A method for extracting astaxanthin from haematococcus pluvialis using na des

Abstract

The application discloses a kind of NADES and method for extracting astaxanthin from haematococcus pluvialis using it, wherein a kind of natural eutectic solvent for extracting astaxanthin from haematococcus pluvialis includes 1 molar part of hydrogen bond acceptor, 2-6 molar parts of hydrogen bond donor;The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,4-butanediol or 1,2-propanediol.The extraction solvent of the application only needs to use the above-mentioned natural eutectic solvent to realize the extraction of astaxanthin in haematococcus pluvialis, which has the advantages of safety, green, higher extraction efficiency, etc., and is very beneficial to the industrialization and large-scale production of astaxanthin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological extraction, in particular to a NADES and a method for extracting astaxanthin from haematococcus pluvialis using the same. BACKGROUND

[0002] Haematococcus pluvialis is a unicellular green microalgae and an important source of astaxanthin. Astaxanthin is known as the king of antioxidants and is widely recognized for its high antioxidant capacity and potential applications in food and cosmetics, and has good effects in preventing cancer, cardiovascular disease, inflammation and neurodegenerative disease. Currently, the methods for extracting natural astaxanthin from haematococcus pluvialis mainly include microwave-assisted extraction, organic solvent extraction, ultrasonic-assisted extraction, enzymatic hydrolysis, supercritical fluid extraction and oil extraction. However, for haematococcus pluvialis, the cell wall has three layers, and the effect of breaking the cell wall is a key factor for astaxanthin extraction.

[0003] Among them, the ultrasonic extraction of astaxanthin from wild haematococcus pluvialis was carried out at different temperatures from 30℃ to 60℃ for 0-90min, and it was found that the astaxanthin recovery rate was the highest when the ultrasonic extraction was carried out at 45℃ and 60min with a power of 18.40w, and the astaxanthin was extracted with acetone.

[0004] In addition, the use of strong acid (HCl) in the prior art can effectively destroy the cell wall, and then the use of acetone for extraction can make the extraction efficiency reach 86-94%, but the use of a large amount of strong acid will pollute the environment and also reduce the quality of astaxanthin. In 2016, Desai et al. treated haematococcus pluvialis with ionic liquid (IL), which can also destroy the cell wall and form micropores, and then ethyl acetate can be used to effectively extract astaxanthin. Among them, the microalgae treated with ionic liquid 1-Ethyl-3methylimidazolium di-butylphosphate ([Emim]DBP) can be extracted with ethyl acetate to achieve an extraction efficiency of more than 70%, but the ionic liquid still has certain environmental pollution. Subsequently, Fan et al. found that the more environmentally friendly protonic ionic liquid Ethanolammonium caproate (EAC) can also effectively dissolve the mannose in the cell wall and effectively extract astaxanthin, but it still has the problem of unsatisfactory extraction effect. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the defects of the extraction method in the prior art that the extraction effect is not ideal and is not environmentally friendly, so as to provide a safe and green method for extracting astaxanthin from haematococcus pluvialis with higher extraction efficiency.

[0006] A natural deep eutectic solvent (NADES) for extracting astaxanthin from Haematococcus pluvialis, comprising 1 mol of a hydrogen bond acceptor and 2-6 mol of a hydrogen bond donor; the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,4-butanediol or 1,2-propanediol.

[0007] When the hydrogen bond donor is 1,4-butanediol, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 6:1.

[0008] When the hydrogen bond donor is 1,2-propanediol, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 2:1.

[0009] A method for extracting astaxanthin from Haematococcus pluvialis using NADES, comprising the following steps:

[0010] Obtaining the natural deep eutectic solvent for extracting astaxanthin from Haematococcus pluvialis described above;

[0011] Mixing the natural deep eutectic solvent with water in a volume ratio of (1-4):1 to prepare an extraction solution;

[0012] Adding Haematococcus pluvialis to the extraction solution, and after constant temperature shock incubation, obtaining an extraction solution by ultrasonic treatment;

[0013] Adding an organic solvent for extraction, obtaining an extraction solution, drying, and obtaining an extraction sample.

[0014] The temperature of the constant temperature shock incubation is 45-70 DEG C, preferably 60 DEG C.

[0015] The ultrasonic treatment time is 50-70 min.

[0016] The extraction time is 5-40 min, preferably 25-40 min.

[0017] The organic solvent used in the extraction is n-hexane, and the extraction is performed at least twice, preferably twice.

[0018] The technical scheme of the present application has the following advantages:

[0019] 1. The natural deep eutectic solvent for extracting astaxanthin from Haematococcus pluvialis provided by the present application uses natural substances with good biocompatibility as hydrogen bond donors and acceptors to form a deep eutectic solvent. Specifically, choline chloride is used as the hydrogen bond acceptor, and 1,4-butanediol or 1,2-propanediol is used as the hydrogen bond donor. The hydrogen bond donor and acceptor themselves have the advantages of low cost, easy preparation, and environmental friendliness. The combination of a specific ratio of hydrogen bond donors and acceptors can achieve more effective extraction of astaxanthin from Haematococcus pluvialis. Compared with other natural deep eutectic solvents in terms of ratio and type, the extraction efficiency of the present application is more significant.

[0020] 2. The natural deep eutectic solvent provided by the application can be recycled, and the recycling effect does not weaken obviously, so it is very beneficial to the industrialization and large-scale production of astaxanthin.

[0021] 3. The method for extracting astaxanthin from Haematococcus pluvialis using NADES provided by the application uses a specific ratio and type of natural deep eutectic solvent which is more environmentally friendly as an extraction solvent, and uses ultrasonic-assisted natural deep eutectic solvent as a pretreatment means for Haematococcus pluvialis cell wall breaking and astaxanthin extraction. Not only can a new green and environmentally friendly method for breaking the cell wall and extracting astaxanthin be developed, but the extraction rate of astaxanthin in the Haematococcus pluvialis can even reach 100%, and the effect is very significant. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 is the extraction rate result graph of astaxanthin extracted by different NADES in Example 1 of the present application;

[0024] Figure 2 is the extraction rate result graph of astaxanthin extracted by NADES and water in different proportions in Example 2 of the present application.

[0025] Figure 3 is the extraction rate result graph of astaxanthin extracted at different extraction temperatures in Example 3 of the present application.

[0026] Figure 4 is the extraction rate result graph of astaxanthin extracted at different ultrasonic times in Example 4 of the present application.

[0027] Figure 5 is the extraction rate result graph of astaxanthin extracted at different extraction times in Example 5 of the present application.

[0028] Figure 6 is the extraction rate result graph of astaxanthin extracted by 3 batches of NADES and the extracted liquid recycled in Example 6 of the present application.

[0029] Figure 7 is the liquid phase comparison graph of astaxanthin and astaxanthin standard after grinding and breaking the cell wall of the extraction sample and the control in Example 1 of the present application. DETAILED DESCRIPTION

[0030] Example 1

[0031] A method for extracting astaxanthin from Haematococcus pluvialis using NADES, comprising:

[0032] The NADES prepared according to Table 1 was mixed with water at a volume ratio of 3:1 to prepare an extraction solution. 10 ml of the extraction solution was taken, 50 mg of Haematococcus pluvialis powder was added and mixed, and then incubated in a 45°C constant temperature water bath at 60 rpm for 1 h. An ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT) was used for ultrasonic treatment for 1 h. An equal volume of n-hexane (10 ml) was added, and the mixture was extracted at 45°C constant temperature water bath at 60 rpm for 30 min. The extraction was repeated once using n-hexane, and the extraction solutions were combined. The extraction solution was rotary evaporated to dryness at 40°C, and then redissolved with 1 ml of methanol to obtain the extraction sample.

[0033] Table 1

[0034] Hydrogen bond acceptor (HBA) Hydrogen bond donor (HBD) HBA:HBD molar ratio Room temperature appearance NA-1 Choline chloride Lactic acid 1:1 Clear, transparent liquid NA-2 Choline chloride Malic acid 1:1 Clear, transparent liquid NA-3 Choline chloride Malonic acid 1:1 Clear, transparent liquid NA-4 Choline chloride 1,4-Butanediol 1:6 Clear, transparent liquid NA-5 Choline chloride 1,2-Propanediol 1:2 Clear, transparent liquid NA-6 Choline chloride Glycerol 1:1 Clear, transparent liquid NA-7 Choline chloride Fructose + water 2:1:1 Clear, transparent liquid NA-8 Choline chloride D-Sorbitol 2:5 Clear, transparent liquid NA-9 Choline chloride Glucose + water 2:1:1 Clear, transparent liquid NA-10 Choline chloride Maltose + water 3:1:2 Pale yellow, transparent liquid NA-11 Choline chloride Urea 1:2 Clear, transparent, viscous liquid NA-12 Choline chloride Xylitol 1:2 Clear, transparent, viscous liquid NA-13 Glycerol L-Proline 3:1 Yellow, transparent liquid NA-14 Glycerol L-Alanine 3:1 Pale yellow, clear, transparent liquid NA-15 Glycerol Glycine 3:1 Orange, clear, transparent liquid NA-16 Glycerol L-Histidine 3:1 White, viscous liquid NA-17 Glycerol L-Threonine 3:1 Clear, transparent liquid NA-18 Glycerol Lysine 4.5:1 Yellow, transparent, viscous liquid NA-19 Glycerol L-Arginine 3:1 White, viscous liquid

[0035] 50 mg of Haematococcus pluvialis powder was weighed and placed in a dry glass homogenizer. 1 mL of dichloromethane-methanol solution was added and the cells were ground to break the cell wall completely. The glass homogenizer was washed with 10 mL of n-hexane solution for 3 times, and then shaken at 200 r / min for 60 min. The mixture was centrifuged at 8000 r / min for 5 min at 5°C, and the supernatant was transferred. The extraction was repeated once using 10 mL of n-hexane solution. The supernatants were combined. The supernatant was rotary evaporated to dryness at 40°C, and then redissolved with 1 ml of methanol to obtain the astaxanthin after grinding and breaking the cell wall.

[0036] The sample was analyzed using a YMC carotenoid chromatographic column with a size of 4.6*250 mm and a particle size of 5 microns. A ternary mobile phase was used, which was composed of methanol, methyl tert-butyl ether and 1% v / v phosphoric acid. The flow rate was 1 ml / min, the detection wavelength was 480 nm, and the column temperature was set at 35°C. The program is shown in Table 2.

[0037] Table 2: Liquid chromatography mobile phase elution conditions

[0038] Time (min) Methanol (%) Methyl tert-butyl ether (%) 1% v / v phosphoric acid (%) 0-10 min 81→71 15→25 4 10 min 81 15 4 10-15 min 81 15 4

[0039] The astaxanthin content in the control astaxanthin after grinding and breaking the cell wall and the astaxanthin content in the extraction sample in the present application were detected by the above method. According to the detection results, the extraction efficiency of astaxanthin in different NADES in Table 1 was calculated using the following calculation formula. Calculation formula: extraction efficiency = astaxanthin content after NADES treatment / astaxanthin content after grinding and breaking the cell wall*100%.

[0040] The extraction rate of astaxanthin extracted by different NADES is shown in Table 2. Figure 1 Figure 1 ​It can be seen that the extraction efficiency of NA-4 and NA-5 is significantly higher than that of other NADES. The extraction efficiency of other NADES is less than 10%, while NA-4 and NA-5 can reach 57.2% and 48.5% respectively, which is very significant.

[0041] Example 2

[0042] A method for extracting astaxanthin from Haematococcus pluvialis using NADES includes:

[0043] Extracts were prepared by mixing NA-4 and water in volume ratios of 1:1, 2:1, 3:1, and 4:1 as described in Example 1. 10 ml of the extract was mixed with 50 mg of Haematococcus pluvialis powder and incubated at 45°C with shaking at 60 rpm for 1 hour. The mixture was then ultrasonically treated for 1 hour using an ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT). An equal volume of n-hexane (10 ml) was added, and the mixture was extracted at 45°C with shaking at 60 rpm for 30 minutes. The extraction was repeated once with n-hexane, and the extracts were combined. The extracts were then rotary evaporated to dryness at 40°C and reconstituted with 1 ml of methanol to obtain the extracted sample.

[0044] The extraction rates of astaxanthin at different water ratios were obtained using the method described in Example 1, and the test results are as follows: Figure 2 As shown, through Figure 2 It can be seen that there is no significant difference in the extraction rate of astaxanthin under volume ratios of 1:1, 2:1, 3:1 and 4:1, but the extraction effect is the best at 2:1, with an extraction efficiency of 61.80±1.24%.

[0045] Example 3

[0046] A method for extracting astaxanthin from Haematococcus pluvialis using NADES includes:

[0047] The extract was prepared by mixing NA-4 with water at a volume ratio of 2:1 as described in Example 1. 10 ml of the extract was mixed with 50 mg of Haematococcus pluvialis powder and incubated in constant temperature water at 25℃, 35℃, 45℃, 60℃, and 80℃ for 1 h with shaking. Then, it was ultrasonically treated for 1 h using an ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT). An equal volume of n-hexane (10 ml) was added, and the mixture was extracted again in constant temperature water at the same temperature with shaking at 60 rpm for 30 min. The extraction was repeated once with n-hexane, and the extracts were combined. The extract was evaporated to dryness at 40℃ and reconstituted with 1 ml of methanol to obtain the extracted sample.

[0048] The extraction rates of astaxanthin at different extraction temperatures were obtained using the method described in Example 1, and the results are as follows: Figure 3 As shown, through Figure 3It can be seen that the appropriate temperature can significantly improve the extraction rate of astaxanthin, especially at 45℃ and 60℃, the extraction efficiency can reach more than 50%, and the extraction efficiency at 60℃ is the best, which can reach 106.71±2.78%.

[0049] Example 4

[0050] A method for extracting astaxanthin from Haematococcus pluvialis using NADES, comprising:

[0051] The NA-4 in Example 1 was mixed with water in a volume ratio of 2:1 to prepare an extraction solution. 10ml of the extraction solution was taken, 50mg of Haematococcus pluvialis algae powder was added and mixed, and then incubated in a constant temperature water bath at 60℃ with 60rpm shaking for 1h. Then the sample was treated with an ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT) for 5min, 10min, 20min, 30min, 45min, 50min, 55min, 60min, 65min, 70min, 75min and 90min. An equal volume of n-hexane (10ml) was added, and then extracted in a constant temperature water bath at 60℃ with 60rpm shaking for 30min. The extraction was repeated once with n-hexane, and the extraction solutions were combined. The extraction solution was rotary evaporated to dryness at 40℃, and then redissolved with 1ml of methanol to obtain the extraction sample.

[0052] The extraction rate of astaxanthin under different ultrasonic times was obtained by the method described in Example 1, and the detection results are shown in Table 2. Figure 4 As shown in Table 2, the extraction rate of astaxanthin under different ultrasonic times was obtained by the method described in Example 1, and the detection results are shown in Table 2. Figure 4 As shown in Table 2, the extraction rate of astaxanthin under different ultrasonic times was obtained by the method described in Example 1, and the detection results are shown in Table 2.

[0053] Example 5

[0054] A method for extracting astaxanthin from Haematococcus pluvialis using NADES, comprising:

[0055] The extract was prepared by mixing NA-4 with water at a volume ratio of 2:1 as described in Example 1. 10 ml of the extract was mixed with 50 mg of Haematococcus pluvialis powder and incubated in a constant-temperature water bath at 60°C with shaking at 60 rpm for 1 hour. Then, it was ultrasonically treated for 60 minutes using an ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT). An equal volume of n-hexane (10 ml) was added, and the mixture was extracted again in a constant-temperature water bath at 60°C with shaking at 60 rpm for 5, 10, 15, 20, 25, 30, 35, and 40 minutes. The extraction was repeated once with n-hexane, and the extracts were combined. The extract was evaporated to dryness at 40°C and reconstituted with 1 ml of methanol to obtain the extracted sample.

[0056] The extraction rates of astaxanthin at different extraction times were obtained using the method described in Example 1, and the results are as follows: Figure 5 As shown, through Figure 5 It can be seen that the extraction rate of astaxanthin is highest when the extraction time is more than 25 minutes, and the extraction rate of astaxanthin can reach 109.60±5.48% when the extraction time is 25 minutes.

[0057] As can be seen from Examples 2-4 above, this invention is based on NA-4, which has the highest extraction efficiency, and after adjusting the water addition ratio (Example 2 and 4), the invention further improves upon this method. Figure 2 Extraction temperature (Example 3 and) Figure 3 ), ultrasound time (Example 4 and Figure 4 Extraction time (Example 5 and) Figure 5 A systematic investigation was conducted, revealing that the optimal extraction efficiency was achieved with a water-to-liquid ratio of 1:1-3:1, a temperature of 45-80℃, an ultrasonic time of 50-70 min, and an extraction time of 25-35 min. Specifically, a water-to-liquid ratio of 2:1, a temperature of 60℃, an ultrasonic time of 60 min, and an extraction time of 30 min resulted in an extraction efficiency exceeding 100%. The extracted samples prepared under the optimal conditions in this example, along with the astaxanthin from Example 1 (after grinding and cell wall disruption) and astaxanthin standards, were analyzed to obtain the corresponding astaxanthin chromatograms, as shown below. Figure 7 As shown, in this Figure 7 a) is the chromatogram of the extracted sample after NA-4 treatment in this invention; b) is the chromatogram of the control astaxanthin obtained by the above-mentioned method of cell wall disruption extraction; c) is the chromatogram of the astaxanthin standard. Figure 7 This further proves the effectiveness of the method of the present invention.

[0058] Example 6

[0059] A method for extracting astaxanthin from Haematococcus pluvialis using NADES, used to verify the effectiveness of NADES recovery, specifically includes:

[0060] The extract was prepared by mixing NA-4 and water at a volume ratio of 2:1 as in Example 1. 10 ml of the extract was mixed with 50 mg of Haematococcus pluvialis powder and incubated in a constant-temperature water bath at 60°C with shaking at 60 rpm for 1 hour. Then, it was ultrasonically treated for 60 minutes using an ultrasonic cleaner (Ningbo Xinzhi, SB25-12DT). An equal volume of n-hexane (10 ml) was added, and the mixture was extracted again in a constant-temperature water bath at 60°C with shaking at 60 rpm for 25 minutes. The extraction with n-hexane was repeated once, and the extracts were combined. The extract was evaporated to dryness at 40°C and reconstituted with 1 ml of methanol to obtain extract sample 1. During the extraction process, the NA-4 and water extract was recovered. 5 ml of this recovered extract was mixed with 25 mg of Haematococcus pluvialis powder and extracted with astaxanthin under the same conditions to obtain extract sample 2.

[0061] Repeat the above steps twice to obtain the test results for three batches of extracted samples. The test results for the three batches of extracted samples are as follows: Figure 6 As shown, the results demonstrate that the efficiency of the recovered NADES did not decrease after being reused three times.

[0062] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting astaxanthin from Haematococcus pluvialis using NADES, characterized in that, Includes the following steps: A natural eutectic solvent for extracting astaxanthin from Haematococcus pluvialis is prepared, comprising a hydrogen bond acceptor and a hydrogen bond donor; wherein the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,4-butanediol or 1,2-propanediol; when the hydrogen bond donor is 1,4-butanediol, the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 6:1; when the hydrogen bond donor is 1,2-propanediol, the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 2:

1. The natural eutectic solvent was mixed with water at a volume ratio of (2-3):1 to prepare an extract. Haematococcus pluvialis was added to the extract, and after constant temperature shaking incubation at 60℃, the extract was obtained by ultrasonic treatment for 50-70 minutes. Add an organic solvent and extract for 25-40 minutes to obtain the extract, dry it, and obtain the extracted sample.

2. The method for extracting astaxanthin from Haematococcus pluvialis using NADES according to claim 1, characterized in that, The organic solvent used in the extraction is n-hexane, and the extraction is performed at least twice.

3. The method for extracting astaxanthin from Haematococcus pluvialis using NADES according to claim 2, characterized in that, The extraction was performed twice.

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