Haematococcus pluvialis culture medium and culture method thereof

Abstract

The invention provides a haematococcus pluvialis culture medium and a culture method thereof. The culture medium comprises a nutrition proliferation culture medium, a stress induction culture medium and an accumulation strengthening culture medium which are used for segmented culture, the nutrition proliferation culture medium comprises sodium nitrate, monopotassium phosphate, potassium chloride, ferric ammonium citrate, sodium citrate and a trace element mixed solution; adding an osmotic stress agent, an oxidation inducer and plant hormones into a stress induction culture medium; and accumulating and strengthening the culture medium to remove sodium nitrate, and adding and supplementing a carbon source, a polymer penetrant and a zwitterionic stabilizer. The culture method comprises the following steps: a nutrition proliferation stage, a stress induction stage and an accumulation strengthening stage. According to the method, through a three-section culture medium and a fine regulation culture process, and through the synergistic effect of a composite stress factor and a dynamic environment signal, on the premise of ensuring relatively high activity of algae cells, bidirectional synergistic efficient improvement of biomass growth and astaxanthin accumulation is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Haematococcus pluvialis culture medium and a culture method thereof. Background Art

[0002] Haematococcus pluvialis occupies an important position in the field of microalgae and is considered the biological source with the strongest ability to accumulate astaxanthin in nature. Astaxanthin is a keto-carotenoid with super antioxidant activity and has extremely broad application prospects in the fields of food additives, nutritional health products, high-grade cosmetics, and aquaculture feeds. However, the large-scale industrial cultivation of Haematococcus pluvialis and the efficient acquisition of astaxanthin still face many technical bottlenecks that need to be solved urgently. One of the core problems is that Haematococcus pluvialis will go through two main stages under natural or conventional culture conditions - the green vegetative cell stage and the red thick-walled spore stage (i.e., the main astaxanthin accumulation stage), but their requirements for environmental conditions are significantly different. Traditional general culture media such as BG-11 often have difficulty in efficiently meeting the dual requirements of its growth and astaxanthin accumulation at the same time. Especially after entering the stress stage, the biomass growth significantly slows down, and the astaxanthin synthesis rate is not ideal.

[0003] In order to promote the accumulation of astaxanthin, the strategy of adding stress factors (such as high light, nutrient deprivation, increased salinity) to the culture medium is commonly adopted in production. Among them, inducing stress by regulating the culture medium components is the most commonly used method. In the prior art, complete or partial nitrogen source deficiency is often used to induce the transformation and accumulation of astaxanthin in algal cells. However, this treatment often leads to a large number of cell deaths or dormancies during the stress stage, resulting in serious biomass loss, and ultimately the total astaxanthin yield per unit volume of the culture solution is not significantly increased. In addition, osmotic stress using a large amount of single high-concentration inorganic salts (such as sodium acetate, sodium chloride) can, to a certain extent, initiate astaxanthin synthesis, but the action mechanism is relatively single, causing a relatively large physiological impact on cells and easily triggering irreversible damage, which limits the effective extension of the stress period and the further improvement of the accumulation efficiency. Therefore, how to finely regulate the composition of the culture medium, while ensuring that the algal cells have high vitality and stress resistance, and more gently, persistently and efficiently induce the activation of the astaxanthin synthesis pathway is the current research focus and difficulty.

[0004] In terms of optimizing the cultivation process, the simple two-stage cultivation (vegetative growth - stress accumulation) often has problems of uncoordinated transitions in actual operation. In the existing technology, when starting the stress stage, there is a lack of precise control over the type of stress factors, the application timing, the action intensity, and the replacement and connection of culture media in different stages, often resulting in insufficient or overloaded stress signal transduction. At the same time, in the crucial post-stress stage, the continuous maintenance of the accumulation environment is ignored, and the algal cells that have initiated the accumulation potential are not effectively supported to continuously and efficiently synthesize astaxanthin, resulting in a relatively low ceiling for the accumulation efficiency.

[0005] In the downstream processing link after harvesting, efficiently releasing astaxanthin from the thick-walled spores of Haematococcus pluvialis with a hard cell wall is another major challenge. Conventional physical cell wall breaking methods (such as high-temperature drying and pulverization, high-pressure homogenization, ball milling) not only consume a large amount of energy but also easily cause the degradation, oxidation, and isomerization of heat-sensitive astaxanthin, reducing the product quality and bioavailability. Although chemical methods (acid and alkali treatment) and enzymatic methods have been explored, chemical methods are prone to introducing harmful residues and destroying the structure of complex natural components, and the efficiency, cost, and economic feasibility of enzymatic methods still need to be greatly improved. Developing a treatment method that can efficiently dissolve or soften the cell wall of Haematococcus pluvialis under mild conditions while maximizing the protection of the integrity of astaxanthin is extremely crucial for improving the yield and quality of astaxanthin extraction.

[0006] To sum up, the main problems in the current large-scale production of Haematococcus pluvialis and its astaxanthin extraction process can be summarized as follows: 1) The general culture medium has weak pertinence for astaxanthin accumulation, the stress induction effect is not ideal, and it is difficult to achieve synergistic and efficient growth of biomass and astaxanthin; 2) The cultivation 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) The existing cell wall breaking and extraction technologies generally have problems such as high energy consumption, high astaxanthin loss rate, heavy environmental burden, or high cost. Therefore, it is necessary to design a culture medium for Haematococcus pluvialis and its cultivation method. Summary of the Invention

[0007] In order to overcome the defects in the existing technology, the present invention provides a culture medium for Haematococcus pluvialis and its cultivation method.

[0008] To achieve the above object, the present invention provides the following technical solutions:

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

[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] Ammonium ferric 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 induction medium is prepared by adding the following to the nutrient proliferation medium: 35-45 mg / L of osmotic stress agent, 0.08-0.12 mg / L of oxidative inducer, and 0.18-0.22 mg / L of plant hormone;

[0018] The accumulation and enrichment medium is obtained by removing sodium nitrate from the nutrient proliferation medium and adding a supplemental carbon source, a polymer osmotic agent, and a zwitterionic stabilizer.

[0019] A core contradiction faced by Haematococcus pluvialis is the huge difference in the requirements of the two key stages of its life cycle. In the green vegetative cell stage, algal cells need sufficient nutrients to proliferate rapidly and accumulate biomass. When it is necessary to transform into the red thick-walled spore stage, that is, the main accumulation period of astaxanthin, appropriate stress signals must be applied to trigger this mechanism. In the prior art, universal culture media, such as BG-11, are commonly used, and stress is induced by simple nitrogen source deficiency. The problem with this approach is that the environment is drastically changed, and cells are forced to accept stress shocks while losing their growth basis, which can easily lead to a large number of cell deaths or dormancy. Even if the cells that survive by chance begin to synthesize astaxanthin, the total biomass loss is too large, and the total astaxanthin obtained in the unit culture medium is difficult to effectively increase. Another common idea is to use a single high-concentration inorganic salt such as sodium chloride to cause osmotic stress to start synthesis, but this mode of action is too simple and crude, and the impact on cell physiology is too strong, often causing irreversible damage, and it is impossible to maintain a long-term and effective mild stress state. The result is that the growth of biomass and the accumulation of astaxanthin conflict with each other, and it is difficult to achieve high efficiency at the same time.

[0020] To solve this difficult problem, we abandoned the general approach and creatively designed a culture medium system that operates in stages, including a nutrient proliferation medium, a stress induction medium, and an accumulation enhancement medium. Each medium is precisely formulated for a specific stage. The core of the nutrient proliferation stage is to make the algal cells grow strong and fast. The nutrient proliferation medium provides a nitrogen source through sodium nitrate at an appropriate concentration, a phosphorus source through potassium dihydrogen phosphate, supplemented with potassium chloride to maintain ionic balance, essential iron elements, and sodium citrate as a carbon source buffer, etc., providing an optimal nutrient environment for the rapid proliferation of cells. After entering the stress induction stage, if the environment suddenly becomes harsh, the cells will not be able to withstand it. Therefore, the stress induction medium does not start from scratch but superimposes carefully selected composite stress factors on the nutrient base of the nutrient proliferation medium, namely the osmotic stress agent sodium salicylate, the oxidative inducer sodium selenite, and the plant hormone methyl jasmonate. These factors are no longer a single strong stimulus but gently activate astaxanthin synthesis by simulating the synergistic action of multiple signaling pathways in natural stress. Sodium salicylate induces moderate osmotic pressure, and sodium selenite provides a controllable oxidative stress signal. Together, they simulate the adversity in the natural environment and initiate the defense response of the cells. Methyl jasmonate plays the role of an internal signaling molecule and is widely involved in the regulation of secondary metabolism in plants and algae. These three work together under the condition of maintaining a specific ratio of sodium salicylate and sodium selenite and an appropriate amount of methyl jasmonate, gently but effectively activating the genes and enzymes related to astaxanthin synthesis in the cells, guiding the cells to gradually and smoothly transform from the vegetative growth state to the thick-walled spore state of accumulating astaxanthin, greatly reducing the risk of cell death or dormancy due to "shock".

[0021] The trace element mixture contains:

[0022] Calculated as boron element, boron compound 2.5 - 4.5 mg / L;

[0023] Calculated as copper element, copper compound 0.004 - 0.006 mg / L;

[0024] Calculated as zinc element, zinc compound 0.06 - 0.08 mg / L;

[0025] Calculated as molybdenum element, molybdenum compound 0.06 - 0.08 mg / L;

[0026] Calculated as manganese element, manganese compound 0.8 - 1.0 mg / L.

[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 phytohormone is methyl jasmonate.

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

[0030] The supplementary carbon source is glucose with an initial concentration of 2 - 3 g / L, the polymeric osmotic agent 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] When the cells successfully initiate the astaxanthin accumulation program, it is not the end of the story. They still need a continuously supportive environment for efficient synthesis. The accumulation enhancement stage is designed for this purpose. Its culture medium is directly derived from the nutrient proliferation medium by removing the nitrogen source sodium nitrate. This retains the basic nutrients and buffer system while removing the nitrogen source to send a continuous "stress" signal to prevent the cells from reverting to the growth mode. The most crucial thing is the addition of the supplementary carbon source glucose, the polymeric osmotic agent polyethylene glycol 6000, and the zwitterionic stabilizer betaine hydrochloride. Removing the nitrogen source tells the cells to stop growing and focus on accumulation, while the supplemented glucose continuously provides the necessary carbon skeletons and energy for the complex biochemical pathway of astaxanthin synthesis. Polyethylene glycol 6000 generates a mild osmotic effect in the cell microenvironment through its polymeric properties, which helps to maintain the osmotic state of the cells. As a compatible solute, betaine hydrochloride can stabilize the protein structure and protect the integrity of cell membranes, especially the organelle membranes, enhancing the tolerance and vitality of the cells under stress pressure. The relay design of the three-stage culture medium in this application has its core principle of precisely matching the different needs of the cells at each physiological transition stage, gently and effectively guiding the life process towards efficient astaxanthin accumulation.

[0032] Although downstream cell disruption is not the core concern of this application, some components selected in the design of the accumulation enhancement culture medium and the characteristics of the entire culture process may also have some potential positive effects on the subsequent cell disruption process. The polymeric osmotic agent polyethylene glycol 6000 contained in the accumulation enhancement culture medium of this application may, under the action of long-term mild osmosis, help to partially soften and loosen the very hard outer wall structure of the Haematococcus pluvialis akinetes. This is equivalent to doing some "preparatory work" for the subsequent cell disruption operation at the microscopic level. The main role of the zwitterionic stabilizer betaine hydrochloride throughout the accumulation enhancement stage is to protect the internal stability of the cells and the integrity of the membranes, which may make the akinete cells obtained at harvest more "robust" and complete in structure.

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

[0034] (1) Nutritional proliferation stage:

[0035] Inoculate algal seeds in the nutritional proliferation medium, control the light intensity at 70 - 80 μmol·m⁻²·s⁻¹, the temperature at 23 - 25 °C, and culture for 14 - 16 days;

[0036] (2) Stress induction stage:

[0037] Concentrate the algal liquid obtained after proliferation in the previous step to 20 - 25% of the original volume, transfer it to the stress induction medium. Before the start of the stress induction stage, add 50% of the total amount of methyl jasmonate when preparing the medium, and add the remaining 50% of methyl jasmonate on the 4th day of the culture in the stress induction stage. In the stress induction stage, the light intensity is increased step by step: 140 - 160 μmol·m⁻²·s⁻¹ for 0 - 3 days, 170 - 190 μmol·m⁻²·s⁻¹ for 4 - 7 days, the temperature is 25 - 27 °C, and culture for 7 - 9 days;

[0038] (3) Accumulation and strengthening stage:

[0039] Transfer the algal liquid obtained in the previous step to the accumulation and strengthening medium, maintain the light intensity at 200 - 210 μmol·m⁻²·s⁻¹, the temperature at 17 - 18 °C, supplement glucose at 0.08 - 0.16 g / L every 24 hours, and culture for 8 - 10 days. In the stress induction stage, the step - by - step increase in light intensity uses blue light with a wavelength range of 450 ± 5 nm.

[0040] After solving the problem of medium adaptability, we further realized that the transition from growth to efficient accumulation is not a switch - like instant switch, but a kinetic process that requires precise regulation. The simple "two - step approach" in the prior art often ignores this point: either the stress factors are added too quickly and strongly, causing signal overload and serious cell damage; or added too slowly or insufficiently, resulting in poor induction effect; and a suitable environment is not continuously provided during the accumulation process, leading to low accumulation efficiency. Therefore, we have made a precise design in the parameter setting of the culture process, especially in the stress induction stage and the accumulation and strengthening stage. In the stress induction stage, a step - by - step increase in light intensity strategy is adopted. In the first three days, it is maintained at a moderately high but bearable light level, and then gradually increased to a higher intensity in the next four days. Particularly importantly, this light uses blue light within a specific wavelength range. Blue light has special significance in the photobiology of algae. Its wavelength can be more effectively recognized by specific photoreceptors, thereby more efficiently activating the gene expression related to the light stress response and astaxanthin synthesis pathway. This not only provides energy but also is a precise light signal.

[0041] The application of methyl jasmonate also follows a strategy, which is to add it in two batches: only 50% of the total amount is added at the beginning of stress induction to initiate the initial stress signal response, and then the remaining 50% is supplemented on the fourth day of the stress induction stage, which is a critical time point when the cells have initially adapted to the initial stress and begin to enter a deeper transformation stage. This time-delayed and slow-release addition method is similar to a "preheating - strengthening" process for the cells. It not only avoids the interference or negative feedback regulation that may be caused by a one-time high-concentration hormone shock but also further strengthens the stimulation signal when the cells are relatively ready, promoting the deepening of the accumulation process.

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

[0043] During the accumulation and strengthening stage, the addition amount of glucose is dynamically adjusted according to the redness rate of algal cells: when the redness rate < 50%, 0.08 - 0.10 g / L of glucose is added every 24 hours; when the redness rate ≥ 50%, 0.12 - 0.16 g / L of glucose is added every 24 hours. When entering the accumulation and strengthening stage, the states of the algal cell population will vary, with some turning red faster and some slower. We observed this, so instead of uniformly supplementing the carbon source, we innovatively adjusted the glucose supplementation rate according to an intuitive indicator, namely the redness rate of algal cells. The redness rate reflects the proportion of cells that have successfully transformed into red spores accumulating astaxanthin. When the redness rate is less than 50%, it means that most cells may still be in the transformation process and the demand for carbon source is relatively stable, so glucose is added at a lower rate. Once the redness rate reaches or exceeds 50%, it indicates that half or more cells have entered the peak period of active astaxanthin synthesis. At this time, the cells are like a fully operating factory, and the demand for carbon source increases sharply. We then correspondingly increase the supplementation rate to meet the requirements of high-intensity anabolic metabolism. This dynamic supply based on the real-time state theoretically ensures that the supply of energy and raw materials exactly matches the demand changes of the cell population, neither wasting nor lacking.

[0044] During the accumulation and enhancement stage, a weak oscillation is applied simultaneously, with the shaker speed at 80 - 100 rpm and the oscillation amplitude at 25 - 35 mm. The weak oscillation introduced during the accumulation and enhancement stage is not for strong agitation. Instead, through gentle shaking, it promotes the uniform mixing and exchange of nutrients, oxygen, and metabolites in the entire culture system, preventing local cells from lacking nutrients or oxygen due to sediment accumulation. This mild physical stimulation also helps maintain cell viability and membrane fluidity, providing a more stable and uniform microenvironment for efficient and long-term astaxanthin synthesis. The integration of these precisely regulated technological steps follows an internal logic of simulating a progressive process where signals are gradually released and strengthened. By means of precise blue light signals, time-controlled slow release of hormones, dynamic carbon source supplementation, and weak oscillation to maintain environmental uniformity, the stability of the stress state and the persistence 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. By designing the Haematococcus pluvialis culture medium and culture method in stages, the present application effectively solves the problems of poor pertinence of the general culture medium, unsatisfactory stress induction effect, and difficulty in synergistically and efficiently increasing biomass and astaxanthin mentioned in the background art. Specifically, we provide a three-stage system of a nutrient proliferation medium, a stress induction medium, and an accumulation and enhancement medium, with each stage optimized according to the life cycle requirements of algal cells. The nutrient proliferation medium contains specific proportions of components such as sodium nitrate, potassium dihydrogen phosphate, potassium chloride, and citrate, aiming to efficiently support the rapid proliferation of algal cells in the green nutrient stage; the stress induction medium adds sodium salicylate as an osmotic stress agent, sodium selenite as an oxidative inducer, and methyl jasmonate as a plant hormone on the basis of nutrient proliferation. These components act synergistically in appropriate proportions to gently activate the astaxanthin synthesis pathway and avoid 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 in a low-stress environment and supporting continuous astaxanthin accumulation. This staged culture medium design avoids forced transitions in traditional methods and can more precisely balance the growth requirements and accumulation requirements of algal cells in principle, reducing cell death and dormancy phenomena.

[0047] 2. In terms of the cultivation process, the present application adopts refined control steps, significantly improving the efficiency and stability of stress initiation and maintenance, and solving the problem of insufficient refinement in the cultivation process of the background art. During the nutritional proliferation stage, biomass accumulation is promoted through light and temperature optimization; during the stress induction stage, a stepped light enhancement and methyl jasmonate batch addition strategy is adopted, that is, only half of the amount is added at the beginning, and the remaining is supplemented after a specific number of days, combined with blue light illumination. This step-by-step action mechanism can slowly release stress signals, avoid irreversible damage caused by overload, and at the same time, blue light can more effectively stimulate light-responsive genes within a specific wavelength range, enhancing the stress response; during the accumulation and strengthening stage, the glucose supplementation amount and weak oscillation are dynamically adjusted according to the redness rate, which can adapt to the astaxanthin synthesis state in cells in real time, and the weak oscillation helps to homogenize nutrients and maintain cell viability, avoiding the yield limitation caused by stress incoordination in the traditional two-stage process. Generally speaking, these process details simulate the natural stress process in principle, and improve the total astaxanthin yield through a gentle and continuous activation method.

[0048] 3. The cultivation method of the present application also introduces a polymer penetrant and an amphoteric ion stabilizer during the accumulation and strengthening stage, which helps to soften the cell wall structure and lay a foundation for subsequent cell wall breaking treatment. Although the downstream extraction link is not the core of the present application, by applying stabilization treatment to cells, it may reduce the subsequent cell wall breaking difficulty and the risk of astaxanthin loss. For example, betaine hydrochloride can relieve osmotic pressure and maintain the integrity of the cell membrane. The whole process strengthens the stress resistance ability and accumulation persistence of algal cells in principle, thus indirectly supporting the efficient acquisition of astaxanthin and making the production of Haematococcus pluvialis more efficient, stable and economical. Detailed implementation manners

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0050] In the present application, the sources of various raw materials are briefly described as follows:

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

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

[0053] The nutrient proliferation medium comprises:

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

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

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

[0057] Ammonium ferric citrate provides an iron element 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 - induced medium is prepared by adding in the nutrient proliferation medium: osmotic stress agent 35 - 45 mg / L, oxidation - inducing agent 0.08 - 0.12 mg / L, phytohormone 0.18 - 0.22 mg / L;

[0061] The accumulation - strengthening medium is obtained by removing sodium nitrate from the nutrient proliferation medium and adding a supplementary carbon source, a polymeric osmotic agent, and an amphoteric ion stabilizer.

[0062] The trace element mixture contains:

[0063] Calculated as boron element, boron compound 2.5 - 4.5 mg / L;

[0064] Calculated as copper element, copper compound 0.004 - 0.006 mg / L;

[0065] Calculated as zinc element, zinc compound 0.06 - 0.08 mg / L;

[0066] Calculated as molybdenum element, molybdenum compound 0.06 - 0.08 mg / L;

[0067] Calculated as manganese element, manganese compound 0.8 - 1.0 mg / L.

[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 - inducing agent is sodium selenite, and the phytohormone is methyl jasmonate.

[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 polymeric osmotic agent is polyethylene glycol 6000 1.7 - 1.9 g / L, and the amphoteric ion stabilizer is betaine hydrochloride 0.08 - 0.12 g / L.

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

[0073] (1)Nutritional proliferation stage:

[0074] Inoculate algal seeds in the nutritional proliferation medium, control the light intensity at 70 - 80 μmol·m⁻²·s⁻¹, the temperature at 23 - 25 °C, and culture for 14 - 16 days;

[0075] (2)Stress induction stage:

[0076] Concentrate the algal liquid obtained in the previous step to 20 - 25% of the original volume, transfer it to the stress induction medium. Before the start of the stress induction stage, add 50% of the total amount of methyl jasmonate when preparing the medium, and add the remaining 50% of methyl jasmonate on the 4th day of the culture in the stress induction stage. In the stress induction stage, the light intensity is increased step by step: 140 - 160 μmol·m⁻²·s⁻¹ for 0 - 3 days, 170 - 190 μmol·m⁻²·s⁻¹ for 4 - 7 days, the temperature is 25 - 27 °C, and culture for 7 - 9 days;

[0077] (3)Accumulation and strengthening stage:

[0078] Transfer the algal liquid obtained in the previous step to the accumulation and strengthening medium, maintain the light intensity at 200 - 210 μmol·m⁻²·s⁻¹, the temperature at 17 - 18 °C, add glucose 0.08 - 0.16 g / L every 24 hours, and culture for 8 - 10 days.

[0079] In the stress induction stage, the step - by - step increase in light intensity uses blue light with a wavelength range of 450 ± 5 nm.

[0080] In the nutritional proliferation stage, introduce a gas containing 2.5 - 3.5% CO2, and the gas flow rate is 0.8 - 1.2 L / min.

[0081] In the accumulation and strengthening stage, the amount of glucose added is dynamically adjusted according to the red - pigmentation rate of algal cells: when the red - pigmentation rate < 50%, add glucose 0.08 - 0.10 g / L every 24 hours; when the red - pigmentation rate ≥ 50%, add glucose 0.12 - 0.16 g / L every 24 hours; in the accumulation and strengthening stage, apply weak oscillation at the same time, the shaker speed is 80 - 100 rpm, and the oscillation amplitude is 25 - 35 mm.

[0082] This application designs a three - stage medium and a finely regulated culture process. Through the synergistic effect of composite stress factors and dynamic environmental signals, on the premise of ensuring the relatively high vitality of algal cells, the two - way synergistic and efficient improvement of biomass growth and astaxanthin accumulation is achieved.

[0083] The core idea of this three-stage cultivation strategy of the present application is to gently induce rather than severely impact with composite signals. The akinetes cultivated in this way have a physiological state closer to the good state during the natural maturation process, higher cell viability, and relatively better integrity of the cell wall and membrane. These akinetes with higher viability and more stable membranes, as the "raw materials" for subsequent cell wall breaking treatment, theoretically can withstand certain processing pressures better than those cells that have been severely stressed and are scarred. This improvement in the native state creates a relatively favorable condition for the development or application of a gentler and more efficient cell wall breaking and extraction method for the starting materials. Overall, from nutritional guarantee, gentle transmission of stress signals to continuous support during the accumulation process, we aim to improve the final yield of astaxanthin in the large-scale cultivation of Haematococcus pluvialis through systematic improvement of the upstream culture medium and process.

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

[0085] Example 1

[0086] The preparation of the culture medium and the cultivation steps are as follows:

[0087] Nutritional proliferation stage:

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

[0089] After inoculating the algal species, control the light intensity at 80 μmol·m⁻²·s⁻¹, the temperature at 25°C, and introduce a gas containing 3.5% CO2 (flow rate 1.0 L / min), and cultivate for 14 days.

[0090] Stress induction stage:

[0091] Concentrate the proliferated algal liquid to 20% of the original volume and transfer it to the stress induction culture medium (adding 45 mg / L of sodium salicylate, 0.10 mg / L of sodium selenite, and 0.18 mg / L of methyl jasmonate on the basis of the nutritional proliferation culture medium).

[0092] Add 50% of the total amount of methyl jasmonate (0.18 mg / L) during the preparation of the culture medium, and add the remaining 50% on the 4th day.

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

[0094] Accumulation and enhancement stage: The algal solution was transferred to an accumulation and enhancement medium (the nutrient proliferation medium with sodium nitrate removed, and 2 g / L glucose, 1.7 g / L polyethylene glycol 6000, and 0.12 g / L betaine hydrochloride added).

[0095] Maintain light at 210 μmol·m⁻²·s⁻¹ and temperature at 18°C, supplement glucose dynamically according to the redness rate: when the redness rate <50%, supplement 0.08 g / L glucose daily, when the redness rate ≥50%, supplement 0.12 g / L, and apply weak oscillation synchronously (shaker speed 100 rpm, amplitude 25 mm), cultured for 10 days.

[0096] Example 2

[0097] In this example, the same parts as in Example 1 will not be elaborated, and the differences are described as follows:

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

[0099] Nutrient proliferation stage:

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

[0101] Light 70 μmol·m⁻²·s⁻¹, temperature 23°C, introduce a gas containing 2.5% CO2 (flow rate 1.2 L / min), cultured for 16 days.

[0102] Stress induction stage:

[0103] Concentrate the algal solution to 25% of the original volume, and the stress induction medium contains 35 mg / L of sodium salicylate, 0.12 mg / L of sodium selenite, and 0.22 mg / L of methyl jasmonate.

[0104] 50% of methyl jasmonate is added initially, and the remaining is added on the 4th day.

[0105] Lighting: 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°C, cultured for 9 days.

[0106] Accumulation and strengthening stage:

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

[0108] Lighting 200 μmol·m⁻²·s⁻¹, temperature 17°C. When the redness rate < 50%, 0.10 g / L of sugar is supplemented daily; when the redness rate ≥ 50%, 0.16 g / L is supplemented. Weak oscillation (80 rpm, amplitude 35 mm), cultured for 8 days.

[0109] Example 3

[0110] In this example, the same parts as in Example 1 will not be elaborated again, and the differences are as follows:

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

[0112] Nutritional proliferation stage:

[0113] Per liter contains 150 mg sodium nitrate, 11 mg potassium dihydrogen phosphate, 50 mg potassium chloride, ammonium ferric citrate (iron 0.10 mg), 2.0 g sodium citrate, 1.2 mL 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, 0.8 mg / L manganese sulfate monohydrate).

[0114] Lighting 75 μmol·m⁻²·s⁻¹, temperature 24°C, introducing a gas containing 3.0% CO2 (flow rate 0.8 L / min), cultured for 15 days.

[0115] Stress induction stage:

[0116] The concentrated algal liquid is concentrated to 22% of the original volume. The stress induction culture medium contains 40 mg / L sodium salicylate, 0.08 mg / L sodium selenite, and 0.20 mg / L methyl jasmonate (mass ratio of sodium salicylate to sodium selenite is 400:1).

[0117] 50% of methyl jasmonate is added initially, and the remaining is added on the 4th day. Lighting: 150 μmol·m⁻²·s⁻¹ (blue light) for 0–3 days, 180 μmol·m⁻²·s⁻¹ (blue light) for 4–7 days, temperature 26°C, cultured for 8 days.

[0118] Accumulation and reinforcement stage:

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

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

[0121] Comparative Example 1

[0122] In this comparative example, the same points as Example 1 are not repeated here, and the differences are as follows:

[0123] Only nutrient proliferation medium (same as in Example 1) was used for 14 days, and then directly transferred to BG-11 medium without nitrogen source (other components were the same), and cultured for 10 days at high light intensity of 190 μmol·m⁻²·s⁻¹ and temperature of 27°C without hormone addition, dynamic sugar supplementation and shaking.

[0124] Comparative Example 2

[0125] In this comparative example, the same points as Example 2 are not repeated here, and the differences are as follows:

[0126] The nutrient proliferation stage was the same as in Example 2. In the stress stage, only the nitrogen source was removed from the nutrient proliferation medium, and sodium salicylate, sodium selenite, and methyl jasmonate were not added. The light intensity 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 same points as Example 3 are not repeated here, and the differences are as follows:

[0129] The vegetative proliferation stage was the same as in Example 3. The stress stage used white light (not blue light) and methyl jasmonate was added at once at 0.20 mg / L (not in steps), and the rest was the same as in Example 3. The accumulation stage was the same as in Example 3.

[0130] Comparative Example 4

[0131] In this comparative example, the same points as Example 1 are not repeated here, and the differences are as follows:

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

[0133] Comparative Example 5

[0134] In this comparative example, the same points as Example 1 are not repeated here, and the differences are as follows:

[0135] The nutrient proliferation and stress phases were the same as in Example 2. The accumulation and strengthening medium was only free of nitrogen source and glucose was added, without polyethylene glycol 6000 and betaine hydrochloride, and other conditions were the same as in Example 2.

[0136] Algae liquid test results and analysis

[0137] The algae liquid at the end of the culture of the above-mentioned examples and comparative examples was tested for indicators, 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 production rate (mg / L) Cell mortality rate (%) Astaxanthin extraction rate after cell wall breaking (%) 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 can be seen from Table 1, the biomass (2.98–3.21 g / L) and total astaxanthin yield (113.8–115.9 mg / L) of Examples 1-3 are significantly higher than those of all comparative examples. In particular, compared with Comparative Example 1 (traditional two-stage method), its astaxanthin yield is less than 40% of that of the example, and the cell mortality rate is as high as 42.3%. This verifies the core advantage of the segmented culture medium of this application: the optimized ratio of the nutrient proliferation culture medium supports high biomass accumulation, laying the foundation for subsequent synthesis.

[0141] In the stress induction stage, the triple synergistic signals of sodium salicylate (osmosis), sodium selenite (oxidation), and methyl jasmonate (hormone) were used. When the three were removed from Example 2, the yield decreased by 46%, gently activating the astaxanthin pathway and avoiding large-scale cell death caused by severe nitrogen deprivation. In the accumulation and strengthening stage, the carbon source was dynamically replenished. When the sugar was fixed, the yield of Example 4 decreased by 16%. The osmotic / membrane stabilizer maintained the cell viability and metabolic efficiency during the synthesis period. When the comparative example 5 was omitted, the extraction rate decreased by 7.2%. It is proved that this application synergistically improves biomass and astaxanthin yield.

[0142] In the examples, the cell mortality rate was lower than 8.5%, while comparative example 3 cancelled the step-by-step addition of blue light and hormones, resulting in a mortality rate of 21.5% and a 28% decrease in astaxanthin yield. This can be explained by the fact that the specific wavelength of blue light significantly enhances the transmission efficiency of light stress signals and promotes astaxanthin synthesis gene expression by activating light receptor proteins. The step-by-step addition of methyl dijasmonate avoids the metabolic inhibition that may be caused by a single high dose, and after the cells initially adapt to the stress, the signal is strengthened for the second time, thereby improving the conversion efficiency. When comparative example 3 is added at one time, the reddening rate is delayed by 50% by 2 days. Weak oscillation promotes 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 is not directly related to the cell wall breaking process, the astaxanthin extraction rates of the 3 examples are significantly higher than those of the 5 comparative examples. In particular, in Comparative Example 5, polyethylene glycol 6000 and betaine hydrochloride were omitted, and the extraction rate decreased to 86.5%, indicating that polyethylene glycol 6000, through mild osmosis during the accumulation stage, partially relaxes the cell wall structure of the chlamydospores, reducing their mechanical strength. Betaine hydrochloride protects the integrity of the cell membrane, reducing the leakage or degradation of intracellular astaxanthin before harvesting (low cell mortality indirectly reduces the number of ruptured cells). The high cell breakage rate under severe stress in Comparative Example 1 can indirectly prove that the spores obtained through the entire three-stage culture in this application have a more robust physiological state, providing high-quality raw materials for subsequent mild cell wall breaking and reducing the energy consumption requirements of strong physical cell wall breaking.

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

[0145] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Haematococcus pluvialis culture medium, characterized in that, The culture medium includes a nutrient proliferation medium, a stress induction medium, and an accumulation enhancement medium for subculture. The nutrient proliferation medium includes: Sodium nitrate 140 - 160 mg / L; Potassium dihydrogen phosphate 11 - 13 mg / L; Potassium chloride 45 - 55 mg / L; Ammonium ferric citrate providing an iron element concentration of 0.08 - 0.12 mg / L; Sodium citrate 1.8 - 2.2 g / L; Trace element mixture 0.8 - 1.2 mL / L; The stress induction medium is prepared by adding in the nutrient proliferation medium: osmotic stress agent 35 - 45 mg / L, oxidation inducer 0.08 - 0.12 mg / L, and phytohormone 0.18 - 0.22 mg / L; The accumulation enhancement medium is obtained by removing sodium nitrate from the nutrient proliferation medium and adding a supplementary carbon source, a polymeric osmotic agent, and an amphoteric ion stabilizer.

2. The Haematococcus pluvialis culture medium according to claim 1, characterized in that, The trace element mixture contains: Boron compound 2.5 - 4.5 mg / L in terms of boron element; Copper compound 0.004 - 0.006 mg / L in terms of copper element; Zinc compound 0.06 - 0.08 mg / L in terms of zinc element; Molybdenum compound 0.06 - 0.08 mg / L in terms of molybdenum element; Manganese compound 0.8 - 1.0 mg / L in terms of manganese element.

3. The Haematococcus pluvialis culture medium according to claim 2, wherein, 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.

4. A Haematococcus pluvialis culture medium according to claim 1, characterized in that, The osmotic stress agent is sodium salicylate, the oxidation inducer is sodium selenite, and the phytohormone is methyl jasmonate.

5. A Haematococcus pluvialis culture medium according to claim 4, characterized in that, The mass ratio of sodium salicylate to sodium selenite is 350 - 450:

1.

6. The Haematococcus pluvialis culture medium according to claim 1, wherein, The supplementary carbon source is glucose with an initial concentration of 2 - 3 g / L, the polymeric osmotic agent is polyethylene glycol 6000 1.7 - 1.9 g / L, and the amphoteric ion stabilizer is betaine hydrochloride 0.08 - 0.12 g / L.

7. A method for culturing Haematococcus pluvialis using the Haematococcus pluvialis culture medium according to any one of claims 1-6, characterized in that, This method includes the following steps: (1) Nutrient proliferation stage: Inoculate the algal species in the nutrient proliferation medium, control the light intensity at 70 - 80 μmol·m⁻²·s⁻¹, the temperature at 23 - 25°C, and culture for 14 - 16 days; (2) Stress induction stage: Concentrate the proliferated algal liquid obtained in the previous step to 20 - 25% of the original volume, transfer it to the stress induction medium. Before the start of the stress induction stage, add 50% of the total amount of methyl jasmonate when preparing the medium, and add the remaining 50% of methyl jasmonate on the 4th day of the culture in the stress induction stage. During the stress induction stage, the light intensity is increased step by step: 140 - 160 μmol·m⁻²·s⁻¹ for 0 - 3 days, 170 - 190 μmol·m⁻²·s⁻¹ for 4 - 7 days, the temperature is 25 - 27°C, and culture for 7 - 9 days; (3) Accumulation enhancement stage: Transfer the algal solution obtained in the previous step into an accumulation and enhancement medium containing 0.9 - 1.1 g / L of glucose, maintain the light intensity at 200 - 210 μmol·m⁻²·s⁻¹, the temperature at 17 - 18 °C, replenish 0.08 - 0.16 g / L of glucose every 24 hours, and culture for 8 - 10 days.

8. A cultivation method of Haematococcus pluvialis according to claim 7, characterized in that, In the stress induction stage, the light intensity is increased step by step using blue light with a wavelength range of 450 ± 5 nm.

9. The culturing method of Haematococcus pluvialis according to claim 7, characterized in that, In the nutritional proliferation stage, a gas containing 2.5 - 3.5% CO2 is introduced at a gas flow rate of 0.8 - 1.2 L / min.

10. A method for culturing Haematococcus pluvialis according to claim 7, characterized in that, In the accumulation and enhancement stage, the amount of glucose replenishment is dynamically adjusted according to the redness rate of algal cells: when the redness rate < 50%, 0.08 - 0.10 g / L of glucose is replenished every 24 hours; when the redness rate ≥ 50%, 0.12 - 0.16 g / L of glucose is replenished every 24 hours; a weak oscillation is applied simultaneously in the accumulation and enhancement stage, with the shaker speed at 80 - 100 rpm and the oscillation amplitude at 25 - 35 mm.