Chemical mutagenesis screening method for high astaxanthin-producing haematococcus pluvialis and application thereof

Abstract

This invention provides a chemical mutagenesis screening method and its application for high-yield astaxanthin-producing Haematococcus pluvialis. The method involves chemically mutagenesizing Haematococcus pluvialis cells in the logarithmic growth phase with ethyl methanesulfonate, followed by single-colony screening to obtain high-yield astaxanthin mutants. After inducing astaxanthin accumulation under suitable culture conditions, the algae are freeze-dried, and then astaxanthin is extracted using a eutectic solvent system formed by thymol and oleic acid. This improves astaxanthin yield and extraction efficiency while reducing environmental pollution caused by traditional organic solvent extraction. This invention is simple to operate, has high extraction efficiency, and shows promising application prospects.

Description

Technical Field

[0001] This invention relates to the fields of microalgae biotechnology and natural product extraction technology, and in particular to a chemical mutagenesis screening method and its application for high astaxanthin production of Haematococcus pluvialis. Background Technology

[0002] Astaxanthin is a natural carotenoid compound with strong antioxidant capabilities, and it has broad application prospects in the food, cosmetics, aquaculture, nutrition and health, and pharmaceutical industries. With the development of the functional food and nutritional supplement market, the demand for astaxanthin is constantly increasing. Artificially synthesized astaxanthin is generally not suitable for human consumption as a food supplement due to the inherent toxicity of its raw materials; therefore, naturally sourced astaxanthin is gradually becoming the main demand in the market.

[0003] Natural astaxanthin mainly comes from biological resources such as microalgae, yeast, and crustacean shells. Among them, astaxanthin from microalgae has high antioxidant activity, and Haematococcus pluvialis is considered one of the microalgae with the highest natural astaxanthin content, reaching up to 5% of the algal dry weight. Therefore, Haematococcus pluvialis has become an important biological resource for the production of natural astaxanthin.

[0004] However, Haematococcus pluvialis cells have a unique structure, with their cell walls typically composed of multiple layers. While this structure facilitates cell survival in adverse environments, it also increases the difficulty of astaxanthin extraction. Current techniques for extracting astaxanthin from Haematococcus pluvialis usually require cell wall disruption followed by extraction with organic solvents. For example, concentrated acids or alkalis are used to treat the algal cells, disrupting their structure, and then volatile organic solvents such as methanol or ethanol are used for extraction. While these methods achieve high extraction efficiency, they have some shortcomings in practical applications. Volatile organic solvents are toxic and easily evaporate during extraction, causing environmental impact and potentially leaving residues in the astaxanthin product, affecting its application in the food and pharmaceutical fields. Furthermore, strong acid or alkali treatments may decrease the stability of astaxanthin, affecting its antioxidant activity. In addition, these extraction processes often involve high energy consumption and complex operating conditions, hindering large-scale green production.

[0005] In recent years, researchers have explored new extraction techniques, such as supercritical carbon dioxide extraction, ultrasound-assisted extraction, microwave-assisted extraction, and ionic liquid extraction. While these methods can improve extraction efficiency to some extent, they still suffer from drawbacks such as high equipment requirements, high production costs, and reagent residues.

[0006] Deep eutectic solvents (DESs) are a new type of solvent system that has attracted attention in recent years. These solvents are typically formed by hydrogen bond acceptors and hydrogen bond donors in a specific ratio, and are characterized by low volatility, simple preparation methods, and good environmental compatibility. In the field of natural product extraction, DESs exhibit excellent solubility and separation performance. For example, choline chloride (ChCl), as a typical DES hydrogen bond acceptor, has shown good application potential in DESs with substances such as lactic acid, glycerol, or oleic acid. Furthermore, hydrophobic DESs formed from terpenes also have certain advantages in the extraction of lipid-soluble active substances. Therefore, using DESs to extract astaxanthin from Haematococcus pluvialis is expected to reduce the use of organic solvents, lower environmental pollution, and improve the safety of the extraction process.

[0007] It is worth noting that in the astaxanthin production process of Haematococcus pluvialis, the astaxanthin accumulation capacity of the algal strain itself has a significant impact on the final yield. There is still room for improvement in the astaxanthin yield of existing algal strains. Therefore, obtaining Haematococcus pluvialis strains with higher astaxanthin content through breeding is of great significance for improving production efficiency.

[0008] Chemical mutagenesis is a commonly used biological breeding method. Ethyl methanesulfonate (EMS) can induce changes in DNA base pairs, thereby generating mutations in the genome and forming mutants with different traits. Screening for Haematococcus pluvialis strains with high astaxanthin accumulation capacity using chemical mutagenesis holds promise for providing new high-yield algal strains for natural astaxanthin production.

[0009] Therefore, how to obtain Haematococcus pluvialis strains with higher astaxanthin yield through chemical mutagenesis, and on this basis, adopt environmentally friendly extraction methods to improve astaxanthin extraction efficiency, reduce the use of organic solvents, and reduce environmental pollution during the extraction process, has become a technical problem that needs to be solved in this field. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a chemical mutagenesis screening method and its application for high-yield astaxanthin-producing Haematococcus pluvialis. This invention involves chemically mutagenesizing Haematococcus pluvialis cells in their logarithmic growth phase using ethyl methanesulfonate (EMS), combined with single-colony screening to obtain mutant algal strains with high astaxanthin accumulation capacity. Based on this, the high-yield algal strains are induced to accumulate astaxanthin under suitable conditions. Stable algal powder is obtained through freeze-drying. Astaxanthin is then extracted from the algae using a eutectic solvent system formed by terpenoids and oleic acid. This method improves astaxanthin yield and extraction efficiency while reducing the use of volatile organic solvents, minimizing environmental pollution caused by traditional extraction processes, and enhancing the safety and stability of the astaxanthin extraction process.

[0011] The technical means employed in this invention are as follows:

[0012] A chemical mutagenesis screening method for high astaxanthin production of Haematococcus pluvialis includes the following steps:

[0013] S11. Algal culture: Haematococcus pluvialis is cultured in a culture medium to the logarithmic growth phase to obtain the algal solution for mutagenesis treatment;

[0014] S12. Chemical mutagenesis treatment: A chemical mutagen is added to the algal solution to induce random mutations in Haematococcus pluvialis cells.

[0015] S13. Termination of mutagenesis and recovery culture: After the mutagenesis treatment is completed, a mutagenesis terminator is added to terminate the mutagenesis reaction, and the mutagenized algal cells are washed and recovered.

[0016] S14. Screening of mutant strains: After recovery culture, algal cells were inoculated into solid culture medium for culture. After single algae were formed, they were selected and high-astaxanthin Haematococcus pluvialis mutant strains were obtained by detecting the astaxanthin content in the algae.

[0017] Furthermore, the chemical mutagen is ethyl methanesulfonate.

[0018] Furthermore, the concentration of the ethyl methanesulfonate is 0.4% to 0.6%.

[0019] Furthermore, the mutagenesis treatment time is 2-4 hours.

[0020] Furthermore, the lethality of the mutagenesis treatment is controlled at 70% to 95%.

[0021] This invention also discloses a method for green extraction of astaxanthin from Haematococcus pluvialis, a high-yield astaxanthin algae obtained using the above-mentioned chemical mutagenesis screening method, comprising the following steps:

[0022] S21. Induction culture: The high-astaxanthin-producing Haematococcus pluvialis mutant strain was cultured under stress conditions to promote astaxanthin accumulation.

[0023] S22. Algal Collection and Freeze-Drying: After induction culture, algal cells are collected and centrifuged. After removing the supernatant, they are freeze-dried at low temperature to obtain algal powder.

[0024] S23, Green Extraction: The freeze-dried algae powder is mixed with a eutectic solvent and extracted by shaking under light-protected conditions to obtain astaxanthin extract.

[0025] Furthermore, the eutectic solvent is formed from terpenoids and fatty acids.

[0026] Furthermore, the eutectic solvent is a eutectic system formed by thymol and oleic acid.

[0027] Furthermore, the molar ratio of thymol to oleic acid is 3:1.

[0028] Furthermore, before the green extraction in the eutectic solvent, the algae powder is subjected to enzymatic hydrolysis. The enzymatic hydrolysis uses a complex enzyme system of cellulase and pectinase with a mass ratio of 2:1.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. This invention utilizes ethyl methanesulfonate (EMS) to chemically mutagenesis of Haematococcus pluvialis in its logarithmic growth phase, combined with single-colony isolation and screening, to obtain a Haematococcus pluvialis mutant strain with significantly increased astaxanthin content. Multiple subcultures verified that this mutant strain exhibits stable genetic traits, with an astaxanthin content reaching 5.03%. Under defined culture conditions, the strain was cultured in BBM medium at 60 μmol / m³. 2 After reaching a stable period by culturing for 11 days under 24°C light intensity, the culture was then subjected to 210 μmol / m 2 High light intensity stress culture for 2 days induces astaxanthin accumulation, which can significantly increase the astaxanthin synthesis level in algae. This mutagenesis screening method can obtain high-astaxanthin-producing algal strains in a short time, providing a stable and reliable source of algal strains for astaxanthin production in Haematococcus pluvialis.

[0031] 2. This invention employs EMS (Enhanced Molecular Subtraction) chemical mutagenesis technology for the mutagenesis breeding of Haematococcus pluvialis. This method can generate a high frequency of point mutations at the DNA level, thereby obtaining diverse genetic variations and providing a rich genetic basis for screening high-yielding algal strains. Compared with physical mutagenesis, EMS mutagenesis causes less damage to chromosome structure, less chromosome breakage or aberration, and the mutants have better genetic stability. Furthermore, this method is relatively simple to operate and easy to implement under laboratory and large-scale cultivation conditions, allowing for the acquisition of a large number of mutant resources in a short time, thus improving the efficiency of screening high-yielding algal strains.

[0032] 3. Regarding astaxanthin extraction, this invention employs a eutectic solvent system composed of oleic acid and DL-menthol, thymol, or geraniol to extract astaxanthin from Haematococcus pluvialis. The TAO system, formed by thymol and oleic acid, exhibits high extraction efficiency. By optimizing the material-liquid ratio, using ultrasonic assistance, and employing enzyme assistance, the astaxanthin extraction efficiency can reach over 97%, significantly improving the extraction efficiency. Furthermore, the raw materials used in this eutectic solvent system are all edible or low-toxicity components, possessing good safety and environmental friendliness. It also provides excellent photostability protection for astaxanthin, maintaining approximately 70% retention after 12 hours of UV irradiation, thus enhancing the stability of the astaxanthin product.

[0033] In summary, this invention obtains high-yield astaxanthin-producing Haematococcus pluvialis strains through chemical mutagenesis screening, and combines freeze-drying with low-eutectic solvent green extraction technology. This significantly improves the astaxanthin yield of algae while reducing environmental pollution problems in traditional organic solvent extraction processes. It provides an effective technical approach for the green preparation of astaxanthin from Haematococcus pluvialis and has good application prospects. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The images show the mutagenesis of Haematococcus pluvialis cells in the logarithmic growth phase using different concentrations of EMS solution for 3 hours in this invention. A is the control group without mutagenesis treatment, B is the group treated with 0.4% EMS, C is the group treated with 0.5% EMS, and D is the group treated with 0.6% EMS.

[0036] Figure 2This is a growth curve diagram of the 33 mutant algal strains obtained by screening in this invention.

[0037] Figure 3 This is a graph showing the astaxanthin content analysis of the mutant algal strains of this invention, which compares the astaxanthin content of 11 algal strains with higher biomass than the original algal strain with that of the original algal strain.

[0038] Figure 4 The graph shows the effect of light intensity on the biomass accumulation of the mutant algal strain, where A represents the mutant strain and B represents the wild algal strain.

[0039] Figure 5 The graph shows the effect of temperature on algal growth, where A represents the mutant strain and B represents the wild-type algal strain.

[0040] Figure 6 This is a comparison chart of astaxanthin extracted from Haematococcus pluvialis using the DESs system of this invention.

[0041] Figure 7 The graph shows the effect of the material-to-liquid ratio on the extraction efficiency and concentration of astaxanthin.

[0042] Figure 8 The effect of ultrasound on the extraction efficiency of astaxanthin is shown in the figure.

[0043] Figure 9 The figure shows the effect of a single enzyme on the extraction of astaxanthin from Haematococcus pluvialis.

[0044] Figure 10 The figure shows the effect of complex enzymes on the extraction of astaxanthin from Haematococcus pluvialis.

[0045] Figure 11 The figure shows the effect of enzyme-assisted ultrasound on the extraction efficiency of astaxanthin from Haematococcus pluvialis.

[0046] Figure 12 This is a graph showing the photostability analysis of astaxanthin. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] This invention provides a method for cultivating a high-yield astaxanthin-producing Haematococcus pluvialis strain. The method involves chemically mutagenesizing Haematococcus pluvialis with ethyl methanesulfonate (EMS) and obtaining a high-yield astaxanthin-producing Haematococcus pluvialis strain through single-colony screening. The specific steps are as follows.

[0049] (1) Preparation of algal solution

[0050] Take 0.5 mL of Haematococcus pluvialis algal solution in the logarithmic growth phase and measure the OD value of the algal solution at a wavelength of 680 nm, which is approximately 0.3. Place the algal solution in a centrifuge tube and centrifuge at 10 kr / min for 5 min at 25 °C to collect algal cells.

[0051] (2) Mutagenesis treatment

[0052] Add 1.8 mL of EMS mutagen solution of different concentrations (0.4%, 0.5%, and 0.6%) to centrifuge tubes. The mixture was then subjected to a light intensity of 40 μmol / m². 2 Mutagenesis was performed under s conditions for 3 hours.

[0053] (3) Termination of mutagenesis

[0054] After the mutagenesis was completed, 0.18 mL of 5% sodium thiosulfate solution was added to the centrifuge tube and allowed to stand for 5 min to terminate the mutagenesis reaction.

[0055] (4) Cell washing

[0056] Centrifuge at 10 kr / min for 5 min at room temperature, discard the supernatant, add 1 mL of sterile BBM liquid culture medium, and gently shake for about 1 min to wash away any residual drugs in the centrifuge tube. Repeat the above washing procedure 2-3 times to remove residual mutagens.

[0057] (5) Restore culture

[0058] After washing, centrifuge at 10 kr / min for 5 min to discard the supernatant, add 1 mL of sterile BBM liquid culture medium, and incubate at 23℃ in the dark for 12 h to allow the algal cells to recover their growth state.

[0059] (6) Plate culture

[0060] After the incubation period ended and the algal culture was restored to its original state, 50 μL of the induced algal culture and the original algal culture were respectively transferred to solid culture plates containing BBM medium and spread evenly using a spreader. The plates were then incubated at 23°C under a light intensity of 25 μmol / m². 2 •s, with a light-to-dark ratio of 12:12.

[0061] (7) Screening of single algal colonies

[0062] After green algae formed on the plate, the number of algal colonies under different mutagenesis conditions was counted and the lethality rate was calculated. Mutagenesis conditions with a lethality rate greater than 80% were selected. Single algal colonies were picked from the plate and transferred to Erlenmeyer flasks containing BBM liquid medium, and incubated at 23°C and a light intensity of 25 μmol / m². 2The cells were cultured under conditions of 12:12 light-to-dark ratio and then passaged.

[0063] (8) Expand cultivation

[0064] After three subcultures, the algal cells were transferred to 250 mL Erlenmeyer flasks for further culture under the following conditions: light intensity 60 μmol / m². 2 •s, temperature 24±1℃, light-dark ratio 12:12.

[0065] Example 1 Mutagenesis Screening

[0066] Following the above method, Haematococcus pluvialis cells in the logarithmic growth phase were subjected to 3-hour mutagenesis treatment with EMS solutions of concentrations of 0.4%, 0.5%, and 0.6%, respectively. The mutagenesis results are as follows: Figure 1 As shown.

[0067] As shown in the figure, all EMS treatment groups at different concentrations were able to form single algal colonies. This is in contrast to the control group without mutagenesis treatment (…). Figure 1 Compared to A), the mortality rate in the 0.4% EMS concentration treatment group was approximately 50%. Figure 1 B), the mortality rate in the 0.5% EMS concentration treatment group was approximately 90%. Figure 1 C), while the 0.6% EMS treatment group only formed a small amount of algal colonies, with a mortality rate of over 99%. Figure 1 D). Considering both mutagenesis efficiency and cell survival, treatment with 0.5% EMS for 3 hours was selected as the optimal condition for mutagenesis of Haematococcus pluvialis.

[0068] Under the above mutagenesis conditions, a total of 35 single algal colonies were selected from the plate for expanded culture. Among them, 2 single algal colonies failed to continue to grow, and finally 33 mutagenized algal strains were obtained for subsequent culture and screening.

[0069] Example 2: Growth Performance Testing

[0070] The 33 mutant algal strains obtained in Example 1 were cultured, and their growth was monitored. Growth curves were plotted as follows: Figure 2 As shown.

[0071] The experimental results showed that the biomass accumulation of each mutant algal strain was slow in the early stage of cultivation. During the first four days of cultivation, the biomass of the mutant algal strains did not show a significant increase, and their biomass accumulation was lower than that of the original algal strains. As the cultivation time increased, the growth rate of some mutant algal strains gradually increased.

[0072] Among them, mutant strains #28 and #33 showed biomass accumulation exceeding that of the original algal strain starting from day 6 of culture; mutant strains #7 and #30 showed biomass accumulation roughly equivalent to that of the original algal strain starting from day 8 of culture. After 10 days of culture, the biomass of mutant algal strains #2, #7, #10, #12, #18, #21, #25, #26, #28, #29, and #33 were all higher than that of the original algal strain.

[0073] Example 3 Astaxanthin Accumulation

[0074] After the mutant algal strains 2#, 3#, 7#, 10#, 12#, 17#, 18#, 21#, 25#, 26#, 28#, 29#, and 33# obtained in Example 2 were cultured to the stable phase, they were subjected to a light intensity of 210 μmol / m². 2 High light stress culture was performed under ·s conditions for 2 days to induce astaxanthin accumulation.

[0075] After the induction culture was completed, the algal cells were recovered, astaxanthin was extracted from the algae, and the astaxanthin content was measured. The results were compared with those of the original algal strain. Figure 3 As shown.

[0076] As shown in the figure, the astaxanthin content of mutant strains #10, #25, #29, and #33 was higher than that of the original strain. Among them, mutant strain #29 had the highest astaxanthin content, reaching 5.08%, which was about 17.6% higher than that of the original strain. In addition, the astaxanthin content of mutant strain #26 was significantly lower than that of the original strain, with a decrease of about 34.0%.

[0077] After multiple subculturings of the selected mutant algal strains, the astaxanthin content was tested again. The results showed that the astaxanthin content of the mutant algal strains remained basically stable without significant changes, indicating that the mutant algal strains did not undergo recovery mutations.

[0078] In summary, by combining EMS chemical mutagenesis with single-algal colony screening, a high-yielding Haematococcus pluvialis strain with astaxanthin content exceeding 5% was obtained.

[0079] Example 4: Optimization of Culture Conditions

[0080] To determine the suitable culture conditions for the mutant algal strains, culture experiments were conducted on the screened mutant algal strains under different light intensities and culture temperatures.

[0081] (1) Determination of optimal lighting conditions

[0082] Light intensity is a crucial factor affecting the growth of Haematococcus pluvialis. In this example, the screened mutant strain #29 and wild-type strains were selected as research subjects and cultured under different light intensity conditions. The set light intensities were 30 μmol / m². 2·s, 60 μmol / m 2 ·s, 90 μmol / m 2 ·s, 120 μmol / m 2 ·s, 150 μmol / m 2 ·s and 180μmol / m 2 ·s.

[0083] Samples were taken on days 1, 3, 5, 7, 9, and 11 during the cultivation process. The algal cell biomass was characterized by the OD value of the algal solution. The results are as follows: Figure 4 As shown.

[0084] from Figure 4 It can be seen that in the early stage of cultivation (the first 5 days), at 90 μmol / m 2 ·s, 120 μmol / m 2 ·s, 150 μmol / m 2 ·s and 180 μmol / m 2 Under light intensity conditions of ·s, the growth rate of the mutant algal strain was significantly higher than that under 30 μmol / m 2 ·s and 60 μmol / m 2 Algal strains under s light conditions. With prolonged culture time, 60 μmol / m 2 Under s-level light conditions, the growth rate of algal cells gradually increased, exhibiting higher biomass accumulation in subsequent culture stages. Starting from day 9, 60 μmol / m 2 The biomass of algal cells cultured under ·s conditions is higher than that under other light conditions.

[0085] Algal cells cultured under all light conditions entered the stationary phase on day 11, at which point biomass essentially ceased to increase. For wild-type algal strains, at 60 μmol / m³... 2 Under s light conditions, its biomass was also highest, but slightly lower than that of the mutagenic algae. Therefore, the optimal light intensity for Haematococcus pluvialis cultivation was determined to be 60 μmol / m². 2 ·s.

[0086] (2) Determination of the optimal culture temperature

[0087] Temperature is also a crucial factor affecting the growth of Haematococcus pluvialis. In this example, mutant and wild-type algal strains were cultured at 21℃, 24℃, and 27℃ to compare the effects of different temperature conditions on algal cell growth. The results are as follows: Figure 5 As shown.

[0088] Depend on Figure 5It can be seen that under the culture condition of 24℃, both algal strains exhibited a faster biomass accumulation rate, with their growth rate significantly higher than that of algal cells under the conditions of 21℃ and 27℃. After 11 days of culture, both the mutant and wild-type algal strains reached their maximum biomass and entered the stationary phase at 24℃. Therefore, the optimal culture temperature for Haematococcus pluvialis was determined to be 24℃.

[0089] (3) Determination of cultivation process

[0090] Based on the above experimental results, the cultivation process for astaxanthin production from Haematococcus pluvialis was determined as follows: Algal seeds were inoculated into BBM medium at a 10% inoculation ratio, and cultured at 60 μmol / m³... 2 The algal cells were cultured for 11 days under light intensity of 24℃ to allow them to reach a stable phase; subsequently, they were cultured at 210 μmol / m 2 High light stress culture was conducted for 2 days under s light intensity conditions to induce astaxanthin accumulation.

[0091] Example 5 Green Extraction

[0092] This embodiment utilizes eutectic solvents (DESs) to extract astaxanthin from Haematococcus pluvialis and optimizes the extraction process.

[0093] (1) Preparation of DESs

[0094] This embodiment utilizes oleic acid with different terpene compounds to prepare eutectic solvent systems, specifically oleic acid with DL-menthol, thymol, and geraniol. The components are prepared according to the following molar ratios:

[0095] MAO:DL-menthol:oleic acid = 2:1;

[0096] TAO: Thymol: Oleic acid = 3:1;

[0097] GAO: Geraniol: Oleic acid = 13:1;

[0098] The above raw materials were directly added to a sealed Erlenmeyer flask and heated at a constant temperature of 60°C with magnetic stirring (500 rpm) until a homogeneous and transparent mixed solution was formed. The resulting solution was then cooled to room temperature and sealed and allowed to stand for 24 hours under dark and dry conditions to observe the stability of the solution system.

[0099] (2) Characterization of the DESs system

[0100] The prepared DESs system was characterized. Characterization was performed by comparing experimental and theoretical solid-liquid phase diagrams. The experimental solid-liquid phase curves were obtained by placing the samples in an ice / NaCl mixture and measuring the melting points of samples with different molar ratios using a thermometer. The theoretical solid-liquid phase curves were calculated using equations representing the solid-liquid equilibrium relationship of the eutectic system. The results show that although the actual eutectic ratio and melting point of the three systems differ somewhat from the theoretical values, they all exhibit significant deviations relative to the ideal activity value (γ=1). Since this type of system is composed of nonionic molecules, it can be classified as a type V DES system.

[0101] Furthermore, this type of DES system exhibits significant hydrophobic properties. The solubility of each component in water is as follows: oleic acid <0.01 mM; DL-menthol <3 mM; thymol 6 mM; geraniol 5 mM; and their water contents are as follows: TAO: 2.9 wt%; MAO: 0.94 wt%; GAO: 2.6 wt%.

[0102] (3) DESs extraction of astaxanthin from Haematococcus pluvialis

[0103] Haematococcus pluvialis cells were collected and centrifuged at 4℃ and 8 kr / min for 5 min to remove the supernatant. The cells were then transferred to a freeze dryer for lyophilization. At room temperature, the lyophilized Haematococcus pluvialis powder was mixed with the prepared DESs solution and placed in a black, light-protected centrifuge tube. After gentle shaking to mix, the tube was incubated at 26℃ and 180 r / min for 24 h. Samples were taken at 1 h, 3 h, 6 h, and 24 h during extraction. Each time, 0.5 mL of the extract was centrifuged at 4℃ and 10 kr / min for 10 min. 0.4 mL of the supernatant was collected and diluted with 3.1 mL of DMSO / methanol solution. After thorough mixing, the absorbance was measured at 470 nm using a UV spectrophotometer to calculate the astaxanthin content in the solution. Three parallel experiments were conducted for each group, and the average value was taken after three replicates.

[0104] Extraction results as follows Figure 6 As shown. By Figure 6 It can be seen that TAO31 showed the best extraction effect for astaxanthin, with an extraction rate of 61% at 6 hours and 83% at 24 hours; MAO21 was second best, with an extraction rate of 55% at 6 hours and 74% at 24 hours; the pure oleic acid system had the lowest extraction efficiency, with an extraction rate of only 21% at 24 hours. Therefore, the TAO31 system (thymol: oleic acid = 3:1) was selected as the solvent system for subsequent astaxanthin extraction studies.

[0105] (4) Optimization of TAO system extraction process

[0106] Although the TAO system has high extraction efficiency under 24-hour conditions, the extraction time is relatively long, so its extraction conditions need to be further optimized.

[0107] A. The effect of the feed-liquid ratio

[0108] Freeze-dried algae powder was mixed with DESs at different material-to-liquid ratios of 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1 (mg / mL). Extraction was performed at room temperature for 24 h. The effects of different material-to-liquid ratios on astaxanthin extraction efficiency and astaxanthin concentration in the extract were investigated. The results are as follows: Figure 7 As shown, with the increase of TAO dosage, the astaxanthin extraction efficiency gradually increases, while the astaxanthin concentration in the extract gradually decreases. Considering both extraction efficiency and astaxanthin concentration, a material-to-liquid ratio of 1:1 is determined to be more suitable.

[0109] B. Ultrasonic-assisted extraction

[0110] A certain amount of Haematococcus pluvialis powder was weighed and added to the TAO system. The mixture was then subjected to ultrasonic treatment at room temperature for 10 min, 20 min, 40 min, and 80 min, respectively, with an ultrasonic power of 500 W. After ultrasonic treatment, the mixture was allowed to stand for 10 min for extraction. The results are as follows: Figure 8 As shown, the extraction efficiency of astaxanthin gradually increased with increasing ultrasonic time, reaching 86.7% when the ultrasonic time was 80 min.

[0111] C. Enzyme-assisted extraction

[0112] To further improve the extraction efficiency of astaxanthin, the enzyme-assisted extraction conditions were studied.

[0113] (a) Single enzyme

[0114] 10 mg of algal powder was weighed out and different amounts of cellulase or pectinase were added: 6000 U, 8000 U, 10000 U, 12000 U, 14000 U, and 16000 U, respectively. Enzymatic hydrolysis was performed at pH 5.0 and 50℃ for 6 hours. After centrifugation to remove the supernatant, 10 mL of TAO system was added for extraction. Extraction was carried out at room temperature in the dark for 10 minutes, and the astaxanthin content was then determined. Three parallel experiments were set up for each group, and the results are as follows: Figure 9 As shown in the figure. The results showed that when 14,000 U of cellulase was added, the extraction rate reached 34% in 10 minutes; when 10,000 U of pectinase was added, the astaxanthin extraction efficiency reached 21.3%.

[0115] (b) Complex enzymes

[0116] Cellulase and pectinase were mixed at ratios of 1:1, 1:2, and 2:1 (U / U) for enzymatic hydrolysis, with enzyme dosage ranging from 6000 to 16000 U. Astaxanthin was then extracted using the method described above, and the results are as follows: Figure 10 As shown, when the ratio of cellulase to pectinase is 2:1 and the enzyme amount is 14000U, the astaxanthin extraction rate reaches 44.6% after 10 minutes.

[0117] D. Enzyme-assisted ultrasonic extraction

[0118] After enzymatic hydrolysis of algal powder under compound enzyme treatment conditions (cellulase:pectinase = 2:1, enzyme amount 14000U), ultrasonic treatment was performed at room temperature for 5 min, 10 min, 15 min, and 20 min, with an ultrasonic power of 500 W. After ultrasonication, extraction was carried out in the dark for 10 min, and the astaxanthin content was measured. The results are as follows: Figure 11 As shown, under the conditions of compound enzyme treatment, the extraction efficiency of astaxanthin can reach over 97% when the ultrasonic time is 10 min.

[0119] In summary, the optimal extraction process for astaxanthin from Haematococcus pluvialis was determined to be: a material-to-liquid ratio of 1:1, enzymatic hydrolysis for 6 hours under the condition of a compound enzyme (cellulase: pectinase = 2:1, enzyme amount 14000U / mg), followed by ultrasonic treatment at room temperature for 10 minutes (500W), and then static extraction for 10 minutes.

[0120] (5) Photostability of astaxanthin extract

[0121] The astaxanthin extract from the TAO system was placed into 5 mL sealed transparent centrifuge tubes and subjected to simulated ultraviolet light irradiation using a UV lamp, with an oleic acid-astaxanthin mixture as a control. The photostability of the TAO system astaxanthin extract was then assessed. 3 mL samples were collected at 1 h, 3 h, 9 h, and 12 h, and the remaining astaxanthin content was determined using a UV spectrophotometer. The photostability of the astaxanthin extract was characterized by the ratio of the remaining astaxanthin content after irradiation to the astaxanthin content before irradiation. Each experiment was conducted in triplicate, with three replicates, and the average value was taken. Results are as follows: Figure 12 As shown. At 3.08 mW / cm 2 Under ultraviolet light, more than 60% of the astaxanthin in the oleic acid-astaxanthin mixture was degraded after 6 hours of light exposure, and almost completely degraded after 12 hours of light exposure; while the astaxanthin extract of the TAO system still maintained a retention rate of nearly 70% after 12 hours of light exposure, indicating that the TAO system can effectively improve the photostability of astaxanthin.

[0122] In summary, this invention, through EMS chemical mutagenesis combined with single-colony screening, obtained a genetically stable, high-astaxanthin-producing Haematococcus pluvialis strain, and further determined suitable culture conditions and astaxanthin-induced accumulation conditions. Based on this, an eutectic solvent system composed of oleic acid and terpenoids was used to extract astaxanthin from Haematococcus pluvialis. Optimization of conditions such as the material-to-liquid ratio, ultrasonic assistance, and enzyme assistance significantly improved the extraction efficiency of astaxanthin. Simultaneously, the eutectic solvent system used provided good stabilizing protection for astaxanthin, effectively reducing photo-oxidative degradation. This invention provides an efficient and green astaxanthin extraction method, achieving the screening and stable cultivation of high-astaxanthin-producing Haematococcus pluvialis strains.

[0123] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A chemical mutagenesis screening method for high astaxanthin-producing Haematococcus pluvialis, characterized in that, Includes the following steps: S11. Algal culture: Haematococcus pluvialis is cultured in a culture medium to the logarithmic growth phase to obtain the algal solution for mutagenesis treatment; S12. Chemical mutagenesis treatment: A chemical mutagen is added to the algal solution to induce random mutations in Haematococcus pluvialis cells. S13. Termination of mutagenesis and recovery culture: After the mutagenesis treatment is completed, a mutagenesis terminator is added to terminate the mutagenesis reaction, and the mutagenized algal cells are washed and recovered. S14. Screening of mutant strains: After recovery culture, algal cells were inoculated into solid culture medium for culture. After single algae were formed, they were selected and high-astaxanthin Haematococcus pluvialis mutant strains were obtained by detecting the astaxanthin content in the algae.

2. The chemical mutagenesis screening method according to claim 1, characterized in that, The chemical mutagen is ethyl methanesulfonate.

3. The chemical mutagenesis screening method according to claim 2, characterized in that, The concentration of ethyl methanesulfonate is 0.4% to 0.6%.

4. The chemical mutagenesis screening method according to claim 1, characterized in that, The mutagenesis treatment time is 2-4 hours.

5. The chemical mutagenesis screening method according to claim 1, characterized in that, The lethality of the mutagenesis treatment was controlled at 70% to 95%.

6. A method for green extraction of astaxanthin from Haematococcus pluvialis, a high-yield astaxanthin-producing algae obtained by the chemical mutagenesis screening method according to any one of claims 1 to 5, characterized in that, Includes the following steps: S21. Induction culture: The high-astaxanthin-producing Haematococcus pluvialis mutant strain was cultured under stress conditions to promote astaxanthin accumulation. S22. Algal Collection and Freeze-Drying: After induction culture, algal cells are collected and centrifuged. After removing the supernatant, they are freeze-dried at low temperature to obtain algal powder. S23, Green Extraction: The freeze-dried algae powder is mixed with a eutectic solvent and extracted by shaking under light-protected conditions to obtain astaxanthin extract.

7. The method according to claim 6, characterized in that, The eutectic solvent is formed from terpenoids and fatty acids.

8. The method according to claim 7, characterized in that, The eutectic solvent is a eutectic system formed by thymol and oleic acid.

9. The method according to claim 8, characterized in that, The molar ratio of thymol to oleic acid is 3:

1.

10. The method according to claim 6, characterized in that, Before green extraction in a eutectic solvent, the algae powder is subjected to enzymatic hydrolysis using a complex enzyme system of cellulase and pectinase in a mass ratio of 2:1.

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