanins of the present invention is simple to operate and has low cost, which is conducive to popularization and application in production practice.

[0008] Preferably, the ultrasonic frequency in step (S.1) is 35 to 45 KHz.

[0009] When the ultrasonic frequency is lower than 35KHz, it is easy to affect the release of anthocyanins in the raw materials, resulting in reduced extraction efficiency. When the ultrasonic frequency is higher than 45KHz, it is easy to damage the anthocyanins in the raw materials, resulting in a reduced anthocyanin yield, and it is also easy to introduce unnecessary impurities, further affecting the extraction purity of anthocyanins.

[0010] Preferably, the pH value of the mixed solution in step (S.1) is 1-2.

[0011] When the pH value of the mixed solution is 1 to 2, the cell structure in the fresh bilberry fruit (raw material) can be effectively prevented from being destroyed, thereby reducing the extraction purity and yield of anthocyanins, and also avoiding the introduction of impurities. At the same time, the appropriate pH value will not affect the activity of the enzyme, which helps to maintain its biological activity, thereby avoiding affecting the type and content of the active ingredients in the extract.

[0012] Preferably, the reaction temperature of the heat preservation extraction process in step (S.1) is 45-55°C, and the extraction time is 1-3h.

[0013] When the reaction temperature of the heat preservation extraction process exceeds 55°C and the extraction time exceeds 3 hours, the effective active ingredients in bilberry are easily destroyed, thereby reducing the quality of the extract. At the same time, high temperature may also promote the dissolution and release of non-target components, resulting in an increase in the impurity content in the extract, further affecting the purity of the extract and subsequent processing. In addition, too long an extraction time will lead to reduced extraction efficiency. When the reaction temperature of the heat preservation extraction process is lower than 45°C and the extraction time is less than 1 hour, it is easy to cause insufficient extraction and a decrease in the extraction rate.

[0014] Preferably, the mass ratio of fresh bilberry fruit to water added in step (S.1) is 1:1-3.

[0015] As a further preference, the mass ratio of fresh bilberry fruit to water added in step (S.1) is 1:2.

[0016] When a small amount of water is added, the mixed solution is too viscous and difficult to disperse evenly; when a large amount of water is added, the amount of mixed solution is too large, which increases the difficulty of subsequent processing and causes a certain amount of resource waste.

[0017] Preferably, the extractant added in step (S.2) is any one or more of a combination of fluorinated carbon nanotubes, aluminum oxide, and silica gel.

[0018] Fluorinated carbon nanotubes, aluminum oxide, and silica gel extractants have a large specific surface area and abundant surface functional groups, which can provide more adsorption sites and enhance the adsorption capacity of anthocyanins, thereby helping to extract anthocyanins from the extract. At the same time, by selecting fluorinated carbon nanotubes, aluminum oxide, silica gel, etc. as extractants, it is helpful to achieve specific extraction of anthocyanins, reduce interference from impurities, and improve the purity of anthocyanins.

[0019] Preferably, the mass of the extractant added in step (S.2) is 0.5-1.5% of the mass of the extract.

[0020] When the mass of the added extractant is too small, it cannot fully contact and adsorb the anthocyanins in the extract, resulting in reduced extraction efficiency and further reduced anthocyanin extraction purity. When the mass of the added extractant is too large, residual problems are likely to occur, which is not conducive to subsequent processing and may also cause environmental pollution. In addition, using too much extractant will also increase production costs and cause a certain degree of resource waste.

[0021] Preferably, the vortex time of the vortex vibration process in step (S.2) is 8 to 12 minutes.

[0022] Preferably, the aqueous solution of ethanol used in the elution process in step (S.2) is 50-95% ethanol.

[0023] Preferably, the mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 20 to 40:1.

[0024] Therefore, the present invention has the following beneficial effects: (1) The present invention uses dispersed micro solid phase extraction to achieve extraction, enrichment and purification in one step. The method has high sensitivity, simple operation, short extraction time, high extraction efficiency, and small amounts of adsorbent and organic reagents, which complies with the principle of "green chemistry"; (2) Compared with the anthocyanin content of about 30% currently on the market, the anthocyanin content obtained by the extraction and purification method of the present invention exceeds 60%, and the yield can reach 90%. The anthocyanin purity is high, and its yield is significantly improved; (3) The anthocyanin extraction and purification method of the present invention is simple to operate and has low cost, which is conducive to popularization and application in production practice. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0026] Example 1 This embodiment provides a method for extracting and purifying anthocyanins from bilberry.

[0027] A method for extracting and purifying anthocyanidins from bilberry comprises the following steps: (S.1) Crush fresh bilberry fruit into a homogenate, add twice the mass of water (i.e., the mass ratio of fresh bilberry fruit to water is 1:2), control the ultrasonic frequency to 40 kHz and perform ultrasound to obtain a mixed solution, adjust the pH to 1.5 with sulfuric acid, and extract at 50°C for 2 h, extract twice, and combine the extracts; (S.2) Add fluorinated carbon nanotubes in an amount of 1% of the mass of the extract to the extract obtained in step (S.1), place in a vortex oscillator and oscillate for 10 minutes, filter with a microfiltration membrane after the reaction is completed, and take the filter residue (i.e., the fluorinated carbon nanotubes adsorbed with anthocyanins). Elute the filter residue with a 95% ethanol aqueous solution (the mass ratio of the 95% ethanol aqueous solution used in the elution process to the filter residue is 30:1), take the eluate and dry it to obtain anthocyanin dry powder.

[0028] Example 2 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the ultrasonic frequency in step (S.1) is controlled to be 35 KHz. The rest is the same as in Example 1.

[0029] Example 3 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the ultrasonic frequency in step (S.1) is controlled to be 45 KHz. The rest is the same as in Example 1.

[0030] Example 4 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.1), the pH is adjusted to 1.0 with sulfuric acid. The rest is the same as in Example 1.

[0031] Example 5 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.1), the pH is adjusted to 2.0 with sulfuric acid. The rest is the same as in Example 1.

[0032] Example 6 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction temperature of the heat preservation extraction process in step (S.1) is 45° C. The rest is the same as in Example 1.

[0033] Example 7 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction temperature of the heat preservation extraction process in step (S.1) is 55° C. The rest is the same as in Example 1.

[0034] Example 8 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction time of the heat preservation extraction process in step (S.1) is 1 hour. The rest is the same as in Example 1.

[0035] Example 9 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction time of the heat preservation extraction process in step (S.1) is 3 hours. Other aspects are the same as those in Example 1.

[0036] Comparative Example 1 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the ultrasonic frequency in step (S.1) is controlled to be 30 KHz. The rest is the same as in Example 1.

[0037] Comparative Example 2 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the ultrasonic frequency in step (S.1) is controlled to be 50 KHz. The rest is the same as in Example 1.

[0038] Comparative Example 3 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.1), the pH is adjusted to 0.5 with sulfuric acid. The rest is the same as in Example 1.

[0039] Comparative Example 4 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.1), the pH is adjusted to 2.5 with sulfuric acid. The rest is the same as in Example 1.

[0040] Comparative Example 5 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction temperature of the heat preservation extraction process in step (S.1) is 40° C. The rest is the same as in Example 1.

[0041] Comparative Example 6 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction temperature of the heat preservation extraction process in step (S.1) is 60° C. The rest is the same as in Example 1.

[0042] Comparative Example 7 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction time of the heat preservation extraction process in step (S.1) is 0.5 h. Other aspects are the same as those in Example 1.

[0043] Comparative Example 8 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the reaction time of the heat preservation extraction process in step (S.1) is 3.5 hours. The rest is the same as in Example 1.

[0044] The extracts were prepared according to the methods of Examples 1 to 9 and Comparative Examples 1 to 8, and the anthocyanin content in the extracts was detected and the extraction rate was calculated. The results are shown in Table 1 below.

[0045] Table 1 Group Extraction rate (%) Example 1 99.21 Example 2 92.67 Example 3 98.02 Example 4 96.87 Example 5 91.07 Example 6 95.75 Example 7 93.25 Example 8 93.36 Example 9 99.01 Comparative Example 1 86.45 Comparative Example 2 88.37 Comparative Example 3 85.29 Comparative Example 4 82.16 Comparative Example 5 84.38 Comparative Example 6 81.87 Comparative Example 7 80.91 Comparative Example 8 83.59 .

[0046] From the data analysis in Table 1, it can be seen that the best extraction process is ultrasound at 40KHz, adjusting the pH to 1.5 with sulfuric acid, keeping at 50℃, extracting for 2h, and extracting twice and combining the extracts.

[0047] Example 10 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.2), aluminum oxide is used to replace carbon fluoride nanotubes. Other steps are the same as those in Example 1.

[0048] Embodiment 11 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein in step (S.2), silica gel is used to replace fluorinated carbon nanotubes. Other steps are the same as those in Example 1.

[0049] Example 12 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the mass of the fluorinated carbon nanotubes added in step (S.2) is 0.5% of the mass of the extract, and the rest is the same as in Example 1.

[0050] Embodiment 13 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the mass of the fluorinated carbon nanotubes added in step (S.2) is 1.5% of the mass of the extract, and the rest is the same as in Example 1.

[0051] Embodiment 14 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the vortexing time of the vortexing vibration process in step (S.2) is 8 minutes. Other conditions are the same as those in Example 1.

[0052] Embodiment 15 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the vortexing time of the vortexing vibration process in step (S.2) is 12 minutes. The rest is the same as in Example 1.

[0053] Comparative Example 9 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the mass of the fluorinated carbon nanotubes added in step (S.2) is 0.25% of the mass of the extract, and the rest is the same as in Example 1.

[0054] Comparative Example 10 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the mass of the fluorinated carbon nanotubes added in step (S.2) is 2% of the mass of the extract, and the rest is the same as in Example 1.

[0055] Comparative Example 11 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the vortexing time of the vortexing vibration process in step (S.2) is 6 minutes. The rest is the same as in Example 1.

[0056] Comparative Example 12 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the vortexing time of the vortexing vibration process in step (S.2) is 14 minutes. Other aspects are the same as those in Example 1.

[0057] Extraction and vortex oscillation reaction were carried out according to the methods in Example 1, Examples 10 to 15 and Comparative Examples 9 to 12, and the anthocyanin content in the liquid and the extractant was detected after the reaction was completed, and the extraction rate was calculated. The results are shown in Table 2 below.

[0058] Table 2 Group Extraction rate (%) Example 1 95.51 Example 10 76.87 Embodiment 11 55.21 Example 12 69.51 Embodiment 13 93.73 Embodiment 14 84.81 Embodiment 15 95.02 Comparative Example 9 47.84 Comparative Example 10 49.36 Comparative Example 11 50.09 Comparative Example 12 52.67 .

[0059] From the data analysis in Table 2, it can be seen that fluorinated carbon nanotubes are the best micro-solid extractant. At the same time, too low a concentration of the micro-solid extractant will lead to a decrease in the extraction rate, and too high a concentration will cause a waste of the extractant without increasing the benefits, so the optimal concentration of the micro-solid extractant is 1% of the mass of the extract. In addition, too short a vortex oscillation time will cause a decrease in the extraction rate. When the vortex oscillation time is greater than 10 minutes, the extraction rate does not change much, so the optimal vortex oscillation time is 10 minutes.

[0060] Example 16 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the aqueous ethanol solution used in the elution process in step (S.2) is a 50% ethanol aqueous solution. Other aspects are the same as those in Example 1.

[0061] Embodiment 17 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the aqueous ethanol solution used in the elution process in step (S.2) is a 70% ethanol aqueous solution. Other aspects are the same as those in Example 1.

[0062] Embodiment 18 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 20:1. Other aspects are the same as those in Example 1.

[0063] Embodiment 19 The difference between this embodiment and embodiment 1 is that: A method for extracting and purifying anthocyanins from bilberry, wherein the mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 40:1. Other aspects are the same as those in Example 1.

[0064] Comparative Example 13 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the aqueous ethanol solution used in the elution process in step (S.2) is a 30% ethanol aqueous solution. Other aspects are the same as those in Example 1.

[0065] Comparative Example 14 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the aqueous ethanol solution used in the elution process in step (S.2) is anhydrous ethanol. Other aspects are the same as those in Example 1.

[0066] Comparative Example 15 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 10:1. Other aspects are the same as those in Example 1.

[0067] Comparative Example 16 The difference between this comparative example and Example 1 is: A method for extracting and purifying anthocyanins from bilberry, wherein the mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 50:1. Other aspects are the same as those in Example 1.

[0068] The eluents were obtained and dried according to the methods in Example 1, Examples 16 to 19 and Comparative Examples 13 to 16, and the content of anthocyanins in the anthocyanin dry powder was detected and the yield was calculated. The results are shown in Table 3 below.

[0069] Table 3 Group Anthocyanin content in dry powder (%) Yield (%) Example 1 61.84 91.62 Example 16 60.37 90.03 Embodiment 17 60.87 90.57 Embodiment 18 61.35 90.23 Embodiment 19 61.43 91.16 Comparative Example 13 21.67 35.75 Comparative Example 14 58.75 88.67 Comparative Example 15 43.56 77.83 Comparative Example 16 54.32 83.51 .

[0070] From the data analysis in Table 3, it can be seen that as an eluent, the higher the ethanol concentration, the better, but the elution effect will decrease when it is pure ethanol (anhydrous ethanol), so the best eluent is 95% ethanol. In addition, when the amount of eluent reaches more than 30 times the mass of the extractant, the yield of anthocyanins no longer increases, indicating that the elution is complete when the amount of eluent reaches 30 times, so the best amount of eluent is 30 times the mass of the extractant.

[0071] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting and purifying anthocyanins from bilberry, characterized in that: The following steps are involved: (S.1) crushing fresh bilberry fruit into a homogenate, adding water, and ultrasonicating to obtain a mixed solution, adjusting the pH, and extracting at a temperature to obtain an extract; (S.2) Add an extractant to the extract obtained in step (S.1), vortex and oscillate, filter with a microfiltration membrane after the reaction is completed, take the filter residue and elute the filter residue with an ethanol aqueous solution, take the eluate and dry it to obtain anthocyanin dry powder.

2. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The ultrasonic frequency in step (S.1) is 35 to 45 KHz.

3. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The pH value of the mixed solution in step (S.1) is 1-2.

4. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The reaction temperature of the heat preservation extraction process in step (S.1) is 45-55°C, and the extraction time is 1-3h.

5. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The mass ratio of fresh bilberry fruit to water added in step (S.1) is 1:1-3.

6. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The extractant added in step (S.2) is any one or more combinations of fluorinated carbon nanotubes, aluminum oxide, and silica gel.

7. The method for extracting and purifying anthocyanins from bilberry according to claim 1 or 6, characterized in that: The mass of the extractant added in step (S.2) is 0.5-1.5% of the mass of the extract.

8. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The vortexing time of the vortexing vibration process in step (S.2) is 8 to 12 minutes.

9. The method for extracting and purifying anthocyanins from bilberry according to claim 1, characterized in that: The aqueous solution of ethanol used in the elution process in step (S.2) is 50-95% ethanol.

10. The method for extracting and purifying anthocyanins from bilberry according to claim 1 or 9, characterized in that: The mass ratio of the ethanol aqueous solution used in the elution process in step (S.2) to the filter residue is 20 to 40:1.