Method for increasing yield of astaxanthin in haematococcus pluvialis

Abstract

The invention relates to the technical field of astaxanthin preparation, and particularly discloses a method for increasing the yield of astaxanthin in haematococcus pluvialis, which comprises the following steps: culturing the haematococcus pluvialis to a growth plateau stage, inoculating the haematococcus pluvialis to a special improved BG-11 culture medium, and carrying out high-light high-salt induction in a columnar photobioreactor; the culture medium is designed for an induction stage and comprises limited sodium nitrate, specific content of salicylic acid, alpha-ketoglutaric acid, sodium chloride and magnesium chloride, and melatonin is not added; in the induction period, step-type increasing illumination intensity is adopted, and white light and ultraviolet light composite light quality treatment is matched. According to the method, the stress tolerance and the astaxanthin synthesis efficiency of cells are remarkably enhanced through the synergistic effect of culture medium components and a light stress procedure, the dry weight of haematococcus pluvialis is 1.69 g L <-1 >, the astaxanthin concentration of haematococcus pluvialis liquid is 105.6 mg L <-1 >, the astaxanthin content of haematococcus pluvialis powder is 6.25% (w / w) within 10 days, the final astaxanthin yield reaches 11.70 mg L <-1 > d <-1 >, and the synchronous great improvement of the astaxanthin yield and the production efficiency is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of astaxanthin production, and particularly relates to a method for improving astaxanthin yield of haematococcus pluvialis. BACKGROUND

[0002] Astaxanthin is a ketocarotenoid with super-antioxidant activity, which has a high application value in the fields of nutrition and health care, cosmetics, aquaculture and medicine. Haematococcus pluvialis is recognized as the strongest biological source of astaxanthin accumulation in nature. Its commercial production usually adopts a two-stage culture mode: in the first stage (green growth stage), the algal cells are promoted to proliferate rapidly under suitable conditions to accumulate biomass; in the second stage (red induction stage), various environmental stresses such as strong light, nutrient deficiency (especially nitrogen), high salt and high osmotic pressure are applied to activate the intracellular astaxanthin synthesis pathway, so that astaxanthin is accumulated in large quantities.

[0003] Although the two-stage strategy is clear in theory, in actual industrial production, the production efficiency of astaxanthin (measured by the mass of astaxanthin produced per unit time and per unit volume) still faces severe challenges, mainly manifested in long induction period (often more than 14 days), limited final astaxanthin content and concentration, and high cell mortality under high-intensity stress leading to biomass loss. The root cause of these problems lies in the fact that the existing technical solutions fail to achieve high coordination and functional matching between the induction medium and the induction stress program.

[0004] At present, the optimization research of the culture medium for Haematococcus pluvialis mainly focuses on promoting the biomass accumulation in the green growth stage. For example, some improved schemes add exogenous antioxidants (such as melatonin), growth regulators or optimize the concentration of macroelements, aiming to protect cells and accelerate division, so as to obtain higher cell density in a shorter time. The core of the design logic of such culture medium is to create and maintain the best growth conditions.

[0005] However, when the cells are transferred from the growth stage to the induction stage, the culture target has undergone a fundamental change: from rapid proliferation to stress tolerance and efficient synthesis of secondary metabolites. If the optimized culture medium serving the growth stage is directly used in the induction stage, a series of internal contradictions will arise: Nutrient signal conflict: high concentrations of nitrogen and other nutrients will continuously transmit signals to the cells that they are in a suitable growth environment, which is antagonistic to the stress signals (such as nitrogen deficiency and high salt) applied in the induction stage, leading to confusion in cell metabolism direction and failure to fully switch to astaxanthin synthesis; Balance disorder between protection and signal: the strong exogenous antioxidants (such as melatonin) added in the growth stage may excessively scavenge reactive oxygen species, which are important stress signal molecules, in the induction stage, thereby weakening the stress intensity and weakening the activation of the astaxanthin synthesis pathway; Lack of support for specific combined stress: conventional or growth-optimized media often do not take into account the extreme environment specific to the induction phase (e.g. the ultra-high light intensity combined with high salt employed in the present invention). For example, high sodium environment can competitively inhibit the uptake of key elements such as magnesium ions, which are crucial for maintaining photosynthetic and metabolic activities of cells under stress.

[0006] Therefore, the prior art lacks a medium specifically designed for the physiological and biochemical needs of the astaxanthin induction phase of Haematococcus pluvialis, which can seamlessly connect and synergize with the high-intensity, combined stress induction procedure, thereby maximizing the synthesis efficiency and final yield of astaxanthin on the basis of ensuring cell survival. The prior art still needs to be improved and developed. SUMMARY

[0007] The primary purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for significantly and efficiently improving the astaxanthin yield of Haematococcus pluvialis.

[0008] The technical solution of the present invention is as follows: A method for improving the astaxanthin yield of Haematococcus pluvialis, comprising the following steps: culturing Haematococcus pluvialis to the growth plateau phase to obtain Haematococcus pluvialis seed liquid; inoculating the Haematococcus pluvialis seed liquid into a columnar photobioreactor containing a special type of improved BG-11 medium for high light and high salt induction; wherein the special type of improved BG-11 medium contains, per liter: sodium nitrate 100-120 mg, magnesium sulfate heptahydrate 100-120 mg, potassium dihydrogen phosphate 30-45 mg, disodium ethylenediaminetetraacetate 15-20 mg, ferric citrate 12.5-14 mg, boric acid 6-8 mg, salicylic acid 4-6 mg, alpha-ketoglutaric acid 3-5 mg, manganese chloride tetrahydrate 1-3 mg, sodium bicarbonate 2.2-3.5 mg, cobalt nitrate hexahydrate 0.05-0.1 mg, sodium molybdate dihydrate 0.3-0.5 mg, copper sulfate pentahydrate 0.05-0.1 mg, zinc sulfate heptahydrate 0.3-0.5 mg, sodium chloride 2000-3000 mg, and magnesium chloride 300-400 mg; during the high light and high salt induction period, 300-320 μmol·m -2 ·s -1 of light treatment on the first day, 380-400 μmol·m -2 ·s -1 of light treatment on the second day, 600-650 μmol·m -2 ·s -1 of light treatment on the third day, 700-750 μmol·m -2 ·s -1 of light treatment on the fourth day, and 800-850 μmol·m-2 ·s -1 Light treatment, using 1000-1100 μmol·m⁻¹ on days 6-10. -2 ·s -1 Lighting processing.

[0009] The method for increasing astaxanthin production from Haematococcus pluvialis includes a special modified BG-11 culture medium containing 110 mg / L sodium nitrate, 5 mg / L salicylic acid, 4 mg / L α-ketoglutarate, 2500 mg / L sodium chloride, and 350 mg / L magnesium chloride.

[0010] The method for increasing astaxanthin production in Haematococcus pluvialis includes Haematococcus pluvialis strain H7, with accession number CGMCCNo.41186.

[0011] The method for increasing astaxanthin production in Haematococcus pluvialis includes the following steps: on day 1, white light with a wavelength of 440-460 nm is used for illumination treatment; from day 2 to day 10, composite light composed of white light with a wavelength of 440-460 nm and ultraviolet light with a wavelength of 360-380 nm is used for illumination treatment according to the ratio of white light LED beads to ultraviolet light LED beads.

[0012] The method for increasing astaxanthin production from Haematococcus pluvialis includes a white light to ultraviolet light lamp ratio of 20:1-5.

[0013] The method for increasing astaxanthin production in Haematococcus pluvialis includes an inoculation density of 1.5 × 10⁻⁶ for the Haematococcus pluvialis seed solution. 5 Up to 2.0×10 5 cells / mL.

[0014] The method for increasing the astaxanthin yield of Haematococcus pluvialis includes the following process: the Haematococcus pluvialis is cultured to a growth plateau in a culture bottle under the following conditions: ambient temperature 24-26℃, and a sterile mixed gas containing 500-800ppm carbon dioxide is continuously introduced into the bottom of the culture bottle at an aeration rate of 1-1.5vvm.

[0015] The method for increasing astaxanthin production from Haematococcus pluvialis includes a high-light, high-salt induction process conducted in a columnar photobioreactor. The induction conditions are: an ambient temperature of 25-30°C, and a sterile mixed gas containing 100-200 ppm carbon dioxide continuously introduced into the bottom of the reactor at a flow rate of 1-1.5 vvm.

[0016] Beneficial Effects: The method for increasing astaxanthin production in Haematococcus pluvialis provided by this invention has significant technical effects, mainly reflected in the following aspects: First, this method achieves a substantial increase in astaxanthin synthesis efficiency through the synergistic effect of a specially designed induction culture medium and a customized light stress program. The appropriate nitrogen restriction in the special culture medium guides cell metabolism towards astaxanthin synthesis, while the removal of potent antioxidants preserves key reactive oxygen species signaling pathways. Combined with stepwise increasing light intensity culminating in ultra-high intensity illumination, and the subsequent introduction of specific ultraviolet light components, a strong and continuous stress signaling network is formed, deeply activating the biological pathway of astaxanthin synthesis, thereby significantly increasing astaxanthin accumulation. Second, while efficiently driving astaxanthin synthesis, this scheme effectively maintains cell viability and biomass. The key ions supplemented in the culture medium for high-salt environments effectively support the core physiological functions of cells under extreme conditions. The stepwise increasing light strategy provides cells with valuable adaptation time, allowing their endogenous protective mechanisms to be gradually established and strengthened. This design avoids mass cell death caused by acute stress, ensuring the stability and sustainability of the production process and laying a solid biomass foundation for high yield. Finally, this invention achieves deep synergy and system optimization among technical elements. Every adjustment to the culture medium component is closely aligned with the design intent of the light stress program. For example, the necessity of specific components added to address high salt content is fully validated under ultra-high light intensity stress; and the positive effects of antioxidants removed to preserve signaling pathways depend on specific signaling molecules generated by subsequent light stress. This non-simple additive, organically integrated technical system simultaneously achieves a comprehensive improvement in astaxanthin yield, content, and production efficiency within a relatively short induction period, solving long-standing technical challenges in the induction phase such as long cycles, low efficiency, and easy cell death, demonstrating outstanding comprehensive advantages. Attached Figure Description

[0017] Figure 1 This is a flowchart of a method for increasing the astaxanthin yield of Haematococcus pluvialis according to the present invention. Detailed Implementation

[0018] This invention provides a method for increasing astaxanthin production in Haematococcus pluvialis. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] Please see Figure 1 , Figure 1 The present invention provides a method for increasing the astaxanthin yield of Haematococcus pluvialis, as shown in the figure, which includes the following steps: S10. Cultivate Haematococcus pluvialis to the growth plateau stage to obtain Haematococcus pluvialis seed culture; S20. The Haematococcus pluvialis seed culture is inoculated into a columnar photobioreactor containing a specially modified BG-11 medium for high-light and high-salt induction; wherein, each liter of the specially modified BG-11 medium contains: sodium nitrate 100-120 mg, magnesium sulfate heptahydrate 100-120 mg, potassium dihydrogen phosphate 30-45 mg, disodium ethylenediaminetetraacetate 15-20 mg, ferric citrate 12.5-14 mg, boric acid 6-8 mg, and salicylic acid 4-6 mg. g, α-ketoglutarate 3-5 mg, manganese chloride tetrahydrate 1-3 mg, sodium bicarbonate 2.2-3.5 mg, cobalt nitrate hexahydrate 0.05-0.1 mg, sodium molybdate dihydrate 0.3-0.5 mg, copper sulfate pentahydrate 0.05-0.1 mg, zinc sulfate heptahydrate 0.3-0.5 mg, sodium chloride 2000-3000 mg, and magnesium chloride 300-400 mg; during the high light and high salt induction period, on the first day, 300-320 μmol·m -2 ·s -1 Light treatment, day 2 using 380-400 μmol·m -2 ·s -1 Light treatment, on the 3rd day, using 600-650 μmol·m -2 ·s -1 Light treatment, with 700-750 μmol·m⁻² on day 4. -2 ·s -1 Light treatment, with 800-850 μmol·m⁻² on day 5. -2 ·s -1 Light treatment, using 1000-1100 μmol·m⁻¹ on days 6-10. -2 ·s -1 Lighting processing.

[0020] This invention provides a specialized improved BG-11 culture medium with components and functions that differ from existing growth media. It is deeply coupled with a step-by-step ultra-high light intensity combined with high salt induction program, forming a new, efficient and synergistic technical system for the induced production of astaxanthin from Haematococcus pluvialis. This effectively solves the technical problems of low astaxanthin yield and long cycle during the induction stage.

[0021] Specifically, compared to the BG-11 culture medium disclosed by the applicant in the published patent CN 121294149 A for promoting the biomass accumulation of Haematococcus pluvialis, the modified BG-11 culture medium of this invention explicitly reduces the amount of sodium nitrate. Nitrogen is an essential element for the synthesis of biomolecules such as proteins and nucleic acids. During the growth stage, a sufficient nitrogen source (such as approximately 1500 mg / L of sodium nitrate in conventional BG-11, and even higher in some optimized growth formulations) is essential for rapid cell proliferation; however, during the astaxanthin induction stage, excessive nitrogen can become a disruptive signal.

[0022] This invention significantly reduces the sodium nitrate concentration to 100-120 mg / L, a concentration far below the requirements for normal growth, but not completely depleted. This moderate nitrogen-limited state produces multiple effects: First, metabolic flux redirection: The relative scarcity of nitrogen means that the carbon fixed by photosynthesis cannot be fully used to synthesize nitrogen-containing compounds (such as proteins), forcing metabolic flux to shift to nitrogen-free synthetic pathways; carbon skeletons such as acetyl-CoA are more directed to the biosynthesis of carotenoids (including astaxanthin), increasing the supply of synthetic precursors from the source; Second, cell cycle regulation: Nitrogen is crucial for cell cycle progression. First, nitrogen restriction signals early environmental resource constraints to cells, helping to arrest the cell cycle in the G1 phase or induce a dormant state. This releases energy and resources from energy-consuming DNA replication and cell division, allowing them to be used for stress adaptation and secondary metabolism. Second, nitrogen restriction itself is a form of stress, and it may have a cross-reinforcing effect with subsequent high salt and high light stresses in the signaling pathway. This can jointly upregulate transcription factors (such as bZIP and the MYB family) related to stress response and secondary metabolism, thereby more strongly activating the astaxanthin synthesis pathway.

[0023] Melatonin is a potent broad-spectrum antioxidant commonly used in plants and microalgae to alleviate oxidative stress and promote growth. The key reverse design of this invention is the complete removal of melatonin, a common component of growth media, from the induction medium: during the stress induction phase, reactive oxygen species (ROS) such as hydrogen peroxide play a dual role, acting as both a damaging factor and an important signaling molecule. A certain level of ROS can act as an alarm signal, activating antioxidant response pathways such as the MAPK kinase cascade and Nrf2 / Keap1, as well as directly regulating the expression of genes related to astaxanthin synthesis. Premature and excessive presence of the exogenous potent antioxidant melatonin can non-selectively and efficiently quench these ROS, potentially leading to insufficient stress signal strength and inadequate activation of the astaxanthin synthesis pathway. This invention does not subject cells to oxidative stress in an unprotected state. The salicylic acid (SA) retained in the culture medium plays a more subtle role. SA primarily acts as a signaling molecule, activating the cell's own regulated antioxidant enzyme system (such as SOD, CAT, and APX). This endogenous system can dynamically adjust in response to ROS levels, allowing signaling ROS to exist and function within a certain threshold while clearing excess toxic ROS. This is a more refined and adaptable antioxidant strategy to dynamic stress processes. This invention removes exogenously added melatonin, reducing the additional metabolic steps that cells may need, allowing cells to focus more on core stress responses and astaxanthin synthesis during resource-intensive induction phases.

[0024] The induced environment of this invention contains a high concentration of sodium chloride (2000-3000 mg / L), creating continuous osmotic and ion stress. Under this premise, the additional addition of magnesium chloride (300-400 mg / L) has important physiological support significance: firstly, it counteracts ion competition and absorption inhibition; the high concentration of sodium chloride... + Possibly related to Mg 2+ Competitive inhibition occurs at cell membrane absorption sites; simultaneously, the hyperosmolar environment may affect the activity of ion transport proteins, which may lead to intracellular Mg... 2+ Insufficient concentration. Secondly, to ensure core metabolic functions, Mg... 2+ It is a key ion in living systems; it is the central atom of the chlorophyll molecule and is directly related to light energy capture in photosynthesis; it is an activator of ribulose-1,5-bisphosphate carboxylase / oxygenase, affecting carbon fixation efficiency; moreover, it is an essential cofactor for hundreds of ATP-dependent enzymes, involved in energy metabolism, nucleic acid synthesis, and certain steps in the astaxanthin synthesis pathway. Maintaining efficient photosynthesis and energy metabolism under ultra-high light stress is fundamental for cell survival and synthetic capacity; thirdly, it plays a synergistic role in osmotic regulation and structural stability: Cl - The introduction of Mg also helps cells maintain ion balance and osmotic pressure, with sufficient Mg 2+It also helps stabilize cellular structures such as ribosomes and membrane systems, enhancing overall stress resistance. Therefore, adding magnesium chloride is a direct reinforcement of the weaknesses in the specific environment induced by high salt, ensuring that key physiological functions of cells do not fail under stress.

[0025] This invention assigns new roles to salicylic acid (4-6 mg / L) and α-ketoglutarate (3-5 mg / L) in the induction system. In the absence of melatonin, salicylic acid's function as a systemic resistance signal initiator is more prominent. It does not rely primarily on direct antioxidant activity, but rather activates the cell's global stress response network, putting the cell in a pre-adaptive state when faced with tiered light stress. Specifically, before induction begins, the salicylic acid (4-6 mg / L) pre-present in the culture medium can be absorbed by the cells and act as a signaling molecule, initiating a systemic acquired resistance (SAR)-like response in advance. It upregulates the expression of a series of antioxidant enzyme genes, including superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, by activating specific transcription factors (such as the NPR1-dependent pathway). This ensures that the cell's endogenous antioxidant enzyme system is already in a pre-activated or highly ready state when faced with progressively increasing light stress, enabling it to clear continuously generated ROS more rapidly and effectively. Under moderate nitrogen-limited conditions, α-ketoglutarate plays a more crucial role as a hub in the tricarboxylic acid cycle and a precursor to glutamate. It provides the carbon skeleton for limited nitrogen assimilation and a precursor for energy metabolism and glutathione (GSH) synthesis, playing a central role in coping with dynamically increasing oxidative stress and maintaining carbon-nitrogen metabolic balance. Specifically, astaxanthin is a carotenoid whose biosynthesis requires a large amount of carbon skeleton (from acetyl-CoA) and reducing power (NADPH). Simultaneously, under high-intensity stress, cellular processes such as maintaining homeostasis, repairing damage, and synthesizing antioxidants all consume large amounts of ATP. Traditional induction media are often carried out under nitrogen-deficient or low-nitrogen conditions, which may lead to impaired carbon metabolic flux or insufficient energy supply. The exogenously added α-ketoglutarate of this invention plays multiple key roles: as a core intermediate in the tricarboxylic acid cycle, α-KG can directly enter the mitochondrial TCA cycle, promoting its operation and efficiently producing NADH and FADH2. This, in turn, generates a large amount of ATP through oxidative phosphorylation, providing ample energy for stress response and astaxanthin synthesis. α-KG can rapidly generate glutamate through transamination. Glutamate is not only a raw material for protein synthesis but also a precursor for glutathione (GSH) synthesis, one of the most important non-enzymatic antioxidants in cells. Therefore, α-KG indirectly ensures the continuous synthesis of GSH, perfectly complementing the salicylic acid-activated enzymatic antioxidant system. During the induction phase (often accompanied by nitrogen limitation), α-KG can act as an amino acid acceptor, participating in nitrogen assimilation and redistribution, preventing feedback inhibition caused by excessive accumulation of carbon metabolites, and ensuring that carbon flow is smoothly directed to the carotenoid (including astaxanthin) synthesis pathway.

[0026] The core of the lighting scheme in this invention lies in its stepped and ultra-high intensity. The physiological significance of the stepped increase is as follows: First, avoid acute photoshock: if 1000 μmol·m⁻¹ is applied directly from day 1... -2 ·s -1 At light intensities of 300-320 μmol·m⁻¹, the vast majority of cells will die due to the inability to withstand the sudden and enormous photo-oxidative stress. This invention provides light intensity from 300-320 μmol·m⁻¹. -2 ·s -1 Initiation is the intensity at which most cells can tolerate and begin to initiate an adaptive response.

[0027] Secondly, induce cellular adaptive acclimatization: daily at approximately 100-200 μmol·m -2 ·s -1The intensity of light increases gradually, giving cells enough time (about 24 hours) to adjust their physiological state. When cells cope with the light intensity of the previous day, they upregulate photoprotective mechanisms (such as increasing lutein cycling, synthesizing more antioxidants, and repairing the photosystem). When the next level of light intensity arrives, the cells are already in a state of stronger defense, thus being able to withstand and utilize higher light energy.

[0028] Third, maximizing the balance between stress response and growth: This gradual strategy keeps cell death at a low level (maintaining a high biomass base), while stress signals are continuously enhanced, guiding more and more cellular resources from growth to the synthesis and accumulation of astaxanthin.

[0029] The induced light intensity of existing technologies is generally between 100 and 250 μmol·m. -2 ·s -1 Order of magnitude. This invention increases the light intensity at the end of the induction phase to 1000-1100 μmol·m. -2 ·s -1 (4-10 times) This is a qualitative leap, not a blind increase, but an inevitable choice based on the customized culture medium support and stepwise adaptation mentioned above. Ultra-high light intensity means a huge increase in the number of photons received per unit cell per unit time, which will produce two key effects: generating extremely strong stress signals: greatly stimulating the cell's adversity survival instinct and maximally activating all rate-limiting enzymes in the astaxanthin synthesis pathway (such as phytoene synthase, β-carotene ketolase, etc.); providing potential extra energy: although most of the excess light energy will be dissipated in the form of heat or fluorescence and generate ROS, under the premise that the cell's antioxidant system and photoprotection mechanism are fully activated and supported (by the culture medium of this invention), more light energy can still be used to drive photosynthesis, providing more reducing power (NADPH) and ATP for astaxanthin synthesis, which is the energy basis for high output.

[0030] The highest level of inventiveness of this invention lies in the precise and functional synergy between the specialized improved BG-11 culture medium and the stepwise ultra-high light intensity induction program. Firstly, the culture medium creates the feasibility and lays the foundation for the induction program: without the pre-adjustment of the specialized culture medium (moderate nitrogen restriction to weaken proliferation impulses, removal of melatonin to preserve the signaling pathway, and addition of magnesium chloride to enhance salt tolerance), cells simply cannot tolerate the rapid increase in temperature from day 3 onwards, ultimately reaching 1000-1100 μmol·m⁻¹. -2 ·s -1The extreme light stress will cause a large-scale collapse during the mid-induction period; secondly, the induction procedure fully stimulates and verifies the design potential of the specialized improved BG-11 medium: the stepwise increasing ultra-high light intensity is a dynamic and continuously increasing stress source, and every design element of the specialized improved BG-11 medium is designed to respond to this dynamic and extreme stress. For example, without the final ultra-high light intensity, the design advantage of removing melatonin (preserving ROS signal) cannot be realized; without a high salt background, the necessity of adding magnesium chloride is greatly reduced; it is the extreme induction conditions that verify and fully release the full design value of this specialized medium; thirdly, the deep synergy ultimately translates into significantly superior technical effects: within a relatively short 10-day induction period, the concentration, content, and production efficiency of astaxanthin were simultaneously and significantly improved, which proves that the present invention is an organic whole, and its effect is far beyond what can be achieved by simply superimposing existing mediums with increased light intensity.

[0031] In some implementations, on the first day, white light with a wavelength of 440-460nm is used for illumination treatment; from the second to the tenth day, composite light composed of white light with a wavelength of 440-460nm and ultraviolet light with a wavelength of 360-380nm is used for illumination treatment according to the ratio of the number of white light chips to the number of ultraviolet light chips.

[0032] In this embodiment, the seed culture was transferred from the vegetative growth stage of green algae to an induction medium containing high salt and low nitrogen, and immediately faced the challenge of light intensity decreasing from the growth light intensity (approximately 25 μmol·m⁻¹). -2 ·s -1 The value jumped to 310 μmol·m -2 ·s -1 The challenge is that the cells are in a dual adaptation period: adapting to the new chemical environment (culture medium) and the new physical environment (high light intensity). Therefore, using pure white light (with a higher proportion of blue light in the 440-460nm range) on the first day can provide sufficient energy: the white light spectrum is relatively broad and can be effectively absorbed by chlorophyll a, b and carotenoids, providing energy (ATP) and reducing power (NADPH) for the cells to carry out basal metabolism and activate stress response genes in the early stage of adaptation; it can also avoid superimposed stress: if ultraviolet light (UV) is introduced on the first day of drastic environmental change, the triple pressure of light intensity stress, chemical stress and UV oxidative stress will be imposed on the cells at the same time, which can easily lead to large-scale photo-oxidative damage and cell death. The first day of pure white light is equivalent to letting the cells adapt to the high light and chemical environment first.

[0033] In this embodiment, the introduction of ultraviolet light (360-380nm UV-A) from day 2 to day 10 has a stress-enhancing and signal-synergistic effect. That is, after the cells have initially adapted, the introduction of UV-A as a powerful and specific co-stress factor, synergistically with visible light stress, deeply activates the astaxanthin synthesis pathway and may induce unique protective metabolism. Specifically, although UV-A does not directly damage DNA (mainly UV-B and UV-C), it can be absorbed by specific intracellular pigments (such as riboflavin and pterin), generating a large amount of reactive oxygen species (ROS), making it a very strong source of oxidative stress. The ROS induced by UV-A, superimposed with the ROS generated by ultra-high intensity white light, forms a more powerful oxidative stress signal storm. This strong signal can more effectively activate key transcription factors regulating astaxanthin synthesis (such as bZIP and MYB) and upregulate rate-limiting enzymes in the astaxanthin synthesis pathway (such as PSY, BKT, etc.). Gene expression of CRTZ (e.g.) pushes the metabolic flow to astaxanthin synthesis to its extreme. The special culture medium of this invention removes the potent antioxidant melatonin in order to retain effective ROS signals. At this time, the introduction of UV-A precisely provides a high-intensity, high-quality ROS signal source under the condition that the cellular antioxidant system (activated by salicylic acid) has been established but not excessively inhibited, ensuring the strength and persistence of the stress signal. Without UV-A, white light alone may not be able to generate a sufficiently strong specific oxidation signal in the later stages to maintain the highest activity of the synthetic pathway.

[0034] The dynamic light quality and stepwise increasing light intensity provided in this embodiment are precisely coordinated in time to construct a dynamic, multi-dimensional stress environment, that is, the light intensity gradually increases from 310 on day 1 to 1000-1100 μmol·m on day 10. -2 ·s -1 The first stress represents a linear increase in quantity; the second stress, introduced with UV light starting on day 2, represents a qualitative upgrade and diversification of the stress. This gradual increase in light intensity and the design of light quality, evolving from single to complex, simulates a constantly changing and increasingly severe natural adversity. Cells are forced to continuously adjust their defense and adaptation strategies, thereby constantly exploring the potential of synthesizing astaxanthin, the ultimate photoprotective mechanism. This dynamic and multidimensional stress is more effective at stimulating the metabolic limits of cells than a single, constant stress.

[0035] In some embodiments, the ratio of the number of white light to ultraviolet light LEDs is 20:1-5.

[0036] In this embodiment, as an example, the number of white light LED beads is kept constant at 20, while the number of ultraviolet light LED beads varies between 1 and 5. This is a key operating parameter that has been optimized and verified. The design of this ratio range is not a random or empirical choice, but is based on profound physiological regulation principles and forms a precise synergy with the overall technical solution of this invention (dedicated culture medium, stepped light intensity). Its main advantage is that it can achieve a dose-controllable effect of UV stress, that is, control the UV-A dose (expressed as relative photon flux density) within a precise window that is both effective and non-lethal.

[0037] UV-A is a strong source of oxidative stress. If the ratio is too low (e.g., below 20:1), the UV signal is too weak to provide sufficient additional ROS signal to deeply activate the astaxanthin synthesis pathway; its effect may be close to that of pure white light, failing to exert a synergistic advantage. If the ratio is too high (e.g., above 20:5), the ROS generated by UV will exceed the cells' clearance and tolerance capacity under the support of the dedicated culture medium, leading to a sharp increase in oxidative damage, increased cell death, and ultimately reduced total yield. The range of 20:1 to 20:5 represents the optimal effective range between the two extremes of insufficient signal and excessive damage.

[0038] In some embodiments, the special modified BG-11 culture medium contains 110 mg / L sodium nitrate, 5 mg / L salicylic acid, 4 mg / L α-ketoglutarate, 2500 mg / L sodium chloride, and 350 mg / L magnesium chloride, but is not limited thereto.

[0039] In some embodiments, the Haematococcus pluvialis strain is Haematococcus pluvialis H7, with accession number CGMCCNo.41186.

[0040] In some embodiments, the inoculation density of the Haematococcus pluvialis seed solution is 1.5 × 10⁻⁶. 5 Up to 2.0×10 5 cells / mL, but not limited to this.

[0041] In some embodiments, the process of culturing Haematococcus pluvialis to the growth plateau phase is carried out in a culture flask under the following conditions: ambient temperature 24-26°C, and a sterile mixed gas containing 500-800 ppm carbon dioxide continuously introduced into the bottom of the culture flask at an aeration rate of 1-1.5 vvm; the high-light and high-salt induction process is carried out in a columnar photobioreactor under the following conditions: ambient temperature 25-30°C, and a sterile mixed gas containing 100-200 ppm carbon dioxide continuously introduced into the bottom of the reactor at an aeration rate of 1-1.5 vvm.

[0042] The present invention will be further explained and illustrated below through specific embodiments: Example 1 A method for increasing astaxanthin production in Haematococcus pluvialis includes the following steps: Seed culture: 5L conical glass flasks were used, equipped with a sterile ventilation system (introducing a mixture of air containing CO2) and an adjustable-intensity LED white light flat panel light source; the temperature was maintained at 25±1℃; continuous white light irradiation was used, with a light intensity of 25±5 μmol·m⁻¹. -2 ·s -1 Sterile air containing 700 ppm CO2 was continuously introduced at a flow rate of 1.2 vvm (volume ratio / min); standard BG-11 medium was used; cell density was monitored daily, and the Haematococcus pluvialis seed culture was used for induction when cell growth reached the plateau phase (usually requiring 5-7 days); the standard BG-11 medium composition (g / L) was: NaNO3, 1.5; K2HPO4·3H2O, 5.24 × 10⁻²; MgSO4·7H2O, 7.5 × 10⁻². -2 CaCl2, 2.72 × 10 -2 Citric acid, 6.562 × 10 -3 Ferric ammonium citrate, 6.0 × 10 -3 ;EDTANa2·2H2O, 1.1×10 -3 Na₂₂CO₃, 2.0 × 10⁻⁶ -2 H3BO3, 2.86 × 10 -3 MnCl2·4H2O, 1.86×10 -3 Na₂MoO₄·2H₂O, 3.9 × 10⁻⁶ -4 ZnSO4·7H2O, 2.2×10 -4 CuSO4·5H2O, 8.0×10 -5 Co(NO3)2·6H2O, 5.0×10 -5 The bacterial strain used was Haematococcus pluvialis strain H7, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41186. Induction and culture: A 3L vertical columnar glass photobioreactor (10 cm inner diameter, 50 cm height) was used. The outer wall of the reactor was surrounded by independently controllable white LED light strips (450 nm main wavelength) and UV-A LED light strips (370 nm main wavelength). The light intensity was calibrated by adjusting the LED drive current and the distance between the reactor and the light source. The temperature was controlled at 28 ± 1℃, and sterile air containing 150 ppm CO2 was continuously introduced into the bottom of the reactor at a flow rate of 1.2 vvm. Haematococcus pluvialis seed culture was inoculated into the columnar photobioreactor containing a special modified BG-11 medium for high-light and high-salt induction. The inoculation density was 1.8 × 10⁻⁶. 5 Cells / mL, each liter of the specially modified BG-11 medium contains: 110 mg sodium nitrate, 110 mg magnesium sulfate heptahydrate, and 350 mg magnesium chloride hexahydrate (equivalent to providing approximately 105 mg Mg). 2+ (Supplementing magnesium source), potassium dihydrogen phosphate 38mg, disodium ethylenediaminetetraacetate 18mg, ferric citrate 13mg, boric acid 7mg, salicylic acid 5mg, α-ketoglutarate 4mg, manganese chloride tetrahydrate 2mg, sodium bicarbonate 3mg, cobalt nitrate hexahydrate 0.08mg, sodium molybdate dihydrate 0.4mg, copper sulfate pentahydrate 0.08mg, zinc sulfate heptahydrate 0.4mg, sodium chloride 2500 mg; During high light and high salt induction: Day 1: 310 μmol·m -2 ·s -1 Day 2: 390 μmol·m -2 ·s -1 Day 3: 625 μmol·m -2 ·s -1 Day 4: 725 μmol·m -2 ·s -1 Day 5: 825 μmol·m -2 ·s -1 Days 6 to 10: 1050 μmol·m -2 ·s -1 On day 1, pure white light was used for illumination. From day 2 to day 10, the light was switched to a combination of white light and ultraviolet light, with the ratio of white light to ultraviolet light LED beads being 20:3.

[0043] Example 2 A method for increasing the astaxanthin yield of Haematococcus pluvialis is the same as that in Example 1, except that the ratio of white light to ultraviolet light LED beads is 20:1.

[0044] Example 3 A method for increasing the astaxanthin yield of Haematococcus pluvialis is the same as that in Example 1, except that the ratio of white light to ultraviolet light LED beads is 20:5. All other steps and formulations are the same as in Example 1.

[0045] Example 4 A method for increasing astaxanthin production from Haematococcus pluvialis, the only difference between this method and Example 1 is that the inoculation density is 1.5 × 10⁻⁶. 5 The modified BG-11 culture medium formula was adjusted to contain the following per liter: sodium nitrate 100 mg, magnesium sulfate heptahydrate 100 mg, magnesium chloride hexahydrate 300 mg, potassium dihydrogen phosphate 30 mg, disodium ethylenediaminetetraacetate 15 mg, ferric citrate 12.5 mg, boric acid 6 mg, salicylic acid 4 mg, α-ketoglutarate 3 mg, manganese chloride tetrahydrate 1 mg, sodium bicarbonate 2.2 mg, cobalt nitrate hexahydrate 0.05 mg, sodium molybdate dihydrate 0.3 mg, copper sulfate pentahydrate 0.05 mg, zinc sulfate heptahydrate 0.3 mg, and sodium chloride 2000 mg. During the high light and high salt induction period, 300 μmol·mL⁻¹ was used on day 1. -2 ·s -1 Light treatment, 380 μmol·m -2 ·s -1 Light treatment, 600 μmol·m -2 ·s -1 Light treatment, 700 μmol·m -2 ·s -1 Light treatment, 800 μmol·m -2 ·s -1 Irradiation treatment, 1000 μmol·m⁻¹ for days 6-10 -2 ·s -1 The light treatment and the remaining steps are the same as in Example 1.

[0046] Example 5 A method for increasing astaxanthin production from Haematococcus pluvialis, the only difference between this method and Example 1 is that the inoculation density is 2 × 10⁻⁶. 5The modified BG-11 culture medium formula was adjusted to contain the following per liter: sodium nitrate 120 mg, magnesium sulfate heptahydrate 120 mg, magnesium chloride hexahydrate 400 mg, potassium dihydrogen phosphate 45 mg, disodium ethylenediaminetetraacetate 20 mg, ferric citrate 14 mg, boric acid 8 mg, salicylic acid 6 mg, α-ketoglutarate 5 mg, manganese chloride tetrahydrate 3 mg, sodium bicarbonate 3.5 mg, cobalt nitrate hexahydrate 0.1 mg, sodium molybdate dihydrate 0.5 mg, copper sulfate pentahydrate 0.1 mg, zinc sulfate heptahydrate 0.5 mg, and sodium chloride 3000 mg. During the high light and high salt induction period, a 320 μmol·mL⁻¹ medium was used on day 1. -2 ·s -1 Light treatment, 400 μmol·m -2 ·s -1 Light treatment, 650 μmol·m⁻² on day 3 -2 ·s -1 Light treatment, 750 μmol·m⁻² on day 4 -2 ·s -1 Light treatment, 850 μmol·m -2 ·s -1 Irradiation treatment, 1100 μmol·m⁻¹ on days 6-10. -2 ·s -1 The light treatment and the remaining steps are the same as in Example 1.

[0047] Comparative Example 1 A method for increasing the astaxanthin yield of Haematococcus pluvialis, the only difference between the steps and those of Example 1 is that pure white light is used for irradiation from day 1 to day 10, that is, the ratio of white light to ultraviolet LED beads is 20:0, and the remaining steps and formulation are the same as those of Example 1.

[0048] Comparative Example 2 A method for increasing the astaxanthin yield of Haematococcus pluvialis, the only difference between the steps and those of Example 1 is that the special improved BG-11 culture medium formula does not contain magnesium chloride hexahydrate; the remaining steps and formula composition are the same as those of Example 1.

[0049] Comparative Example 3 A method for increasing the astaxanthin yield of Haematococcus pluvialis is the same as that in Example 1, except that the sodium nitrate content in the modified BG-11 culture medium is increased to 600 mg. All other steps and formulations are the same as in Example 1.

[0050] Comparative Example 4 A method for increasing astaxanthin production in Haematococcus pluvialis differs from Example 1 only in that, during the induction period, instead of using a stepwise increase in light intensity, a constant, moderate light intensity of 400 μmol·m⁻¹ is maintained from day 1 to day 10. -2 ·s -1 The remaining steps and formulation are the same as in Example 1.

[0051] The dry weight and astaxanthin content of the products obtained in Examples 1-5 and Comparative Examples 1-4 were determined, and the results are shown in Table 1. For the dry weight determination, the algal mud was dried in an oven at 85°C until constant weight to obtain algal powder dry matter. Methods for determining astaxanthin content include: a. Sample processing: Take an appropriate amount of algal solution, centrifuge to collect algal mud, wash with distilled water, and freeze-dry to obtain algal powder; b. Extraction: Accurately weigh approximately 10 mg of algal powder into a centrifuge tube, add 10 mL of dimethyl sulfoxide (DMSO); place in a 55°C water bath and extract for 30 minutes in the dark, vortexing for 30 seconds every 10 minutes during the extraction. c. Measurement: Centrifuge the extract at 8000 rpm for 5 minutes, take the supernatant, dilute it appropriately, and use a UV-Vis spectrophotometer to measure the absorbance value at a wavelength of 480 nm. d. Calculation: Astaxanthin concentration was calculated based on a pre-plotted standard curve using astaxanthin standards (Sigma-Aldrich); Astaxanthin content (%) = (mass of extracted astaxanthin / mass of algal powder used for extraction) × 100%. Astaxanthin yield (mg·L) -1 ·d -1 = Astaxanthin concentration at the end of induction (mg / L) / number of induction days (10 days).

[0052] Table 1 Test Results

[0053] As can be seen from the data in Table 1, Examples 1-5 all used the special improved BG-11 culture medium of the present invention combined with stepwise increasing ultra-high light intensity and composite light quality. The final astaxanthin yield of all examples (9.47-11.70 mg / L / d) was significantly higher than that of traditional induction methods (usually less than 5 mg / L / d), and the astaxanthin content (5.39-6.25%) was also at a relatively high level in the industry. This set of data as a whole proves the universality and effectiveness of the technical solution of the present invention and the tolerance of the process parameters.

[0054] In Example 1, the dry weight was 1.69 g / L, the astaxanthin concentration was 105.60 mg / L, the content was 6.25%, and the yield was 11.70 mg / L / d. All four indicators were the highest among all examples. This group used optimal values ​​for the culture medium composition and the light program (e.g., median light intensity gradient, UV ratio 20:3), representing the optimal implementation of the present invention. The results showed that under these parameters, cell biomass accumulation and astaxanthin synthesis reached an optimal balance, with sufficient Mg... 2+ It supports high biomass under high light; moderate nitrogen restriction and precise light stress work together to direct metabolic flow to astaxanthin synthesis, achieving simultaneous maximization of yield, content and efficiency.

[0055] In Examples 2 and 3, the dry weight was the same (1.58 g / L), and the astaxanthin concentration, content, and yield were very similar, all significantly lower than in Example 1 and slightly higher than in Comparative Example 1 (without UV). These two groups only changed the ratio of white light to UV light bulbs (20:1 and 20:5), with highly similar results, though slightly lower than in Example 1 (20:3). This indicates that the introduction of UV is necessary: ​​the yields of the two groups (~9.5 mg / L / d) were higher than in Comparative Example 1 (8.73 mg / L / d) without UV, confirming that adding UV-A as an auxiliary stress factor from days 2 to 10 effectively enhances astaxanthin synthesis. The data also show that there is an optimal range for UV dosage: at a ratio of 20:1, the UV signal may be slightly weak, resulting in insufficient additive effect on ROS signaling and pathway activation; at a ratio of 20:5, the UV may be too strong, causing oxidative damage beyond the cell's tolerance range, leading to partial cell death or excessive metabolic burden, thus offsetting its signal enhancement effect. The 20:3 ratio in Example 1 is likely the optimal stress dose for this system, achieving the best balance between signal intensity and cell damage.

[0056] The performance indicators of Examples 4 (lower limit of parameters) and 5 (upper limit of parameters) fall between those of Examples 1 and Examples 2 / 3, with Example 5 showing slightly better overall performance than Example 4. Specifically, Example 4 (lower limit group): lower nitrogen source (100 mg / L), cofactors (SA, α-KG), and stress intensity (light intensity and salinity both at the lower limit) may have placed the cells in a relatively mild stress environment. While this ensured a high survival rate (1.56 g / L dry weight is acceptable), the driving force for astaxanthin synthesis was slightly insufficient, resulting in the final concentration and yield (9.80 mg / L / d) failing to reach their maximum. Example 5 (upper limit group): higher nitrogen source (120 mg / L), cofactors, salinity, and light intensity constituted the strongest stress combination. Biomass (1.61 g / L) remained good, indicating that the cells tolerated stronger stress under the support of the specialized culture medium. The astaxanthin yield (10.25 mg / L / d) was better than the lower limit group, but still did not surpass the preferred group (Example 1). This could be because the nitrogen source is at the upper limit of its range, slightly increasing interference with metabolic shifts; or the marginal effect of oxidative damage from the highest light intensity (1100 μmol) is beginning to appear. This suggests that not all parameters are better the higher they are; the optimal combination is the result of a fine balance.

[0057] Comparative Examples 1-4, by applying the principle of a single variable, eliminated a key element from the present invention. The results, compared with Example 1, reversely verified the necessity of each technical element and its irreplaceable role in the synergistic system of the present invention.

[0058] In Comparative Example 1, which used only pure white light, the astaxanthin yield (8.73 mg / L / d) was approximately 25% lower than that of Example 1 (11.70 mg / L / d). Dry weight, concentration, and content were all significantly lower. This gap directly demonstrates the immense value of introducing UV-A composite light quality in the later stages of induction. As described in the mechanism above, UV-A is a high-quality, high-intensity specific oxidative stress source. Without UV-A, the ROS signal generated by white light alone may be insufficient in intensity and type to sustain the peak activation state of the astaxanthin synthesis pathway in the later stages of induction (days 6-10). The results of Comparative Example 1 are equivalent to a high-performance version of traditional photoinduction. Although it is superior to ordinary methods due to the use of the special culture medium and stepwise white light of this invention, the lack of UV enhancement prevented it from reaching its maximum production capacity. This confirms the description in the specification: "Without UV-A, white light alone may not be able to generate a sufficiently strong specific oxidative signal in the later stages to maintain the highest activity of the synthesis pathway."

[0059] The data for Comparative Example 2 (without additional MgCl2) are disastrous, with a yield (5.10 mg / L / d) less than half that of Example 1, a significantly reduced astaxanthin content (3.40%), and the most severe biomass loss (only 1.35 g / L dry weight). This comparison most sharply reveals the extreme importance of magnesium supplementation under high salt stress, without additional MgCl2. 2+ Cells in high concentrations of Na + Facing ion competition and absorption inhibition in the environment, intracellular Mg 2+ Deficiency; while Mg 2+ Magnesium chloride is the chlorophyll center, an activator of key enzymes such as Rubisco, and an essential cofactor for ATP. Its absence directly leads to: photosynthetic collapse (inability to effectively utilize progressively increasing light intensity, resulting in an energy crisis); metabolic network paralysis (insufficient ATP supply for synthesis and repair); and cell death (inability to maintain basic physiological functions, leading to large-scale cell death under extremely high light intensity and a sharp drop in biomass). Ultimately, cells struggle to survive, making astaxanthin synthesis impossible. The results of Comparative Example 2 validate the prediction made in the background section: a high-sodium environment may competitively inhibit the absorption of key elements such as magnesium ions, while magnesium is crucial for maintaining photosynthetic and metabolic activity under stress. The addition of magnesium chloride is not merely icing on the cake, but a vital component ensuring cell survival in high-salt, high-light adverse conditions.

[0060] Comparative Example 3 (high nitrogen: 600 mg / L NaNO3) presented a contradictory result: its biomass (1.70 g / L) was even slightly higher than that of Example 1, but its astaxanthin content (3.70%) and yield (6.99 mg / L / d) were significantly reduced (yield decreased by about 40%). This comparison convincingly demonstrates the core logic of moderate nitrogen restriction during the induction phase. Although the higher nitrogen concentration (600 mg / L) did not reach the level of the growth medium (usually 1500 mg / L), it was sufficient to send confusing signals to the cells. Metabolic conflict: Cells simultaneously receive two conflicting instructions: nitrogen for growth and high light and high salt stress response. Metabolic flux is fragmented, with some resources still being used for the synthesis of growth-related nitrogenous compounds, rather than being fully utilized for the synthesis of astaxanthin, a nitrogen-free secondary metabolite. Cell state mismatch: Relatively sufficient nitrogen may cause more cells to try to maintain a dividing or active metabolic state, rather than enter a dormant or quiescent phase suitable for accumulating secondary metabolites; The result was that cells grew more, but were less willing to produce astaxanthin, precisely confirming the nutrient signaling conflict problem pointed out in the background art. Comparative Example 3 was essentially a failed optimization attempt to protect cells or increase biomass by providing more nitrogen, but it severely sacrificed the production target (astaxanthin). The moderately low nitrogen (110 mg / L) of Example 1 successfully locked cellular metabolism in the direction of astaxanthin synthesis.

[0061] The yield of Comparative Example 4 (constant medium light) (7.33 mg / L / d) was about 37% lower than that of Example 1. Although its biomass (1.55 g / L) was comparable to some examples, its astaxanthin synthesis efficiency was severely insufficient. This comparison highlights the decisive advantage of dynamically escalating ultra-high light intensity programs over static, medium light intensity programs. Lacking domestication and with a low upper limit of tolerance: From day one, the cells are subjected to a fixed light intensity of 400 μmol. Although this intensity itself is not fatal, the cells can only adapt to this level. They do not have the opportunity, as in Example 1, to gradually build up a strong endogenous photoprotection mechanism (such as antioxidant enzyme system, lutein cycle, cell wall thickening, etc.) to cope with the extreme light intensity of 800-1100 μmol through daily increasing challenges. Insufficient stress intensity leads to inadequate activation of the astaxanthin synthesis pathway: The ROS signal intensity generated by a constant 400 μmol light intensity is fixed and finite. It cannot create a stress gradient that increases over time like a stepped light intensity, thus failing to continuously and progressively stimulate and upregulate all rate-limiting enzyme genes in the astaxanthin synthesis pathway. Therefore, although the cells in Comparative Example 4 were still viable, they remained in an under-stimulated state, and their astaxanthin synthesis potential was not fully realized. The stepped light intensity in Example 1 was a sophisticated acclimatization-challenge process that both protected the cells and gradually pushed their potential to its limits.

[0062] The differences between each comparative example and Example 1 are far greater than the simple summation of individual technical elements. For example, Comparative Example 2 (Mg deficiency) yielded the worst results, indicating that if cell survival is problematic, any light optimization is ineffective. Comparative Examples 3 (high nitrogen) and 4 (constant light) show that even if cells survive, the results are still unsatisfactory if the metabolic direction is incorrect or the stress intensity is insufficient. This profoundly reveals the tight logical connection and functional coupling between the various technical elements of this invention: the specialized culture medium provides the physiological basis for implementing extreme light stress; and extreme light stress, in turn, verifies and amplifies the design value of the specialized culture medium. This deep synergy results in a significant leap in overall technical effectiveness, solving the systemic problem of long induction cycles, low efficiency, and easy cell death pointed out in the background art.

[0063] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for increasing astaxanthin production from Haematococcus pluvialis, characterized in that, Includes the following steps: Haematococcus pluvialis was cultured to a growth plateau to obtain Haematococcus pluvialis seed culture; The Haematococcus pluvialis seed culture was inoculated into a columnar photobioreactor containing a specially modified BG-11 medium for high-light and high-salt induction. The specially modified BG-11 medium, per liter, contained: 100-120 mg sodium nitrate, 100-120 mg magnesium sulfate heptahydrate, 30-45 mg potassium dihydrogen phosphate, 15-20 mg disodium ethylenediaminetetraacetate, 12.5-14 mg ferric citrate, 6-8 mg boric acid, and 4-6 mg salicylic acid. α-Ketoglutarate 3-5 mg, manganese chloride tetrahydrate 1-3 mg, sodium bicarbonate 2.2-3.5 mg, cobalt nitrate hexahydrate 0.05-0.1 mg, sodium molybdate dihydrate 0.3-0.5 mg, copper sulfate pentahydrate 0.05-0.1 mg, zinc sulfate heptahydrate 0.3-0.5 mg, sodium chloride 2000-3000 mg, and magnesium chloride 300-400 mg; during the high light and high salt induction period, on the first day, 300-320 μmol·m -2 ·s -1 Light treatment, day 2 using 380-400 μmol·m -2 ·s -1 Light treatment, on the 3rd day, using 600-650 μmol·m -2 ·s -1 Light treatment, with 700-750 μmol·m⁻² on day 4. -2 ·s -1 Light treatment, with 800-850 μmol·m⁻² on day 5. -2 ·s -1 Light treatment, using 1000-1100 μmol·m⁻¹ on days 6-10. -2 ·s -1 Lighting processing.

2. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, The modified BG-11 culture medium contains 110 mg / L sodium nitrate, 5 mg / L salicylic acid, 4 mg / L α-ketoglutarate, 2500 mg / L sodium chloride, and 350 mg / L magnesium chloride.

3. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, The Haematococcus pluvialis strain mentioned is Haematococcus pluvialis H7, with accession number CGMCCNo.41186.

4. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, On day 1, white light with a wavelength of 440-460 nm was used for illumination treatment; from day 2 to day 10, composite light consisting of white light with a wavelength of 440-460 nm and ultraviolet light with a wavelength of 360-380 nm was used for illumination treatment according to the ratio of white light LED beads to ultraviolet light LED beads.

5. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 4, characterized in that, The ratio of white light to ultraviolet light LEDs is 20:1-5.

6. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, The inoculation density of the Haematococcus pluvialis seed solution was 1.5 × 10⁻⁶. 5 Up to 2.0×10 5 cells / mL.

7. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, The process of culturing Haematococcus pluvialis to the growth plateau phase is carried out in a culture bottle under the following conditions: ambient temperature 24-26℃, and a sterile mixed gas containing 500-800ppm carbon dioxide is continuously introduced into the bottom of the culture bottle at an aeration rate of 1-1.5vvm.

8. The method for increasing astaxanthin production from Haematococcus pluvialis according to claim 1, characterized in that, The high-light, high-salt induction process is carried out in a columnar photobioreactor. The induction conditions are: ambient temperature 25-30℃, and a sterile mixed gas containing 100-200ppm carbon dioxide is continuously introduced into the bottom of the reactor at an aeration rate of 1-1.5vvm.

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