Process for the fermentative production of astaxanthin

Abstract

The application provides a method for fermenting astaxanthin. The method comprises fermenting astaxanthin by using a Schizochytrium sp. with a preservation number of CCTCC M 2021564 as a fermentation strain. The strain is a mutant strain with high astaxanthin content, which is obtained by using a Schizochytrium sp. screened and separated from seawater as a starting strain and by mutagenesis screening, and the astaxanthin obtained by the strain contains a large amount of DHA astaxanthin ester, and has high economic value and application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a fermentation production method for astaxanthin. Background Technology

[0002] Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-diketoyl) is a type of carotenoid. Natural astaxanthin is mainly derived from Haematococcus pluvialis, Rhodotorula rubrum, shrimp, crab, and other crustaceans. However, animals cannot synthesize astaxanthin themselves and must obtain it from plants or algae through the food chain. Astaxanthin can quench singlet oxygen, scavenge free radicals, and prevent or terminate chain reactions caused by singlet oxygen and free radicals. Therefore, it possesses anti-cancer, anti-inflammatory, anti-aging, and anti-fatigue activities, prevents cardiovascular diseases, protects the optic nerve, enhances immunity, and effectively reduces the incidence of diabetes.

[0003] Docosahexaenoic acid (DHA) is an essential building block for brain cell formation, development, and function. Human memory and cognitive abilities rely on DHA for maintenance and enhancement. Supplementing with DHA can promote full brain cell development, slow cognitive decline, alleviate forgetfulness, and prevent Alzheimer's disease (senile dementia).

[0004] Recent studies have shown that astaxanthin esters have a higher absorption and utilization rate than free astaxanthin, especially astaxanthin esters with medium- and long fatty acid chains. Schizochytrium is an important strain for the industrialization of DHA (docosahexaenoic acid). It grows rapidly, can undergo large-scale liquid submerged fermentation, and possesses an intracellular astaxanthin biosynthetic pathway. These advantages provide a convenient foundation for the industrial production of astaxanthin. The conversion of the precursor acetyl-CoA to astaxanthin within Schizochytrium cells involves as many as 14 enzymatic reactions (…). Figure 1 The main destination of acetyl-CoA is the production of fatty acids, which makes it difficult to increase the content of astaxanthin through traditional fermentation processes, thus restricting the industrialization of astaxanthin production using Schizochytrium. Summary of the Invention

[0005] The purpose of this invention is to provide a fermentation production method for astaxanthin.

[0006] To achieve the objective of this invention, in a first aspect, this invention provides a fermentation production method for astaxanthin, the method comprising: using Schizochytrium glomeratum with preservation number CCTCC NO: M 2021564 as a fermentation strain to produce astaxanthin through fermentation.

[0007] In this invention, the astaxanthin includes DHA astaxanthin monoester and DHA astaxanthin diester, with structures shown in formula (I) and formula (II), respectively:

[0008]

[0009] The aforementioned method involves centrifuging to collect the bacterial cells after fermentation, followed by cell wall disruption and extraction with an extractant to obtain microbial oil containing carotenoids, including astaxanthin.

[0010] DHA (including free DHA and DHA in astaxanthin) accounts for more than 30% of the total fatty acids in astaxanthin.

[0011] Astaxanthin accounts for more than 1.0% of the total oil mass, preferably more than 1.5%, and more preferably more than 2.5%.

[0012] The carotenoids also include β-carotene, wherein the mass ratio of astaxanthin to β-carotene is greater than 1, preferably greater than 3.

[0013] After fermentation, the microbial oil (total oil) can reach more than 4% of the fermentation broth by mass.

[0014] The aforementioned method involves using alkaline protease to disrupt the cell walls of the bacteria, followed by repeated extraction with n-hexane. The mixed oil obtained after each extraction is filtered and desolventized to obtain microbial oil.

[0015] Secondly, the present invention provides a fermented product of Schizochytrium sp., the preparation method of which includes: using Schizochytrium sp. CABIO-A-2-III with preservation number CCTCC NO: M 2021564 as the fermentation strain to produce astaxanthin, and collecting the cell bodies by centrifugation after fermentation; or, extracting the cell bodies with an extractant after cell wall disruption to obtain microbial oil containing astaxanthin.

[0016] Schizochytrium sp. CABIO-A-2-III is now deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, China, accession number CCTCC NO: M 2021564, deposited on May 19, 2021.

[0017] The astaxanthin includes DHA astaxanthin monoester and DHA astaxanthin diester, the structures of which are shown in formula (I) and formula (II), respectively.

[0018] Astaxanthin accounts for more than 1.0% of the total oil mass, preferably more than 1.5%, and more preferably more than 2.5%. DHA (including free DHA and DHA in astaxanthin) accounts for more than 30% of the total fatty acids in astaxanthin.

[0019] The fermented product also includes β-carotene, and the mass ratio of astaxanthin to β-carotene is greater than 1, preferably greater than 3.

[0020] After fermentation, the microbial oil (total oil) can reach more than 4% of the fermentation broth by mass.

[0021] Thirdly, the present invention provides a carotenoid obtained by fermentation of Schizochytrium (preferably Schizochytrium with preservation number CCTCC NO: M2021564); the carotenoid includes astaxanthin and β-carotene, wherein the mass ratio of astaxanthin to β-carotene is greater than 1, preferably greater than 3.

[0022] The astaxanthin includes DHA astaxanthin monoester and DHA astaxanthin diester, the structures of which are shown in formula (I) and formula (II), respectively.

[0023] In this invention, the *Schizochytridactylogyrus* strain with accession number CCTCC NO: M 2021564 is a high-astaxanthin mutant strain obtained through mutagenesis screening using *Schizochytridactylogyrus* isolated from seawater as the starting strain. The astaxanthin content of this mutant strain is more than 7000 times higher than that of the starting strain, and the astaxanthin content in its carotenoids is more than three times that of β-carotene. Unexpectedly, its astaxanthin contains esterified astaxanthin, with more than 30% of the fatty acids in the esterified astaxanthin being DHA. This invention discloses for the first time the presence of esterified forms of polyunsaturated fatty acids and carotenoids in *Schizochytridactylogyrus*, namely, the DHA astaxanthin ester form.

[0024] In one specific embodiment of the present invention, the fermentation method of the Schizochytrium fungus with accession number CCTCC NO: M 2021564 includes:

[0025] 1. Seed activation culture: The Schizochytrium mutant strain was inoculated into the activation medium and cultured at 28℃ with a shaking speed of 200 r / min for 48 h to obtain the activated seed culture solution. The activation medium consisted of: glucose 10 g / L, sodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 20 g / L, magnesium sulfate 0.5 g / L, and natural pH.

[0026] 2. Seed Culture Expansion: The activated seed culture solution from step 1 was inoculated into a shake flask containing expansion medium at an inoculation rate of 10% (volume ratio) for cultivation (200 mL / L). The culture temperature was 28℃, and the culture time was 48 h. The shaking speed was 200 r / min to obtain the expanded seed culture solution. The expansion medium consisted of: glucose 40 g / L, monosodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 10 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.5 g / L, and natural pH.

[0027] 3. Fermentation in flasks: The expanded seed culture from step 2 was inoculated into fermentation flasks containing fermentation medium at a 10% (volume ratio) inoculation rate (40 mL / 250 mL). Fermentation was carried out at 28℃ for 120 hours using a shaker at 220 rpm. The fermentation medium consisted of: glucose 40 g / L, yeast extract 4 g / L, monosodium glutamate 30 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, calcium chloride 0.5 g / L, sodium bicarbonate 0.5 g / L, sodium sulfate 8 g / L, ammonium sulfate 6 g / L, potassium chloride 0.5 g / L, and natural pH.

[0028] After fermentation, the bacterial cells were collected by centrifugation and dried to obtain a bacterial powder containing 1.25% astaxanthin and 0.06% β-carotene. DHA accounted for 34.3% of the total fatty acids in the astaxanthin. After optimization of fermentation conditions, the astaxanthin content in the total lipids could reach over 2.5%. Under the same fermentation conditions, the astaxanthin content in the powder of the *Schizochytrium* starting strain (without mutagenesis) was only 0.0002% of the total oil and 0.005% of the total oil, and DHA was not detected in the astaxanthin.

[0029] 4. Post-processing: Take the fermentation broth obtained from fermentation culture and add a compound enzyme solution to break the cell wall (refer to the cell wall breaking method in paragraph 76 of the instruction manual in CN111378699A for enzymatic hydrolysis and cell wall breaking, then add the extractant n-hexane for extraction. The extraction process is protected by nitrogen purging. The solid phase obtained after extraction is transferred to the extraction vessel for repeated extraction. This process is repeated until there is no oil in the extract. After each extraction, filter and separate the mixed oil, remove solvent, and the obtained microbial oil contains carotenoids; the carotenoids include astaxanthin and β-carotene, and the astaxanthin includes DHA astaxanthin esters (including DHA astaxanthin monoester and DHA astaxanthin diester).

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] The astaxanthin of this invention is obtained by fermentation of Schizochytrium, wherein astaxanthin forms astaxanthin ester with DHA, which can give astaxanthin higher nutritional value and further expand its scope of application and effect.

[0032] The Schizochytrium strain provided by this invention, with accession number CCTCC NO: M 2021564, has a highly efficient ability to biosynthesize carotenoids. Using it for astaxanthin production can overcome the current production bottleneck of astaxanthin from Schizochytrium strains. This strain can be used to produce oil products with high astaxanthin content as well as deep-processed products, and has good application prospects. Attached Figure Description

[0033] Figure 1 This describes the astaxanthin synthesis pathway.

[0034] Figure 2 This is the mass spectrometry detection result of DHA astaxanthin monoester in this invention.

[0035] Figure 3 This is the mass spectrometry detection result of DHA astaxanthin diester in this invention. Detailed Implementation

[0036] This invention provides a strain for producing DHA astaxanthin ester, and a method for producing DHA astaxanthin ester.

[0037] 1. Methods for strain mutagenesis and high-throughput screening

[0038] 1.1 Mutagenesis

[0039] (1) The starting strain was Schizochytrium, which was isolated from seawater.

[0040] (2) Inoculate the bacterial strain into the activation medium and culture at a temperature of 28°C. Shake the incubator at a speed of 200 r / min for 48 h until the logarithmic growth phase.

[0041] (3) Take 1 ml of the activated seed culture medium from step (2) and air dry it under sterile conditions to form bacterial plaques. Aseptically transfer the culture dish containing the bacterial plaques into a high-energy particle beam injector and pass it through a high-energy N2D beam injector with an energy of 20 keV. + Ion beam implantation mutagenesis, N + Ion beam implantation dose 10 17 ions / cm 2 .

[0042] (4) Wash the bacterial film after the above mutagenesis treatment with sterile water, inoculate it into PDA medium, and culture at 28°C with a shaking speed of 200 r / min for 60 h until the stable period.

[0043] 1.2 Screening

[0044] The culture medium in step (4) was sterilely centrifuged, physiological saline was added to adjust the cell density and 1 mg / L Nile Red was added for staining. The mixture was then dispensed into the sample tubes of the flow cytometer. The algal cell resuspension was detected by the flow cytometer equipped with an argon ion laser and single-cell sorting was performed in the FL1 channel. The single colony with the strongest fluorescence signal superposition was selected.

[0045] The above steps are repeated multiple times. Due to the powerful and efficient sorting function of flow cytometry, manual operation is greatly reduced, and strains with high oil content and high astaxanthin concentration can be screened from a large number of mutant strains.

[0046] 2. Fermentation culture

[0047] The process was scaled up in shake flasks, 50L tanks, and 12-ton fermenters, and the changes in astaxanthin content were measured sequentially.

[0048] 3. Isolation of carotenoids

[0049] Carotenoids were separated using thin-layer chromatography (TLC). The TLC conditions were as follows: Silica gel was activated in a 110℃ oven for 2 hours and then packed into a column using a wet packing method. The column was then compacted with 3 column volumes of petroleum ether. 2 g of the microbial oil extracted after fermentation was dissolved in 5 mL of petroleum ether and loaded onto the column. Unreacted free fatty acids were first eluted with petroleum ether, then DHA astaxanthin diester was eluted with a petroleum ether / acetone (95:5, V / V) solution, and finally DHA astaxanthin monoester was eluted with a petroleum ether / acetone (90:10, V / V) solution. After elution, the monoester and diester fractions were collected separately and evaporated to dryness under reduced pressure to obtain DHA astaxanthin mono / diesters.

[0050] 4. Detection Method

[0051] 4.1 Determination of Carotenoid Content in Oils

[0052] Take 1 mL of bacterial culture into a 10 mL centrifuge tube, centrifuge at 13000 rpm for 1 min, and discard the supernatant; add 800 μL of ethyl acetate and 500 μL of grinding beads (1 mm), and freeze at -20℃ for 15 min to cool the bacterial culture; grind twice at 5000 rpm for 15 s, with a 20 s interval; repeat the above steps until the extract is colorless; blow dry with nitrogen, add 5 mL of 0.5 mol / L sodium hydroxide methanol solution, and shake well. Heat in a 60℃ water bath for 30-60 min, shaking every 5 min. Then remove, add 5 mL of BF3 methanol solution, and shake well. Heat in a 60℃ water bath for 30 min, shaking every 5 min. After the water bath, remove, cool to room temperature, add 1-10 mL of saturated saline, and allow to stand for separation. Transfer the sample tube to a centrifuge at 12,000 rpm for 5 minutes; extract the upper organic layer with a disposable syringe, pass it through a membrane (0.2 μm), and then test the sample on the centrifuge.

[0053] Carotenoids were detected by reversed-phase liquid chromatography (RP-HPLC) using a C18-filled YMC column. The chromatographic conditions were modified from the reference conditions provided by the column. Quantitative analysis was performed using the external standard method. HPLC conditions: a) Mobile phase A: methanol / MTBE / water (8:1 / 15 / 4, v / v); Mobile phase B: methanol / MTBE / water (7:90 / 3, v / v); b) Gradient elution: Mobile phase A from 0-90 min gradually from 100% to 0%; Mobile phase B from 0-90 min gradually from 0% to 100%; c) Total flow rate 1.0 mL / min; Column temperature 30℃ (maximum 50℃); Maximum column pressure 20 MPa; d) Detection wavelength 450 nm.

[0054] 4.2 Determination of DHA content in astaxanthin

[0055] Accurately weigh 0.1 g of astaxanthin sample into a 25 mL volumetric flask, and add 5 mL of tridecylglycerol internal standard solution. Add 5 mL of 0.5 mol / L sodium hydroxide methanol solution and shake well. Heat in a 60 °C water bath for 30-60 min, shaking every 5 min. Then remove from the heat, add 5 mL of BF3 methanol solution, and shake well. Heat in a 60 °C water bath for 30 min, shaking every 5 min. After the water bath, remove from the heat, cool to room temperature, add 1-10 mL of saturated saline solution, and allow to stand for phase separation. Filter using a disposable syringe and a 0.22 μm organic phase needle filter, and inject the supernatant into a gas chromatograph at a volume of 0.5-2.0 μL.

[0056] Chromatographic conditions for sample detection: Column: DB-23, 30m × 0.25mm, 0.25μm; Carrier gas: High-purity nitrogen (99.999%); Flow control: Constant pressure mode 12.1psi or 12.3psi; Injector temperature: 250℃; Detector temperature: 280℃ or 300℃; Column oven temperature program: Initial temperature 90℃, hold for 1 min, ramp to 240℃ at 9℃ / min, hold at 240℃ for 5 min, ramp to 250℃ at 3℃ / min, hold at 250℃ for 4 min; Injection mode: Split mode.

[0057] 4.3 Mass spectrometry detection method for DHA astaxanthin esters

[0058] DHA astaxanthin esters were detected using liquid chromatography-mass spectrometry (LC-MS). The LC conditions were as described in section 4.1, and the mass spectrometry conditions were as follows: APCI source, positive ion mode, primary MS mass scan range of m / z 300–1400. Nebulizer gas pressure 60 psi; drying gas (N2) flow rate 5 L / min, drying gas temperature 350 °C, ion source temperature 450 °C. Capillary pressure 3.7 kV, corona current 4 μA. Secondary MS used precursor and daughter ion scanning modes, fragmentation voltage 60 V, collision energy 25 eV, mass scan range of m / z 100–1500.

[0059] In this invention, the microbial oil comprises polyunsaturated fatty acids and fat-soluble components such as carotenoids and DHA. The polyunsaturated fatty acids may be in the form of free fatty acids, fatty acid salts, fatty acid esters, monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), and / or phospholipids (PL).

[0060] This invention discloses for the first time the presence of esterified forms of polyunsaturated fatty acids and carotenoids in Schizochytrium, namely the form of DHA astaxanthin ester.

[0061] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0062] Unless otherwise specified, the percentage sign "%" used in this invention refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0063] Example 1: Obtaining Schizochytrium, a fungus that produces astaxanthin esters

[0064] 1. Mutagenesis

[0065] (1) The starting strain was Schizochytrium isolated from seawater, see CN111235035A.

[0066] (2) Inoculate the bacterial strain into the activation medium and culture at a temperature of 28°C. Shake the incubator at a speed of 200 r / min for 48 h until the logarithmic growth phase.

[0067] (3) Take 1 ml of the activated seed culture medium from step (2) and air dry it under sterile conditions to form bacterial plaques. Aseptically transfer the culture dish containing the bacterial plaques into a high-energy particle beam injector and pass it through a high-energy N2D beam injector with an energy of 20 keV. + Ion beam implantation mutagenesis, N + The ion beam implantation dose is 10 17 ions / cm 2 .

[0068] (4) The bacterial film after the above mutagenesis treatment was washed off with sterile water and inoculated into a low-nitrogen, low-sugar Schizochytrium culture medium. The culture temperature was 28℃, the shaking speed of the shaker was 200r / min, and the culture was carried out for 60h until the stationary phase.

[0069] 2. Screening

[0070] Centrifuge the culture medium in step (4) aseptically, and add physiological saline to adjust the cell density to 1×10⁻⁶. 5 -1×10 6 The concentration of the algal cell suspension was between CFU / mL (cells / mL), and Nile red staining was added (the final concentration of Nile red was 1 mg / L). The suspension was then dispensed into the sample tubes of the flow cytometer. The flow cytometer, equipped with an argon ion laser, was used to detect the single-cell sorting of the algal cell resuspension in the FL1 channel. The single colony with the strongest fluorescence signal was selected for further verification.

[0071] The above steps were repeated multiple times. Due to the powerful and efficient sorting function of the flow cytometer, manual operation was greatly reduced. Mutants with high oil content and high astaxanthin concentration could be screened from a large number of mutant strains (preservation number CCTCC NO: M 2021564).

[0072] Example 2: Shake Flask Fermentation Culture

[0073] (1) Seed activation culture: The Schizochytrium mutant strain was inoculated into the activation medium and cultured at a temperature of 28℃, a shaking speed of 200r / min, and a culture time of 48h. The activation medium consisted of: glucose 10g / L, sodium glutamate 25g / L, yeast extract 10g / L, sodium chloride 20g / L, magnesium sulfate 0.5g / L, and natural pH.

[0074] (2) Seed expansion culture: The activated seed culture solution from step (1) was inoculated into a shake flask containing expansion culture medium at an inoculation rate of 10% (volume ratio) for culture (200 mL / L). The culture temperature was 28℃, the culture time was 48 h, and the shaking speed of the shaker was 200 r / min. The expansion culture medium consisted of: glucose 40 g / L, sodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 10 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.5 g / L, and natural pH.

[0075] (3) Fermentation in a fermentation bottle: The expanded seed culture broth from step (2) was inoculated into a fermentation bottle containing fermentation medium at an inoculation rate of 10% (volume ratio) (40 mL / 250 mL). The culture temperature was 28℃, the culture time was 120 h, and the shaking speed on the shaker was 220 r / min. The fermentation medium consisted of: glucose 40 g / L, yeast extract 4 g / L, monosodium glutamate 30 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, calcium chloride 0.5 g / L, sodium bicarbonate 0.5 g / L, sodium sulfate 8 g / L, ammonium sulfate 6 g / L, potassium chloride 0.5 g / L, and natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by adding glucose continuously.

[0076] After fermentation, microbial oil accounted for 3.8% of the fermentation broth by mass.

[0077] After fermentation, the bacterial cells are collected by centrifugation and dried to obtain bacterial powder (dried bacterial cells) with a total oil content of 45%.

[0078] (4) Post-processing: Take the fermentation broth and add the compound enzyme solution to break the cell wall (refer to the cell wall breaking method in paragraph 76 of the instruction manual in CN111378699A). Then add the extractant n-hexane for extraction. Nitrogen protection is provided during the extraction process. The solid phase obtained after extraction is transferred to the extraction vessel for repeated extraction. This process is repeated until there is no oil in the extract. After each extraction, filter the mixed oil obtained after extraction, desolvent it, and obtain the microbial oil.

[0079] (5) Detection

[0080] The obtained microbial oil and its carotenoids were detected using the aforementioned method. The astaxanthin content in the oil was 1.25%, and the β-carotene content was 0.06%. The astaxanthin contained astaxanthin esters (including DHA astaxanthin monoester and DHA astaxanthin diester). Their mass spectrometry detection results are as follows: Figure 2 and Figure 3 As shown in the figure, DHA accounted for 34.3% (mass percentage) of the total fatty acid content in astaxanthin.

[0081] Example 3: Expanded Culture

[0082] 1. 50L tank fermentation

[0083] The seed activation and seed expansion culture methods are the same as in Example 2.

[0084] Fermentation culture: The expanded seed culture broth from step (2) was inoculated at a rate of 10% (volume ratio) into a 50L fermenter containing 30L of fermentation medium for fermentation culture. The culture temperature was 28℃, the culture time was 120h, and the rotation speed was 220r / min. The fermentation medium consisted of: glucose 40g / L, yeast extract 4g / L, monosodium glutamate 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 5g / L, calcium chloride 0.5g / L, sodium bicarbonate 0.5g / L, sodium sulfate 8g / L, ammonium sulfate 6g / L, potassium chloride 0.5g / L, and pH was natural. During fermentation, the carbon source concentration in the fermentation broth was controlled at 10g / L by adding glucose, and the pH of the fermentation broth was controlled between 6.8 and 7.2 by adding citric acid.

[0085] The obtained microbial oil and its carotenoids were detected using the aforementioned method. The total oil content in the dried bacterial cells was 43.5%, the astaxanthin content in the oil was 2.50%, the β-carotene content was 0.1%, and the astaxanthin contained astaxanthin ester. The DHA content in the astaxanthin was found to account for 36.4% (mass percentage) of the total fatty acid content.

[0086] Comparative Example 1:

[0087] (1) Seed activation culture: Unmutated Schizochytrium fungi were inoculated into activation culture medium and cultured at 28℃ with a shaking speed of 200r / min for 48h. The activation culture medium consisted of 10g / L glucose, 25g / L sodium glutamate, 10g / L yeast extract, 20g / L sodium chloride, 0.5g / L magnesium sulfate, and natural pH.

[0088] (2) Seed expansion culture: The activated seed culture solution from step (1) was inoculated into a shake flask containing expansion culture medium at an inoculation rate of 10% (volume ratio) for culture (200 mL / L). The culture temperature was 28℃, the culture time was 48 h, and the shaking speed of the shaker was 200 r / min. The expansion culture medium consisted of: glucose 40 g / L, sodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 10 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.5 g / L, and natural pH.

[0089] (3) Fermentation in a fermentation bottle: The expanded seed culture broth from step (2) was inoculated into a fermentation bottle containing fermentation medium at an inoculation rate of 10% (volume ratio) (40 mL / 250 mL). The culture temperature was 28℃, the culture time was 120 h, and the shaking speed on the shaker was 220 rpm. The fermentation medium consisted of: 40 g / L glucose, 4 g / L yeast extract, 30 g / L monosodium glutamate, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.5 g / L sodium bicarbonate, 8 g / L sodium sulfate, 6 g / L ammonium sulfate, 0.5 g / L potassium chloride, and natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by adding glucose continuously.

[0090] After fermentation, microbial oil accounted for 4.3% of the fermentation broth by mass, and the total oil content in the dry cells was 49.3%.

[0091] (4) Post-processing: The same cell wall breaking method as in Example 2 was used.

[0092] The astaxanthin content in the oil obtained above was 0.0002%, the β-carotene content was 0.005%, and DHA was not detected in the astaxanthin.

[0093] Example 4 50m 3 Large-scale fermentation

[0094] Seed propagation: The propagated seed culture solution obtained from step (2) of Example 2 was successively selected at a volume ratio of 10% to 50L, 100L, and 1.7m³. 3 12m 3 The seed culture medium was gradually expanded in the seed tank (60% full), cultured at 28℃ for 120 hours, and shaken at 220 rpm. The fermentation medium consisted of: glucose 40 g / L, yeast extract 4 g / L, monosodium glutamate 30 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, calcium chloride 0.5 g / L, sodium bicarbonate 0.5 g / L, sodium sulfate 8 g / L, ammonium sulfate 6 g / L, potassium chloride 0.5 g / L, and natural pH.

[0095] Fermentation culture: The above-mentioned expanded seed culture solution was inoculated into containers of 30 ml at an inoculation rate of 10% (v / v). 3 50m of fermentation medium 3Fermentation was carried out in a fermenter at a temperature of 28℃ for 120 hours with a stirring speed of 220 rpm. During fermentation, the carbon source concentration in the fermentation broth was controlled at 10 g / L by adding glucose, and the pH of the fermentation broth was controlled between 6.8 and 7.2 by adding citric acid. The fermentation medium consisted of: 40 g / L glucose, 4 g / L yeast extract, 30 g / L monosodium glutamate, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.5 g / L sodium bicarbonate, 8 g / L sodium sulfate, 6 g / L ammonium sulfate, and 0.5 g / L potassium chloride, with a natural pH.

[0096] After fermentation, the microbial oil accounted for 5.0% of the fermentation broth by mass.

[0097] The aforementioned method was used to extract and detect the obtained microbial oil and its carotenoids. The oil content in the dried bacterial cells was 44.3%, the astaxanthin content was 2.72%, the β-carotene content was 0.12%, and the astaxanthin contained astaxanthin ester. The DHA content in the astaxanthin was found to be 36.5% (mass percentage) of the total fatty acid content.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A fermentation production method for astaxanthin, characterized in that, The Schizochytrium fungus with accession number CCTCC M 2021564 ( Schizochytrium sp.) is used as a fermentation strain for the fermentation production of astaxanthin.

2. The method according to claim 1, characterized in that, The astaxanthin comprises DHA astaxanthin monoester and DHA astaxanthin diester, with structures shown in formula (I) and formula (II), respectively: Equation (I) Equation (II).

3. The method according to claim 2, characterized in that, DHA accounts for more than 30% of the total fatty acids in astaxanthin.

4. The method according to claim 2, characterized in that, After fermentation, the cells were collected by centrifugation, and after cell wall disruption, they were extracted with an extractant to obtain microbial oil containing carotenoids, including astaxanthin.

5. The method according to claim 4, characterized in that, Astaxanthin accounts for more than 1.0% of the total oil mass.

6. The method according to claim 5, characterized in that, Astaxanthin accounts for more than 1.5% of the total oil mass.

7. The method according to claim 6, characterized in that, Astaxanthin accounts for more than 2.5% of the total oil mass.

8. The method according to claim 4, characterized in that, Carotenoids also include β-carotene, with the mass ratio of astaxanthin to β-carotene greater than 1.

9. The method according to claim 8, characterized in that, The mass ratio of astaxanthin to β-carotene in carotenoids is greater than 3.

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