Method for extracting and purifying astaxanthin from yarrowia lipolytica fermentation liquor

Abstract

The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting and purifying astaxanthin from yarrowia lipolytica fermentation liquor. The method comprises the following steps: firstly, carrying out engineering transformation on yarrowia lipolytica to obtain yarrowia lipolytica engineering bacteria capable of producing astaxanthin, culturing and fermenting the engineering bacteria to obtain astaxanthin, and then carrying out wall breaking on yeast fermentation liquor containing astaxanthin by utilizing a high-pressure homogenization method, so that astaxanthin components in the yeast fermentation liquor are fully dissolved out, and the yield of the astaxanthin is improved. And extracting and separating by adopting an organic solvent, and purifying the obtained crude astaxanthin extracting solution by using macroporous adsorption resin to finally obtain the astaxanthin with high yield and high purity. By adopting the method disclosed by the invention, not only can the astaxanthin with higher content be obtained, but also the purity of the astaxanthin is 99.2% or above, and the method is very suitable for large-scale production of the astaxanthin and has very good economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth. Background Art

[0002] Astaxanthin (3,3′-dihydroxy-4,4′-diketo-β,β′-carotene, AST) is an oxidized carotenoid that has potential benefits for a variety of diseases including cancer, hypertension, diabetes, cardiovascular disease, gastrointestinal disease, liver disease, neurodegenerative diseases, skin diseases, etc. It is often used to combat oxidative damage to diseased cells. For this reason, its demand in the food industry, medicine, cosmetics and animal feed (such as salmon and prawns) is rapidly increasing.

[0003] Astaxanthin was first discovered in 1938 and used as a pigment in aquaculture for the first time. At present, the sources of astaxanthin are mainly chemical synthesis and biological extraction. Since the antioxidant capacity of chemically synthesized astaxanthin is lower than that of biologically extracted astaxanthin, and the intermediates and by-products synthesized by chemical synthesis have potential residues, synthetic astaxanthin has not yet been approved for human use. Therefore, astaxanthin is still mainly obtained by biological extraction, mainly through extraction from aquatic products, microalgae and astaxanthin-producing yeast cells.

[0004] In the existing related technologies, there are many studies on the methods of astaxanthin extraction and purification. For example, patent CN107805215A provides a method of low-temperature grinding, enzymatic low-temperature wall breaking, organic solvent multi-stage extraction and combined macroporous adsorption resin purification to extract and purify astaxanthin from Haematococcus pluvialis; patent CN109053520A uses an acid-heat method to break the wall of red yeast to extract astaxanthin; patent CN111170915A uses ultrasound to break the wall of aquatic products, and uses supercritical carbon dioxide technology to extract astaxanthin; patent CN116836098A uses an organic solvent to extract the fermentation broth, and then uses supercritical carbon dioxide for secondary extraction.

[0005] However, most of the technologies related to astaxanthin extraction and purification, including the above patents, have the following problems: First, the cell wall breaking method of astaxanthin fermentation broth is complicated, the cell wall breaking rate is low, and it is not easy to achieve large-scale production; second, a large amount of acid or alkaline solution is used in the cell wall breaking process, which not only causes environmental pollution, but also causes loss of the quality and content of astaxanthin; third, the astaxanthin extraction process is cumbersome, the purification cost is high, it is difficult to form a large-scale industry, and the purity of the purified astaxanthin is limited. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth.

[0007] The present invention firstly performs engineering modification on Yarrowia lipolytica to obtain an engineered Yarrowia lipolytica bacteria capable of producing astaxanthin (this operation is reflected in the patent CN118272421A applied for by the inventor's unit and will not be repeated here), obtains astaxanthin by culturing and fermenting the engineered bacteria, and then uses a high-pressure homogenization method to break the cell wall of the yeast fermentation broth containing astaxanthin so that the astaxanthin component therein is fully dissolved, and further uses an organic solvent for extraction and separation, and the obtained crude astaxanthin extract is eluted and purified by a macroporous adsorption resin, and finally a high-content, high-purity astaxanthin can be obtained.

[0008] More specifically, the present invention provides a method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, which specifically comprises the following steps:

[0009] (1) Preparation of Yarrowia lipolytica fermentation broth containing astaxanthin

[0010] The plasmid containing β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ was transformed into Yarrowia lipolytica IF015 to construct a recombinant engineering bacterium, and the fermentation culture was carried out to obtain a Yarrowia lipolytica fermentation broth containing astaxanthin;

[0011] (2) Extraction of astaxanthin

[0012] The yeast fermentation broth obtained in (1) is broken by a high-pressure homogenization method, and the mixture is centrifuged, washed, extracted and separated by an organic solvent to obtain crude astaxanthin;

[0013] (3) Purification of astaxanthin

[0014] The crude astaxanthin extract obtained in (2) is purified by using a macroporous adsorption resin to obtain purified astaxanthin, wherein the macroporous adsorption resin is selected from at least one of HP-20, AB-8, DM130 and HPD-722.

[0015] In the above-mentioned astaxanthin extraction and purification method provided by the present invention, preferably, in (1), the culture medium used comprises:

[0016] YPD medium: tryptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L;

[0017] Fermentation medium: glucose 80 g / L, soy peptone 20 g / L, beef extract 10 g / L, ammonium sulfate 15 g / L, magnesium sulfate heptahydrate 12 g / L, pH 6.8.

[0018] Preferably, the yeast fermentation broth described in (2) has a bacterial concentration of 30-40%.

[0019] During the research process, the inventors found that in the process of high-pressure homogenization and cell wall breaking of the yeast fermentation broth obtained by fermentation, if the bacterial concentration is too high, the high-pressure homogenization will easily clog the device and the homogenization cell wall breaking rate will be seriously reduced; in addition, if the bacterial concentration is maintained at a low level, although it has a good cell wall breaking effect to a certain extent, the energy consumption is too high, which is not conducive to the large-scale production of astaxanthin. Only when the bacterial concentration is within the range of 30% to 40% can a good cell wall breaking rate and low energy consumption be achieved at the same time.

[0020] Preferably, the pressure of the high-pressure homogenization described in (2) is 500-600 bar, and the number of homogenization times is 2-3 times.

[0021] More preferably, the pressure of the high-pressure homogenization described in (2) is 500 bar, and the number of homogenization times is 2 times.

[0022] Preferably, the organic solvent described in (2) is at least one selected from anhydrous ethanol, n-hexane, acetone, dichloromethane and ethyl acetate.

[0023] More preferably, the organic solvent described in (2) is at least one selected from anhydrous ethanol, dichloromethane and ethyl acetate.

[0024] Preferably, the weight of the organic solvent in (2) is 250 to 20,000 times the weight of astaxanthin, and the extraction temperature is 25 to 70°C.

[0025] More preferably, the weight of the organic solvent in (2) is 300 to 16,000 times the weight of astaxanthin, and the extraction temperature is 25 to 65°C.

[0026] In fact, the inventors used liquid chromatography to calculate the specific content of astaxanthin that could be produced by fermentation during the initial fermentation process, and further selected different types of organic reagents to extract astaxanthin according to the solubility of astaxanthin in different types of organic reagents. The solubility of astaxanthin in ethanol was 16.7 g / mg, the solubility in ethyl acetate was 1.7 g / mg, and the solubility in dichloromethane was 0.5 g / mg.

[0027] In the present invention, by calculating the content of astaxanthin and then according to the solubility of astaxanthin in the organic solvent, an appropriate amount of organic solvent can be more reasonably used to extract and separate it, which can not only save extraction reagents and avoid waste of reagents, but also can extract the astaxanthin component to the greatest extent.

[0028] Preferably, the macroporous adsorption resin described in (3) is HP-20 macroporous adsorption resin.

[0029] More preferably, the macroporous adsorption resin described in (3) is HP-20 macroporous adsorption resin, and the adsorption capacity of HP-20 resin is 1.2 mg / g dry resin.

[0030] As a further preferred embodiment, in (3), the specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: a mixture of n-hexane and ethanol is used as the eluent for gradient elution, the volume ratio of n-hexane to ethanol in the eluent is 0-2h: 5:1; 2-4h: 5:3; 4-5h: 5:2; 5-6h: 1:2, the loading rate is 1.5-2.5Bv / h, the elution rate is 0.8-1.5Bv / h, and the eluate is collected starting from the volume of 1.5Bv.

[0031] Alternatively, in (3), the specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: using a mixture of n-hexane and ethyl acetate as an eluent for gradient elution, the volume ratio of n-hexane to ethyl acetate in the eluent is 0-1h: 7:2; 1-2h: 7:3; 2-3h: 5:3; 3-4h: 4:3, the loading rate is 1.5-2.5Bv / h, the elution rate is 1.0-1.5Bv / h, and the eluate is collected starting from the volume of 1.5Bv.

[0032] Alternatively, in (3), the specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: using a mixture of n-hexane and acetone as an eluent for gradient elution, the volume ratio of n-hexane to acetone in the eluent is 0-1h: 6:1; 1-2h: 5:1; 2-3h: 3:1, the loading rate is 0.5-1.5Bv / h, the elution rate is 0.4-0.8Bv / h, and the eluate is collected starting from a volume of 1.0Bv.

[0033] The beneficial effects of the present invention are:

[0034] Provided is a complete process for the biological preparation, extraction and purification of astaxanthin. On the one hand, Yarrowia lipolytica IF015 is engineered and then fermented and cultured with the engineered bacteria to obtain a crude astaxanthin fermentation liquid with a large yield. On the other hand, a simple physical wall-breaking method is used to achieve wall-breaking extraction of crude astaxanthin. In addition, a process for purifying the crude astaxanthin extract is provided, that is, HP-20 macroporous adsorption resin is used and organic solvents such as n-hexane, ethanol, ethyl acetate, etc. are used as eluents for elution. The purity of the obtained astaxanthin is improved compared with that in the prior art, and the highest purity can reach 99.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The HPLC spectrum of astaxanthin purified by the method of Example 1 of the present invention;

[0036] Figure 2 The HPLC spectrum of astaxanthin purified by the method of Example 2 of the present invention;

[0037] Figure 3 The HPLC spectrum of astaxanthin purified by the method of Example 3 of the present invention;

[0038] Figure 4 The HPLC spectrum of astaxanthin purified by the method of Comparative Example 1 of the present invention;

[0039] Figure 5 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 2 of the present invention;

[0040] Figure 6 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 3 of the present invention;

[0041] Figure 7 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 4 of the present invention;

[0042] Figure 8 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 5 of the present invention;

[0043] Fig. 9 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 6 of the present invention;

[0044] Fig.10 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 7 of the present invention;

[0045] Fig.11 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 8 of the present invention;

[0046] Fig.12 This is the HPLC spectrum of astaxanthin purified by the method of Comparative Example 9 of the present invention. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0048] The culture medium formula adopted in the present invention is as follows:

[0049] YPD medium: tryptone 20g / L, yeast extract 10g / L, glucose 20g / L. Fermentation medium: glucose 80g / L, soy peptone 20g / L, beef extract 10g / L, ammonium sulfate 15g / L, magnesium sulfate heptahydrate 12g / L, pH 6.8.

[0050] HPLC detection conditions: chromatographic column, YMC-Carotenoid-C18 chromatographic column (4.6 mm × 250 mm, 5 μm); column temperature, 30°C; detection wavelength, 474 nm;

[0051] The mobile phase was a mixture of acetonitrile: methanol: isopropanol: water (v / v / v / v) = 9:6:4:1; flow rate, 1.5 mL / min; detector, UV detector; injection volume, 20 μL.

[0052] Example 1

[0053] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0054] (1) Preparation of Yarrowia lipolytica fermentation broth containing astaxanthin

[0055] The plasmid containing β-carotene ketolase CrtW (accession number: ADN43075.1) and β-carotene hydroxylase CrtZ (accession number: UAW09501.1) constructed in our laboratory (patent CN118272421A) was transformed into Yarrowia lipolytica IF015 to obtain transformants. The correct transformants were screened by gene sequencing to obtain Yarrowia lipolytica engineered bacteria. The engineered bacteria were inoculated into YPD liquid culture medium, shake-cultured at 30°C and 220rpm for 14h, and then transferred to fermentation medium, and shake-cultured at 30°C and 220rpm for 96h to obtain Yarrowia lipolytica fermentation broth containing astaxanthin.

[0056] (2) Extraction of astaxanthin

[0057] First, the pressure of homogenization wall breaking is screened, and the specific operations are as follows:

[0058] Three portions of the Yarrowia lipolytica fermentation broth containing astaxanthin obtained in (1) were taken respectively, with a bacterial broth concentration of 30%, each portion of 100 g, and homogenized twice under pressure conditions of 400 bar, 500 bar, and 600 bar, respectively, and the cell wall breaking rate was calculated by microscopic counting method.

[0059] The results show that when the homogenization pressure is 400 bar, 500 bar, and 600 bar, the wall breaking rates are 70%, 90%, and 92%, respectively. Considering the wall breaking rate and energy consumption, the present invention selects a homogenization pressure of 500 bar for wall breaking.

[0060] Under the above premise, 500 g of the Yarrowia lipolytica fermentation broth containing astaxanthin obtained in (1) was taken with a bacterial concentration of 30%. The mixture was homogenized twice at a pressure of 500 bar and centrifuged at a speed of 6000 rpm for 10 min to obtain a heavy phase and a light phase, respectively. The heavy phase was washed with water and centrifuged. The astaxanthin content in the light phase and the heavy phase was detected by high performance liquid chromatography (HPLC). The results showed that the astaxanthin content in the light phase was 0 mg, and the astaxanthin content in the heavy phase was 635 mg.

[0061] Finally, 9.5 kg of anhydrous ethanol was added to the heavy phase (the astaxanthin content was 635 mg) obtained by centrifugation, stirred and extracted at 65°C and 230 rpm for 30 min, and filtered with filter paper; 200 g of anhydrous ethanol was continued to be added to the filter cake, extracted at 65°C and 230 rpm for 30 min, filtered with filter paper, and the two extracts were combined to obtain the crude astaxanthin extract, and the astaxanthin content in the extract was detected by HPLC.

[0062] The results showed that the astaxanthin content in the extract was 624 mg and the extraction rate was 98.3%.

[0063] (3) Purification of astaxanthin

[0064] The crude astaxanthin extract obtained in (2) was purified using HP-20 macroporous adsorption resin, and the adsorption capacity of HP-20 resin was 1.2 mg / g dry resin. The eluent during purification was a mixture of n-hexane-ethanol, the loading rate during elution was 2 Bv / h, the elution rate was 1.0 Bv / h, and the gradient elution was performed according to Table 1 below.

[0065] Table 1 Elution ratio of eluent n-hexane-ethanol (v / v)

[0066] Time (h) 0～1 1～2 2～3 3～4 4～5 5～6 n-Hexane 5 5 5 5 5 1 Ethanol 1 1 3 3 2 2

[0067] During the purification process of astaxanthin, the eluate was collected starting from a volume of 1.5 Bv, and a total of 6.3 kg of the eluate was collected. The eluate was concentrated in a water bath at 50° C. and vacuum dried to obtain 510 mg of astaxanthin with a yield of 80.3%.

[0068] The purity of astaxanthin was tested by HPLC. Figure 1 As shown, the retention time of astaxanthin was 3.313 min and the purity was 99.6%.

[0069] Example 2

[0070] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0071] (1) Exactly the same as Example 1;

[0072] (2) Different from Example 1, the solvent used for extracting astaxanthin in this example is ethyl acetate, and the specific operation is as follows:

[0073] Take the heavy phase obtained after centrifugation in step (2) of Example 1 (the astaxanthin content is 635 mg), add 0.95 kg of ethyl acetate, stir and extract at 55° C. and 230 rpm for 25 min, and filter with filter paper; continue to add 150 g of ethyl acetate to the filter cake, stir and extract at 55° C. and 230 rpm for 30 min, filter with filter paper, and combine the two extracts to obtain a crude astaxanthin extract. The astaxanthin content in the extract is detected by HPLC.

[0074] The results showed that the astaxanthin content in the extract was 630 mg and the extraction rate was 99.2%.

[0075] (3) The crude astaxanthin extract obtained in (2) was purified using HP-20 macroporous adsorption resin. The eluent during purification was a mixture of n-hexane and ethyl acetate. The loading rate during elution was 2 Bv / h, the elution rate was 1.0 Bv / h, and the gradient elution was performed according to Table 2.

[0076] Table 2 Eluent ratio of n-hexane-ethyl acetate (v / v)

[0077]

[0078]

[0079] In the above elution process, the eluate was collected starting from the volume of 1.5 Bv, and a total of 500 g of the eluate was collected. The eluate was concentrated in a water bath under vacuum at 50° C. and dried under vacuum to obtain 515 mg of astaxanthin with a yield of 81.1%.

[0080] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 2 As shown, the retention time of astaxanthin was 3.317 min and the purity was 99.8%.

[0081] Example 3

[0082] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0083] (1) Exactly the same as Example 1;

[0084] (2) Different from Example 1, the solvent used for extracting astaxanthin in this example is dichloromethane, and the specific operation is as follows:

[0085] To the heavy phase (astaxanthin content: 635 mg) obtained after centrifugation in step (2) of Example 1, 203 g of dichloromethane was added, and the mixture was stirred and extracted at 25 ° C. and 230 rpm for 30 min, and filtered with filter paper; 50 g of dichloromethane was continued to be added to the filter cake, and the mixture was stirred and extracted at 25 ° C. and 230 rpm for 30 min, and filtered with filter paper. The two extracts were combined, and the astaxanthin content in the extract was detected by HPLC.

[0086] The results showed that the astaxanthin content in the extract was 632 mg and the extraction rate was 99.5%.

[0087] (3) The crude astaxanthin extract obtained in (2) was purified using HP-20 macroporous adsorption resin. The eluent during purification was a mixture of n-hexane and acetone. The loading rate during elution was 1.0 Bv / h, the elution rate was 0.5 Bv / h, and gradient elution was performed according to Table 3.

[0088] Table 3 n-hexane-acetone (v / v) eluent ratio

[0089] time 1 h 2 h 3 h n-Hexane 6 5 3 acetone 1 1 1

[0090] In the above elution process, the eluate was collected starting from 1 Bv volume, and a total of 120 g of the eluate was collected. The eluate was concentrated in a water bath under vacuum at 30° C. and dried under vacuum to obtain 513 mg of astaxanthin with a yield of 80.8%.

[0091] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 3 As shown, the retention time of astaxanthin was 3.315 min and the purity was 99.2%.

[0092] Comparative Example 1

[0093] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0094] (1)-(2) are the same as in Example 1, except that:

[0095] The macroporous adsorption resin used in the purification of crude astaxanthin in (3) of this comparative example is AB-8 macroporous adsorption resin, the eluent is a mixture of n-hexane-ethanol, the elution rate is 0.5 Bv / h, and the collection starts from 2 Bv volume. After multiple elution experiments, the yield of astaxanthin is 56.3%.

[0096] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 4 As shown, the retention time of astaxanthin was 3.310 min and the purity was 70.8%.

[0097] Comparative Example 2

[0098] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0099] (1)-(2) Same as Example 2;

[0100] The difference from Example 2 is that the macroporous adsorption resin used in the purification of crude astaxanthin in (3) of this comparative example is AB-8 macroporous adsorption resin, the elution rate is 0.8 Bv / h, and the collection starts from the volume of 2 Bv. After multiple elution experiments, the yield of astaxanthin is 65.8%.

[0101] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 5 As shown, the retention time of astaxanthin was 3.320 min and the purity was 68.4%.

[0102] Comparative Example 3

[0103] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0104] (1)-(2) Same as Example 3;

[0105] The difference from Example 3 is that the macroporous adsorption resin used in the purification of crude astaxanthin in (3) of this comparative example is AB-8 macroporous adsorption resin, the loading rate during elution is 1.2 Bv / h, the elution rate is 0.3 Bv / h, and the collection starts from the volume of 1.5 Bv. After multiple experiments, the astaxanthin yield is 39.8%.

[0106] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 6 As shown, the retention time of astaxanthin was 3.302 min and the purity was 88.4%.

[0107] Comparative Example 4

[0108] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0109] (1)-(2) Same as Example 1;

[0110] The difference from Example 1 is that the macroporous adsorption resin used in (3) of this comparative example to purify crude astaxanthin is DM130 macroporous adsorption resin, the eluent is the same as that of Example 1, the loading rate during elution is 2.0 Bv / h, the elution rate is 0.8 Bv / h, and the collection starts from 4.0 Bv volume. After multiple experiments, the astaxanthin yield is 40.6%.

[0111] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 7 As shown, the retention time of astaxanthin was 3.332 min and the purity was 66.3%.

[0112] Comparative Example 5

[0113] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0114] (1)-(2) Same as Example 2;

[0115] The difference from Example 2 is that the macroporous adsorption resin used in (3) of this comparative example to purify crude astaxanthin is DM130 macroporous adsorption resin, the loading rate during elution is 2.0 Bv / h, the elution rate is 1.0 Bv / h, and the collection starts from 3.0 Bv volume. After multiple experiments, the astaxanthin yield is 62.2%.

[0116] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Figure 8 As shown, the retention time of astaxanthin was 3.314 min and the purity was 82.4%.

[0117] Comparative Example 6

[0118] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0119] (1)-(2) Same as Example 3;

[0120] The difference from Example 3 is that the macroporous adsorption resin used in the purification of crude astaxanthin in (3) of this comparative example is DM130 macroporous adsorption resin, the loading rate during elution is 2.0 Bv / h, the elution rate is 1.5 Bv / h, and the collection starts from 3.0 Bv volume. After multiple experiments, the astaxanthin yield is 30.3%.

[0121] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Fig. 9 As shown, the retention time of astaxanthin was 3.309 min and the purity was 84.4%.

[0122] Comparative Example 7

[0123] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0124] (1)-(2) Same as Example 1;

[0125] The difference from Example 1 is that the macroporous adsorption resin used in (3) of this comparative example to purify crude astaxanthin is HPD-722 macroporous adsorption resin, the loading rate during elution is 3.0 Bv / h, the elution rate is 1.0 Bv / h, and the collection starts from 3.0 Bv volume. After multiple experiments, the astaxanthin yield is 35%.

[0126] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Fig.10 As shown, the retention time of astaxanthin was 3.307 min and the purity was 60.4%.

[0127] Comparative Example 8

[0128] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0129] (1)-(2) Same as Example 2;

[0130] The difference from Example 2 is that the macroporous adsorption resin used in (3) of this comparative example to purify crude astaxanthin is HPD-722 macroporous adsorption resin, the loading rate during elution is 1.5 Bv / h, the elution rate is 1.0 Bv / h, and the collection starts from 3.0 Bv volume. After multiple experiments, the astaxanthin yield is 48%.

[0131] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Fig.11 As shown, the retention time of astaxanthin was 3.303 min and the purity was 74.5%.

[0132] Comparative Example 9

[0133] A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, specifically comprising the following steps:

[0134] (1)-(2) Same as Example 3;

[0135] The difference from Example 3 is that the macroporous adsorption resin used in the purification of crude astaxanthin in (3) of this comparative example is HPD-722 macroporous adsorption resin, the loading rate during elution is 1.5 Bv / h, the elution rate is 0.5 Bv / h, and the collection starts from 3.0 Bv volume. After multiple experiments, the astaxanthin yield is 20%.

[0136] The purity of astaxanthin obtained was tested by HPLC. The test results are as follows: Fig.12 As shown, the retention time of astaxanthin was 3.287 min and the purity was 81.4%.

[0137] It can be seen from the above experimental results of the present invention that the method of fermentation using engineered bacteria in the present invention can obtain a large yield of astaxanthin in a short time, and for the fermentation broth containing astaxanthin obtained after fermentation, good dissolution of astaxanthin therein can be achieved only by a simple physical wall breaking method, with a wall breaking rate of up to 90%, without the need to add additional chemical reagents such as acids and alkalis, thereby avoiding the damage of astaxanthin to the additives.

[0138] In addition, in the present invention, HP-20 macroporous adsorption resin is used to purify the crude astaxanthin after extraction and separation. The yield and purity of astaxanthin in each embodiment are relatively high, with the highest yield reaching 81.1% and the purity reaching 99.8%.

[0139] However, after the inventors adjusted the macroporous adsorption resin for astaxanthin purification, that is, using macroporous adsorption resins of models such as AB-8, DM130, and HPD-722 to purify astaxanthin, the yield and purity of the obtained astaxanthin were significantly low, making it difficult to achieve good technical effects.

Claims

1. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth, characterized in that: The steps include: (1) Preparation of Yarrowia lipolytica fermentation broth containing astaxanthin The plasmid containing β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ was transformed into Yarrowia lipolytica IF015 to construct a recombinant engineering bacterium, and the fermentation culture was carried out to obtain a Yarrowia lipolytica fermentation broth containing astaxanthin; (2) Extraction of astaxanthin The yeast fermentation broth obtained in (1) is broken by a high-pressure homogenization method, and the mixture is centrifuged, washed, extracted and separated by an organic solvent to obtain a crude astaxanthin extract; (3) Purification of astaxanthin The crude astaxanthin extract obtained in (2) is purified by using a macroporous adsorption resin to obtain purified astaxanthin, wherein the macroporous adsorption resin is selected from at least one of HP-20, AB-8, DM130, and HPD-722.

2. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth as claimed in claim 1, characterized in that: The pressure of the high-pressure homogenization described in (2) is 500~600 bar, and the number of homogenization times is 2~3 times.

3. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth as claimed in claim 1, characterized in that: The organic solvent described in (2) is at least one selected from anhydrous ethanol, n-hexane, acetone, dichloromethane and ethyl acetate.

4. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth according to claim 1, characterized in that: The weight of the organic solvent described in (2) is 250 to 20,000 times the weight of astaxanthin, and the extraction temperature is 25 to 70°C.

5. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth as claimed in claim 1, characterized in that: The macroporous adsorption resin described in (3) is HP-20 macroporous adsorption resin.

6. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth according to claim 1, characterized in that: (3) The specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: gradient elution is performed using a mixture of n-hexane and ethanol as the eluent, the volume ratio of n-hexane to ethanol in the eluent is 0~2 h: 5:1; 2~4 h: 5:3; 4~5 h: 5:2; 5~6 h: 1:2, the loading rate is 1.5~2.5 Bv / h, the elution rate is 0.8~1.5 Bv / h, and the eluate is collected starting from the volume of 1.5 Bv.

7. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth according to claim 1, characterized in that: (3) The specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: gradient elution is performed using a mixture of n-hexane and ethyl acetate as the eluent, the volume ratio of n-hexane to ethyl acetate in the eluent is 0~1 h: 7:2; 1~2 h: 7:3; 2~3 h: 5:3; 3~4 h: 4:3, the loading rate is 1.5~2.5 Bv / h, the elution rate is 1.0~1.5 Bv / h, and the eluate is collected starting from the volume of 1.5 Bv.

8. A method for extracting and purifying astaxanthin from Yarrowia lipolytica fermentation broth according to claim 1, characterized in that: (3) The specific operation of purifying astaxanthin using macroporous adsorption resin HP-20 is as follows: gradient elution is performed using a mixture of n-hexane and acetone as the eluent, the volume ratio of n-hexane to acetone in the eluent is 0~1 h: 6:1; 1~2 h: 5:1; 2~3 h: 3:1, the loading rate is 0.5~1.5 Bv / h, the elution rate is 0.4~0.8 Bv / h, and the eluate is collected starting from the volume of 1.0 Bv.

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