METHOD FOR PREPARING WATER-SOLUBLE ASTAXANTHIN COMPLEX AND AQUEOUS SOLUTION OF ASTAXANTHIN PREPARED THEREBY

Abstract

Provided is a method for preparing water-soluble astaxanthin complex. The method includes mixing a raw material containing astaxanthin with a solution containing an organic acid and performing cell disruption and leaching and can enable natural astaxanthin to interact with components such as proteins, nucleic acids, or polysaccharides that naturally exist in the raw material, so as to directly prepare water-soluble astaxanthin complex without modifying astaxanthin. The method requires a natural source of astaxanthin, has low equipment requirements and production costs, is simple to operate, green and safe with no organic solvent residues, and easy to industrialize.

The representative figure is FIG. 1.

Claims

1. A method for preparing water-soluble astaxanthin complex, comprising: (1) mixing a raw material containing astaxanthin with a solution containing an organic acid and performing cell disruption to obtain a leachate; and (2) stirring and leaching the leachate in step (1) and performing solid-liquid separation to obtain water-soluble astaxanthin complex.

2. The method according to claim 1, wherein the raw material containing astaxanthin comprises any one selected from the group consisting of Haematococcus pluvialis, Phaffia rhodozyma, crustaceans, chlorella, and a combination of at least two selected therefrom.

3. The method according to claim 1, wherein the organic acid in the solution containing an organic acid in step (1) has a mass concentration of 0.1 to 2.0 moL/L.

4. The method according to claim 1, wherein the solution containing an organic acid has a pH of 3.0 to 6.5, preferably 5.0 to 6.3.

5. The method according to claim 1, wherein the organic acid comprises any one selected from the group consisting of malic acid, tartaric acid, glycine, oxalic acid, citric acid, and a combination of at least two selected therefrom.

6. The method according to claim 1, wherein the solution containing an organic acid is a pH buffer solution of an organic acid.

7. The method according to claim 6, wherein the pH buffer solution of an organic acid comprises any one selected from the group consisting of a buffer solution of malic acid and sodium malate, a buffer solution of tartaric acid and sodium tartrate, a buffer solution of glycine and HCl, a buffer solution of oxalic acid and sodium oxalate, a buffer solution of citric acid and sodium citrate, and a combination of at least two selected therefrom.

8. The method according to claim 1, wherein a material-to-liquid ratio of the raw material containing astaxanthin to the solution containing an organic acid is 0.1-10 g: 10-40 mL.

9. The method according to claim 1, wherein in step (1) and/or step (2), an auxiliary agent is added to assist in leaching.

10. The method according to of claim 1, wherein the stirring and leaching in step (2) is performed for 6 to 72 hours.

11. The method according to claim 1, wherein the solid-liquid separation in step (2) comprises centrifugal separation.

12. The method according to claim 1, further comprising: (3) re-leaching raw material residues containing astaxanthin obtained through the solid-liquid separation in step (2) at least once.

13. The method according to claim 1, comprising: (1) mixing a raw material containing astaxanthin with a solution containing an organic acid and performing cell disruption to obtain a leachate, wherein the organic acid has a mass concentration of 0.1 to 2.0 moL/L, the solution containing an organic acid is a pH buffer solution of an organic acid with a pH of 3.0 to 6.5, and a material-to-liquid ratio of the raw material containing astaxanthin to the solution containing an organic acid is 0.1-10 g: 10-40 mL; (2) stirring and leaching the leachate in step (1) for 6 to 72 h at 20 to 35° C. under a closed condition without light, performing centrifugal separation for 30 to 60 min at 2 to 5° C. and 7000 to 9000 r/min, and filtering a supernatant obtained through the centrifugal separation with a filter to obtain water-soluble astaxanthin complex; and (3) mixing raw material residues containing astaxanthin obtained through solid-liquid separation in step (2) with a solution containing an organic acid to obtain a leachate and repeating step (2), wherein the re-leaching is performed at least once.

14. An aqueous solution of astaxanthin prepared by the method for preparing water-soluble astaxanthin complex according to claim 1.

15. A lyophillized powder of astaxanthin prepared by lyophillizing the aqueous solution of astaxanthin according to claim 14.

16. The method according to claim 9, wherein the auxiliary agent comprises any one selected from the group consisting of proteins, nucleic acids, polysaccharides, and a combination of at least two selected therefrom.

17. The method according to claim 1, wherein the stirring and leaching is performed at a temperature of 20 to 35° C.

18. The method according to claim 1, wherein the stirring and leaching is performed under a closed condition without light.

19. The method according to claim 11, wherein the centrifugal separation is performed at a temperature of 2 to 5° C.

20. The method according to claim 11, wherein after the centrifugal separation, a supernatant obtained through the centrifugal separation is filtered with a filter to obtain a solution of water-soluble astaxanthin complex.

21. The method according to claim 12, wherein the re-leaching comprises: mixing the raw material residues containing astaxanthin with a solution containing an organic acid to obtain a leachate and repeating step (2).

22. The method according to claim 21, wherein the re-leaching adopts the same solution containing an organic acid as step (1) and/or a different solution containing an organic acid from step (1).

23. The method according to claim 21, wherein the re-leaching adopts the same solution containing an organic acid as step (1) and then a different solution containing an organic acid from step (1).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a flowchart of a method for preparing water-soluble astaxanthin complex according to the present application.

[0065] FIG. 2 is a diagram of water-soluble astaxanthin complex prepared in Example 1 of the present application under a transmission electron microscope.

DETAILED DESCRIPTION

[0066] The technical solutions of the present application are further described below through embodiments in conjunction with drawings.

[0067] The present application is further described in detail below. The examples described below are merely simple examples of the present application and not intended to represent or limit the scope of the present application. The protection scope of the present application is defined by the claims.

[0068] As shown in FIG. 1 which is a flowchart of a method for preparing water-soluble astaxanthin complex according to the present application, the method specifically includes the following steps:

[0069] (1) mixing a raw material containing astaxanthin with a solution containing an organic acid and performing cell disruption to obtain a leachate;

[0070] (2) stirring and leaching the leachate in step (1) and performing solid-liquid separation to obtain water-soluble astaxanthin complex; and

[0071] (3) mixing raw material residues containing astaxanthin obtained through the solid-liquid separation in step (2) with the solution containing an organic acid to obtain a leachate and repeating step (2), where re-leaching is performed at least once.

I. EXAMPLES

Example 1

[0072] This example provides a method for preparing water-soluble astaxanthin complex. The method includes the following steps:

[0073] (1) Haematococcus pluvialis (wet algae) was taken out from a refrigerator of −20° C. and thawed, 1 g of Haematococcus pluvialis algae mud was weighed and added with 20 mL of a buffer solution of citric acid/sodium citrate with a concentration of 0.1 mol/L and a pH of 6.0, they were mixed thoroughly, and the mixed solution of Haematococcus pluvialis was disrupted for 30 min with an ultrasonic disruptor to obtain a leachate;

[0074] (2) the leachate in step (1) was transferred to a 50 mL brown finger-shaped bottle, placed on a magnetic stirrer, and stirred and leached at 25° C. for 24 h in a closed environment without light; and

[0075] (3) the mixed solution after leaching in step (2) was transferred to a centrifuge tube and centrifuged at 4° C. and 8000 rpm for 30 min with a centrifuge, and a supernatant after centrifugation was filtered with a disposable filter with a pore size of 3 μm to obtain water-soluble astaxanthin complex that was uniform, stable, orange-red and translucent and had a concentration of 72.66 μg/mL.

[0076] The morphological characterization of water-soluble astaxanthin complex prepared in this example was carried out. A drop of freshly prepared water-soluble astaxanthin complex was dropped on a clean paraffin board and a copper mesh was gently placed on the surface of the drop so that the drop was immersed in the copper mesh. The copper mesh was removed after 10 min, the liquid on the surface of the copper mesh was wiped off with a filter paper, and the copper mesh was then placed with the surface with the liquid up. The front face of the copper mesh was dyed with 1% phosphotungstic acid for 10 min, dried, and observed with a transmission electron microscope (JEM-1400 transmission electron microscope produced by Japan JEOL) whose field of view was adjusted.

[0077] The diagram of water-soluble astaxanthin complex prepared in this example under the transmission electron microscope is shown in FIG. 2. The prepared water-soluble astaxanthin complex has a regular and complete shape and a spherical shape with good dispersibility, and most astaxanthin particles have a diameter within a range of 40 to 100 nm, do not agglomerate, and have relatively high stability.

Example 2

[0078] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate with a pH of 6.0” in step (1) was replaced with “a buffer solution of citric acid/sodium citrate with a pH of 3.0”.

Example 3

[0079] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate with a pH of 6.0” in step (1) was replaced with “a buffer solution of citric acid/sodium citrate with a pH of 4.0”.

Example 4

[0080] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate with a pH of 6.0” in step (1) was replaced with “a buffer solution of citric acid/sodium citrate with a pH of 5.0”.

Example 5

[0081] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate” in step (1) was replaced with “a buffer solution of glycine/HCl”.

Example 6

[0082] This example provides a method for preparing water-soluble astaxanthin complex. The method performs re-extraction six times on raw material residues containing astaxanthin obtained through the centrifugal separation in step (3) in Example 1, which specifically includes the following steps:

[0083] the raw material residues containing astaxanthin obtained through the centrifugal separation in step (3) were mixed with a buffer solution of citric acid/sodium citrate with a concentration of 0.1 mol/L and a pH of 6.0 at a material-to-liquid ratio of 1 g:20 mL to obtain a leachate, step (2) was repeated, and the re-leaching was repeated six times.

Example 7

[0084] This example provides a method for preparing water-soluble astaxanthin complex. The method performs re-extraction 10 times on raw material residues containing astaxanthin obtained through the centrifugal separation in step (3) in Example 1, which specifically includes the following steps:

[0085] (4) the raw material residues containing astaxanthin obtained through the centrifugal separation in step (3) were mixed with a buffer solution of citric acid/sodium citrate with a concentration of 0.1 mol/L and a pH of 6.0 at a material-to-liquid ratio of 1 g:20 mL to obtain a leachate, step (2) was repeated, and the re-leaching was repeated six times; and

[0086] (5) the raw material residues containing astaxanthin obtained through the centrifugal separation in step (4) were mixed with a buffer solution of citric acid/sodium citrate with a concentration of 0.1 mol/L and a pH of 5.0 at a material-to-liquid ratio of 1 g:20 mL to obtain a leachate, step (2) was repeated, and the re-leaching was repeated four times.

Example 8

[0087] This example provides a method for preparing water-soluble astaxanthin complex. The method includes the following steps:

[0088] (1) Chlorella (wet algae) was taken out from a refrigerator of −20° C. and thawed, 10 g of Chlorella algae mud were weighed and added with 40 mL of a buffer solution of citric acid/sodium citrate with a concentration of 0.1 mol/L and a pH of 3.0, they were mixed thoroughly, and the mixed solution of Chlorella was disrupted for 60 min with an ultrasonic disruptor to obtain a leachate;

[0089] (2) the leachate in step (1) was transferred to a 50 mL brown finger-shaped bottle, placed on a magnetic stirrer, and stirred and leached at 35° C. for 12 h in a closed environment without light; and

[0090] (3) the mixed solution after leaching in step (2) was transferred to a centrifuge tube and centrifuged at 3° C. and 7000 rpm for 60 min with a centrifuge, and a supernatant after centrifugation was filtered with a disposable filter with a pore size of 2.5 μm to obtain water-soluble astaxanthin complex that was uniform, stable, orange-red and translucent.

Example 9

[0091] This example provides a method for preparing water-soluble astaxanthin complex. The method includes the following steps:

[0092] (1) Rhodotorula fafu was taken out from a refrigerator of −20° C. and thawed, 0.5 g of Rhodotorula fafu mud were weighed and added with 10 mL of a buffer solution of malic acid/sodium malate with a concentration of 0.15 mol/L and a pH of 6.5, they were mixed thoroughly, and the mixed solution of Rhodotorula fafu was disrupted for 60 min with an ultrasonic disruptor to obtain a leachate;

[0093] (2) the leachate in step (1) was transferred to a 50 mL brown finger-shaped bottle, placed on a magnetic stirrer, and stirred and leached at 20° C. for 48 h in a closed environment without light; and

[0094] (3) the mixed solution after leaching in step (2) was transferred to a centrifuge tube and centrifuged at 5° C. and 9000 rpm for 40 min with a centrifuge, and a supernatant after centrifugation was filtered with a disposable filter with a pore size of 3 μm to obtain water-soluble astaxanthin complex that was uniform, stable, orange-red and translucent.

Example 10

[0095] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate” in step (1) was replaced with “a buffer solution of tartaric acid/sodium tartrate”.

Example 11

[0096] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 1 except that “a buffer solution of citric acid/sodium citrate” in step (1) was replaced with “a solution of citric acid”.

Example 12

[0097] This example provides a method for preparing water-soluble astaxanthin complex, which is the same as that in Example 7 except that 0.001 g of ATP synthase β-subunit were added in last four re-extractions in step (5).

[0098] II. Test and Result

[0099] 1. Extraction Rate

[0100] The content of astaxanthin in a solution of water-soluble astaxanthin complex is tested by the following method: astaxanthin is extracted from water-soluble astaxanthin complex by use of methanol and chloroform. 1 mL of water-soluble astaxanthin complex was extracted with organic solvents and 1 mL of methanol and 2 mL of chloroform were added in sequence. Water-soluble astaxanthin complex was extracted until an aqueous phase was almost colorless, and organic phases were collected, dried with nitrogen, and passed through an organic filter membrane of 0.22 μm by use of 0.5 mL of methanol and 0.5 mL of methyl t-butyl ether (1/1, v/v) to collect samples for a later analysis. Standard astaxanthin (1 mg) was fully dissolved in 5 mL of methanol and 5 mL of methyl t-butyl ether (1/1, v/v) and passed through an organic filter membrane of 0.22 μm to collect samples for a later analysis. The content of astaxanthin was determined by HPLC.

[0101] Chromatographic column: YMC-Carotenoid-C30 chromatographic column (4.6 mm×250 mm, 5 μm); mobile phase: A is methanol and B is methyl t-butyl ether; linear gradient elution: B is 10% from 0 to 15 min, B increases from 10% to 60% from 15 to 25 min, and B decreases from 60% back to 10% from 25 to 35 min; flow rate: 1 mL/min; DAD detection wavelength: 476 nm; column oven temperature: 35° C.; sample volume: 20 μL. The content of astaxanthin in 1 mL of water-soluble astaxanthin complex is calculated according to formula (1):

[00001]$\begin{array}{cc}{\mu }_g=\frac{S_1\times m\times A}{S_2}& \left(1\right)\end{array}$

[0102] In formula (1), μ.sub.g denotes the mass of astaxanthin in 1 mL of water-soluble astaxanthin complex, S.sub.1 denotes the peak area of astaxanthin in water-soluble astaxanthin complex, S.sub.2 denotes the peak area of standard astaxanthin, m denotes the mass of astaxanthin in 20 μL of standard solution of astaxanthin and is 2 mg, and A denotes a conversion factor and is 50.

[0103] The extraction rate is calculated as follows: all astaxanthin is extracted from a raw material containing astaxanthin according to the above experimental method, the total content of astaxanthin in the raw material is obtained, and the extraction rate of astaxanthin is calculated according to formula (2):

[00002]$\begin{array}{cc}\mathrm{Extraction}\mathrm{rate}\%=\frac{\begin{array}{c}\mathrm{Content}\mathrm{of}\mathrm{astaxanthin}\mathrm{in}\mathrm{water}-\\ \mathrm{soluble}\mathrm{astaxanthin}\end{array}}{\begin{array}{c}\mathrm{Total}\mathrm{content}\mathrm{of}\mathrm{astaxanthin}\mathrm{in}\\ \mathrm{the}\mathrm{raw}\mathrm{material}\mathrm{containing}\mathrm{astaxanthin}\end{array}}\times 100\%& \left(2\right)\end{array}$

[0104] The extraction rates of astaxanthin in Examples 1 to 5 are calculated according to the above method and shown in Table 1.

TABLE-US-00001 TABLE 1 Example Example 1 Example 2 Example 3 Example 4 Example 5 Extraction 37.45 ± 0.87 3.93 ± 0.34 14.17 ± 1.22 24.13 ± 1.41 1.44 ± 0.25 Rate (wt %)

[0105] The extraction rates of six re-extractions in Example 6 are calculated according to the above method. The results of the first extraction in Example 1 and the six re-extractions in Example 6 are shown in Table 2.

TABLE-US-00002 TABLE 2 Extraction Times Extraction Rate (wt %) First extraction 37.45 ± 0.87  First re-extraction 11.66 ± 0.98  Second re-extraction 8.11 ± 0.55 Third re-extraction 3.71 ± 1.29 Fourth re-extraction 1.89 ± 0.49 Fifth re-extraction 0.52 ± 0.22 Sixth re-extraction 0.54 ± 0.17

[0106] The extraction rates of the last four re-extractions in Example 7 are calculated according to the above method. The results of the re-extractions are shown in Table 3.

TABLE-US-00003 TABLE 3 Example 7 Extraction Times Extraction Rate (wt %) Seventh re-extraction 1.67 ± 0.44 Eighth re-extraction 1.31 ± 0.37 Ninth re-extraction 0.61 ± 0.24 Tenth re-extraction 0.58 ± 0.11

[0107] 2. Antioxidant Property

[0108] Method for analyzing the antioxidant property: an antioxidant capacity of water-soluble astaxanthin complex is determined through the scavenging rate of ABTS.sup.+ free radicals. A solution of potassium persulfate with a concentration of 2.6 mmol/L and a solution of ABTS with a concentration of 7.4 mmol/L were mixed in a brown finger-shaped bottle in equal amounts and reacted at room temperature for 12 h without light to obtain an operating solution. The overnight ABTS.sup.+ solution was diluted with absolute ethanol until the absorbance at 734 nm was 0.700±0.20 for use. 10 μL of water-soluble astaxanthin complex were placed in a 96-well plate and added with 200 μL of diluted ABTS.sup.+ solution. The mixed solution was mixed thoroughly and shaken uniformly and reacted for 1 h at normal temperature with no light. A microplate reader was used for measuring the absorbance at a wavelength of 734 nm and the absorbance was recorded as A1. 10 μL of water-soluble astaxanthin complex were mixed thoroughly with 200 μL of absolute ethanol and reacted for 1 h at normal temperature with no light. The microplate reader was used for measuring the absorbance at a wavelength of 734 nm and the absorbance was recorded as A2. 10 μL of absolute ethanol were mixed thoroughly with 200 μL of ABTS.sup.+ solution and reacted for 1 h at normal temperature with no light. The microplate reader was used for measuring the absorbance at a wavelength of 734 nm and the absorbance was recorded as A0.

[0109] The above steps were repeated three times and data was recorded respectively. The scavenging rate of ABTS.sup.+ free radicals by water-soluble astaxanthin complex is calculated according to formula (3):

[00003]$\begin{array}{cc}\mathrm{Scavenging}\mathrm{rate}\%=\frac{A_0-\left(A_1-A_2\right)}{A_0}\times 100\%& \left(3\right)\end{array}$

[0110] In formula (3), A1 denotes the absorbance after the sample reacts with ABTS.sup.+ free radicals, A2 denotes the absorbance of the sample itself, and A0 denotes the absorbance of the blank group.

[0111] The antioxidant property of water-soluble astaxanthin complex in Examples 1 to 5 was calculated according to the above method and expressed as the scavenging rate of ABTS.sup.+ free radicals. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Example Scavenging Rate (wt %) Example 1 82.99 ± 2.67 Example 2 32.51 ± 1.98 Example 3 50.48 ± 2.34 Example 4 68.89 ± 2.55 Example 5 25.32 ± 1.56

[0112] The following points can be seen from Table 1 and Table 4:

[0113] (1) It can be seen from Examples 1 to 4 that the effect of the buffer system of citric acid/sodium citrate in preparing water-soluble astaxanthin complex is related to the pH of the system. The extraction rate of astaxanthin increases with an increase of the pH within the pH range of 3.0 to 6.0, and the antioxidant property of the obtained water-soluble astaxanthin complex increases with an increase of the pH. When the pH is 5.0 to 6.0, the effect in preparing water-soluble astaxanthin complex is better, the highest extraction rate of astaxanthin can reach 37.45%, and the antioxidant property can be up to 82.99 wt % in terms of the scavenging rate of ABTS.sup.+ free radicals.

[0114] (2) It can be seen from Examples 1 and 5 that the buffer system of citric acid/sodium citrate is used in Example 1, and the buffer solution of glycine/HCl is used in Example 5; in Example 1, the extraction rate is 37.45 wt %, and the scavenging rate of ABTS.sup.+ free radicals is 82.99 wt %, while in Example 5, the extraction rate is only 1.44 wt % and the scavenging rate of ABTS.sup.+ free radicals is only 25.32 wt %, which shows that the present application improves the extraction rate and antioxidant property of water-soluble astaxanthin complex by selecting a particular buffer solution system.

[0115] It can be seen from Table 2 and Table 3 that the raw material residues of astaxanthin after leaching and extraction still contain astaxanthin and can be extracted multiple times to improve the overall extraction rate of the process, and the extraction rate of astaxanthin gradually decreases when the same pH buffer solution is used for leaching and extraction multiple times and can be improved again by changing the ratio or pH of the pH buffer solution. Due to mild conditions and simple operations of leaching and extraction, the extraction rate can be improved through multiple times of leaching and extraction, which is very easy for industrial production. The overall extraction rate reaches 68.05 wt % after 10 re-extractions in Example 7, which has a high industrial application value.

[0116] Moreover, Examples 8 and 9 can achieve similar extraction effects to those of Example 1 and can also prepare water-soluble astaxanthin complex in one step. Details are not repeated here.

[0117] Example 10 that uses a buffer solution of tartaric acid/sodium tartrate has a lower extraction rate and a poorer antioxidant property of the prepared water-soluble astaxanthin complex than Example 1 that uses a buffer solution of citric acid/sodium citrate. In the present application, the astaxanthin extraction effect is intermediate and a relatively good extraction rate and antioxidant property can be achieved when a system of malic acid/sodium malate is used.

[0118] In Example 11, it is difficult to ensure the stability of the pH in the extraction process when a solution of citric acid is used instead of a pH buffer system. Relative to the use of a buffer solution of citric acid/sodium citrate buffer, the extraction rate decreases.

[0119] In Example 12, proteins are added in the re-extraction process so that the assembly of astaxanthin in the solution is promoted and the extraction rate of astaxanthin is improved.

[0120] In summary, the method for preparing water-soluble astaxanthin complex provided by the present application includes mixing a raw material containing astaxanthin with a solution containing an organic acid and performing cell disruption and leaching and can enable natural astaxanthin to interact with components such as proteins, nucleic acids, or polysaccharides that naturally exist in the raw material, where the extraction rate of the first extraction is 1.44 wt % or more and can be 37.45 wt % or more at most and the overall extraction rate of multiple extractions can reach 68.05 wt %. The method can obtain water-soluble astaxanthin complex in one step without solvent residues, requires a natural source of astaxanthin, and is simple to operate and easy to industrialize.

[0121] The applicant has stated that although the detailed structure characteristics of the present application are described through the examples described above, the present application is not limited to the detailed structure characteristics described above, which means that the implementation of the present application does not necessarily depend on the detailed structure characteristics described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of units selected in the present application and addition of auxiliary units thereof, and selections of specific methods, etc., all fall within the protection scope and the disclosed scope of the present application.