Haematococcus pluvialis mutant strain for efficiently producing astaxanthin and application thereof

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a highly efficient Haematococcus pluvialis mutant strain for astaxanthin production and its applications. This invention achieves efficient and stable astaxanthin production from Haematococcus pluvialis through targeted strain modification and optimized culture process. The invention obtained a highly efficient astaxanthin-producing Haematococcus pluvialis mutant strain by ARTP mutagenesis, β-ionone domestication, and glycerol domestication. Furthermore, this invention also employs lightless heterotrophic growth, short-term weak light activation, and lightless salt stress to induce astaxanthin production in the Haematococcus pluvialis mutant strain. The Haematococcus pluvialis mutant strain provided by this invention can achieve high-density biomass accumulation and high astaxanthin production in a closed fermenter, with a cell dry weight exceeding 20 g / L and an astaxanthin content reaching 11%. It also has the advantages of a short fermentation cycle and strong batch-to-batch stability, making it feasible for industrial-scale application.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and relates to a Haematococcus pluvialis mutant strain that produces astaxanthin and its application, specifically to a Haematococcus pluvialis CO1 strain that produces astaxanthin and its application. Background Technology

[0002] Astaxanthin is a ketocarotenoid, chemically named 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene. Astaxanthin is a natural antioxidant that effectively scavenge free radicals within cells, slowing down the cellular aging process. It possesses antioxidant, anti-inflammatory, immune-boosting, and tumor-inhibiting effects, and is widely used in cosmetics and health products. Currently, natural astaxanthin is mainly extracted from crustaceans, yeast, or microalgae, with Haematococcus pluvialis (Haematococcus pluvialis) being a prime example. Haematococcus Pluvialis Haematococcus pluvialis is the microorganism with the highest natural astaxanthin content in nature. Therefore, it is considered one of the best raw materials for producing astaxanthin.

[0003] Currently, the traditional method of producing astaxanthin from Haematococcus pluvialis mainly relies on open-air raceway ponds for photosynthetic autotrophic cultivation. This photosynthetic autotrophic cultivation method is susceptible to weather changes and contamination by other microorganisms, and the yield is extremely unstable between batches, with a long production cycle and high management costs. To overcome these limitations, it is necessary to screen superior Haematococcus pluvialis strains and improve cultivation methods during the cultivation process to increase astaxanthin yield.

[0004] Patent document CN104893978A discloses a strain of Haematococcus pluvialis ENN71 and its cultivation method. This Haematococcus pluvialis ENN71 exhibits advantages in astaxanthin production, including a short growth cycle, high natural astaxanthin content, and stable genetic performance. Compared to wild-type Haematococcus pluvialis strains, the growth rate of this mutant ENN71 is more than 40% higher, and the induced astaxanthin content is more than twice that of the wild-type strains. Experiments have shown that this mutant Haematococcus pluvialis ENN71 produces astaxanthin with a content exceeding 4.09%, demonstrating promising prospects for industrial production.

[0005] Patent document CN110343616A discloses a high-astaxanthin-producing Haematococcus pluvialis JNU35, its cultivation method, and its applications. This high-astaxanthin-producing Haematococcus pluvialis JNU35 can effectively balance the simultaneous and rapid accumulation of biomass and astaxanthin content, greatly increasing both biomass and astaxanthin content, and significantly reducing the cost of astaxanthin production. Experiments have shown that during astaxanthin production, the biomass of this Haematococcus pluvialis JNU35 can reach as high as 13.89 g / L, and the astaxanthin content can reach a maximum of 4.35%, demonstrating significant application value in practical production.

[0006] However, the efficiency and yield of astaxanthin produced by fermentation of Haematococcus pluvialis are generally low, which cannot meet the production requirements of industrialization. Therefore, researching and developing a method for the industrial production of astaxanthin from Haematococcus pluvialis with low energy consumption and high biomass and astaxanthin content remains a pressing challenge. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a highly efficient Haematococcus pluvialis mutant strain for astaxanthin production and its applications. This invention solves the problems of high carbon source costs and high induction energy consumption in the industrial production of astaxanthin from Haematococcus pluvialis through targeted strain modification and optimized culture processes. Furthermore, the invention addresses the high operating costs associated with photoheterotrophic induction techniques, which often rely on acetate as a carbon source and require high dissolved oxygen control.

[0008] This invention utilizes ARTP mutagenesis, β-ionone domestication, and glycerol domestication to obtain a highly efficient astaxanthin-producing Haematococcus pluvialis mutant strain. Simultaneously, this invention employs lightless heterotrophic growth, short-term low-light activation, and lightless salt stress to induce astaxanthin production in the Haematococcus pluvialis mutant strain. The culture method provided by this invention uses glycerol as the carbon source, is independent of glucose, high-concentration acetic acid stress, and high dissolved oxygen induction, and achieves efficient accumulation by activating astaxanthin synthesis through short-term low-light activation combined with salt stress and nitrogen starvation. The astaxanthin production process of the Haematococcus pluvialis mutant strain provided by this invention can achieve high-density biomass accumulation and high astaxanthin production in a closed fermenter, while also possessing the advantages of stable production process, strong controllability, and low cost, making it suitable for large-scale industrial production.

[0009] The technical solution provided by this invention is as follows: This invention provides a highly efficient method for producing astaxanthin from Haematococcus pluvialis CO1, wherein Haematococcus pluvialis CO1 ( Haematococcus pluvialis C01) was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number: CCTCC NO: M 2026603.

[0010] The Haematococcus pluvialis CO1, which provides efficient astaxanthin production according to this invention, was obtained through ARTP mutagenesis, β-ionone domestication, and glycerol domestication. The specific modification method is as follows: (1) ARTP mutagenesis: Haematococcus pluvialis was first subjected to ARTP mutagenesis, and a basic mutant with high astaxanthin production was obtained by screening. (2) β-ionone domestication: The ARTP mutant strain selected in step (1) is used as the starting strain. Through β-ionone gradient domestication, the negative feedback regulation of key enzymes in carotenoid synthesis is gradually eliminated, and a mutant strain that can relieve the feedback inhibition mechanism of the intracellular astaxanthin synthesis pathway is constructed, thereby breaking through its own metabolic bottleneck and enabling the strain to have high production potential. (3) Glycerol domestication: Using glycerol of different concentration gradients as carbon sources, the strain domesticated by β-ionone in step (2) was continuously subcultured and domesticated. A mutant strain that can tolerate 20 g / L glycerol was screened out. Glycerol, an industrial by-product, was used to replace acetic acid or glucose, thereby getting rid of the dependence on high-priced organic carbon sources, reducing the cost of culture medium from the source, and the content of astaxanthin was also significantly increased.

[0011] This invention yielded a highly efficient astaxanthin-producing mutant strain of *Haematococcus pluvialis* through ARTP mutagenesis, β-ionone domestication, and glycerol domestication. Specifically, ARTP mutagenesis combined with β-ionone gradient domestication successfully overcame the feedback inhibition of the intracellular astaxanthin synthesis pathway, enabling the strain to overcome its own metabolic limitations. Furthermore, the introduction of a glycerol domestication strategy allowed the selected mutant strain to adapt to a high-concentration glycerol environment, effectively reducing culture costs.

[0012] Furthermore, the present invention also claims protection for the use of the above-mentioned highly efficient astaxanthin-producing Haematococcus pluvialis CO1 in the preparation of astaxanthin.

[0013] In addition, the present invention also provides a process for producing astaxanthin from Haematococcus pluvialis, which uses the above-mentioned Haematococcus pluvialis CO1 to induce production.

[0014] This invention optimizes the culture process for inducing astaxanthin production in Haematococcus pluvialis CO1, employing a three-stage process combining lightless heterotrophic growth, short-term low-light activation, and lightless salt stress induction. The short-term low-light treatment serves only as a signal stimulus to initiate the metabolic pathway, eliminating the need for a continuous lighting system and significantly reducing light energy consumption and equipment investment. The induction stage, using sodium chloride salt stress combined with low to moderate dissolved oxygen control, avoids the high energy consumption associated with maintaining high dissolved oxygen levels, reducing the aeration load on the fermenter. These mild induction conditions promote cell survival and product accumulation, improving process controllability.

[0015] Specifically, the process for producing astaxanthin from Haematococcus pluvialis provided by the present invention includes the following steps: Step S1: Inoculate the above-mentioned Haematococcus pluvialis CO1 into liquid fermentation medium II and culture it for 2-4 days under dark conditions at a temperature of 25-30 ℃; Step S2: After the dark heterotrophic culture is completed, stop nitrogen supplementation, adjust the culture temperature to 25~28 ℃, and then irradiate with white light for 0.5~6 h. Step S3: After the light exposure ends, resume dark culture, add sodium chloride, and culture at 22~25 ℃ for 4~6 days to obtain the product.

[0016] Furthermore, the liquid fermentation medium II in step S1 consists of the following components and their concentrations: 15~25 g / L glycerol, 2~3 g / L sodium nitrate, 1~3 g / L yeast extract, 0.1~0.5 g / L dipotassium hydrogen phosphate, 0.1~0.5 g / L magnesium sulfate, 0.01~0.05 g / L ferric ammonium citrate, 1~3 g / L sodium bicarbonate, 1~3 mL trace element stock solution.

[0017] Furthermore, the trace element mother liquor is composed of the following components and their contents: 2~3 g / L boric acid, 1~2 g / L manganese chloride tetrahydrate, 0.1~0.5 g / L zinc sulfate heptahydrate, 0.01~0.1 g / L copper sulfate pentahydrate, 0.1~0.5 g / L sodium molybdate dihydrate, 0.01~0.1 g / L cobalt nitrate hexahydrate.

[0018] Furthermore, the light-free culture conditions in step S1 are as follows: culture for 2 to 4 days under light-free conditions with a temperature of 25 to 30 ℃, an aeration rate of 0.1 to 0.4 vvm, a dissolved oxygen concentration of 10 to 30%, and a pH of 6.0 ± 0.5.

[0019] Furthermore, the intensity of white light in step S2 is 5~30 μmol / m² / s.

[0020] Further, the light-free culture conditions in step S3 are as follows: sodium chloride is added and its final concentration is adjusted to 0.2~0.6% (m / V), and cultured for 4~6 days at a temperature of 22~25 ℃, a dissolved oxygen concentration of 10~30%, and a sodium acetate concentration of 1.0~1.2 g / L.

[0021] Furthermore, the process for producing astaxanthin from Haematococcus pluvialis provided by this invention includes three stages: a lightless heterotrophic growth stage, a short-term weak light activation stage, and a lightless salt stress induction stage. The specific steps are as follows: Step S1, Heterotrophic Growth Stage Without Light: High-density cultivation under completely dark conditions with sufficient nitrogen sources allows for rapid accumulation of biomass.

[0022] (1) Streak the strain onto BG11 solid medium and incubate at 28 °C until a single colony appears. Pick a single colony and inoculate it into a 500 mL shake flask containing 300 mL of BG11 liquid medium. Incubate at 28 °C with shaking until the cell density reaches 102. 6 Approximately [number] cells / mL.

[0023] (2) Inoculate the bacterial culture into a 5 L fermenter containing 3 L of liquid fermentation medium II at a 10% inoculation rate and culture at 28 °C in the dark.

[0024] (3) Adjust the ventilation rate to 0.1~0.4 vvm, control the stirring speed to 100~300 rpm / min, control the dissolved oxygen to 20~30%, control the pH to 6.0, and culture for 3 days.

[0025] Step S2, Short-term weak light activation stage: Nitrogen supplementation was stopped, and the culture temperature was adjusted to 25–28 °C. A low-intensity (5–30 μmol / m² / s) short-term light treatment for 30 min–6 h was applied to activate astaxanthin synthesis gene expression through photo-signaling molecular regulation, thereby initiating the astaxanthin biosynthetic pathway. The light conditions at this stage were insufficient to support net carbon assimilation, and the algal cells did not experience significant biomass growth during this period. This prevented energy transfer towards cell division, ensuring that the metabolic flux in the subsequent induction phase was focused on the efficient accumulation of astaxanthin. Simultaneously, the culture temperature at this stage, in conjunction with the weak light signal, further optimized the catalytic efficiency of related synthases.

[0026] Step S3, Light-free salt stress induction stage: After restoring complete darkness, NaCl was added to the fermentation broth and its final concentration was adjusted to 0.4% (m / V) to induce cell transformation and astaxanthin synthesis using salt stress signals. At this point, the fermentation system mainly relied on glycerol as the carbon source, and the sodium acetate concentration only needed to be maintained at 1.0–1.2 g / L. The dissolved oxygen concentration was controlled at 15–20%, and this dissolved oxygen level was maintained by adjusting the air ventilation rate and stirring speed in real time. The cells were continuously cultured at 22–25 °C for 5 days to continuously induce efficient accumulation of astaxanthin.

[0027] In summary, compared with the prior art, the Haematococcus pluvialis mutant strain for high-efficiency astaxanthin production provided by the present invention has the following advantages: (1) This invention uses Haematococcus pluvialis CO1 for astaxanthin production, which can rapidly accumulate the biomass and astaxanthin content of the Haematococcus pluvialis mutant strain. Testing revealed that the Haematococcus pluvialis CO1, in a closed fermentation tank, achieved a cell dry weight of over 20 g / L and an astaxanthin content as high as 11%, significantly increasing the astaxanthin content of Haematococcus pluvialis and greatly reducing the production cost of astaxanthin.

[0028] (2) The present invention uses Haematococcus pluvialis CO1 for the production of astaxanthin, which has the advantages of high stability and short production cycle. Testing revealed that the fermentation cycle of Haematococcus pluvialis CO1 in a closed fermenter is about 6 days. At the 200 L fermenter level, the astaxanthin content of 5 batches remained stable between 10.92% and 11.25%, and the cell dry weight remained stable between 24.85% and 25.61 g / L, demonstrating strong batch-to-batch stability and suitability for large-scale industrial production.

[0029] Microbial information of Haematococcus pluvialis CO1 provided by this invention: Haematococcus pluvialis CO1 ( Haematococcus pluvialis C01 was deposited on April 3, 2026, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2026603, address: Wuhan University, Wuhan, China, 430072, China. Attached Figure Description

[0030] Figure 1 The graph shows the astaxanthin content and cell dry weight of different fermentation batches of Haematococcus pluvialis C01. Detailed Implementation

[0031] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0032] Example 1: Preparation of Culture Medium The culture media involved in this invention are shown in Table 1.

[0033] Table 1 Preparation of culture medium name Ingredients and their content BG11 liquid culture medium 1.5 g / L sodium nitrate, 0.04 g / L dipotassium hydrogen phosphate, 0.075 g / L magnesium sulfate, 0.036 g / L calcium chloride, 0.006 g / L citric acid, 0.006 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL of trace element stock solution; the trace element stock solution consists of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. BG11 solid culture medium The following ingredients were used: 1.5 g / L sodium nitrate, 0.04 g / L dipotassium hydrogen phosphate, 0.075 g / L magnesium sulfate, 0.036 g / L calcium chloride, 0.006 g / L citric acid, 0.006 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, 1 mL trace element stock solution, and 15 g / L agar powder; the trace element stock solution consisted of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Screening solid culture medium I The following ingredients were used: 1.23 g / L sodium acetate, 2.50 g / L sodium chloride, 0.01 g / L ferrous sulfate, 0.15 g / L sodium nitrate, 0.04 g / L dipotassium hydrogen phosphate, 0.075 g / L magnesium sulfate, 0.036 g / L calcium chloride, 0.006 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, 1 mL trace element stock solution, and 15 g / L agar powder; the trace element stock solution consisted of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Screening solid culture medium II The formula consists of 20 g / L glycerol, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, 2 mL trace element stock solution, 500 μmol / L β-ionone, and 15 g / L agar powder. The trace element stock solution is composed of 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Liquid fermentation medium I The following ingredients were used: 5 g / L sodium acetate, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution. The trace element stock solution consisted of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Liquid fermentation medium II 20 g / L glycerol, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution; the trace element stock solution consists of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Primary acclimatization culture medium 2 g / L glycerol, 4 g / L sodium acetate, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution; the trace element stock solution consists of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Secondary acclimatization culture medium The formula consists of 5 g / L glycerol, 3 g / L sodium acetate, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution. The trace element stock solution is composed of 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Tertiary acclimatization culture medium The formula consists of 10 g / L glycerol, 2 g / L sodium acetate, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution. The trace element stock solution is composed of 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Level IV acclimatization culture medium The formula consists of 15 g / L glycerol, 1 g / L sodium acetate, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution. The trace element stock solution is composed of 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Five-level acclimatization culture medium 20 g / L glycerol, 2.5 g / L sodium nitrate, 2 g / L yeast extract, 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L ferric ammonium citrate, 1 g / L sodium bicarbonate, and 2 mL of trace element stock solution; the trace element stock solution consists of: 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate. Example 2: Astaxanthin Detection Method After the Haematococcus pluvialis culture was completed, 1 mL of Haematococcus pluvialis cell culture medium was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded to obtain Haematococcus pluvialis cell precipitate. 5 mL of a mixture of 5% KOH and 30% CH3OH was added, and the mixture was incubated at 70 ℃ for 5 min to destroy chlorophyll. The mixture was then centrifuged at 5000 rpm for 5 min, and the supernatant was discarded to obtain the Haematococcus pluvialis cell precipitate. Next, 25 μL of glacial acetic acid and 1 mL of DMSO were added, and the mixture was incubated at 70 ℃ for 10 min, shaking constantly during incubation. The mixture was then centrifuged at 5000 rpm for 5 min, and the supernatant was collected.

[0034] Repeat the above steps, extracting the algal cells at least three times until the algae turn white. Dilute the algae 2-10 times with DMSO to a concentration of 100 mg / L, and determine the astaxanthin content (g astaxanthin / g cell dry weight) using a Waters 2996 liquid chromatograph at 492 nm.

[0035] Example 3: ARTP Mutagenesis and Screening Haematococcus pluvialis can synthesize astaxanthin under conditions of high light, high salinity, and nitrogen deficiency, but the production cycle is long and the yield is greatly affected by the environment. Using ARTP (Atmospheric and Room Temperature Plasma) multi-round mutagenesis technology, Haematococcus pluvialis was mutated, and high-yield astaxanthin mutant strains were screened under specific conditions.

[0036] (1) Preparation of bacterial suspension: Haematococcus pluvialis (from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences) was used to prepare the bacterial suspension. Haematococcus pluvialis Cells (labeled FACHB-876) were inoculated into BG11 liquid medium and cultured at 28°C until the cell concentration reached 10⁻⁶. 6 The cells were collected by centrifugation at approximately 100 cells / mL, then washed twice with physiological saline and resuspended in an equal volume of physiological saline. (2) ARTP mutagenesis treatment: The bacterial suspension from step (1) was evenly spread onto the surface of a sterile slide and transferred to the ARTP operating chamber. The position of the sample was adjusted so that the distance between it and the jet outlet of the plasma generator was about 5 mm. The gas flow rate was set to 10 SLM, the power was set to 120 W, and the time was controlled at 60 s. The treated sample was eluted into BG11 liquid medium and incubated in the dark at 28 ℃ for 24 hours. The bacterial suspension was then serially diluted and spread onto screening solid medium I and incubated until a single colony grew.

[0037] (3) Preliminary screening: Select single colonies from the plates in step (2), select independent single colonies one by one, activate them into a new screening solid medium I for further separation, obtain single colonies with higher purity, and select colonies with redder color for fermentation culture.

[0038] (4) Liquid fermentation verification: The reddish colonies screened in step (3) were inoculated into liquid fermentation medium I and cultured at 28°C in the dark for 10 days. 7 The concentration was approximately cells / mL. The temperature was then lowered to 26 °C, and the cells were irradiated with weak light (15 μmol / m² / s) for 2 hours under white light. Afterward, the cells were returned to a dark state, and sodium chloride was added to a final concentration of 4 g / L. Acetic acid was controlled at 1.2 g / L, and the cells were incubated at 24 °C for 5 days. The bacterial cells were collected, and the pigment was extracted. The astaxanthin concentration was determined by HPLC, following the specific detection method described in Example 2. Initial screening revealed that 8 bacterial strains had high astaxanthin content.

[0039] (5) Secondary screening: The strains obtained in the initial screening in step (4) are subjected to stability tests. The fermentation performance of the strains is evaluated through multiple fermentations, and finally a strain with better stability is selected.

[0040] (6) Multiple rounds of mutagenesis: Following the above operation, the strains obtained from the secondary screening were subjected to a second round of ARTP mutagenesis. After primary and secondary screening, a better strain was obtained. Then, a third round of ARTP mutagenesis was carried out, and finally a strain A01 with high astaxanthin content was selected, with an astaxanthin content of 2.95% and a cell dry weight of 15.58 g / L.

[0041] Example 3 obtained a mutant strain A01 that was independent of high light induction through ARTP mutagenesis, which provided a foundation for the domestication of subsequent strains.

[0042] Example 4: β-Ionone domestication The astaxanthin synthesis pathway involves negative feedback regulation by endogenous products. For example, excessively high β-carotene levels can inhibit the activity or expression of a key enzyme in carotenoid synthesis (phytoene synthase CrtYB, PSY) through an inhibitory mechanism. To eliminate this inhibitory effect, ARTP mutagenesis strains were domesticated using the β-carotene analog β-ionone, resulting in a gradual increase in astaxanthin content.

[0043] (1) The mutant strain A01 from Example 3 was inoculated onto BG11 solid medium and cultured at 28 °C until a single colony appeared. A single colony was picked and inoculated into BG11 liquid medium and cultured at 28 °C with shaking until the cell density reached 102. 6 Approximately [number] cells / mL.

[0044] (2) Dilute the bacterial solution from step (1) tenfold to 10. 3 10 4 The diluted solutions were spread onto BG11 solid medium containing different concentrations of β-ionone (20, 50, 100 μmol / L) and placed at 28 ℃ for acclimatization culture. The colony color was observed after 5-6 days of culture.

[0045] (3) Select the reddish colonies from step (2), ferment them using liquid fermentation medium I, extract the pigment, and detect the astaxanthin content. Refer to Example 2 for the specific detection method. The astaxanthin content of strain B01 was the highest, reaching 3.84%, and the cell dry weight was 13.96 g / L.

[0046] (4) Based on strain B01 in step (3), a second domestication was carried out, with β-ionone content of 100, 200, and 300 μmol / L respectively. Colonies with a redder color were selected for liquid fermentation culture, pigments were extracted, and the astaxanthin content was detected. The specific detection method is described in Example 2. The results showed that the astaxanthin content reached a maximum of 5.34%, and this strain was designated B02 with a cell dry weight of 11.36 g / L.

[0047] (5) Based on the B02 strain from step (4), a third domestication was performed, with β-ionone contents of 300, 400, and 500 μmol / L, respectively. Colonies with a redder color were selected for liquid fermentation culture, pigments were extracted, and the astaxanthin content was detected. The specific detection method is described in Example 2. The results showed that the highest astaxanthin content reached 6.91%, and this strain was designated B03 with a cell dry weight of 10.95 g / L.

[0048] (6) Subculture the B03 strain from step (5) 10 times on BG11 solid medium containing 500 μmol / L β-ionone until the strain can grow stably at this concentration.

[0049] Using β-ionone as the selection pressure for high-yield astaxanthin Haematococcus pluvialis, after multiple rounds of gradual domestication at different gradients, mutant strains that can relieve feedback inhibition were screened, and this performance can be stably inherited, providing a foundation for subsequent domestication of carbon sources.

[0050] Example 5: Autonomous domestication of carbon source glycerol To better apply the domesticated strain B03 to industrial production, glycerol was used as a carbon source to further increase the astaxanthin content.

[0051] (1) The strain B03 domesticated in Example 4 was placed on BG11 solid medium and cultured at 28 °C until a single colony appeared. A single colony was picked and inoculated into BG11 liquid medium and cultured at 28 °C with shaking until the cell density reached 102. 6 Approximately [number] cells / mL.

[0052] (2) Transfer 10% to liquid fermentation medium I and culture under dark conditions at a temperature of 28 °C.

[0053] (3) When the cell density in step (2) increases significantly, transfer the cells to the primary acclimatization medium at a 10% inoculation rate and culture them in the dark at 28 °C. After growth, continue to passage them three times in fresh primary acclimatization medium.

[0054] (4) Continue to transfer the bacterial culture to the secondary acclimatization medium and subculture it 4 times.

[0055] (5) Following the above strategy, the bacterial culture was continuously passaged and acclimatized in the three-level acclimatization medium, the four-level acclimatization medium and the five-level acclimatization medium, and each level was passaged 4 times.

[0056] (6) Dilute the last generation of bacterial culture from step (5) to 10. 3 10 4 The sample was spread onto screening solid medium II and incubated at 28 °C until a single colony grew.

[0057] (7) Select the redder, more prominent, and faster-growing colonies from step (6) and ferment them in liquid fermentation medium II. Extract the pigment and detect the astaxanthin content. Refer to Example 2 for the specific detection method. The results showed that the highest astaxanthin yield reached 7.59%. The strain was labeled as C01 and the cell dry weight was 19.28 g / L.

[0058] By using glycerol as a carbon source for domestication, Haematococcus pluvialis gradually adapted to the growth environment of glycerol, and the astaxanthin content was further increased. Strain C01 can be regarded as the best candidate strain for small-scale fermentation and pilot-scale amplification.

[0059] Example 6: 5 L fermenter culture test of Haematococcus pluvialis mutant strain C01 1. Fermentation culture: The strain C01 obtained from domestication in Example 5 was tested on a 5 L fermenter using a three-stage fermentation strategy, including a lightless heterotrophic growth stage, a short-term weak light activation stage, and a lightless salt stress induction stage.

[0060] Step S1, Heterotrophic Growth Stage Without Light: (1) Streak strain C01 onto BG11 solid medium and incubate at 28 °C until single colonies appear. Pick a single colony and inoculate it into a 500 mL shake flask containing 300 mL of BG11 liquid medium. Incubate at 28 °C with shaking until the cell density reaches 102. 6 Approximately [number] cells / mL.

[0061] (2) Inoculate the bacterial culture into a 5 L fermenter containing 3 L of liquid fermentation medium II at a 10% inoculation rate and culture at 28 °C in the dark.

[0062] (3) Adjust the ventilation rate to 0.1~0.4 vvm, control the stirring speed to 100~300 rpm / min, control the dissolved oxygen to 20~30%, control the pH to 6.0, and culture for 3 days.

[0063] Step S2, Short-term weak light activation stage: After the end of the lightless heterotrophic growth stage, the addition of nitrogen source to the fermenter was immediately stopped, and the lighting system was then turned on to enter the short-term weak light activation treatment step. In this stage, white light with an intensity of 15 μmol / m² / s was used for irradiation, and the single irradiation time was 2 h. The culture temperature was adjusted to 26 ℃.

[0064] Step S3, Light-free salt stress induction stage: After the light exposure ends and the fermentation broth is returned to a completely dark state, NaCl is added to the fermentation broth and its final concentration is adjusted to 0.4% (m / V). The concentration of sodium acetate is maintained at 1.0~1.2 g / L, and the dissolved oxygen concentration is controlled at 15~20%. The broth is then cultured at 24 ℃ for 5 days to obtain the final product.

[0065] 2. Test Results: After cultivation, pigments were extracted, and the astaxanthin content was detected. The specific detection method is described in Example 2. The results showed that the dry weight of Haematococcus pluvialis cells during the lightless heterotrophic growth stage was 20.43 g / L, and the astaxanthin content was 9.81%, indicating that this Haematococcus pluvialis mutant strain can achieve high-density cell fermentation and high astaxanthin production after three stages of cultivation.

[0066] Example 7: Scale-up culture test of Haematococcus pluvialis mutant strain C01 in a 200 L fermenter 1. Fermentation culture: Based on the control strategy of a 5 L fermenter, strain C01 was scaled up to a 200 L fermenter.

[0067] Step S1, Heterotrophic Growth Stage Without Light: 1.1 Seed liquid preparation: (1) Streak strain C01 onto BG11 solid medium and incubate at 28 °C until single colonies appear. Pick a single colony and inoculate it into a 500 mL shake flask containing 300 mL of BG11 liquid medium. Incubate at 28 °C with shaking until the cell density reaches 102. 6 Approximately [number] cells / mL.

[0068] (2) Inoculate the bacterial culture into a 5 L fermenter containing 3 L of liquid fermentation medium II at a 10% inoculation rate and culture at 28°C in the dark.

[0069] (3) Adjust the aeration rate to 0.1~0.4 vvm, the stirring speed to 100~300 rpm / min, the dissolved oxygen to 20~30%, the pH to 6.0, and incubate for 3 days.

[0070] 1.2. Heterotrophic growth in the absence of light: (1) Transfer the seed culture at a 10% inoculation rate to a 200 L fermenter containing 120 L of liquid fermentation medium II and culture at 28 °C in the dark for 3 days.

[0071] (2) Adjust the aeration rate to 0.1~0.4 vvm, the stirring speed to 100~300 rpm / min, the dissolved oxygen to 20~30%, the pH to 6.0, and incubate for 3 days.

[0072] Step S2, Short-term weak light activation stage: After the end of the lightless heterotrophic growth stage, the addition of nitrogen source to the fermenter was immediately stopped, and the lighting system was then turned on to enter the short-term weak light activation treatment step. In this stage, white light with an intensity of 15 μmol / m² / s was used for irradiation, and the single irradiation time was 2 h. The culture temperature was adjusted to 26 ℃.

[0073] Step S3, Light-free salt stress induction stage: After the light exposure ends and the fermentation broth is returned to a completely dark state, NaCl is added to the fermentation broth and its final concentration is adjusted to 0.4% (m / V). The concentration of sodium acetate is maintained at 1.0~1.2 g / L, and the dissolved oxygen concentration is controlled at 15~20%. The broth is then cultured at 24 ℃ for 5 days to obtain the final product.

[0074] 2. Test Results: After cultivation, pigments were extracted, and the astaxanthin content was detected. The specific detection method is described in Example 2. The results showed that the dry weight of Haematococcus pluvialis cells in the lightless heterotrophic growth stage was 25.58 g / L, and the astaxanthin content reached 11.06% after short-term weak light activation and lightless salt stress induction.

[0075] Example 8: Fermentation stability test of Haematococcus pluvialis mutant strain C01 To verify the stability of different fermentation batches of Haematococcus pluvialis C01, five batches were continuously produced in a 200 L fermenter.

[0076] Step S1, Heterotrophic Growth Stage Without Light: 1.1 Seed liquid preparation: (1) Streak strain C01 onto BG11 solid medium and incubate at 28 °C until single colonies appear. Pick a single colony and inoculate it into a 500 mL shake flask containing 300 mL of BG11 liquid medium. Incubate at 28 °C with shaking until the cell density reaches 102. 6 Approximately [number] cells / mL.

[0077] (2) Inoculate the bacterial culture into a 5 L fermenter containing 3 L of liquid fermentation medium II at a 10% inoculation rate and culture at 28°C in the dark.

[0078] (3) Adjust the aeration rate to 0.1~0.4 vvm, the stirring speed to 100~300 rpm / min, the dissolved oxygen to 20~30%, the pH to 6.0, and incubate for 3 days.

[0079] 1.2. Heterotrophic growth in the absence of light: (1) Transfer the seed culture at a 10% inoculation rate to a 200 L fermenter containing 120 L of liquid fermentation medium II and culture at 28 °C in the dark for 3 days.

[0080] (2) Adjust the aeration rate to 0.1~0.4 vvm, the stirring speed to 100~300 rpm / min, the dissolved oxygen to 20~30%, the pH to 6.0, and incubate for 3 days.

[0081] Step S2, Short-term weak light activation stage: After the end of the lightless heterotrophic growth stage, the addition of nitrogen source to the fermenter was immediately stopped, and the lighting system was then turned on to enter the short-term weak light activation treatment step. In this stage, white light with an intensity of 15 μmol / m² / s was used for irradiation, and the single irradiation time was 2 h. The culture temperature was adjusted to 26 ℃.

[0082] Step S3, Light-free salt stress induction stage: After the light exposure ends and the fermentation broth is returned to a completely dark state, NaCl is added to the fermentation broth and its final concentration is adjusted to 0.4% (m / V). The concentration of sodium acetate is maintained at 1.0~1.2 g / L, and the dissolved oxygen concentration is controlled at 15~20%. The broth is then cultured at 24 ℃ for 5 days to obtain the final product.

[0083] Five batches were produced through continuous fermentation using the above cultivation method.

[0084] 2. Test Results: After cultivation, the pigment was extracted, and the astaxanthin content was detected. The specific detection method is described in Example 2, and the results are as follows: Figure 1 As shown. Figure 1 This image shows the astaxanthin content and cell dry weight of different fermentation batches of Haematococcus pluvialis C01. Figure 1 It can be seen that the astaxanthin content is stable between 10.92% and 11.25% and the cell dry weight is stable between 24.85% and 25.61 g / L between different batches, which indicates that the stability between different batches is strong and suitable for industrial production.

[0085] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A highly efficient Haematococcus pluvialis C01 for astaxanthin production, characterized in that, The Haematococcus pluvialis C01 ( Haematococcus pluvialis C01) was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number: CCTCC NO: M 2026603.

2. The application of Haematococcus pluvialis CO1, as described in claim 1, for the efficient production of astaxanthin in the preparation of astaxanthin.

3. A process for producing astaxanthin from Haematococcus pluvialis, characterized in that, It is induced by Haematococcus pluvialis CO1 as described in claim 1.

4. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 3, characterized in that, Includes the following steps: Step S1: Inoculate the Haematococcus pluvialis CO1 as described in claim 1 into liquid fermentation medium II and culture it for 2-4 days under dark conditions at a temperature of 25-30 ℃; Step S2: After the dark heterotrophic culture is completed, stop nitrogen supplementation, adjust the culture temperature to 25~28 ℃, and then irradiate with white light for 0.5~6 h. Step S3: After the light exposure ends, resume dark culture, add sodium chloride, and culture at 22~25 ℃ for 4~6 days to obtain the product.

5. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 4, characterized in that, The liquid fermentation medium II in step S1 consists of the following components and their concentrations: 15~25 g / L glycerol, 2~3 g / L sodium nitrate, 1~3 g / L yeast powder, 0.1~0.5 g / L dipotassium hydrogen phosphate, 0.1~0.5 g / L magnesium sulfate, 0.01~0.05 g / L ferric ammonium citrate, 1~3 g / L sodium bicarbonate, 1~3 mL trace element stock solution.

6. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 5, characterized in that, The trace element mother liquor consists of the following components and their contents: 2~3 g / L boric acid, 1~2 g / L manganese chloride tetrahydrate, 0.1~0.5 g / L zinc sulfate heptahydrate, 0.01~0.1 g / L copper sulfate pentahydrate, 0.1~0.5 g / L sodium molybdate dihydrate, 0.01~0.1 g / L cobalt nitrate hexahydrate.

7. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 4, characterized in that, The light-free culture conditions in step S1 are as follows: culture for 2 to 4 days under light-free conditions with a temperature of 25 to 30 ℃, an aeration rate of 0.1 to 0.4 vvm, a dissolved oxygen concentration of 10 to 30%, and a pH of 6.0 ± 0.

5.

8. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 4, characterized in that, The intensity of white light in step S2 is 5~30 μmol / m² / s.

9. The process for producing astaxanthin from Haematococcus pluvialis as described in claim 4, characterized in that, The light-free culture conditions in step S3 are as follows: sodium chloride is added and its final concentration is adjusted to 0.2-0.6%, and cultured for 4-6 days at a temperature of 22-25 ℃, a dissolved oxygen concentration of 10-30%, and a sodium acetate concentration of 1.0-1.2 g / L.

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