Breeding method of all-trans astaxanthin
By employing pre-adaptation and gradient stress induction methods, the problems of low all-trans astaxanthin ratio and severe cell damage were solved, achieving efficient astaxanthin production, improving the purity and yield of all-trans astaxanthin, and reducing cell mortality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies suffer from problems such as low all-trans astaxanthin ratio, severe cell damage, and long culture cycles.
The method of pre-adaptation domestication-gradient stress induction-dynamic nutrient regulation was adopted. The Haematococcus pluvialis mutant strain YYAM001 was screened, and the algae were activated, pre-adapted domesticated, and subjected to gradient stress induction. The culture medium conditions were regulated by an online monitoring system. Finally, the algae were harvested and astaxanthin was extracted.
It significantly improves the purity and yield of all-trans astaxanthin, reduces cell mortality, ensures algal harvesting efficiency and astaxanthin synthesis stability, and shortens the culture cycle.
Smart Images
Figure CN121759563A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of shrimp farming technology, specifically to a method for farming all-trans astaxanthin. Background Technology
[0002] Astaxanthin is a carotenoid with powerful antioxidant activity, widely used in food, health products, cosmetics, and animal feed. All-trans astaxanthin is the most active astaxanthin variant, exhibiting significantly higher antioxidant capacity and bioavailability than the cis isomer. Therefore, increasing the yield and proportion of all-trans astaxanthin in aquaculture is one of the core objectives of industrial astaxanthin production.
[0003] Currently, the main method of astaxanthin cultivation is microalgae (such as Haematococcus pluvialis). Existing technologies mostly adopt a two-stage cultivation model of "vegetative growth-stress induction": the first stage optimizes nutritional conditions to promote microalgae cell proliferation, and the second stage induces astaxanthin synthesis through strong light, nitrogen deficiency, salt stress and other methods.
[0004] Existing stress-induced conditions easily lead to the large-scale generation of cis isomers, with all-trans astaxanthin typically accounting for only 60%-70%, which is insufficient to meet the needs of high-end applications. Extreme stresses such as strong light and high salt can damage the cell membrane structure of microalgae, leading to increased cell mortality (usually exceeding 30%) and reducing the actual harvesting efficiency of astaxanthin. The connection between vegetative growth and stress induction is unreasonable, resulting in a slow transition of cells from the proliferative state to the astaxanthin synthesis state, with the overall culture cycle lasting as long as 15-20 days. Summary of the Invention
[0005] The purpose of this invention is to provide a method for cultivating all-trans astaxanthin, in order to solve the problems of low all-trans astaxanthin ratio, severe cell damage during the induction stage, and long culture cycle in the prior art.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for cultivating all-trans astaxanthin, comprising the following steps:
[0007] S1. Algal strain screening and activation: The Haematococcus pluvialis mutant strain YYAM001 was screened and inoculated into activation medium. It was cultured for 3-5 days at a temperature of 22-25℃, a light intensity of 3000-5000 lx, a photoperiod of 12h:12h, and a pH of 7.0-7.5, until the cell concentration reached 1.0 × 10⁻⁶ cells / day. 6 -2.0×10 6 cells / mL, to obtain activated algal solution;
[0008] S2. Pre-acclimatization culture: Inoculate the activated algal solution into the pre-acclimatization culture medium at a volume ratio of 5%-10%. Gradually decrease the temperature from 25℃ to 20℃, gradually increase the light intensity from 5000 lx to 8000 lx, and add 0.05-0.1 g / L β-ionone. Culture for 4-6 days until the cell concentration reaches 5.0 × 10⁻⁶ cells / day. 6 -8.0×10 6 The pre-adapted algal solution was obtained by adding 0.03 g / L proline and 0.02 g / L betaine to the activated medium.
[0009] S3. Gradient stress induction culture: The pre-adapted algal solution is transferred to an induction culture device, and three stages of induction are performed sequentially:
[0010] The first stage was conducted at a temperature of 20℃, a light intensity of 12000lx, a light cycle of 14h:10h, with the addition of a nitrogen source limiter and methyl jasmonate, and a salinity of 10‰.
[0011] The second stage involved a temperature of 18℃, a light intensity of 18000lx, a light cycle of 16h:8h, the addition of salicylic acid, and a salinity of 20‰.
[0012] In the third stage, the temperature was 18℃, the light intensity was 15000lx, the photoperiod was 14h:10h, 0.05g / L glucose was added, and the salinity was 20‰; after 9 days of cultivation, an algal solution rich in all-trans astaxanthin was obtained.
[0013] S4. Algal harvesting and astaxanthin extraction: The algal cells were harvested by centrifugation, freeze-dried, and then extracted with ultrasonic-assisted extraction using a mixed solvent of ethanol and ethyl acetate. The purified all-trans astaxanthin was obtained by silica gel column chromatography.
[0014] Further, the activation culture medium in S1 consists of: sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.075 g / L, calcium chloride 0.02 g / L, disodium EDTA 0.005 g / L, ferric sulfate 0.001 g / L, and trace element stock solution 1 mL / L; the trace element stock solution consists of: zinc sulfate 0.022 g / L, copper sulfate 0.008 g / L, manganese chloride 0.018 g / L, and sodium molybdate 0.005 g / L.
[0015] Furthermore, the amount of β-ionone added in S2 is 0.08 g / L.
[0016] Furthermore, in S2, the temperature decrease rate is 1℃ / day, the light intensity increase rate is 1000lx / day, and the pH is maintained at 7.2-7.8 during the pre-adaptation and acclimatization process by adding 5% sodium bicarbonate solution.
[0017] Furthermore, the nitrogen source limiting agent in S3 is a mixture of ammonium nitrate and urea, with a mass ratio of 1:2 and an addition amount of 0.1 g / L.
[0018] Furthermore, the amount of methyl jasmonate added in S3 is 0.02-0.03 g / L, and the amount of salicylic acid added is 0.01-0.02 g / L.
[0019] Furthermore, during the gradient stress induction culture process in S3, the dissolved oxygen concentration in the culture medium is adjusted to 5-8 mg / L in real time through an online monitoring system, and deionized water is added once a day to replenish the evaporation loss.
[0020] Furthermore, the conditions for ultrasonic-assisted extraction in S4 are: power 200W, frequency 40kHz, temperature 40℃, and time 30min; the volume ratio of the ethanol-ethyl acetate mixed solvent is 3:1.
[0021] Compared with existing technologies, the present invention provides a method for cultivating all-trans astaxanthin, which effectively promotes cell proliferation and significantly improves the purity and yield of all-trans astaxanthin through pre-adaptation domestication, gradient stress induction and dynamic nutritional regulation. At the same time, it effectively reduces cell mortality, avoids cell damage caused by extreme stress, ensures algal harvesting efficiency and astaxanthin synthesis stability, and exhibits excellent performance under different parameter combinations. Attached Figure Description
[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0023] Figure 1 This is a schematic diagram of the overall process provided for an embodiment of the present invention. Detailed Implementation
[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.
[0025] As attached Figure 1 As shown:
[0026] This invention provides a method for cultivating all-trans astaxanthin, comprising the following steps:
[0027] S1. Algal strain screening and activation: The mutant strain YYAM001 of Haematococcus pluvialis (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 45099) was screened, inoculated into activation medium, and cultured for 3-5 days at a temperature of 22-25℃, a light intensity of 3000-5000 lx, a photoperiod of 12h:12h, and a pH of 7.0-7.5 until the cell concentration reached 1.0×106-2.0×106 cells / mL to obtain activated algal solution;
[0028] The activation culture medium consisted of: sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.075 g / L, calcium chloride 0.02 g / L, disodium EDTA 0.005 g / L, ferric sulfate 0.001 g / L, and trace element stock solution 1 mL / L.
[0029] The trace element mother liquor composition is as follows: zinc sulfate 0.022 g / L, copper sulfate 0.008 g / L, manganese chloride 0.018 g / L, sodium molybdate 0.005 g / L.
[0030] S2. Pre-acclimatization culture: The activated algal solution is inoculated into the pre-acclimatization medium at a volume ratio of 5%-10%. The temperature is gradually reduced from 25℃ to 20℃, and the light intensity is gradually increased from 5000 lx to 8000 lx. 0.05-0.1 g / L β-ionone is added, and the culture is carried out for 4-6 days until the cell concentration reaches 5.0×106-8.0×106 cells / mL to obtain the pre-acclimatized algal solution. The pre-acclimatization medium is the activated medium supplemented with 0.03 g / L proline and 0.02 g / L betaine. The amount of β-ionone added is 0.08 g / L.
[0031] In S2, the temperature decrease rate is 1℃ / day, the light intensity increase rate is 1000lx / day, and the pH is maintained at 7.2-7.8 during the pre-acclimatization culture by adding 5% sodium bicarbonate solution.
[0032] S3. Gradient stress induction culture: The pre-adapted algal solution is transferred to an induction culture device, and three stages of induction are performed sequentially:
[0033] The first stage involved a temperature of 20°C, a light intensity of 12000 lx, a light cycle of 14h:10h, and the addition of a nitrogen source limiter and methyl jasmonate, with a salinity of 10‰. The nitrogen source limiter was a mixture of ammonium nitrate and urea in a mass ratio of 1:2, added at a rate of 0.1 g / L. The amount of methyl jasmonate added was 0.02–0.03 g / L, and the amount of salicylic acid added was 0.01–0.02 g / L.
[0034] The second stage involved a temperature of 18℃, a light intensity of 18000lx, a light cycle of 16h:8h, the addition of salicylic acid, and a salinity of 20‰.
[0035] In the third stage, the temperature was 18℃, the light intensity was 15000lx, the photoperiod was 14h:10h, 0.05g / L glucose was added, and the salinity was 20‰; after 9 days of cultivation, an algal solution rich in all-trans astaxanthin was obtained.
[0036] During the gradient stress induction culture process, the dissolved oxygen concentration in the culture medium was adjusted to 5-8 mg / L in real time through an online monitoring system, and deionized water was added once a day to replenish the evaporation loss.
[0037] S4. Algal harvesting and astaxanthin extraction: The algal cells were harvested by centrifugation, freeze-dried, and then extracted with ultrasonic-assisted extraction using an ethanol-ethyl acetate mixed solvent. The purified all-trans astaxanthin was obtained by silica gel column chromatography. The ultrasonic-assisted extraction conditions were: power 200W, frequency 40kHz, temperature 40℃, and time 30min. The volume ratio of the ethanol-ethyl acetate mixed solvent was 3:1.
[0038] Example 1:
[0039] S1. The mutant strain YYAM001 of *Haematococcus pluvialis* (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 45099) was screened and inoculated into 10 L of activation medium. The activation medium consisted of: sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.075 g / L, calcium chloride 0.02 g / L, disodium EDTA 0.005 g / L, ferric sulfate 0.001 g / L, and a trace element stock solution of 1 mL / L (trace element stock solution: zinc sulfate 0.022 g / L, copper sulfate 0.008 g / L, manganese chloride 0.018 g / L). / L, sodium molybdate 0.005g / L); pH was adjusted to 7.5 by adding 0.1mol / L sodium hydroxide solution. Culture conditions: temperature 25℃, light intensity 5000lx, photoperiod 12h:12h (light:dark), sterile air was continuously introduced through an aeration device at a rate of 0.5L / (L·min) and cultured for 4 days. The cell concentration reached 1.8×106 cells / mL by hemocytometer counting, and activated algae solution was obtained.
[0040] S2. Pre-acclimatization culture: Inoculate the activated algal solution at a volume ratio of 8% into 50L of pre-acclimatization medium (add 0.03g / L proline and 0.02g / L betaine to the activated medium), and accurately add 0.08g / L β-ionone (4g β-ionone for 50L medium); control culture conditions: gradually decrease the temperature from 25℃ to 20℃ (cooling rate 1℃ / day, adjusting the temperature control device at the same time every day), and increase the light intensity from 5000 L / L... The x was gradually increased to 8000 lx (increase rate of 1000 lx / day, increased daily via a dimmer), the light cycle was 12h:12h, the pH was maintained at 7.5-7.8 by intermittently adding 5% sodium bicarbonate solution (pH was monitored every 8 hours, and 0.1-0.2 L of sodium bicarbonate solution was added when it was below 7.5), and the aeration rate was continuously 0.8 L / (L·min) for 5 days. The cell concentration reached 7.2 × 106 cells / mL, and the pre-adapted algal solution was obtained.
[0041] S3. Gradient stress induction culture: Transfer all pre-acclimatized algal solution to a 200L induction culture device, turn on the online monitoring system (real-time monitoring of dissolved oxygen, pH, and cell concentration), and perform three stages of induction sequentially:
[0042] First induction phase (1-3 days): Temperature constant at 20℃, light intensity increased to 12000 lx, photoperiod 14h:10h (6 am to 8 pm), add 0.1 g / L ammonium nitrate-urea mixture (mass ratio 1:2, 20 g mixture added to 200 L of culture medium, including 6.67 g ammonium nitrate and 13.33 g urea) and 0.025 g / L methyl jasmonate (add 5 g methyl jasmonate), adjust salinity to 10‰ by adding sodium chloride (add 2 kg sodium chloride), maintain pH at 7.8, and control dissolved oxygen concentration to 6 mg / L by aeration and stirring at 150 r / min and aeration rate of 1.0 L / (L·min)).
[0043] Second induction phase (4-6 days): Temperature drops to 18℃, light intensity increases to 18000lx, photoperiod is 16h:8h (light from 5 am to 9 pm), nitrogen source is stopped, 0.015g / L salicylic acid is added (add 3g salicylic acid), sodium chloride is added to increase salinity to 20‰ (add an additional 2kg sodium chloride), pH is maintained at 8.0, dissolved oxygen is adjusted to 7mg / L, stirring speed is 180r / min, and aeration rate is 1.2L / (L·min)).
[0044] The third induction phase (7-9 days): maintain the temperature at 18℃, reduce the light intensity to 15000 lx, and use a 14h:10h photoperiod (light from 6 am to 8 pm). Add 0.05 g / L glucose (add 10 g glucose), maintain the salinity at 20‰, maintain the pH at 8.2, and maintain the dissolved oxygen at 7 mg / L. Add deionized water once a day at 9 am to replenish evaporation loss (the amount of replenishment is 1%-2% of the culture medium volume). After 9 days of culture, the algal cell concentration in the culture medium reaches 12.5 × 106 cells / mL.
[0045] S4. Algal Harvesting and Astaxanthin Extraction: The induced algal culture solution was transferred in batches to a high-speed centrifuge at 5000 rpm for 10 minutes to collect the algal sludge. The sludge was washed three times with deionized water (5 L of deionized water added each time, stirred thoroughly, and then centrifuged). The washed sludge was then transferred to a freeze dryer at -50℃ and 10 Pa vacuum for 12 hours until the moisture content reached 4%. The dried algal cells were pulverized to 80 mesh using a high-speed pulverizer. 100 g of the dry powder was added to 1 L of an ethanol-ethyl acetate mixture (volume ratio 3:1, 750 mL ethanol + 250 mL ethyl acetate) and placed in an ultrasonic extractor at 200 W power and 40 Hz frequency. Extract at kHz and 40℃ for 30 min; after extraction, centrifuge (4000 r / min, 5 min), collect the supernatant, repeat the extraction once with the residue, and combine the two supernatants; transfer the combined extract to a rotary evaporator, set the temperature to 50℃ and the vacuum degree to 50 Pa, and concentrate under reduced pressure to a volume of 50 mL to obtain crude astaxanthin; load the crude product onto a silica gel column chromatography apparatus (silica gel column specifications: diameter 5 cm, height 50 cm, silica gel type 200-300 mesh), elute with a hexane-ethyl acetate mixed solvent (volume ratio 5:1), control the flow rate at 2 mL / min, collect the fraction corresponding to the elution peak, concentrate and dry again under reduced pressure to obtain pure all-trans astaxanthin. High-performance liquid chromatography (HPLC) analysis (detection conditions: C18 column, mobile phase methanol-acetonitrile-dichloromethane = 45:45:10, flow rate 1.0 mL / min, detection wavelength 474 nm) showed that the proportion of all-trans astaxanthin was 92.3%, the astaxanthin yield was 28.5 mg / g dry algae, and the cell mortality rate was 8.7% (detected by trypan blue staining method).
[0046] Example 2:
[0047] S1. Algal strain screening and activation: The Haematococcus pluvialis mutant strain YYAM001 (CGMCC No. 45099) was screened and inoculated into 15L of activation medium. The composition of the activation medium was the same as in Example 1. The pH was adjusted to 7.0 by adding 0.1mol / L hydrochloric acid solution. The culture conditions were: temperature 22℃, light intensity 3000lx, photoperiod 12h:12h, aeration rate 0.4L / (L·min). After 5 days of culture, the cell concentration reached 1.0×106 cells / mL, and the activated algal solution was obtained.
[0048] S2. Pre-acclimatization culture: The activated algal solution was inoculated into 60L of pre-acclimatization culture medium (same composition as in Example 1) at a volume ratio of 5%, and 0.05g / L β-ionone (3g added) was added. Culture conditions were controlled as follows: the temperature was gradually reduced from 25℃ to 20℃ (1℃ / day), the light intensity was gradually increased from 5000lx to 8000lx (1000lx / day), the photoperiod was 12h:12h, the pH was maintained at 7.2-7.5 by adding 5% sodium bicarbonate solution, and the aeration rate was 0.7L / (L·min). After 6 days of culture, the cell concentration reached 5.0×106 cells / mL, and the pre-acclimatized algal solution was obtained.
[0049] S3. Gradient stress induction culture: Transfer the pre-acclimatized algal solution to a 250L induction culture device, turn on the online monitoring system, and implement three-stage induction conditions:
[0050] First induction phase (1-3 days): temperature 20℃, light intensity 12000lx, photoperiod 14h:10h, addition of 0.1g / L ammonium nitrate-urea mixture (25g added per 250L, ammonium nitrate 8.33g, urea 16.67g) and 0.02g / L methyl jasmonate (5g added), salinity 10‰ (2.5kg sodium chloride added), pH 7.5, dissolved oxygen 5mg / L, stirring speed 140r / min, aeration rate 0.9L / (L·min));
[0051] Second induction phase (4-6 days): Temperature 18℃, light intensity 18000lx, photoperiod 16h:8h, stop adding nitrogen source, add 0.01g / L salicylic acid (add 2.5g), increase salinity to 20‰ (add an additional 2.5kg sodium chloride), pH 7.8, dissolved oxygen 6mg / L, stirring speed 170r / min, aeration rate 1.1L / (L·min));
[0052] Third induction stage (7-9 days): temperature 18℃, light intensity 15000lx, photoperiod 14h:10h, add 0.05g / L glucose (12.5g added), salinity 20‰, pH 8.0, dissolved oxygen 6mg / L; add deionized water at 10 am daily (1%-2%), culture for 9 days, and the algal cell concentration reaches 10.2×106 cells / mL.
[0053] S4. Algal Harvesting and Astaxanthin Extraction: Harvesting and extraction were performed as in Example 1. 100g of dried algal powder was weighed and extracted twice using 1L of a 3:1 mixture of ethanol and ethyl acetate (200W, 40kHz, 40℃, 30min) via ultrasonic extraction. The extracts were combined, concentrated, and purified by silica gel column chromatography. HPLC analysis showed that all-trans astaxanthin accounted for 90.5%, the astaxanthin yield was 25.3mg / g of dry algal body, and the cell mortality rate was 9.2%.
[0054] Example 3:
[0055] S1. Algal strain screening and activation: The Haematococcus pluvialis mutant strain YYAM001 (CGMCC No. 45099) was screened and inoculated into 20L of activation medium. The composition of the activation medium was the same as in Example 1, and the pH was adjusted to 7.3. The culture conditions were: temperature 24℃, light intensity 4000lx, photoperiod 12h:12h, aeration rate 0.6L / (L·min). After 3 days of culture, the cell concentration reached 2.0×106 cells / mL, and the activated algal solution was obtained.
[0056] S2. Pre-acclimatization culture: The activated algal solution was inoculated into 80L of pre-acclimatization culture medium (same composition as in Example 1) at a volume ratio of 10%, and 0.1g / L β-ionone (8g added) was added; the culture conditions were controlled as follows: the temperature was gradually reduced from 25℃ to 20℃ (1℃ / day), the light intensity was gradually increased from 5000lx to 8000lx (1000lx / day), the photoperiod was 12h:12h, the pH was maintained at 7.6-7.8 by adding 5% sodium bicarbonate solution, the aeration rate was 0.9L / (L·min), and the culture was carried out for 4 days until the cell concentration reached 8.0×106 cells / mL, thus obtaining the pre-acclimatized algal solution.
[0057] S3, Gradient stress induction culture: Transfer the pre-adapted algal solution to a 300L induction culture device, turn on the online monitoring system, and implement three-stage induction conditions:
[0058] First induction phase (1-3 days): temperature 20℃, light intensity 12000lx, photoperiod 14h:10h, addition of 0.1g / L ammonium nitrate-urea mixture (30g added per 300L, ammonium nitrate 10g, urea 20g) and 0.03g / L methyl jasmonate (9g added), salinity 10‰ (3kg sodium chloride added), pH 8.0, dissolved oxygen 8mg / L, stirring speed 160r / min, aeration rate 1.3L / (L·min));
[0059] Second induction phase (4-6 days): Temperature 18℃, light intensity 18000lx, photoperiod 16h:8h, stop adding nitrogen source, add 0.02g / L salicylic acid (6g added), increase salinity to 20‰ (add an additional 3kg sodium chloride), pH 8.2, dissolved oxygen 7mg / L, stirring speed 190r / min, aeration rate 1.4L / (L·min));
[0060] Third induction phase (7-9 days): temperature 18℃, light intensity 15000lx, photoperiod 14h:10h, add 0.05g / L glucose (15g added), salinity 20‰, pH 8.5, dissolved oxygen 7mg / L; add deionized water at 11 am daily (1%-2%), culture for 9 days, and the algal cell concentration reaches 14.8×106 cells / mL.
[0061] S4. Harvesting of algae and extraction of astaxanthin: The harvesting and extraction steps are the same as in Example 1. After extraction and purification, 100g of dried algae powder was analyzed by HPLC. The proportion of all-trans astaxanthin was 91.8%, the astaxanthin yield was 27.6mg / g of dry algae, and the cell mortality rate was 8.1%.
[0062] Comparative Example 1: Traditional Two-Stage Culture Method
[0063] A two-stage culture method using existing technology was employed: Haematococcus pluvialis (a common strain, not the YYAM001 mutant) was inoculated into 10L of activation medium (same composition as in Example 1) and cultured at 25℃, 5000 lx, 12h:12h for 5 days (vegetative growth stage), reaching a cell concentration of 2.1 × 10⁶ cells / mL; then, it was directly transferred to a 200L culture device and induced to grow under strong light (20000 lx), high salt (25‰), and nitrogen-deficient conditions for 10 days (stress induction stage), with a photoperiod of 16h:8h and a temperature of 20℃; subsequent harvesting and extraction of S were the same as in Example 1. HPLC analysis showed that the all-trans astaxanthin content was 68.5%, the astaxanthin yield was 15.2 mg / g dry algae, and the cell mortality rate was 32.1%.
[0064] A two-stage culture method using existing technology was employed: A common strain of Haematococcus pluvialis was inoculated into an activation medium and cultured at 25°C, 5000 lx, 12h:12h for 5 days (vegetative growth stage); then directly transferred to strong light (20000 lx), high salt (25‰), and nitrogen-deficient conditions for 10 days of induction culture (stress induction stage); subsequent harvesting and extraction of S were the same as in Example 1. Analysis showed that the all-trans astaxanthin content was 68.5%, the astaxanthin yield was 15.2 mg / g dry algae, and the cell mortality rate was 32.1%.
[0065] The data comparison is shown in the table below:
[0066] project Example 1 Example 2 Example 3 Comparative Example 1 (Traditional Two-Stage Method) <![CDATA[Cell concentration after algal strain activation (×10 6 cells / mL)]]> 1.8 1.0 2.0 2.1 <![CDATA[Cell concentration after preconditioning (×10 6 cells / mL)]]> 7.2 5.0 8.0 (No pre-adaptation phase) <![CDATA[Cell concentration after induction (×10 6 cells / mL)]]> 12.5 10.2 14.8 8.3 (Post-induction) Percentage of all-trans astaxanthin (%) 92.3 90.5 91.8 68.5 Astaxanthin production (mg / g dry algae) 28.5 25.3 27.6 15.2 Cell death rate (%) 8.7 9.2 8.1 32.1
[0067] The data is summarized as follows:
[0068] After pre-adaptation and gradient stress induction, the cell concentrations in all three groups of examples reached over 10.2 × 10⁶ cells / mL, with Example 3 having the highest concentration (14.8 × 10⁶ cells / mL). However, Comparative Example 1, lacking a pre-adaptation phase, was subjected to direct extreme stress, resulting in inhibited cell proliferation and a cell concentration of only 8.3 × 10⁶ cells / mL after induction, significantly lower than the example groups.
[0069] The proportion of all-trans astaxanthin in Examples 1-3 was consistently above 90%, with an average of 91.5%, which was 33.6% higher than that in Comparative Example 1 (68.5%). This demonstrates that the three-stage culture mode of the present invention can effectively promote the synthesis of all-trans isomers and inhibit the formation of cis isomers.
[0070] The astaxanthin yield in the example group was 25.3-28.5 mg / g dry thallus, with an average of 27.1 mg / g dry thallus, which was 78.3% higher than that in Comparative Example 1 (15.2 mg / g dry thallus). The core reason is that gradient stress reduced cell damage and improved thallus harvesting efficiency. At the same time, the precursor substances accumulated during the pre-adaptation stage promoted astaxanthin synthesis.
[0071] The cell mortality rate in the example group was controlled below 10% (8.1%-9.2%), which was much lower than the 32.1% in Comparative Example 1. This indicates that gradient stress induction avoided the sudden impact of extreme conditions on cells, effectively protected the cell membrane integrity, and provided a guarantee for the stable synthesis and efficient harvesting of astaxanthin.
[0072] In summary, this aquaculture method, through the "pre-adaptation domestication-gradient stress induction-dynamic nutritional regulation" model, is significantly superior to the traditional two-stage method in terms of cell proliferation, all-trans astaxanthin purity, yield, and cell survival stability. Moreover, the examples under different parameter combinations all showed stable and excellent performance, and have industrial promotion value.
[0073] The comparative results show that the culture methods of the three embodiments of the present invention are significantly better than the traditional two-stage culture method in terms of all-trans astaxanthin ratio, yield and cell survival rate. Moreover, high all-trans ratio and low cell damage can be stably achieved under different parameter combinations, proving the stability and superiority of the technical solution of the present invention.
[0074] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A method for cultivating all-trans astaxanthin, characterized in that, Includes the following steps: S1. Screen the Haematococcus pluvialis mutant strain YYAM001, inoculate it into activation medium, and culture it for 3-5 days at a temperature of 22-25℃, a light intensity of 3000-5000 lx, a photoperiod of 12h:12h, and a pH of 7.0-7.5 until the cell concentration reaches 1.0×10⁻⁶. 6 -2.0×10 6 cells / mL, to obtain activated algal solution; S2. Inoculate the activated algal solution into the pre-adaptation medium at a volume ratio of 5%-10%. Gradually decrease the temperature from 25℃ to 20℃, gradually increase the light intensity from 5000 lx to 8000 lx, add 0.05-0.1 g / L β-ionone, and culture for 4-6 days until the cell concentration reaches 5.0 × 10⁻⁶ cells / day. 6 -8.0×10 6 The pre-adapted algal solution was obtained by adding 0.03 g / L proline and 0.02 g / L betaine to the activated medium. S3. Transfer the pre-adapted algal solution into the induction culture device and perform three-stage induction sequentially: The first stage was conducted at a temperature of 20℃, a light intensity of 12000 lx, a light cycle of 14h:10h, with the addition of a nitrogen source limiter and methyl jasmonate, and a salinity of 10‰. The second stage involved a temperature of 18℃, a light intensity of 18000lx, a light cycle of 16h:8h, the addition of salicylic acid, and a salinity of 20‰. In the third stage, the temperature was 18℃, the light intensity was 15000lx, the photoperiod was 14h:10h, 0.05g / L glucose was added, and the salinity was 20‰. After culturing for 9 days, an algal solution rich in all-trans astaxanthin was obtained. S4. The algae were collected by centrifugation, freeze-dried, and then extracted with ultrasonic-assisted extraction using a mixed solvent of ethanol and ethyl acetate. The purified all-trans astaxanthin was obtained by silica gel column chromatography.
2. The method for cultivating all-trans astaxanthin according to claim 1, characterized in that, The activation culture medium in S1 consists of: sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.075 g / L, calcium chloride 0.02 g / L, disodium EDTA 0.005 g / L, ferric sulfate 0.001 g / L, and trace element stock solution 1 mL / L; the trace element stock solution consists of: zinc sulfate 0.022 g / L, copper sulfate 0.008 g / L, manganese chloride 0.018 g / L, and sodium molybdate 0.005 g / L.
3. The method for cultivating all-trans astaxanthin according to claim 1, characterized in that, The amount of β-ionone added in S2 is 0.08 g / L.
4. The method for cultivating all-trans astaxanthin according to claim 1, characterized in that, The temperature in S2 decreases at a rate of 1°C / day, the light intensity increases at a rate of 1000 lx / day, and the pH is maintained at 7.2-7.8 during the pre-acclimatization and acclimatization process by adding 5% sodium bicarbonate solution.
5. The method for cultivating all-trans astaxanthin according to claim 1, characterized in that, The nitrogen source limiting agent in S3 is a mixture of ammonium nitrate and urea in a mass ratio of 1:2, with an addition amount of 0.1 g / L.
6. The method for cultivating all-trans astaxanthin according to claim 5, characterized in that, The amount of methyl jasmonate added in S3 is 0.02-0.03 g / L, and the amount of salicylic acid added is 0.01-0.02 g / L.
7. A method for cultivating all-trans astaxanthin according to claim 6, characterized in that, During the gradient stress induction culture process in S3, the dissolved oxygen concentration in the culture medium is controlled in real time to be 5-8 mg / L through an online monitoring system, and deionized water is added once a day to replenish the evaporation loss.
8. The method for cultivating all-trans astaxanthin according to claim 1, characterized in that, The conditions for ultrasonic-assisted extraction described in S4 are: power 200W, frequency 40kHz, temperature 40℃, and time 30min; the volume ratio of the ethanol-ethyl acetate mixed solvent is 3:1.