Methods for extracting astaxanthin oil and astaxanthin from bacterial cells

Abstract

This invention discloses a method for extracting astaxanthin oil and astaxanthin from microorganisms, comprising the following steps: (1) adding a lysozyme to the wet residue of astaxanthin-producing microorganisms and performing an enzymatic hydrolysis reaction under suitable conditions to obtain an enzymatic hydrolysate; (2) drying the enzymatic hydrolysate to obtain a dry residue; (3) extracting the dry residue with a first solvent and separating the solid and liquid to obtain extract A and residue A; (4) concentrating extract A under reduced pressure to remove the solvent to obtain an oily substance, filtering to remove solids to obtain astaxanthin oil; (5) extracting residue A with a second solvent and separating the solid and liquid to obtain extract B and residue B; (6) concentrating extract B under reduced pressure, adding a third solvent, crystallizing at low temperature, filtering to obtain astaxanthin crystals. The method of this invention can extract astaxanthin and astaxanthin oil separately from microorganisms, with a total astaxanthin yield of over 95% and an astaxanthin purity of over 93%.

Description

Technical Field

[0001] This invention belongs to the fields of chemical engineering and food engineering, and specifically relates to a method for simultaneously extracting astaxanthin oil and astaxanthin from bacterial cells. Background Technology

[0002] Pharborg yeast (Xanthophyllomyces dendrorhous, formerly known as Phaffia rhodozyma) can metabolize and produce an important physiologically active substance—astaxanthin, a small amount of which exists in an esterified form. In addition, Pharborg yeast cells are rich in protein, fatty acids, and vitamins. Fatty acids account for approximately 30% of the dry mycelium, including various unsaturated fatty acids such as oleic acid and linoleic acid.

[0003] Astaxanthin molecules contain 11 conjugated double bonds, giving it superior antioxidant capabilities. Based on its antioxidant function, astaxanthin can protect the skin, preventing wrinkles and worsening pigmentation. Human aging, inflammation, cancer, and arteriosclerosis are all caused by cellular oxidative deficiencies. Astaxanthin, with its strong antioxidant properties, can effectively scavenge intracellular free radicals, enhance cell regeneration, and maintain bodily balance. Therefore, astaxanthin can protect cell health from the inside out, delay aging, prevent cancer, and resist disease. Astaxanthin also has strong coloring properties and is widely used in aquaculture feed, such as in the farming of whiteleg shrimp, salmon, grouper, and salmonids, giving farmed aquatic products the same color and quality as wild-caught products.

[0004] In recent years, the market demand for astaxanthin has been increasing, particularly in the aquatic feed, cosmetics, and health and pharmaceutical sectors. Currently, astaxanthin is mainly produced through artificial synthesis, extraction from aquatic waste, and natural fermentation. Natural fermentation is considered the safest and most environmentally friendly method, making it more popular in the market. Natural fermentation involves the cultivation of fungi such as Haematococcus pluvialis and Pharfovia rubescens. Pharfovia rubescens exhibits faster propagation rates and lower cultivation costs, making it a promising candidate for the market.

[0005] Existing astaxanthin purification processes are complex, resulting in low purity and low extraction efficiency. There is a lack of research on the extraction of oil from Phaefuer's red yeast, neglecting the extraction and utilization of fatty acids from Phaefuer's red yeast cells, leading to resource waste.

[0006] Patent CN104030957B describes a method for obtaining astaxanthin with a purity greater than 99% and a recovery rate greater than 60% by washing bacterial cells, disrupting the cell walls with dimethyl sulfoxide (DMSO) solvent, extracting with anhydrous ethanol, then extracting with n-hexane and deionized water, followed by silica gel column chromatography. However, the use of DMSO solvent for cell disruption, due to its high boiling point and potential residue, results in a low yield unsuitable for industrial application. Patent CN111995880B describes a method for mechanically disrupting dried bacterial cells, followed by saponification with sodium hydroxide and ethanol, further solvent extraction, and concentration and crystallization to obtain astaxanthin. However, saponification in this method damages the astaxanthin oil, leading to a low yield and low utilization of the saponified product, resulting in oil waste. Patent CN114751848A describes an acid-based method for disrupting the cell walls of *Phaeff's red yeast*, followed by extraction of crude astaxanthin with an alcohol solvent, and then extraction with oil and a suitable amount of water to obtain astaxanthin essential oil. Because astaxanthin is extremely unstable under acidic and alkaline conditions, acid-based cell disruption can lead to a certain degree of astaxanthin degradation. Furthermore, the addition of soybean oil and sunflower oil further increases the process cost. Patent CN103848769B describes using dimethyl sulfoxide (DMSO) to disrupt the cell walls of *Phaeff's erythrosaccharide* cells, followed by acetone extraction to obtain a crude extract. This crude extract is then defatted and purified using petroleum ether extraction to obtain a secondary crude extract. Saturated brine at 4°C is added, and DMSO is extracted again with ethyl acetate. The extract is then evaporated at low temperature to obtain astaxanthin powder, dissolved in dichloromethane, crystallized with methanol, and centrifuged to obtain astaxanthin crystals. This method uses DMSO for cell disruption, which is difficult to completely remove. Additionally, it uses five solvents: acetone, petroleum ether, ethyl acetate, dichloromethane, and methanol. The large number of solvents makes subsequent solvent recovery extremely difficult, resulting in a complex process with high costs, making it unsuitable for industrial production.

[0007] Currently, astaxanthin extraction suffers from several problems, including complex extraction processes, high costs, low extraction rates, and neglect of the fatty acid value in Rhodopsin cells, leading to resource waste. Therefore, developing a novel process capable of simultaneously extracting fatty acids and astaxanthin from Rhodopsin cells has significant economic value. Summary of the Invention

[0008] To fully recover and utilize both fatty acids (including oleic acid, linoleic acid, and polyunsaturated fatty acids) and astaxanthin from astaxanthin-producing microorganisms (such as Haematococcus pluvialis, Pharfthyrium rubrum, Rhodotorula glutinis, Rhodotorula spp., and marine Rhodotorula), and to reduce the types and amounts of organic solvents used in the separation and extraction steps, thus avoiding the use of expensive reagents, the inventors, through repeated experiments, developed a new process for simultaneously extracting astaxanthin oil and astaxanthin from fermentation cells, achieving maximum utilization of resources. Specifically, this invention includes the following technical solutions.

[0009] A method for extracting astaxanthin oil and astaxanthin from bacterial cells includes the following steps:

[0010] (1) Add wall-lysing enzyme to the wet bacterial residue obtained by centrifuging the microbial fermentation broth that produces astaxanthin, and carry out enzymatic hydrolysis under suitable conditions to obtain enzymatic hydrolysate.

[0011] (2) The enzymatic hydrolysate obtained in step (1) is dried to obtain a dry bacterial residue with a low water content, for example, not more than 5%.

[0012] (3) The dried fungal residue obtained in step (2) is extracted with the first solvent, and the solid and liquid are separated to obtain extract A and fungal residue A;

[0013] (4) The solvent in the extract A obtained in step (3) is removed by vacuum concentration to obtain an oily substance. After filtration, the solids are removed to obtain astaxanthin oil.

[0014] (5) Extract the bacterial residue A obtained in step (3) with a second solvent, and separate the solid and liquid to obtain extract B and bacterial residue B;

[0015] (6) After concentrating the extract B obtained in step (5) under reduced pressure to a certain volume, add the third solvent, i.e. the crystallization solvent, crystallize at low temperature, preferably under stirring, filter, and obtain astaxanthin crystals.

[0016] In one embodiment, the microorganism is Pharfogel's red yeast.

[0017] Preferably, the above method further includes the following steps:

[0018] (7) After crystallization filtration in step (6), the filter cake is washed with a top-washing solvent, preferably twice or more, and dried under low temperature and reduced pressure to obtain astaxanthin crystals. The top-washing solvent used is preferably the same as the third solvent, i.e., the crystallization solvent.

[0019] In one embodiment, the low-temperature vacuum drying temperature in step (7) is 30±5℃, preferably about 30℃, and the drying time is 3-5h.

[0020] Preferably, in step (5), after extracting the fungal residue A with the second solvent to obtain extract B and fungal residue B, the fungal residue B is further extracted with the second solvent, and after solid-liquid separation, extract B is obtained. The extracts B are then combined for the next step of processing.

[0021] In one embodiment, the lysozyme described in step (1) is a yeast lysozyme derived from Arthrobacter luteus.

[0022] Optionally, the lysozyme is a pH 4.0-6.5 buffer solution containing the lysozyme, such as a phosphate buffer solution with a pH 4.5-6.5, with an enzyme dosage of 10 U / mL to 100 U / mL, and the weight-volume ratio of wet bacterial residue to the lysozyme buffer is 1:0.5 to 1.5, more preferably about 1:1.

[0023] It should be understood that in this article, when describing numerical characteristics, the terms "approximately", "about", or "around" mean that the expressed number may have an error range or fluctuation range of ±10%, ±8%, ±6%, ±4%, or ±2%.

[0024] Preferably, the suitable conditions for the enzyme reaction in step (5) are pH 4.5 to 6.5, temperature 30 to 50°C, and enzyme hydrolysis time 1 to 3 hours.

[0025] The drying process in step (2) above is selected from spray drying, fluidized bed drying, oven drying, and blow drying, with spray drying being preferred.

[0026] Preferably, the moisture content of the dried fungal residue obtained after drying in step (2) is not higher than 5%.

[0027] In one embodiment, the first solvent in step (3) is a mixture of alkane and alcohol in a volume ratio of 1 to 5:1, wherein the alkane is selected from the group consisting of petroleum ether, n-hexane, cyclohexane, and mixtures of two or more thereof; and the alcohol is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, sec-butanol, tert-butanol, 3-pentanol, 2-pentanol, tert-pentanol, 2-methylbutanol, 3-methyl-3-pentanol, isopentanediol, and mixtures of two or more thereof.

[0028] The second solvent in step (5) is selected from the group consisting of dichloromethane, trichloromethane, and mixtures thereof.

[0029] The third solvent in step (6), i.e. the crystallization solvent, is selected from the group consisting of ketones, alcohols, and mixtures of two or more of them; the ketones are selected from acetone, 2-butanone, penta-2-one, penta-3-one, hex-2-one or hex-3-one, preferably acetone.

[0030] Preferably, the top washing solvent in step (7) is the same as the third solvent, i.e., the crystallization solvent, and is selected from the group consisting of ketones, alcohols, and mixtures of two or more of them. The ketones are selected from acetone, 2-butanone, penta-2-one, penta-3-one, hex-2-one, or hex-3-one, with acetone being preferred.

[0031] In one embodiment, the weight-volume ratio of the dried bacterial residue to the first solvent in step (3) is 1:5 to 10, and the soaking and stirring temperature is 40 to 50°C.

[0032] The extraction described in steps (3) and (5) above can refer to constant temperature stirring extraction, with an extraction time sufficient to cause more than 80%, preferably more than 85%, more preferably more than 90%, and most preferably more than 95% of the target product to be separated from the bacterial residue, for example, about 1 hour.

[0033] The solid-liquid separation described in steps (3) and (5) above can be filtration or centrifugation.

[0034] In one embodiment, the reduced pressure concentration temperature in step (6) is 40-50°C, and the concentration factor is 20-30 times; the low-temperature stirring temperature is controlled at -5-5°C. The crystallization solvent is cold acetone at -5-5°C, and it is added while stirring at a constant speed, preferably slowly and at a constant speed. The addition time of the crystallization solvent is controlled to be 20-30 minutes, and the amount used is 1-3 times the volume of the concentrated liquid.

[0035] Preferably, the wet fungal residue described in step (1) can be treated by the following process before the enzyme reaction:

[0036] The microbial fermentation broth is centrifuged to obtain microbial sludge, which is then resuspended in water (e.g., deionized water or pure water) or a buffer solution (e.g., pH 4.5-6.5 phosphate buffer solution) and centrifuged again to remove residual impurities from the fermentation broth; or the microbial fermentation broth is diluted with water to more than twice its volume and centrifuged to remove residual impurities from the fermentation broth.

[0037] In one embodiment, the astaxanthin oil obtained in step (4) is preferably further enriched with an antioxidant such as esterified vitamin C, and then sealed with nitrogen. This product can be used or sold as commercial astaxanthin oil.

[0038] The amount of the aforementioned antioxidants, such as esterified vitamin C, can be 0.5% to 1.5% of the weight of astaxanthin oil.

[0039] Another aspect of the present invention provides an astaxanthin crystal form A, which is prepared by the method described above, and whose XRPD spectrum, expressed in 2θ angles, using CuKα radiation, exhibits diffraction peaks at least at 8.381°±0.2°, 13.693°±0.2°, 16.841°±0.2°, 19.754°±0.2°, 25.892°±0.2°, and 27.683°±0.2°.

[0040] Furthermore, the crystal form A also exhibits diffraction peaks at 2θ values ​​of 10.261°±0.2°, 12.325°±0.2°, 21.521°±0.2°, and 22.863°±0.2°.

[0041] Astaxanthin crystal form A has significantly higher chemical stability than existing astaxanthin products, which is reflected in its higher antioxidant properties. After being exposed to air for 7 days, the purity of existing astaxanthin crystals has decreased to less than 70% of the original purity, while the purity of crystal form A can still be maintained at at least 80%, thus making it more suitable for preservation and storage.

[0042] The organic solvents used in this invention are inexpensive and readily available bulk chemical raw materials. Targeting astaxanthin-producing microorganisms such as *Phaefflera heliotropium* fermentation cells, a specific process route allows for the separate extraction of economically valuable astaxanthin and astaxanthin oil from the cells. The total astaxanthin yield exceeds 95%, and the astaxanthin purity exceeds 93%. The extracted astaxanthin oil can be used as a feed additive, directly sprayed onto the feed surface for feeding, turning waste into treasure, fully utilizing the intracellular oils, avoiding resource waste, and significantly enhancing the economic value of the microorganisms. This invention first extracts astaxanthin oil from the microbial cells, greatly reducing the impact of lipid-soluble impurities on astaxanthin extraction, purification, and crystallization, promoting the subsequent crystallization of high-purity, high-quality astaxanthin crystals. The obtained high-quality astaxanthin crystal form A exhibits good stability and strong antioxidant properties, and can be used in the medical, health, and cosmetic industries. Because the new process of this invention is simple to operate, safe and reliable, and low in cost, and can simultaneously prepare two application forms of astaxanthin (astaxanthin and astaxanthin oil), it meets the needs of different fields and is suitable for industrial production and widespread application. Attached Figure Description

[0043] Figure 1 This is the X-ray powder diffraction pattern (XPRD) of astaxanthin crystal form A prepared in this invention. Detailed Implementation

[0044] The purity of astaxanthin extracted from red phaflycium using existing technologies is less than 90%, and it contains fat-soluble impurities such as fatty acids (astaxanthin oil components). When exposed to air, it is easily oxidized, rancid, and produces an off-odor, which seriously reduces the quality of astaxanthin and consequently its use and economic value. One important reason for this is that there are many unsaturated bonds in the molecular structure of astaxanthin.

[0045] Astaxanthin, also known as astaxanthin, is the only known natural 2-thioimidazolium amino acid. It belongs to the carotenoid family and its chemical name is 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene. Its structural formula is as follows.

[0046]

[0047] Astaxanthin is a terpene unsaturated compound with a six-membered ring structure consisting of four isoprene units linked by conjugated double bonds and two isoprene units at each end. The conjugated double bonds, the unsaturated ketone groups at the ends of the conjugated double bonds, and the hydroxyl groups can attract unpaired electrons from free radicals or donate electrons to free radicals, thus scavenging free radicals and exerting an antioxidant effect. This gives astaxanthin its extremely strong antioxidant capacity, but it is also prone to oxidation itself.

[0048] Experimental comparisons revealed that removing impurities and astaxanthin oil components from astaxanthin allows it to form a specific crystal form, which increases the oxidation reaction cycle of astaxanthin and significantly reduces the rate of oxidation and deterioration.

[0049] In some implementations, the terms “(deterioration rate) reduction,” “decrease,” or “reduction” can mean a reduction of at least 10% compared to a reference level (existing astaxanthin products), such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a reduction of 100%.

[0050] In the field of chemistry, it is well known that during the crystallization of certain chemical substances, various factors can alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms in the crystal lattice and forming different crystal structures. Different crystal forms often possess different physical and chemical properties, such as differences in melting point, hardness, stability, and dissolution rate. Crystal forms can be characterized by X-ray powder diffraction (XPRD) spectra; different crystal forms exhibit different XPRD patterns, with variations in the positions of diffraction peaks, or absorption peaks, represented by the 2θ angle.

[0051] X-ray powder diffraction (XPRD) is also known as powder X-ray diffraction (PXRD).

[0052] The specific crystal form A of the present invention preferably has diffraction peaks at least at 8.381°±0.1°, 13.693°±0.1°, 16.841°±0.1°, 19.754°±0.1°, 25.892°±0.1° and 27.683°±0.1°; more preferably, it has diffraction peaks at least at 8.381°, 13.693°, 16.841°, 19.754°, 25.892° and 27.683°.

[0053] X-ray powder diffraction (XPRD) is also known as powder X-ray diffraction (PXRD).

[0054] Furthermore, crystal form A preferably also has diffraction peaks at 2θ values ​​of 10.261°±0.1°, 12.325°±0.1°, 21.521°±0.1° and 22.863°±0.1°; more preferably, it also has diffraction peaks at 2θ values ​​of 10.261°, 12.325°, 21.521° and 22.863°.

[0055] Based on this research, we designed a process to first separate astaxanthin oil from the bacteria and then extract and purify astaxanthin, so that both substances can be recycled and reused, and the astaxanthin oil that was originally discarded as an impurity can be fully utilized as a feed additive.

[0056] Experimental comparisons revealed that using alkanes alone could not extract most fatty acids from the fungal residue, and using alcohol solvents alone could not distinguish between fatty acids (astaxanthin oil) and astaxanthin for separate extraction. However, when alkanes (such as petroleum ether) and alcohols (such as ethanol) are mixed in an appropriate ratio, the vast majority of fatty acids (astaxanthin oil) can be extracted from the fungal residue, while the vast majority of astaxanthin is retained within the residue.

[0057] As used herein, the terms “mainly,” “mostly,” or “absolutely most” mean 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more of the total content of a component.

[0058] In this processing step, the extraction of astaxanthin oil greatly reduces the impact of fat-soluble impurities on astaxanthin crystallization and improves the purity of yeast astaxanthin crystals obtained in the later crystallization. Experiments have shown that the preliminary extraction of astaxanthin oil is the key to improving the yield of subsequent astaxanthin crystal extraction, and it also greatly improves the purity of astaxanthin powder. The purity of crystal A is at least 93%, which can be used in the medical, health care, and cosmetic industries.

[0059] A crucial step in improving the recovery rate of astaxanthin oil and astaxanthin from microbial cells is cell disruption. Various cell disruption methods exist, each with its advantages and disadvantages. Studies have found that organic solvent disruption, such as dimethyl sulfoxide (DMSO), is fast but leaves residues that hinder astaxanthin oil separation, significantly impacting astaxanthin quality. Acid or alkaline disruption methods easily cause reactions in astaxanthin, reducing yield and damaging the astaxanthin oil, leading to waste and insufficient recovery. Mechanical disruption methods, such as homogenization, often result in insufficient extraction of astaxanthin and astaxanthin oil from the homogenate, reducing the yield of both products. Enzymatic disruption is slower but yields a higher overall yield of astaxanthin and astaxanthin oil, making it the primary method chosen in this invention.

[0060] In our study on enzymatic cell wall disruption, we compared over thirty enzymes, including lysozyme (bacterial lysin, such as Bacillus circulatory systemicus cell wall lysin), cellulase (yeast cellulase, yeast lysin), cellulase, alkaline protease, cellulase / alkaline protease complex, β-glucanase, chitinase, and β-glucanase / chitinase complex, for Pharfogel's yeast. We found that the yeast cellulase derived from Arthrobacter luteus exhibited the best catalytic effect, relatively fast cell wall disruption speed, high cell wall disruption rate, and the highest recovery rate of astaxanthin and astaxanthin oil. Therefore, the yeast cellulase derived from Arthrobacter luteus was used in all the following examples.

[0061] The process of this invention uses relatively few types of organic solvents; for example, only petroleum ether, ethanol, acetone, dichloromethane, or trichloromethane are needed to implement the technical solution of this invention. These organic solvents are all inexpensive, readily available, and commonly used bulk chemical reagents.

[0062] To avoid unnecessary oxidation side reactions of astaxanthin, it is preferable to control the temperature of the entire process below 50°C. The inventors also optimized the operating conditions of each step. For example, in order to improve the yield of crystalline astaxanthin, in the acetone crystallization of step (6), in addition to pre-cooling the acetone to -5 to 5°C, the feeding speed should be strictly controlled so as not to be too fast. Cold acetone should be added slowly and uniformly while stirring.

[0063] The present invention is further illustrated below by way of examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the invention. Various changes or modifications made by those skilled in the art based on the concept of the present invention should fall within the protection scope of the present invention.

[0064] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0065] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0066] Example

[0067] The main instruments and equipment used in this embodiment are: an Agilent 1200 high-performance liquid chromatograph (HPLC), Agilent Technologies, Inc., USA; and an SHJ-6AB constant temperature water bath, Jintan Liangyou Instrument Co., Ltd.

[0068] The content of astaxanthin was determined by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: column: Agilent ZORBAX SB-C18, detection wavelength: 478 nm, column temperature: 30 °C, mobile phase: 100% methanol, injection volume: 10 μL, flow rate: 1 mL / min.

[0069] The cell wall disruption rate was determined by the release of intracellular astaxanthin. The cell wall disruption rate was calculated as: (Astaxanthin content after ethanol soaking / Total astaxanthin content obtained by the dimethyl sulfoxide method) × 100%. Specifically: For the ethanol method, astaxanthin content in *Phaefflera rubra* was determined by adding 15 mL of ethanol to 1 mL of the disrupted *Phaefflera rubra* solution, ultrasonically soaking for 20 min, bringing the volume to a final volume, filtering, and then determining the astaxanthin content. For the dimethyl sulfoxide method, astaxanthin content in *Phaefflera rubra* was determined by adding 4 mL of dimethyl sulfoxide to 1 mL of the disrupted *Phaefflera rubra* solution, ultrasonically soaking for 20 min, bringing the volume to a final volume, diluting 1 mL of the solution five times with ethanol, filtering, and then determining the astaxanthin content by high-performance liquid chromatography (HPLC).

[0070]

[0071] V-oil: Volume of yeast astaxanthin oil

[0072] β-oil: Astaxanthin concentration in yeast astaxanthin oil

[0073] m: Weight of astaxanthin crystals

[0074] W: Astaxanthin content in crystals

[0075] V: Fermentation broth volume

[0076] β-fermentation broth: Astaxanthin concentration in the fermentation broth.

[0077] Example 1

[0078] 10 L of *Phaefflera heliotropium* cell fermentation broth (475 μg / ml) was added to 10 L of water, stirred thoroughly, and centrifuged to obtain wet cell residue. 5 L of phosphate buffer containing yeast lysozyme (20 U / mL) was added, pH 5.0, and the mixture was hydrolyzed at 45℃ for 3 h, resulting in a cell wall rupture rate of approximately 97.6%. The residue was spray-dried at an inlet air temperature of 190℃ and an outlet air temperature of 85℃ to obtain dry cell residue with a moisture content of 3.5%. 4 L of petroleum ether and 2 L of ethanol were added to the dry cell residue, and the mixture was stirred and soaked at 50℃ for 1 h. The soaking solution was filtered and concentrated under reduced pressure at 50℃ to remove the solvent, yielding yeast astaxanthin oil. Impurities were removed by filtration, and 2 g of esterified vitamin C was added. The mixture was then stored under nitrogen and sealed.

[0079] The remaining filter cake was added to 5L of dichloromethane and stirred at 50℃ for 1 hour. The soaking liquid was obtained by filtration. The bacterial residue was further extracted with 5L of dichloromethane for a second extraction. The soaking liquids were combined and concentrated under reduced pressure to 0.5L. 1L of acetone at 0℃ was slowly and uniformly added using a peristaltic pump at a rate of 3L / h. The mixture was stirred at 0℃ and 100rpm for 1 hour to crystallize. The filter cake was obtained by filtration and washed twice with 50mL of acetone at 0℃ each time. The filter cake was dried under reduced pressure at 30℃ for 4 hours, and the astaxanthin crystals were collected.

[0080] Using this method, 0.32 L of yeast astaxanthin oil was prepared, with an astaxanthin content of 2.1 g / L as determined by HPLC. 4.2 g of yeast astaxanthin crystals were also prepared, with a content of 93.6% as determined by HPLC. The total astaxanthin yield was 96.8%.

[0081] Comparative Example 1

[0082] Take 10L of red Pharbitis yeast cell fermentation broth (470μg / ml), add 10L of water, stir well, centrifuge to obtain wet cell residue, add 5L of phosphate buffer containing yeast cell wall lysin with an enzyme activity of 20U / mL, pH 5.0, and enzymatically hydrolyze for 3h at 45℃, with a cell wall rupture rate of approximately 97%. Spray dry the residue at 190℃ inlet air temperature and 85℃ outlet air temperature to obtain dry cell residue with a water content of 3.4%. Add 4L of petroleum ether and 2L of ethanol to the residue, stir and soak at 50℃ for 1h, filter the soaking liquid, concentrate under reduced pressure at 50℃ to remove the solvent, and obtain yeast astaxanthin oil. Filter to remove impurities, add 2g of esterified vitamin C, and store under nitrogen and airtight conditions.

[0083] The remaining filter cake was added to 5L of dichloromethane and stirred at 50℃ for 1 hour. The solution was filtered to obtain the soaking liquid. The bacterial residue was further extracted with 5L of dichloromethane. The soaking liquids were combined and concentrated under reduced pressure to 0.5L. Immediately, 1L of acetone at 0℃ was added, and the mixture was stirred at 0℃ and 100rpm for 1 hour to crystallize. The filter cake was filtered and washed twice with 50mL of acetone at 0℃ each time. The filter cake was dried under reduced pressure at 30℃ for 4 hours, and the astaxanthin crystals were collected.

[0084] Using this method, 0.324 L of yeast astaxanthin oil was prepared, with an astaxanthin content of 2.0 g / L as determined by HPLC. 4.9 g of yeast astaxanthin crystals were also prepared, with a content of 78.8% as determined by HPLC. The total astaxanthin yield was 96.0%.

[0085] Comparing Comparative Example 1 with Example 1, we can conclude that if the acetone flow rate is too fast, the purity of astaxanthin crystals is low, suggesting that other impurities or solvent mother liquor may be present in the crystals.

[0086] Example 2

[0087] Take 15L of red Pharbitis yeast cell fermentation broth (491μg / ml), add 15L of water, stir well, centrifuge to collect wet cell residue, add 5L of phosphate buffer containing yeast cell wall lysing enzyme with an activity of 50U / mL, pH 5.0, and enzymatically hydrolyze at 40℃ for 3h. The cell wall rupture rate is 98.1%. Spray dry the residue to obtain dry cell residue with a water content of 3.5%. Add 5L of petroleum ether and 5L of methanol to the residue, stir and soak at 40℃ for 1h, filter the soaking liquid, concentrate under reduced pressure at 45℃ to remove the solvent, and obtain yeast astaxanthin oil. Filter to remove impurities, add 4.5g of esterified vitamin C, and store under nitrogen and airtight conditions.

[0088] The remaining filter cake was added to 10L of chloroform and stirred at 40℃ for 1 hour. The soaking liquid was obtained by filtration. The bacterial residue was further extracted with 10L of chloroform for a second extraction. The soaking liquids were combined and concentrated under reduced pressure to 1L. 3L of acetone at -5℃ was slowly and uniformly added using a peristaltic pump at a rate of 6L / h. The mixture was stirred at -5℃ and 200rpm for 1 hour to crystallize. The filter cake was obtained by filtration and washed twice with 50mL of acetone at -5℃ each time. The filter cake was dried under reduced pressure at 30℃ for 5 hours, and the yeast astaxanthin crystals were collected.

[0089] Using this method, 0.450 L of yeast astaxanthin oil was prepared, with an astaxanthin content of 2.3 g / L as determined by HPLC. 6.4 g of yeast astaxanthin crystals were also prepared, with a content of 94.0% as determined by HPLC. The total astaxanthin yield was 95.7%.

[0090] X-ray powder diffraction pattern (XPRD pattern) of astaxanthin crystals is shown below. Figure 1 As shown, it is defined as astaxanthin crystal form A.

[0091] XRPD spectra of crystal form A, expressed in 2θ angles using CuKα radiation, show diffraction peaks at 8.381°±0.2°, 10.261°±0.2°, 12.325°±0.2°, 13.693°±0.2°, 16.841°±0.2°, 19.754°±0.2°, 21.521°±0.2°, 22.863°±0.2°, 25.892°±0.2°, and 27.683°±0.2°.

[0092] The extraction of yeast astaxanthin oil in the first step of this process is the key to the crystallization in the second step. By extracting yeast astaxanthin oil, the influence of fat-soluble impurities on astaxanthin crystallization is reduced, resulting in yeast astaxanthin crystals with high purity.

[0093] Comparative Example 2

[0094] Take 15L of *Phaefflera heliotropium* cell fermentation broth (473 μg / ml), add 15L of water, stir well, centrifuge to collect wet cell residue, add 5L of phosphate buffer containing yeast lysozyme (50 U / mL), pH 5.0, and enzymatically hydrolyze for 3 hours at 40℃. The cell wall rupture rate was 97.9%. The spray tower inlet temperature was 190℃ and the outlet temperature was 88℃. Spray dry to obtain dry cell residue with a water content of 3.2%. Add 10L of chloroform to the cell residue, stir and soak for 1 hour at 40℃, filter to obtain the soaking liquid, add another 10L of chloroform to the cell residue for a second extraction, combine the soaking liquids, concentrate under reduced pressure to 1L, slowly and uniformly add 3L of acetone at -5℃ using a peristaltic pump, control the rate at 6L / h, stir at -5℃ and 200rpm, and crystallize for 1 hour. Filter to obtain filter cake, wash twice with 50mL of acetone at -5℃ each time, dry the filter cake under reduced pressure at 30℃ for 5 hours, and collect yeast astaxanthin crystals.

[0095] 9.5g of yeast astaxanthin crystals were prepared, and the content was 66.8% astaxanthin as determined by HPLC, with a total astaxanthin yield of 89.4%.

[0096] Comparing Comparative Example 2 with Example 2, we can conclude that the initial yeast astaxanthin oil extraction can greatly improve the purity of astaxanthin crystals. Therefore, the first step of yeast astaxanthin oil extraction is the key to the second step of crystallization.

[0097] Comparative Example 3

[0098] The crystallization process in patent CN103848769B was repeated. 3.0g of the crystal powder from Example 1 was dissolved in dichloromethane (100mL volume), and methanol was slowly added until crystals precipitated. The mixture was allowed to stand for 16 hours to allow the dichloromethane to evaporate naturally. The mixture was then centrifuged at 4°C and dried under reduced pressure.

[0099] 2.5g of yeast astaxanthin crystals were prepared, and the content was 88.5% astaxanthin as determined by HPLC, with a total astaxanthin yield of 78.8%.

[0100] Comparative Example 4

[0101] The crystallization process in patent CN103848769B was repeated. 3.0g of the crystal powder from Example 2 was dissolved in dichloromethane (100mL volume), and methanol was slowly added until crystals precipitated. The mixture was allowed to stand for 6 hours to allow the dichloromethane to evaporate naturally. The mixture was then centrifuged at 4°C and dried under reduced pressure.

[0102] 2.0 g of yeast astaxanthin crystals were prepared, and the content was 94.1% astaxanthin as determined by HPLC, with a total astaxanthin yield of 66.7%.

[0103] Example 3

[0104] The chemical stability of crystal form A from Example 2 was compared with that of crystals from Comparative Examples 3 and 4 under air exposure. The results are shown in Table 1.

[0105] Table 1. Comparison of stability between crystal form A and astaxanthin crystals in Example 2 (Patent CN103848769B)

[0106]

[0107]

[0108] As shown in Table 1, after being exposed to air for 7 days, the purity of the astaxanthin crystals in the prior art has decreased to less than 70% of the original purity, but the purity of crystal form A can still be maintained at more than 80%, indicating that astaxanthin crystal form A has higher antioxidant properties and significantly higher chemical stability than the astaxanthin crystals in the prior art.

[0109] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims. These modifications or variations do not affect the essence of the present invention and should all be included within the scope of the present invention.

Claims

1. A method for simultaneously extracting astaxanthin oil and astaxanthin from bacterial cells, characterized in that, Includes the following steps: (1) Add a cell wall lysing enzyme to the wet bacterial residue of astaxanthin-producing microorganisms and carry out an enzymatic hydrolysis reaction under suitable conditions to obtain an enzymatic hydrolysate. The cell wall lysing enzyme is a yeast cell wall lysing enzyme derived from *Arthrobacter cuspidatum*. (2) The enzymatic hydrolysate obtained in step (1) is dried to obtain dried bacterial residue; (3) The dried fungal residue obtained in step (2) is extracted with the first solvent, and the solid and liquid are separated to obtain extract A and fungal residue A; (4) The solvent in the extract A obtained in step (3) is removed by vacuum concentration to obtain an oily substance. After filtration, the solids are removed to obtain astaxanthin oil. (5) Extract the fungal residue A obtained in step (3) with a second solvent, and separate the solid and liquid to obtain extract B and fungal residue B; (6) After concentrating the extract B obtained in step (5) under reduced pressure, add the third solvent, i.e., the crystallization solvent, and crystallize at -5~5℃. Filter to obtain astaxanthin crystals. The microorganism in question is Pharfogel's rubrum.

2. The method according to claim 1, characterized in that, It also includes the following steps: (7) After the crystallization filtration in step (6), the filter cake is washed with a top washing solvent and dried under reduced pressure to obtain astaxanthin crystals.

3. The method according to claim 1, characterized in that, The lysozyme mentioned in step (1) is a pH 4.0-6.5 buffer containing lysozyme, with an enzyme dosage of 10 U / mL to 100 U / mL, and a weight-volume ratio of wet bacterial residue to lysozyme buffer of 1:0.5 to 1.

5.

4. The method according to claim 1, characterized in that, The drying process in step (2) is selected from spray drying, fluidized bed drying, and blow drying.

5. The method according to claim 1, characterized in that, The first solvent in step (3) is a mixture of alkane and alcohol in a volume ratio of 1 to 5:1, wherein the alkane is selected from the group consisting of petroleum ether, n-hexane, cyclohexane, and mixtures of two or more thereof; and the alcohol is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, sec-butanol, tert-butanol, 3-pentanol, 2-pentanol, tert-pentanol, 2-methylbutanol, 3-methyl-3-pentanol, isopentanediol, and mixtures of two or more thereof. The second solvent in step (5) is selected from the group consisting of: dichloromethane, trichloromethane, and mixtures thereof; The third solvent in step (6) is selected from the group consisting of ketones, alcohols, and mixtures of two or more thereof; wherein the ketones are selected from acetone, 2-butanone, penta-2-one, penta-3-one, hex-2-one, or hex-3-one; The top wash solvent in step (7) is the same as the third solvent and is selected from the following group: ketones, alcohols, and mixtures of two or more of them.

6. The method according to claim 1, characterized in that, The solid-liquid separation described in steps (3) and (5) is filtration or centrifugation.

7. The method according to claim 1, characterized in that, The extraction described in steps (3) and (5) refers to constant temperature stirring extraction.

8. The method according to claim 1, characterized in that, Antioxidants were further added to the astaxanthin oil obtained in step (4), and the mixture was then sealed with nitrogen.

9. The method according to claim 1, characterized in that, The temperature of the entire process is controlled below 50℃.

10. The method according to claim 1, characterized in that, The astaxanthin crystal has a purity of over 93%, and is of astaxanthin crystal form A. Using CuKα radiation, the XRPD spectrum, expressed in 2θ angles, exhibits diffraction peaks at at least 8.381°±0.2°, 13.693°±0.2°, 16.841°±0.2°, 19.754°±0.2°, 25.892°±0.2°, and 27.683°±0.2°.

11. The method according to claim 10, characterized in that, The astaxanthin crystal form A also exhibits diffraction peaks at 2θ values ​​of 10.261°±0.2°, 12.325°±0.2°, 21.521°±0.2° and 22.863°±0.2°.

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