Method for improving extraction efficiency of astaxanthin in yeast by using ethanol as solvent
By mixing anhydrous ethanol with glycerol as a solvent and stirring in a water bath, the problem of poor solubility when extracting astaxanthin in yeast is solved, which improves the extraction efficiency and safety and reduces the cost.
Patent Information
- Application Number
- CN202411835008.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-07
AI Technical Summary
In the prior art, when ethanol extracts as a solvent for astaxanthin in yeast, the solubility is poor, resulting in low extraction efficiency, high cost, and safety hazards.
The extraction efficiency of astaxanthin is improved by mixing anhydrous ethanol with glycerol as the extraction solvent and sealing and stirring in a water bath.
The extraction efficiency of astaxanthin was improved, and the astaxanthin content in the solution increased by 10-27%, reducing the extraction cost and reducing the risk of using toxic solvents.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for improving the extraction efficiency of astaxanthin in yeast by using ethanol as a solvent, and belongs to the field of biotechnology. Background Art
[0002] Astaxanthin is a keto-carotenoid. Due to its multiple biological functions such as coloring, anti-oxidation, light protection, promoting reproduction, enhancing immunity, and maintaining the health of the central nervous system, it has attracted much attention from domestic and foreign food, medicine, cosmetics, aquaculture and other industries in recent years. Astaxanthin is a purple-red crystal, insoluble in water, and easily soluble in organic solvents such as chloroform, acetone, benzene and carbon disulfide. At present, the common astaxanthin extraction methods are: oil dissolution method, solvent extraction method, enzymatic method, supercritical extraction method, etc. Among them, the oil dissolution method extract is not easy to separate from the high-boiling oil, resulting in a high separation cost; the price of enzymatic enzyme preparations is high, and the enzymatic hydrolysis takes a long time. Supercritical extraction is a green and safe extraction and separation method, but the purchase and maintenance of its instruments and equipment require high investment costs, so there is still a distance from industrialization. Common extraction solvents in solvent extraction are acetone, methanol, isopropanol, petroleum ether, hexane, etc. Studies have shown that when ethanol, acid, alkali, acetone and dichloromethane are used to extract astaxanthin from discarded shrimp heads and shells, dichloromethane has the highest extraction rate. Among the organic solvents (methanol, ethanol, acetonitrile, etc.) used to extract astaxanthin from Haematococcus pluvialis, acetone has the highest extraction rate. It can be seen that different solvents have different extraction effects, the specific components extracted are also different, and the most suitable extractants are different for different raw materials. In addition, since organic solvents such as acetone have low boiling points, are volatile, and have certain toxicity, there are safety and health issues in the processing process, and therefore they are subject to certain restrictions. In comparison, using ethanol as an extraction solvent can avoid the disadvantages of other organic solvents being toxic to the human body, but astaxanthin has poor solubility in ethanol, and the volume of ethanol required is too large, which increases the extraction cost.
[0003] In addition, most of the existing literature is inclined to the extraction method of astaxanthin from Haematococcus pluvialis, and most methods require separate crushing before further extraction. There are few methods that specifically extract astaxanthin from yeast, but as far as the current astaxanthin market is concerned, the production of astaxanthin using yeast will be a hot spot. Therefore, how to efficiently extract astaxanthin from yeast has become an urgent problem to be solved. Summary of the invention
[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent, thereby improving the extraction efficiency of astaxanthin.
[0005] In order to solve the above technical problems, the technical solution provided by the present invention is: A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh the extract, suspend it in anhydrous ethanol, and stir it in a sealed water bath for 20-30 minutes; (2) Add glycerin and continue to seal and stir; (3) Centrifuge and take the supernatant to obtain a solution containing astaxanthin.
[0006] Furthermore, in step (1), the extract may be in the form of: ① bacterial cells obtained after centrifugation of the fermentation liquid: the fermentation liquid is centrifuged in a centrifuge at a speed of 10,000 rpm for 5 minutes, and the supernatant is removed to obtain wet bacterial cells; ② powder after the fermentation liquid is spray-dried: the fermentation liquid is directly fed into a spray tower, or the fermentation liquid is washed with water through a ceramic membrane and then fed into a spray tower, and the spray tower parameters are set to 300 L per hour, an inlet air temperature of 180°C, and an outlet air temperature of 95°C to obtain a dry powder; ③ a product obtained by using a physical method to break the wall of the fermentation liquid after spray drying: the powder after the fermentation liquid is spray-dried is rolled by a roller press to obtain a flake material, and the flake material is crushed by a grinder to obtain a powder.
[0007] Furthermore, the fermentation broth is prepared according to patent CN118441007A.
[0008] Furthermore, in step (1), if the extract is measured as wet weight, the mass ratio of the extract to anhydrous ethanol is 1:10-15; if the extract is measured as dry weight, the mass ratio of the extract to anhydrous ethanol is 1:43-48.
[0009] Furthermore, in step (1), the temperature of the water bath is 50-55°C.
[0010] Furthermore, the volume ratio of anhydrous ethanol in step (1) to glycerol in step (2) is 4:1.
[0011] Anhydrous ethanol is easy to volatilize. When it volatilizes into the air and reaches a certain concentration, it will explode when it encounters an open flame, which is very dangerous. The use of glycerol mixed with anhydrous ethanol can not only increase the yield of anhydrous ethanol extraction of astaxanthin, but also reduce the risk factor. In addition, glycerol can replace part of ethanol. The price of glycerol is lower than that of anhydrous ethanol, which can reduce the cost of extracting astaxanthin.
[0012] Furthermore, in step (2), the sealed stirring time is 1-2 hours.
[0013] Furthermore, in step (3), the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 2-5 min.
[0014] Furthermore, in step (3), the astaxanthin content in the astaxanthin-containing solution is increased by 10-27%. Under the same time, equal weight of extracts, and equal volume of extraction solutions, after a single extraction, the astaxanthin content in the solution of the method of the present invention is increased by 10-27% compared with extraction with anhydrous ethanol.
[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The method of the present invention can improve the extraction efficiency of astaxanthin in Yarrowia lipolytica by using anhydrous ethanol. Under the conditions of the same time, equal weight of extract and equal volume of extraction solution, after a single extraction, the astaxanthin content in the solution increases by 10-27% compared with extraction with anhydrous ethanol, that is, the astaxanthin content in the same volume of extractant increases, thereby reducing the number of extraction times and improving the astaxanthin extraction efficiency.
[0016] (2) The method of the present invention uses glycerol and anhydrous ethanol as extraction solvents, and uses glycerol to replace part of the ethanol. The price of glycerol is lower than that of anhydrous ethanol, which can reduce the cost of extracting astaxanthin. In addition, the selected anhydrous ethanol and glycerol are safe and non-toxic, and will not affect the use of astaxanthin.
[0017] (3) The method of the present invention can improve the yield of astaxanthin. The astaxanthin that cannot be completely extracted by anhydrous ethanol in the discarded extract residue after multiple extractions with ethanol can be recovered by the method of the present invention. DETAILED DESCRIPTION
[0018] Example 1 A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in 4 mL of anhydrous ethanol, and stir them in a sealed container at 50°C in a water bath for 30 min; (2) Add 1 mL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0019] Note: The density of anhydrous ethanol is 0.789-0.791 g / mL.
[0020] Example 2 A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in 4 mL of anhydrous ethanol, and stir them in a sealed container at 55°C in a water bath for 20 min. (2) Add 1 mL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 12,000 rpm for 2 min and collect the supernatant to obtain a solution containing astaxanthin.
[0021] Example 3 A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh 50 mg of Yarrowia lipolytica wet cells after centrifugation of the fermentation broth, suspend them in 800 μL of anhydrous ethanol, and seal and stir them in a 52°C water bath for 25 min; (2) Add 200 μL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 10,000 rpm for 5 min and collect the supernatant to obtain a solution containing astaxanthin.
[0022] Example 4 A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh 50 mg of the residue after the astaxanthin is extracted from Yarrowia lipolytica with anhydrous ethanol, suspend it in 800 μL of anhydrous ethanol, and seal and stir it in a 52°C water bath for 25 min; (2) Add 200 μL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 12,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0023] Example 5 A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent comprises the following steps: (1) Weigh 50 mg of the residue after the astaxanthin is extracted from Yarrowia lipolytica with anhydrous ethanol, suspend it in 800 μL of anhydrous ethanol, and seal and stir it in a 55°C water bath for 20 min; (2) Add 200 μL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 11,000 rpm for 4 min and collect the supernatant to obtain a solution containing astaxanthin.
[0024] Comparative Example 1 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in 5 mL of anhydrous ethanol, and stir them in a sealed container at 50°C in a water bath for 30 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0025] Comparative Example 2 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in a mixed solution of 4 mL of anhydrous ethanol and 1 mL of glycerol, and seal and stir in a 50°C water bath for 30 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0026] Comparative Example 3 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in a mixed solution of 3.34 mL of anhydrous ethanol and 1.67 mL of glycerol, and seal and stir in a 50°C water bath for 30 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0027] Comparative Example 4 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in 3.34 mL of anhydrous ethanol, and stir them in a sealed container at 50°C in a water bath for 30 min; (2) Add 1.67 mL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0028] Comparative Example 5 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in 2.5 mL of anhydrous ethanol, and stir them in a sealed container at 50°C in a water bath for 30 min; (2) Add 2.5 mL of glycerol and continue to seal and stir for 1.5 h; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0029] Comparative Example 6 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 70 mg of broken Yarrowia lipolytica cells, suspend them in a mixed solution of 2.5 mL of anhydrous ethanol and 2.5 mL of glycerol, and seal and stir in a 50°C water bath for 30 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 10,000 rpm for 3 min and collect the supernatant to obtain a solution containing astaxanthin.
[0030] Comparative Example 7 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 50 mg of Yarrowia lipolytica cells after centrifugation of the fermentation broth, suspend them in 1 mL of anhydrous ethanol, and seal and stir them in a 52°C water bath for 25 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 10,000 rpm for 5 min and collect the supernatant to obtain a solution containing astaxanthin.
[0031] Comparative Example 8 A method for extracting astaxanthin from yeast comprises the following steps: (1) Weigh 50 mg of the residue after the astaxanthin is extracted from Yarrowia lipolytica with anhydrous ethanol, suspend it in 1 mL of anhydrous ethanol, and seal and stir it in a 55°C water bath for 20 min; (2) Continue to seal and stir for 1.5 hours; (3) Centrifuge at 11,000 rpm for 4 min and collect the supernatant to obtain a solution containing astaxanthin.
[0032] Experimental Example 1 The astaxanthin content in the astaxanthin-containing solutions of Example 1 and Comparative Examples 1-2 was detected using an ELISA instrument. The results are listed in Table 1.
[0033] Astaxanthin content detection: The astaxanthin solution (mixed extract of anhydrous ethanol and glycerol) is treated as follows: after the anhydrous ethanol is evaporated, an acetone solution with a volume of 2 times that of glycerol is added to dissolve the residue, and the liquid is poured into a separatory funnel to retain the red solution to obtain an acetone solution containing astaxanthin, and acetone is added to the acetone solution to make it equal to the volume of the astaxanthin solution. A standard curve of astaxanthin content is prepared by an enzyme marker, and the absorbance of the solution at 478nm is detected by an enzyme marker, and the astaxanthin content is calculated according to the standard curve.
[0034] Table 1. Astaxanthin content As can be seen from Table 1, Example 1 and Comparative Examples 1-2 both use the same volume of solvent to extract astaxanthin. Comparative Example 1 uses anhydrous ethanol to extract astaxanthin, and the solution contains 343.98 mg / L of astaxanthin; Comparative Example 2 uses anhydrous ethanol and glycerol in a mixed solution with a volume ratio of 4:1 to extract astaxanthin, and the solution contains 257.08 mg / L of astaxanthin; and the solution of the method of the present invention contains 436.85 mg / L of astaxanthin. It can be seen that compared with Comparative Example 1 using only ethanol to extract astaxanthin, the astaxanthin content extracted by the present invention is increased by 27.0%; Comparative Example 2 uses anhydrous ethanol and glycerol in a mixed solution with a volume ratio of 4:1 to extract astaxanthin, and the astaxanthin content is not increased.
[0035] Experimental Example 2 The astaxanthin content in the solutions containing astaxanthin in Example 1, Comparative Example 1, and Comparative Examples 3-4 was detected using an ELISA instrument. The results are listed in Table 2.
[0036] Table 2. Astaxanthin content As can be seen from Table 2, Example 1, Comparative Example 1 and Comparative Examples 3-4 all use the same volume of solvent to extract astaxanthin. Comparative Example 1 uses anhydrous ethanol to extract astaxanthin, and the solution contains 343.98 mg / L of astaxanthin. Comparative Example 3 uses anhydrous ethanol and glycerol in a mixed solution with a volume ratio of 2:1 to extract astaxanthin, and the solution contains 89.35 mg / L of astaxanthin. Comparative Example 4 first extracts with 3.34 mL of anhydrous ethanol and then adds 1.67 mL of glycerol to extract astaxanthin. The volume ratio of the two is 2:1, and the solution contains 283.65 mg / L of astaxanthin. The solution of the method of the present invention contains 436.85 mg / L of astaxanthin. It can be seen that the extraction of astaxanthin using anhydrous ethanol and glycerol in a volume ratio of 2:1 as solvent does not improve the extraction content.
[0037] Experimental Example 3 The astaxanthin content in the astaxanthin-containing solutions of Example 1, Comparative Example 1 and Comparative Examples 5-6 was detected using an ELISA instrument. The results are listed in Table 3.
[0038] Table 3. Astaxanthin content As can be seen from Table 3, Example 1, Comparative Example 1 and Comparative Examples 5-6 all use the same volume of solvent to extract astaxanthin. Comparative Example 1 uses anhydrous ethanol to extract astaxanthin, and the solution contains 343.98 mg / L of astaxanthin; Comparative Example 5 first extracts with 2.5 mL of anhydrous ethanol and then adds 2.5 mL of glycerol to extract astaxanthin, the volume ratio of the two is 1:1, and the solution contains 98.98 mg / L of astaxanthin; Comparative Example 6 extracts astaxanthin with anhydrous ethanol and glycerol in a mixed solution with a volume ratio of 1:1, and the solution contains 27.12 mg / L of astaxanthin. The solution of the method of the present invention contains 436.85 mg / L of astaxanthin. It can be seen that the extraction of astaxanthin with anhydrous ethanol and glycerol in a volume ratio of 1:1 as solvent does not improve the extraction content.
[0039] Experimental Example 4 The astaxanthin content in the astaxanthin-containing solutions of Example 3 and Comparative Example 7 was detected using an ELISA instrument. The results are listed in Table 4.
[0040] Table 4. Astaxanthin content As can be seen from Table 4, Example 3 and Comparative Example 7 both used the same volume of solvent to extract astaxanthin. Comparative Example 7 used anhydrous ethanol to extract astaxanthin, and the solution contained 154.25 mg / L astaxanthin. The solution of the method of the present invention contained 169.81 mg / L astaxanthin. Compared with the astaxanthin extracted with ethanol in Comparative Example 7, the content of astaxanthin extracted by the method of the present invention increased by 10.08%.
[0041] Experimental Example 5 The astaxanthin content in the astaxanthin-containing solutions of Example 5 and Comparative Example 8 was detected using an ELISA instrument. The results are listed in Table 5.
[0042] Table 5. Astaxanthin content As can be seen from Table 5, Example 5 and Comparative Example 8 both used the same volume of solvent to extract astaxanthin from the residue. Comparative Example 8 used anhydrous ethanol to extract astaxanthin, and the solution contained 16.99 mg / L astaxanthin, while the solution of the method of the present invention contained 32.05 mg / L astaxanthin. It can be seen that the content of astaxanthin extracted by the method of the present invention is 1.8 times that of astaxanthin extracted by Comparative Example 8, indicating that the method of the present invention can improve the astaxanthin yield and recover part of the astaxanthin that cannot be completely extracted when anhydrous ethanol is present.
Claims
1. A method for improving the extraction efficiency of astaxanthin in yeast using ethanol as a solvent, characterized in that: The steps include: (1) Weigh the extract, suspend it in anhydrous ethanol, and stir it in a sealed water bath for 20-30 minutes; (2) Add glycerin and continue to seal and stir; (3) Centrifuge and take the supernatant to obtain a solution containing astaxanthin.
2. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: In step (1), the extract comprises: ① bacterial cells obtained after centrifugation of the fermentation liquid: the fermentation liquid is centrifuged in a centrifuge at a speed of 10000 rpm for 5 minutes, and the supernatant is removed to obtain wet bacterial cells; ② powder after the fermentation liquid is spray-dried: the fermentation liquid is directly fed into a spray tower, or the fermentation liquid is washed with water through a ceramic membrane and then fed into a spray tower, and the spray tower parameters are set to 300 L per hour, the inlet air is 180°C, and the outlet air is 95°C to obtain dry powder; ③ the fermentation liquid is spray-dried, and the wall is broken by a physical method to obtain a product: the powder after the fermentation liquid is spray-dried is rolled by a roller press to obtain a flake material, and the flake material is crushed by a grinder to obtain a powder.
3. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: In step (1), if the extract is measured as wet weight, the mass ratio of the extract to anhydrous ethanol is 1:10-15; if the extract is measured as dry weight, the mass ratio of the extract to anhydrous ethanol is 1:43-48.
4. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: In step (1), the temperature of the water bath is 50-55°C.
5. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: The volume ratio of anhydrous ethanol in step (1) to glycerol in step (2) is 4:
1.
6. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: In step (2), the sealed stirring time is 1-2 hours.
7. The method for improving the extraction efficiency of astaxanthin in yeast using ethanol as solvent according to claim 1, characterized in that: In step (3), the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 2-5 min.