Haematococcus pluvialis culture medium and culture method thereof

Abstract

The application provides a haematococcus pluvialis culture medium and a culture method thereof, and the culture medium comprises a nutrition proliferation culture medium used in a segmented culture, a stress induction culture medium and an accumulation strengthening culture medium; the nutrition proliferation culture medium comprises sodium nitrate, potassium dihydrogen phosphate, potassium chloride, ferric ammonium citrate, sodium citrate and a trace element mixed solution; the stress induction culture medium is added with an osmotic stress agent, an oxidation inducer and a plant hormone; and the accumulation strengthening culture medium removes sodium nitrate and is added with a carbon source supplement, a polymer osmotic agent and a zwitterionic stabilizer. The culture method comprises the following steps: a nutrition proliferation stage, a stress induction stage and an accumulation strengthening stage. Through the three-segmented culture medium and the fine regulation and control culture process, and through the synergistic effect of the composite stress factors and the dynamic environmental signals, the bidirectional synergistic and efficient improvement of the biomass growth and astaxanthin accumulation is realized under the premise of ensuring the high activity of algal cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium for Haematococcus pluvialis and its culture method. Background Technology

[0002] Haematococcus pluvialis holds a significant position in the field of microalgae, considered the most potent natural source of astaxanthin. Astaxanthin is a ketocarotenoid with exceptional antioxidant activity, showing immense potential in food additives, nutritional supplements, high-end cosmetics, and aquaculture feed. However, large-scale industrial cultivation of Haematococcus pluvialis and efficient astaxanthin extraction still face numerous unresolved technical bottlenecks. One core challenge lies in the fact that Haematococcus pluvialis undergoes two main stages under natural or conventional culture conditions—the green vegetative cell stage and the red thick-walled spore stage (the primary astaxanthin accumulation stage)—but their environmental requirements differ significantly. Traditional general-purpose culture media, such as BG-11, often fail to simultaneously and efficiently meet the dual demands of growth and astaxanthin accumulation, especially during the stress stage, when biomass growth slows significantly and astaxanthin synthesis rates become unsatisfactory.

[0003] To promote astaxanthin accumulation, a common strategy in production is to add stress factors (such as high light intensity, nutrient deprivation, and increased salinity) to the culture medium. Among these, inducing stress by regulating the culture medium composition is the most frequently used method. Current techniques often employ complete or partial nitrogen deprivation to induce algal cell transformation and astaxanthin accumulation. However, this treatment often leads to mass cell death or dormancy during the stress phase, resulting in significant biomass loss, and ultimately, no significant increase in the total astaxanthin yield per unit volume of culture medium. Furthermore, the extensive use of single high-concentration inorganic salts (such as sodium acetate and sodium chloride) for osmotic stress, while initiating astaxanthin synthesis to some extent, has a relatively simple mechanism of action, causes significant physiological shock to cells, and can easily lead to irreversible damage, limiting the effective extension of the stress cycle and further improvement in accumulation efficiency. Therefore, how to finely regulate the culture medium composition to induce the activation of the astaxanthin synthesis pathway more gently, persistently, and efficiently while ensuring high algal cell viability and stress resistance is currently a key research focus and challenge.

[0004] Regarding the optimization of cultivation processes, the simple two-stage cultivation (vegetative growth – stress accumulation) often suffers from transitional inconsistencies in actual operation. Existing technologies lack precise control over the type, timing, intensity, and transition of stress factors during the stress initiation phase, frequently leading to insufficient or excessive stress signal transduction. Furthermore, in the critical post-stress phase, the continuous maintenance of the accumulation environment is neglected, failing to effectively support the continued efficient synthesis of astaxanthin by algal cells that have activated their accumulation potential, resulting in a low ceiling for accumulation efficiency.

[0005] In the downstream processing stage after harvest, efficiently releasing astaxanthin from the thick-walled spores of Haematococcus pluvialis with its hard cell walls is another major challenge. Conventional physical cell wall disruption methods (such as high-temperature drying and pulverization, high-pressure homogenization, and ball milling) are not only energy-intensive but also prone to degradation, oxidation, and isomerization of heat-sensitive astaxanthin, reducing product quality and bioavailability. Although chemical methods (acid and alkali treatment) and enzymatic hydrolysis have been explored, chemical methods are prone to introducing harmful residues and damaging the complex structure of natural components, while the efficiency, cost, and economic feasibility of enzymatic hydrolysis still need significant improvement. Developing a processing method that can efficiently dissolve or soften the cell walls of Haematococcus pluvialis under mild conditions while maximally protecting the integrity of astaxanthin is crucial for improving the yield and quality of astaxanthin extraction.

[0006] In summary, the main problems in the current large-scale production of Haematococcus pluvialis and its astaxanthin extraction process can be summarized as follows: 1) General-purpose culture media are not highly targeted to astaxanthin accumulation, the stress induction effect is not ideal, and biomass and astaxanthin are difficult to grow synergistically and efficiently; 2) The culture process is not refined enough, and the stress initiation and maintenance strategies are not efficient, stable, and mild enough, resulting in limited total astaxanthin yield; 3) Existing cell wall disruption extraction technologies generally suffer from high energy consumption, high astaxanthin loss rate, heavy environmental burden, or high cost. Therefore, it is necessary to design a Haematococcus pluvialis culture medium and its culture method. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, a culture medium for Haematococcus pluvialis and its culture method are provided.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A culture medium for Haematococcus pluvialis, comprising a nutrient proliferation medium for segmented culture, a stress induction medium, and an accumulation and enhancement medium;

[0010] The nutrient proliferation culture medium includes:

[0011] Sodium nitrate 140-160 mg / L;

[0012] Potassium dihydrogen phosphate 11-13 mg / L;

[0013] Potassium chloride 45-55 mg / L;

[0014] Ferric ammonium citrate provides an iron concentration of 0.08-0.12 mg / L;

[0015] Sodium citrate 1.8-2.2 g / L;

[0016] Trace element mixture: 0.8-1.2 mL / ;

[0017] The stress-inducing medium is a nutrient proliferation medium supplemented with: 35-45 mg / L of osmotic stress agent, 0.08-0.12 mg / L of oxidation inducer, and 0.18-0.22 mg / L of plant hormone;

[0018] The accumulation and enhancement medium is composed of the nutrient proliferation medium with sodium nitrate removed and supplemented with a carbon source, polymer penetrant, and zwitterionic stabilizer.

[0019] A core contradiction facing Haematococcus pluvialis is the significant difference in requirements between its two critical life cycle stages. During the green vegetative cell stage, algal cells require ample nutrients for rapid proliferation and biomass accumulation. However, when transitioning to the red thick-walled spore stage, the primary accumulation period of astaxanthin, appropriate stress signals must be applied to trigger this mechanism. Current techniques commonly use universal culture media, such as BG-11, relying on simple nitrogen deprivation to induce stress. This approach leads to a drastic change in environment; cells, while losing their growth foundation, are forced to undergo stress, easily resulting in mass cell death or dormancy. Even if the surviving cells begin synthesizing astaxanthin, the total biomass loss is too great, making it difficult to effectively increase the total astaxanthin obtained per unit of culture medium. Another common approach is to use a single high-concentration inorganic salt, such as sodium chloride, to create osmotic stress and initiate synthesis. However, this method is too simplistic and harsh, exerting excessive stress on cell physiology, often causing irreversible damage and failing to maintain a long-term, effective, and mild stress state. Consequently, biomass growth and astaxanthin accumulation conflict, making it difficult to achieve efficient results simultaneously.

[0020] To address this challenge, we abandoned a generic approach and creatively designed a phased culture medium system, comprising a nutrient proliferation medium, a stress induction medium, and an accumulation and enhancement medium. Each medium is precisely formulated for a specific phase. The core of the nutrient proliferation phase is to promote rapid and robust algal cell growth. The nutrient proliferation medium provides nitrogen through an appropriate concentration of sodium nitrate, phosphorus through potassium dihydrogen phosphate, and is supplemented with potassium chloride to maintain ion balance, essential iron, and sodium citrate as a carbon source buffer, providing an optimal nutrient environment for rapid cell proliferation. In the stress induction phase, if the environment suddenly becomes harsh, the cells will not be able to cope. Therefore, the stress induction medium is not a separate system, but rather a carefully selected composite stress factor superimposed on the nutrient base of the nutrient proliferation medium: the osmotic stress agent sodium salicylate, the oxidation inducer sodium selenite, and the plant hormone methyl jasmonic acid. These factors are no longer single, strong stimuli, but rather gently activate astaxanthin synthesis by mimicking the synergistic effects of multiple signaling pathways in natural stress. Sodium salicylate induces moderate osmotic pressure, while sodium selenite provides controlled oxidative stress signals. Together, they mimic stress in the natural environment, initiating a cellular defense response. Methyl jasmonic acid acts as an internal signaling molecule, widely involved in secondary metabolic regulation in plants and algae. These three components work synergistically, under specific ratios of sodium salicylate and sodium selenite, and with an appropriate amount of methyl jasmonic acid, to gently yet effectively activate genes and enzymes related to astaxanthin synthesis within cells. This guides cells from a vegetative growth state to a thick-walled spore state where astaxanthin accumulates, significantly reducing the risk of cell death or dormancy due to "shock."

[0021] The trace element mixture contains:

[0022] Based on elemental boron, boron compounds are present in concentrations of 2.5-4.5 mg / L.

[0023] Based on elemental copper, copper compounds are present at concentrations of 0.004-0.006 mg / L.

[0024] Zinc compounds, calculated as elemental zinc, are present at concentrations of 0.06-0.08 mg / L.

[0025] Molybdenum compounds, calculated as elemental molybdenum, are present in concentrations of 0.06-0.08 mg / L.

[0026] Manganese compounds are present in concentrations of 0.8-1.0 mg / L based on elemental manganese.

[0027] The boron compound is boric acid, the copper compound is copper sulfate pentahydrate, the zinc compound is zinc sulfate heptahydrate, the molybdenum compound is sodium molybdate dihydrate, and the manganese compound is manganese sulfate monohydrate.

[0028] The osmotic stress agent is sodium salicylate, the oxidation inducer is sodium selenite, and the plant hormone is methyl jasmonic acid.

[0029] The mass ratio of sodium salicylate to sodium selenite is 350-450:1.

[0030] The supplementary carbon source is glucose, with an initial concentration of 2-3 g / L; the polymer penetrant is polyethylene glycol 6000 at 1.7-1.9 g / L; and the zwitterionic stabilizer is betaine hydrochloride at 0.08-0.12 g / L.

[0031] Once cells successfully initiate the astaxanthin accumulation process, the process is not yet complete; they still require a continuously supporting environment for efficient synthesis. The accumulation enhancement phase is designed for this purpose. Its culture medium is derived directly from a nutrient proliferation medium with the nitrogen source sodium nitrate removed. This preserves basic nutrients and a buffer system while simultaneously removing the nitrogen source to send a continuous "stress" signal, preventing cells from reverting to growth mode. Crucially, it includes the addition of glucose as a supplementary carbon source, polyethylene glycol 6000 as a polymeric permeabilizer, and betaine hydrochloride as an amphoteric stabilizer. The removal of the nitrogen source signals to cells to stop growing and focus on accumulation, while the supplemented glucose continuously provides the necessary carbon skeleton and energy for the complex biochemical pathway of astaxanthin synthesis. Polyethylene glycol 6000, through its polymer properties, produces a mild osmotic effect in the cellular microenvironment, helping to maintain the cell's osmotic state. Betaine hydrochloride, as a compatible solute, stabilizes protein structures and protects the integrity of cell membranes, especially organelle membranes, enhancing the cell's tolerance and viability under stress. The core principle of the three-stage culture medium relay design in this application is to precisely match the different needs of cells at each physiological transition stage, and gently and effectively guide the life process toward efficient accumulation of astaxanthin.

[0032] While downstream cell wall disruption is not the core focus of this application, the components selected in designing the accumulation and enhancement medium, as well as the characteristics of the entire culture process, may have some potential positive effects on subsequent cell wall disruption. The polymeric penetrant polyethylene glycol 6000 contained in the accumulation and enhancement medium of this application may, under long-term, gentle permeation, help to partially soften and loosen the very hard outer wall structure of *Rhodochophora* thick-walled spores. This is equivalent to doing some "preparatory work" at the microscopic level for subsequent cell wall disruption operations. The zwitterionic stabilizer betaine hydrochloride mainly plays a role in protecting the stability of the cell interior and the integrity of the membrane throughout the accumulation and enhancement stage, which may make the thick-walled spore cells obtained at harvest structurally more "robust" and intact.

[0033] A method for culturing Haematococcus pluvialis using a culture medium, the method comprising the following steps:

[0034] (1) Nutritional proliferation stage:

[0035] Inoculate algae into the nutrient proliferation medium, control the light intensity at 70-80 μmol·m⁻²·s⁻¹, the temperature at 23-25℃, and culture for 14-16 days;

[0036] (2) Stress-induced phase:

[0037] Concentrate the proliferated algal solution obtained in the previous step to 20-25% of its original volume and transfer it to the stress-inducing medium. Before the start of the stress-inducing stage, add 50% of the total amount of methyl jasmonate when preparing the medium. On the 4th day of the stress-inducing stage, add the remaining 50% of methyl jasmonate. During the stress-inducing stage, increase the light intensity stepwise: 140-160 μmol·m⁻²·s⁻¹ for 0-3 days, 170-190 μmol·m⁻²·s⁻¹ for 4-7 days, and maintain the temperature at 25-27℃ for 7-9 days.

[0038] (3) Accumulation and reinforcement stage:

[0039] The algal solution obtained in the previous step was transferred to the accumulation and strengthening medium, maintaining a light intensity of 200-210 μmol·m⁻²·s⁻¹ and a temperature of 17-18℃. Glucose was added every 24 hours at a rate of 0.08-0.16 g / L, and the culture was carried out for 8-10 days. During the stress induction phase, blue light with a wavelength range of 450±5 nm was used to gradually increase the light intensity.

[0040] After resolving the culture medium compatibility issue, we further realized that the transition from growth to efficient accumulation is not an instantaneous on / off switch, but a kinetic process requiring precise control. Existing technologies often employ a simple "two-step" approach that overlooks this: either the stress factor is added too quickly and aggressively, causing signal overload and severe cell damage; or it is added too slowly or insufficiently, resulting in poor induction. Furthermore, a suitable environment is not consistently provided during accumulation, leading to low accumulation efficiency. Therefore, we meticulously designed the parameters of the culture process, especially during the stress induction and accumulation enhancement phases. During the stress induction phase, a stepwise increase strategy was adopted for light intensity. For the first three days, the light level was maintained at a moderately high but tolerable level, gradually increasing to a higher intensity over the next four days. Crucially, this light used blue light within a specific wavelength range. Blue light holds special significance in algal photobiology; its wavelength is more effectively recognized by specific photoreceptors, thereby more efficiently activating gene expression related to light stress response and astaxanthin synthesis pathways. This not only provides energy but also a precise light signal.

[0041] The application of methyl jasmonate also follows a strategic approach, employing a two-stage addition method: 50% of the total amount is added at the start of stress induction to initiate the initial stress signal response. Then, on the fourth day of the stress induction phase, a critical time point when cells have initially adapted to the initial stress and begun to enter a deeper transition phase, the remaining 50% is added. This time-phased, sustained-release approach is similar to giving cells a "warm-up" process. It avoids the interference or negative feedback regulation that might result from a single high-concentration hormone shock, while further amplifying the stimulus signal when cells are relatively ready, thus promoting the deepening of the accumulation process.

[0042] During the nutrient proliferation stage, a gas containing 2.5-3.5% CO2 is introduced at a flow rate of 0.8-1.2 L / min.

[0043] During the accumulation and enhancement phase, the glucose supplementation rate is dynamically adjusted based on the algal cell reddening rate: when the reddening rate is <50%, 0.08-0.10 g / L of glucose is added every 24 hours; when the reddening rate is ≥50%, 0.12-0.16 g / L of glucose is added every 24 hours. During the accumulation and enhancement phase, the state of the algal cell population varies, with some turning red faster than others. Observing this, we did not uniformly supplement carbon sources, but innovatively adjusted the glucose supplementation rate dynamically based on a direct indicator: the algal cell reddening rate. The reddening rate reflects the proportion of cells that have successfully converted into astaxanthin-accumulating red spores. When the reddening rate is below 50%, it means that most cells are likely still in the conversion process, and the demand for carbon sources is relatively stable; therefore, glucose is supplemented at a lower rate. Once the reddening rate reaches or exceeds 50%, it indicates that half or more of the cells have entered the peak period of active astaxanthin synthesis. At this time, the cells are like a fully operational factory, and the demand for carbon sources increases dramatically. We then correspondingly increase the supplementation rate to meet the needs of high-intensity anabolic metabolism. This dynamic replenishment based on real-time status ensures, in principle, that the supply of energy and raw materials precisely matches the changing needs of the cell population, resulting in neither waste nor shortage.

[0044] During the accumulation and enhancement phase, weak oscillations are applied simultaneously, with the shaker speed at 80-100 rpm and the oscillation amplitude at 25-35 mm. The purpose of this gentle oscillation is not vigorous stirring, but rather to promote the uniform mixing and exchange of nutrients, oxygen, and metabolites throughout the culture system, preventing localized cell deprivation or hypoxia due to sediment buildup. This mild physical stimulation also helps maintain cell viability and membrane fluidity, providing a more stable and homogeneous microenvironment for efficient and long-lasting astaxanthin synthesis. These finely regulated process steps are integrated to simulate a gradual process of signal release and enhancement. Through precise blue light signaling, timed slow-release hormone addition, dynamic carbon source replenishment, and weak oscillations to maintain environmental homogeneity, the stability of the stress state and the sustainability of the efficient accumulation process are jointly maintained.

[0045] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0046] 1. This application effectively solves the problems mentioned in the background art, such as the lack of specificity of general culture media, unsatisfactory stress induction effects, and difficulty in achieving synergistic and efficient growth of biomass and astaxanthin, by designing a phased culture medium and cultivation method for Haematococcus pluvialis. Specifically, we provide a three-stage system: a nutrient proliferation medium, a stress induction medium, and an accumulation and enhancement medium, each stage optimized for the life cycle needs of algal cells. The nutrient proliferation medium contains specific proportions of sodium nitrate, potassium dihydrogen phosphate, potassium chloride, and citrate, designed to efficiently support the rapid proliferation of algal cells during the green nutrient stage; the stress induction medium adds sodium salicylate as an osmotic stress agent, sodium selenite as an oxidation inducer, and methyl jasmonic acid as a plant hormone to the nutrient proliferation medium. These components work synergistically in appropriate proportions to gently activate the astaxanthin synthesis pathway, avoiding cell damage caused by traditional single stress; the accumulation and enhancement medium removes the nitrogen source and supplements glucose, polyethylene glycol 6000, and betaine hydrochloride, thereby maintaining cell viability and osmotic balance under low stress conditions and supporting continuous astaxanthin accumulation. This phased culture medium design avoids the forced transition in traditional methods, and in principle can more accurately balance the growth and accumulation needs of algal cells, reducing cell death and dormancy.

[0047] 2. Regarding the culture process, this application employs refined control steps, significantly improving the efficiency and stability of stress initiation and maintenance, and solving the problem of insufficient refinement in the culture process of the prior art. In the nutrient proliferation stage, biomass accumulation is promoted through optimized light and temperature. In the stress induction stage, a stepwise light enhancement and batch addition strategy of methyl jasmonate is used, i.e., only half the amount is added at the beginning, with the remainder added after a specific number of days, combined with blue light illumination. This stepwise mechanism can alleviate the release of stress signals and avoid irreversible damage caused by overload. Simultaneously, blue light can more effectively stimulate photoresponsive genes within a specific wavelength range, enhancing the stress response. In the accumulation and enhancement stage, the amount of glucose supplementation is dynamically adjusted according to the reddening rate, and weak oscillations are applied. This allows for real-time adaptation to the intracellular astaxanthin synthesis state, while weak oscillations help homogenize nutrients and maintain cell viability, avoiding the yield limitations caused by stress incoordination in traditional two-stage processes. Overall, these process details simulate the natural stress process in principle, improving the total astaxanthin yield through a gentle and continuous activation method.

[0048] 3. The cultivation method of this application also introduces polymer permeabilizers and zwitterionic stabilizers during the accumulation and enhancement stage, which helps soften the cell wall structure and lays the foundation for subsequent cell wall disruption. Although the downstream extraction step is not the core of this application, applying stabilization treatment to the cells may reduce the difficulty of subsequent cell wall disruption and the risk of astaxanthin loss. For example, betaine hydrochloride can alleviate osmotic pressure and maintain cell membrane integrity. In principle, the entire process enhances the stress resistance and accumulation sustainability of algal cells, thereby indirectly supporting efficient astaxanthin acquisition and making the production of Haematococcus pluvialis more efficient, stable, and economical. Detailed Implementation

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0050] The sources of various raw materials in this application are briefly described as follows:

[0051] Sodium nitrate: Purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 7631-99-4, AR grade. Potassium dihydrogen phosphate: Purchased from Tianjin Kemei Chemical Reagent Co., Ltd., CAS No. 7778-77-0, analytical grade. Potassium chloride: Purchased from Xilong Scientific Co., Ltd., CAS No. 7447-40-7, superior grade. Ferrous ammonium citrate: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS No. 1185-57-5, biological reagent grade. Sodium citrate: Purchased from Aladdin Biochemical Technology Co., Ltd., CAS No. 6132-04-3, food grade. Boric acid: Purchased from Sigma-Aldrich, CAS No. 10043-35-3, ACS reagent grade. Copper sulfate pentahydrate: Purchased from Guangdong Guanghua Technology Co., Ltd., CAS No. 7758-99-8, industrial grade. Zinc sulfate heptahydrate: Purchased from Tianjin Yongda Chemical Reagent Co., Ltd., CAS No. 7446-20-0, chemically pure. Sodium molybdate dihydrate: Purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No. 10102-40-6, laboratory grade. Manganese sulfate monohydrate: Purchased from Beijing Huawirui Chemical Co., Ltd., CAS No. 10034-96-5, high purity. Sodium salicylate: Purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., CAS No. 54-21-7, pharmaceutical grade. Sodium selenite: Purchased from Sigma-Aldrich, CAS No. 10102-18-8, biotechnology grade. Methyl jasmonate: Purchased from Shanghai Jianglai Industrial Co., Ltd., CAS No. 39924-52-2, plant culture specific grade. Glucose: Purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 50-99-7, injection grade. Polyethylene glycol 6000: Purchased from Aladdin Biochemical Technology Co., Ltd., CAS No. 25322-68-3, molecular biology grade. Betaine hydrochloride: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS No. 590-46-5, cell culture grade.

[0052] A culture medium for Haematococcus pluvialis, comprising a nutrient proliferation medium for segmented culture, a stress induction medium, and an accumulation and enhancement medium;

[0053] The nutrient proliferation culture medium includes:

[0054] Sodium nitrate 140-160 mg / L;

[0055] Potassium dihydrogen phosphate 11-13 mg / L;

[0056] Potassium chloride 45-55 mg / L;

[0057] Ferric ammonium citrate provides an iron concentration of 0.08-0.12 mg / L;

[0058] Sodium citrate 1.8-2.2 g / L;

[0059] Trace element mixture: 0.8-1.2 mL / .

[0060] The stress-inducing medium is a nutrient proliferation medium supplemented with: 35-45 mg / L of osmotic stress agent, 0.08-0.12 mg / L of oxidation inducer, and 0.18-0.22 mg / L of plant hormone;

[0061] The accumulation and enhancement medium is composed of the nutrient proliferation medium with sodium nitrate removed and supplemented with a carbon source, polymer penetrant, and zwitterionic stabilizer.

[0062] The trace element mixture contains:

[0063] Based on elemental boron, boron compounds are present in concentrations of 2.5-4.5 mg / L.

[0064] Based on elemental copper, copper compounds are present at concentrations of 0.004-0.006 mg / L.

[0065] Zinc compounds, calculated as elemental zinc, are present at concentrations of 0.06-0.08 mg / L.

[0066] Molybdenum compounds, calculated as elemental molybdenum, are present in concentrations of 0.06-0.08 mg / L.

[0067] Manganese compounds are present in concentrations of 0.8-1.0 mg / L based on elemental manganese.

[0068] The boron compound is boric acid, the copper compound is copper sulfate pentahydrate, the zinc compound is zinc sulfate heptahydrate, the molybdenum compound is sodium molybdate dihydrate, and the manganese compound is manganese sulfate monohydrate.

[0069] The osmotic stress agent is sodium salicylate, the oxidation inducer is sodium selenite, and the plant hormone is methyl jasmonic acid.

[0070] The mass ratio of sodium salicylate to sodium selenite is 350-450:1.

[0071] The supplementary carbon source is glucose, with an initial concentration of 2-3 g / L; the polymer penetrant is polyethylene glycol 6000 at 1.7-1.9 g / L; and the zwitterionic stabilizer is betaine hydrochloride at 0.08-0.12 g / L.

[0072] A method for culturing Haematococcus pluvialis using a culture medium, the method comprising the following steps:

[0073] (1) Nutritional proliferation stage:

[0074] Inoculate algae into the nutrient proliferation medium, control the light intensity at 70-80 μmol·m⁻²·s⁻¹, the temperature at 23-25℃, and culture for 14-16 days;

[0075] (2) Stress-induced phase:

[0076] Concentrate the proliferated algal solution obtained in the previous step to 20-25% of its original volume and transfer it to the stress-inducing medium. Before the start of the stress-inducing stage, add 50% of the total amount of methyl jasmonate when preparing the medium. On the 4th day of the stress-inducing stage, add the remaining 50% of methyl jasmonate. During the stress-inducing stage, increase the light intensity stepwise: 140-160 μmol·m⁻²·s⁻¹ for 0-3 days, 170-190 μmol·m⁻²·s⁻¹ for 4-7 days, and maintain the temperature at 25-27℃ for 7-9 days.

[0077] (3) Accumulation and reinforcement stage:

[0078] The algal solution obtained in the previous step was transferred into the accumulation and strengthening medium, and the light intensity was maintained at 200-210 μmol·m⁻²·s⁻¹, the temperature at 17-18℃, and glucose was added at 0.08-0.16 g / L every 24 hours for 8-10 days.

[0079] In the stress-induced phase, the light intensity is stepped up using blue light with a wavelength range of 450±5 nm.

[0080] During the nutrient proliferation stage, a gas containing 2.5-3.5% CO2 is introduced at a flow rate of 0.8-1.2 L / min.

[0081] During the accumulation and enhancement phase, the amount of glucose supplemented is dynamically adjusted according to the reddening rate of algal cells: when the reddening rate is <50%, 0.08-0.10 g / L of glucose is added every 24 hours; when the reddening rate is ≥50%, 0.12-0.16 g / L of glucose is added every 24 hours. During the accumulation and enhancement phase, weak oscillation is applied at the same time, with the shaking speed at 80-100 rpm and the oscillation amplitude at 25-35 mm.

[0082] This application designs a three-stage culture medium and a finely controlled culture process. Through the synergistic effect of complex stress factors and dynamic environmental signals, it achieves a bidirectional and efficient enhancement of biomass growth and astaxanthin accumulation while ensuring high algal cell viability.

[0083] The core idea of ​​this three-stage culture strategy is to utilize gentle induction rather than drastic shock through complex signals. This results in thick-walled spores that are physiologically closer to their optimal state during natural maturation, exhibiting higher cell viability and better cell wall and membrane integrity. These thick-walled spores, with their higher viability and more stable membranes, are theoretically better able to withstand processing stress than cells damaged by severe stress. This improvement in their native state creates a relatively favorable condition for developing or applying gentler and more efficient cell-wall breaking and extraction methods. Overall, from nutritional support and gentle stress signal transmission to continuous support for the accumulation process, we aim to improve the final yield of astaxanthin in large-scale Haematococcus pluvialis culture through systematic improvements to upstream culture media and processes.

[0084] The technical solutions of the present invention are further illustrated below through examples and comparative examples, but the scope of protection of the present invention is not limited thereto.

[0085] Example 1

[0086] The culture medium preparation and culturing steps are as follows:

[0087] Nutritional proliferation stage:

[0088] Prepare a nutrient proliferation medium containing 160 mg sodium nitrate, 12 mg potassium dihydrogen phosphate, 45 mg potassium chloride, ferric ammonium citrate (providing 0.12 mg iron), 2.2 g sodium citrate, and 1.0 mL of a trace element mixture (containing 4.5 mg / L boric acid, 0.005 mg / L copper sulfate pentahydrate, 0.07 mg / L zinc sulfate heptahydrate, 0.07 mg / L sodium molybdate dihydrate, and 0.9 mg / L manganese sulfate monohydrate).

[0089] After inoculating with algae, the light intensity was controlled at 80 μmol·m⁻²·s⁻¹, the temperature at 25℃, and a gas containing 3.5% CO₂ was introduced (flow rate 1.0 L / min) for 14 days.

[0090] Stress-induced phase:

[0091] The algal growth solution was concentrated to 20% of its original volume and transferred to a stress-inducing medium (sodium salicylate 45 mg / L, sodium selenite 0.10 mg / L, and methyl jasmonate 0.18 mg / L were added to the nutrient growth medium).

[0092] 50% of the total methyl jasmonate (0.18 mg / L) was added when the culture medium was prepared, and the remaining 50% was added on the 4th day.

[0093] Light intensity: 160 μmol·m⁻²·s⁻¹ (wavelength 450±5 nm blue light) for 0–3 days, increased to 190 μmol·m⁻²·s⁻¹ (same wavelength) for 4–7 days, temperature 27℃, culture for 7 days.

[0094] Accumulation and reinforcement stage: The algal solution was transferred to the accumulation and reinforcement medium (the nutrient proliferation medium was modified by removing sodium nitrate and adding 2 g / L glucose, 1.7 g / L polyethylene glycol 6000 and 0.12 g / L betaine hydrochloride).

[0095] Maintain a light intensity of 210 μmol·m⁻²·s⁻¹ and a temperature of 18℃. Supplement glucose dynamically based on the reddening rate: 0.08 g / L glucose daily when the reddening rate is <50%, and 0.12 g / L daily when the reddening rate is ≥50%. Simultaneously apply weak shaking (100 rpm, 25 mm amplitude) and culture for 10 days.

[0096] Example 2

[0097] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0098] The culture medium preparation and culturing steps are as follows:

[0099] Nutritional proliferation stage:

[0100] Each liter contains 140 mg sodium nitrate, 13 mg potassium dihydrogen phosphate, 55 mg potassium chloride, ferric ammonium citrate (0.08 mg iron), 1.8 g sodium citrate, and 0.8 mL of a trace element mixture (containing 2.5 mg / L boric acid, 0.006 mg / L copper sulfate pentahydrate, 0.08 mg / L zinc sulfate heptahydrate, 0.06 mg / L sodium molybdate dihydrate, and 1.0 mg / L manganese sulfate monohydrate).

[0101] Irradiation was 70 μmol·m⁻²·s⁻¹, temperature was 23℃, and gas containing 2.5% CO₂ was introduced (flow rate 1.2 L / min) for 16 days.

[0102] Stress-induced phase:

[0103] The algal solution was concentrated to 25% of its original volume. The stress-inducing medium contained 35 mg / L sodium salicylate, 0.12 mg / L sodium selenite, and 0.22 mg / L methyl jasmonate.

[0104] 50% methyl jasmonate was added initially, with the remainder added on day 4.

[0105] Light: 140 μmol·m⁻²·s⁻¹ (blue light) for 0–3 days, 170 μmol·m⁻²·s⁻¹ (blue light) for 4–7 days, temperature 25℃, culture for 9 days.

[0106] Accumulation and reinforcement phase:

[0107] The culture medium contained 2.5 g / L glucose, 1.9 g / L polyethylene glycol 6000, and 0.08 g / L betaine hydrochloride.

[0108] Irradiation was 200 μmol·m⁻²·s⁻¹, temperature was 17℃. When the reddening rate was <50%, sugar was supplemented daily at 0.10 g / L, and when the reddening rate was ≥50%, sugar was supplemented at 0.16 g / L. The mixture was cultured with weak shaking (80 rpm, amplitude 35 mm) for 8 days.

[0109] Example 3

[0110] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0111] The culture medium preparation and culturing steps are as follows:

[0112] Nutritional proliferation stage:

[0113] Each liter contains 150 mg sodium nitrate, 11 mg potassium dihydrogen phosphate, 50 mg potassium chloride, ferric ammonium citrate (0.10 mg iron), 2.0 g sodium citrate, and 1.2 mL of a trace element mixture (containing 3.5 mg / L boric acid, 0.004 mg / L copper sulfate pentahydrate, 0.06 mg / L zinc sulfate heptahydrate, 0.08 mg / L sodium molybdate dihydrate, and 0.8 mg / L manganese sulfate monohydrate).

[0114] Irradiation was 75 μmol·m⁻²·s⁻¹, temperature was 24℃, and gas containing 3.0% CO₂ was introduced (flow rate 0.8 L / min) for 15 days.

[0115] Stress-induced phase:

[0116] The algal solution was concentrated to 22% of its original volume. The stress-inducing medium contained 40 mg / L sodium salicylate, 0.08 mg / L sodium selenite, and 0.20 mg / L methyl jasmonate (sodium salicylate to sodium selenite mass ratio 400:1).

[0117] Methyl jasmonate was added initially at 50%, with the remainder added on day 4. Light: 150 μmol·m⁻²·s⁻¹ (blue light) for days 0–3, and 180 μmol·m⁻²·s⁻¹ (blue light) for days 4–7. Temperature: 26℃. Cultured for 8 days.

[0118] Accumulation and reinforcement phase:

[0119] The culture medium contained 3 g / L glucose, 1.8 g / L polyethylene glycol 6000, and 0.10 g / L betaine hydrochloride.

[0120] Irradiation was 205 μmol·m⁻²·s⁻¹, temperature was 17.5℃. When the reddening rate was <50%, sugar was supplemented daily at 0.09 g / L, and when the reddening rate was ≥50%, sugar was supplemented at 0.14 g / L. The mixture was cultured with weak shaking (90 rpm, amplitude 30 mm) for 9 days.

[0121] Comparative Example 1

[0122] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0123] The culture was carried out in nutrient proliferation medium (same as in Example 1) for 14 days, and then directly transferred to nitrogen-free BG-11 medium (other components are the same), and cultured for 10 days under high light of 190 μmol·m⁻²·s⁻¹ and at a temperature of 27℃, without hormone addition, dynamic sugar supplementation or shaking.

[0124] Comparative Example 2

[0125] In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows:

[0126] The nutrient proliferation stage was the same as in Example 2. During the stress stage, only the nitrogen source was removed from the nutrient proliferation medium; sodium salicylate, sodium selenite, and methyl jasmonate were not added. The light and temperature were the same as in Example 2. The accumulation stage was the same as in Example 2.

[0127] Comparative Example 3

[0128] In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows:

[0129] The nutrient proliferation stage is the same as in Example 3. During the stress stage, white light (not blue light) and methyl jasmonate were added at a single dose of 0.20 mg / L (not in stages), otherwise the same as in Example 3. The accumulation stage is the same as in Example 3.

[0130] Comparative Example 4

[0131] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0132] The nutritional proliferation and stress phases were the same as in Example 1. The accumulation phase was changed to a fixed daily glucose supplementation of 0.12 g / L (ignoring the reddening rate), without any shaking.

[0133] Comparative Example 5

[0134] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0135] The nutrient proliferation and stress phases were the same as in Example 2. The accumulation-enhancing medium was prepared by removing the nitrogen source and adding glucose, without polyethylene glycol 6000 and betaine hydrochloride, and other conditions were the same as in Example 2.

[0136] Algal Solution Test Results and Analysis

[0137] The algal solutions of the above-described embodiments and comparative examples were subjected to index measurements at the end of the culture period, and the results are shown in Table 1.

[0138] Table 1 Measurement Results

[0139] Group Biomass dry weight (g / L) Astaxanthin content (mg / g) Total astaxanthin yield (mg / L) Cell death rate (%) Astaxanthin extraction rate (%) after cell wall disruption Example 1 3.21 35.6 114.3 8.1 92.4 Example 2 2.98 38.2 113.8 7.5 93.0 Example 3 3.15 36.8 115.9 7.8 93.7 Comparative Example 1 1.87 24.3 45.4 42.3 76.1 Comparative Example 2 2.05 29.7 60.9 34.8 84.2 Comparative Example 3 2.64 31.5 83.2 21.5 88.9 Comparative Example 4 3.02 32.1 97.0 11.2 90.3 Comparative Example 5 2.91 35.0 101.9 9.8 86.5

[0140] As shown in Table 1, the biomass (2.98–3.21 g / L) and total astaxanthin yield (113.8–115.9 mg / L) of Examples 1-3 were significantly higher than those of all comparative examples. In particular, compared to Comparative Example 1 (traditional two-stage method), its astaxanthin yield was less than 40% of that of the Examples, and its cell death rate was as high as 42.3%. This verifies the core advantage of the segmented culture medium of this application: the optimized formulation of the nutrient proliferation medium supports high biomass accumulation, laying the foundation for subsequent synthesis.

[0141] During the stress induction phase, the astaxanthin pathway was gently activated through a triple synergistic signaling pathway involving sodium salicylate (osmotic), sodium selenite (oxidative), and methyl jasmonate (hormonal). In Comparative Example 2, the yield decreased by 46% when all three were removed. This process avoids large-scale cell death caused by severe nitrogen deprivation. During the accumulation and enhancement phase, dynamic carbon source replenishment was observed. In Comparative Example 4, fixed glucose supplementation reduced the yield by 16%. The osmotic / membrane stabilizer maintained cell viability and metabolic efficiency during the synthesis phase. In Comparative Example 5, omitting this stabilizer resulted in a 7.2% decrease in extraction rate. This demonstrates that the present application synergistically enhances biomass and astaxanthin yield.

[0142] In the examples, the cell mortality rate was less than 8.5%, while in Comparative Example 3, the elimination of the stepwise addition of blue light and hormones resulted in a mortality rate of 21.5% and a 28% decrease in astaxanthin yield. This indicates that a specific wavelength of blue light significantly enhances the transmission efficiency of photo-stress signals, promoting the expression of astaxanthin synthesis genes by activating photoreceptor proteins. The stepwise addition of methyl jasmonate avoids the metabolic inhibition that may be caused by a single high dose, and enhances the signal a second time after the cells have initially adapted to the stress, improving the conversion efficiency. In contrast, when Comparative Example 3 was added in a single dose, the reddening rate was delayed by 2 days to 50%. Weak oscillation promotes the homogeneous distribution of nutrients / gases, reduces local environmental stress, and indirectly supports high astaxanthin content, confirming that the refined process of this application stabilizes the stress response.

[0143] Although this application does not directly involve the cell wall disruption process, the astaxanthin extraction rates of the three examples are significantly higher than those of the five comparative examples. In particular, comparative example 5, which omits polyethylene glycol 6000 and betaine hydrochloride, saw its extraction rate drop to 86.5%, indicating that polyethylene glycol 6000, through gentle osmosis during the accumulation phase, partially relaxes the cell wall structure of thick-walled spores, reducing their mechanical strength. Betaine hydrochloride protects the membrane integrity, reducing leakage or degradation of intracellular astaxanthin before harvest (low cell mortality indirectly reduces cell rupture). The high cell breakage rate under severe stress in comparative example 1 indirectly proves that the spores obtained through the entire three-stage culture process of this application are physiologically more robust, providing high-quality raw materials for subsequent gentle cell wall disruption and reducing the energy consumption requirements of strong physical cell wall disruption.

[0144] In summary, the culture medium design and process control of this invention systematically solve the core problems of biomass-astaxanthin synergistic enhancement and stress response stability through three innovations: stage adaptability optimization, synergistic slow release of stress signals, and dynamic maintenance of accumulation environment, providing an efficient and reliable path for industrial production.

[0145] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method of cultivating Haematococcus pluvialis using a Haematococcus pluvialis cultivation medium, characterized in that, The Haematococcus pluvialis culture medium comprises a nutrition proliferation culture medium used in a segmented culture, a stress induction culture medium, and an accumulation strengthening culture medium; The nutrition proliferation culture medium comprises: Sodium nitrate 140-160 mg / L; Potassium dihydrogen phosphate 11-13 mg / L; Potassium chloride 45-55 mg / L; Iron element concentration provided by ferric ammonium citrate 0.08-0.12 mg / L; Sodium citrate 1.8-2.2 g / L; Trace element mixed solution 0.8-1.2 mL / L; The stress induction culture medium is obtained by adding, in the nutrition proliferation culture medium, an osmotic stress agent 35-45 mg / L, an oxidation inducer 0.08-0.12 mg / L, and a plant hormone 0.18-0.22 mg / L; The accumulation strengthening culture medium is obtained by removing sodium nitrate from the nutrition proliferation culture medium and adding a supplemental carbon source, a polymer osmotic agent, and a zwitterionic stabilizer; The trace element mixed solution comprises: Boric acid 2.5-4.5 mg / L; Copper sulfate pentahydrate 0.004-0.006 mg / L; Zinc sulfate heptahydrate 0.06-0.08 mg / L; Sodium molybdate dihydrate 0.06-0.08 mg / L; Manganese sulfate monohydrate 0.8-1.0 mg / L; The osmotic stress agent is sodium salicylate, the oxidation inducer is sodium selenite, and the plant hormone is methyl jasmonate; The supplemental carbon source is glucose with an initial concentration of 2-3 g / L, the polymer osmotic agent is polyethylene glycol 6000 1.7-1.9 g / L, and the zwitterionic stabilizer is betaine hydrochloride 0.08-0.12 g / L; The method comprises the following steps: (1) Nutrition proliferation stage: Inoculate the algal seed in the nutrient propagation medium, control the light intensity 70-80 μmol·m -2 ·s -1 , temperature 23-25℃, and culture for 14-16 days; (2) Stress induction stage: The algal liquid obtained after proliferation in the previous step is concentrated to 20-25% of the original volume, transferred to the stress induction medium, and 50% of the total amount of methyl jasmonate is added at the time of preparation of the medium before the start of the stress induction phase. The remaining 50% of methyl jasmonate is added on the 4th day of cultivation during the stress induction phase, and the light intensity is increased in stages during the stress induction phase: 0-3 days 140-160 μmol·m -2 ·s -1 , 4-7 days 170-190 μmol·m -2 ·s -1 , temperature 25-27°C, cultivation for 7-9 days; (3) Accumulation strengthening stage: The algal liquid obtained in the previous step was transferred into the accumulation-enhanced medium containing glucose 0.9-1.1 g / L, maintaining the light intensity 200-210 μmol·m -2 ·s -1 -2, the temperature 17-18℃, and the glucose 0.08-0.16 g / L was supplemented every 24 hours, and the culture was carried out for 8-10 days. In the stress induction stage, the stepwise increase in light intensity uses blue light with a wavelength range of 450±5 nm; In the accumulation strengthening stage, the glucose supplement amount is dynamically adjusted according to the red rate of the algal cells: when the red rate is <50%, 0.08-0.10 g / L of glucose is supplemented every 24 hours; when the red rate is ≥50%, 0.12-0.16 g / L of glucose is supplemented every 24 hours; and in the accumulation strengthening stage, weak oscillation is applied simultaneously, with a shaker speed of 80-100 rpm and an oscillation amplitude of 25-35 mm.

2. The method of claim 1, wherein, In the nutrition proliferation stage, 2.5-3.5% CO2-containing gas is introduced at a flow rate of 0.8-1.2 L / min.

3. The method of claim 1, wherein, The mass ratio of sodium salicylate to sodium selenite is 350-450:1.