A fermentation method for the extraction and purification of astaxanthin

Abstract

The invention discloses an extraction and purification process for producing astaxanthin by a fermentation method, wherein Yarrowia solani is used as a strain for fermentation, and extraction and purification are performed through wall breaking, filtration, supercritical extraction, activated carbon or white clay decolorization, chromatographic separation, evaporation concentration, organic solvent crystallization, and centrifugal drying processes to obtain astaxanthin crystals with a content of more than 98.5%. At the same time, astaxanthin powder with a purity of 15% and astaxanthin powder with a content of 50% can also be obtained during the production process.

Description

Technical Field

[0001] The present invention relates to an extraction and purification process for producing astaxanthin by fermentation, and specifically belongs to an extraction and purification process for astaxanthin. Background Art

[0002] Astaxanthin is the fat-soluble carotenoid compound with the strongest antioxidant activity in nature, and is usually found in various marine bacteria, algae and aquatic animals. Astaxanthin can protect cells, lipids and membrane lipoproteins from oxidative damage; astaxanthin also has anti-aging, anti-inflammatory, anti-cancer properties, inhibits Helicobacter pylori infection, activates immune response, improves memory, etc. It has been widely used in food, medicine, health products, cosmetics and feed. Its global annual demand has reached 250 tons, with sales of nearly US$450 million, and is expected to reach US$2.5 billion by 2025, with extremely high economic and application value.

[0003] At present, the fermentation method for producing astaxanthin in the market is mainly based on Phaffia rubra, but the purity of astaxanthin obtained by fermentation is too low to meet production needs.

[0004] Yarrowia lipolytica contains abundant acetyl-CoA, a precursor raw material for synthesizing carotenoid compounds, and a large amount of lipids are accumulated in its cells without producing endotoxins. These characteristics make Yarrowia lipolytica an excellent base bacteria for biosynthesizing astaxanthin. The present invention uses the extraction and purification process of Yarrowia lipolytica or Saccharomyces cerevisiae to produce astaxanthin, and the purity of astaxanthin is as high as 98.5%, and at the same time, it can meet the requirements of products with 15% purity and 50% purity. The production process is simple and can be applied to large-scale production. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an extraction and purification process for producing astaxanthin by fermentation, which can simultaneously meet the production of astaxanthin products with a purity of 15%, 50% and above 98.5%, and ultimately produce high-purity astaxanthin crystals with a content of above 98.5% by crystallization.

[0006] The technical solution of the present invention is as follows:

[0007] The object of the present invention is to provide an extraction and purification process for producing astaxanthin by a fermentation method, wherein the Yarrowia yeast expression and culture biological fermentation method is used for extraction and purification through wall breaking, filtration, supercritical extraction, decolorization, chromatographic separation, evaporation concentration, organic solvent crystallization and centrifugal drying, and finally astaxanthin crystals with a content of more than 98.5% are obtained.

[0008] Furthermore, the extraction and purification process of astaxanthin produced by the fermentation method comprises the following steps:

[0009] (1) fermenting astaxanthin with Yarrowia lipolytica as the strain, and performing cell wall breaking treatment on the fermented liquid after the fermented liquid is placed in a tank, and simultaneously adding butylated hydroxyanisole antioxidant during cell wall breaking;

[0010] (2) filtering and sterilizing the astaxanthin fermentation liquid after cell wall breaking;

[0011] (3) extracting the filtered fermentation broth with supercritical carbon dioxide to increase the purity of astaxanthin to more than 15%, and spray drying to produce astaxanthin powder with a purity of more than 15%;

[0012] (4) the astaxanthin liquid after supercritical extraction is further purified by simple decolorization treatment;

[0013] (5) The astaxanthin liquid after simple decolorization is further purified by a carbon 18 reverse phase chromatography device, and the purity is increased to more than 50%;

[0014] (6) removing water, organic solvent and volatile impurities from the astaxanthin liquid with a purity of more than 50% obtained after chromatographic separation by evaporation;

[0015] (7) the evaporated astaxanthin liquid is put into a crystallization tank, and petroleum ether or dichloromethane with a mass fraction of 98% or more is added for cooling and crystallization, and the crystallization temperature is reduced to below 10° C., and astaxanthin crystals with a purity of less than 95% are added as seeds during crystallization, and the crystallization is carried out for more than 24 hours;

[0016] (8) The crystallized astaxanthin is centrifuged and then dried to obtain the final astaxanthin crystals.

[0017] Furthermore, in the step (1), the fermentation broth is broken by chemical, mechanical or biological methods, wherein the chemical method uses acid to adjust the pH of the fermentation broth to 4.0-4.5; the mechanical method uses high-pressure homogenization at above 100MP; and the biological method uses lysozyme to add to the fermentation broth.

[0018] Furthermore, the amount of antioxidant butylated hydroxyanisole added in step (1) is 0.2% of the total astaxanthin content in the fermentation broth.

[0019] Furthermore, the filtration and sterilization in step (2) is performed by ceramic membrane filtration or plate and frame filtration.

[0020] Furthermore, in step (5), the simple decolorization is performed by using activated carbon or activated clay for decolorization and purification.

[0021] Compared with the prior art, the beneficial effects of the present invention are astaxanthin production by Yarrowia lycopersicum culturing and biological fermentation, and extraction and purification by cell wall breaking, filtration, supercritical extraction, activated carbon or clay decolorization, chromatographic separation, evaporation concentration, organic solvent crystallization, and centrifugal drying processes, so as to obtain astaxanthin crystals with a content of more than 98.5%. In addition, 15% purity astaxanthin powder and 50% astaxanthin powder can also be produced and processed at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a flowchart of the astaxanthin production process. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0024] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0025] In the present application, Yarrowia lipolytica (ATCC No. MAY-2613, purchased from ATCC) is used.

[0026] Example 1

[0027] The extraction and purification process of astaxanthin produced by fermentation method specifically includes the following steps:

[0028] (1) fermenting astaxanthin using Yarrowia lipolytica as a strain, placing the fermented liquid in a tank, stirring, adding hydrochloric acid or sulfuric acid to adjust the pH to 4.0-4.5 for chemical cell wall breaking, and adding butylated hydroxyanisole (BHA) antioxidant during cell wall breaking, wherein the amount of butylated hydroxyanisole added is 0.2% of the total astaxanthin content in the fermented liquid;

[0029] (2) the astaxanthin fermentation liquid after cell wall breaking is sterilized by plate and frame filtration;

[0030] (3) The filtered fermentation broth is extracted by supercritical carbon dioxide to increase the purity of astaxanthin to more than 15%. At this time, if no further purification is required, astaxanthin powder with a purity of more than 15% can be produced by spray drying;

[0031] (4) If further purification is required, the astaxanthin liquid after supercritical carbon dioxide extraction is subjected to activated carbon decolorization treatment;

[0032] (5) The astaxanthin liquid after activated carbon decolorization is further purified by a carbon 18 reverse chromatography device, at which point the purity can be increased to more than 50%. If subsequent crystallization is not required, spray drying can be performed to obtain a 50% astaxanthin powder;

[0033] (6) removing water, organic solvent and volatile impurities from the astaxanthin liquid obtained after chromatographic separation by evaporation;

[0034] (7) the evaporated astaxanthin liquid is put into a crystallization tank, and petroleum ether with a mass fraction of 98% or more is added to cool and crystallize, the crystallization temperature is reduced to below 10° C., and astaxanthin crystals with a purity of less than 95% are added as seeds during crystallization, and the crystallization is carried out for more than 24 hours;

[0035] (8) The crystallized astaxanthin is centrifuged and then dried to obtain final astaxanthin crystals. The purity of the astaxanthin crystals can be as high as 98.5% or more.

[0036] Example 2

[0037] The extraction and purification process of astaxanthin produced by fermentation method specifically includes the following steps:

[0038] (1) fermenting astaxanthin using Yarrowia lipolytica as the strain, placing the fermented liquid in a tank, stirring, adding lysozyme to biologically break the fermented liquid, and simultaneously adding butylated hydroxyanisole (BHA) antioxidant, wherein the amount of butylated hydroxyanisole added is 0.2% of the total astaxanthin content in the fermented liquid; in addition, a mechanical breaking method can be used to create a pressure of more than 100 MP in the fermenter for high-pressure homogenization breaking;

[0039] (2) The fermented liquid after cell wall breaking is filtered and sterilized by ceramic membrane;

[0040] (3) The filtered fermentation broth is extracted by supercritical carbon dioxide to increase the purity of astaxanthin to more than 15%. At this time, if no further purification is required, astaxanthin powder with a purity of more than 15% can be produced by spray drying;

[0041] (4) decolorizing the astaxanthin after supercritical extraction using activated white clay;

[0042] (5) The decolorized astaxanthin liquid is further purified by a carbon 18 reverse phase chromatography device, and the purity can be increased to more than 50%. If subsequent crystallization is not required, spray drying can be performed to obtain a 50% astaxanthin powder;

[0043] (6) after chromatographic separation, the astaxanthin liquid is concentrated by evaporation to remove water, organic solvents and volatile impurities;

[0044] (7) the evaporated astaxanthin liquid is put into a crystallization tank, and dichloromethane with a mass concentration of 98% or more is added to perform cooling crystallization, the crystallization temperature is below 10° C., high-purity astaxanthin crystals with a purity of 95% or less are added as crystal seeds during crystallization, and the crystallization is performed for more than 24 hours;

[0045] (8) The crystallized astaxanthin is centrifuged and then dried to obtain high-purity astaxanthin.

[0046] The above shows and describes the basic principles and main process features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

[0047] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A fermentation method for extracting and purifying astaxanthin, characterized in that: The Yarrowia solani expression culture biological fermentation method produces astaxanthin through cell wall breaking, filtration, supercritical extraction, decolorization, chromatographic separation, evaporation concentration, organic solvent crystallization and centrifugal drying to extract and purify, and finally obtain astaxanthin crystals with a content of more than 98.5%, including the following steps: (1) fermenting astaxanthin with Yarrowia lipolytica as the strain, and performing cell wall breaking treatment on the fermented liquid after the fermented liquid is placed in a tank, and simultaneously adding butylated hydroxyanisole antioxidant during cell wall breaking; (2) filtering and sterilizing the astaxanthin fermentation liquid after cell wall breaking; (3) extracting the filtered fermentation broth with supercritical carbon dioxide to increase the purity of astaxanthin to more than 15%, and spray drying to produce astaxanthin powder with a purity of more than 15%; (4) the astaxanthin liquid after supercritical extraction is further purified by simple decolorization treatment; (5) The astaxanthin liquid after simple decolorization is further purified by a carbon 18 reverse phase chromatography device, and the purity is increased to more than 50%; (6) removing water, organic solvent and volatile impurities from the astaxanthin liquid with a purity of more than 50% obtained after chromatographic separation by evaporation; (7) the evaporated astaxanthin liquid is put into a crystallization tank, and petroleum ether or dichloromethane with a mass fraction of 98% or more is added for cooling and crystallization, and the crystallization temperature is reduced to below 10° C., and astaxanthin crystals with a purity of less than 95% are added as seeds during crystallization, and the crystallization is carried out for more than 24 hours; (8) The crystallized astaxanthin is centrifuged and then dried to obtain the final astaxanthin crystals.

2. The extraction and purification process for producing astaxanthin by fermentation as claimed in claim 1, characterized in that: In the step (1), the cell wall breaking treatment of the fermentation liquid can be carried out by chemical cell wall breaking, mechanical cell wall breaking or biological cell wall breaking. The chemical cell wall breaking uses acid to adjust the pH value of the fermentation liquid to 4.0-4.

5. The mechanical cell wall breaking is carried out by high pressure homogenization at above 100MP. The biological cell wall breaking is carried out by adding lysozyme to the fermentation liquid.

3. The extraction and purification process for producing astaxanthin by fermentation as claimed in claim 1, characterized in that: The amount of antioxidant butylated hydroxyanisole added in step (1) is 0.2% of the total astaxanthin content in the fermentation broth.

4. The extraction and purification process for producing astaxanthin by fermentation as claimed in claim 1, characterized in that: The filtration and sterilization in step (2) is performed by ceramic membrane filtration or plate and frame filtration.

5. The extraction and purification process for producing astaxanthin by fermentation as claimed in claim 1, characterized in that: In the step (5), the simple decolorization is performed by using activated carbon or activated clay for decolorization and purification.

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