Chlorella sorokiniana M6, application thereof and method for synchronously synthesizing lutein and protein

Abstract

The invention belongs to the technical field of microorganisms, and discloses chlorella sorokiniana M6, application of the chlorella sorokiniana M6 and a method for synchronously synthesizing xanthophyll and protein. The preservation number of the Chlorella sorokiniana M6 provided by the invention is GDMCC NO.66205, the Chlorella sorokiniana M6 has an excellent capability of co-producing lutein and protein, and is almost free of chlorophyll synthesis, so that the Chlorella sorokiniana M6 has very important economic value and practical significance for realizing commercial application of microalgae protein and lutein production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to Chlorella sorokiniana M6 and application thereof and a method for simultaneously synthesizing lutein and protein. BACKGROUND

[0002] Lutein is widely distributed in plants, algae and certain microorganisms, contains multiple conjugated double bonds and hydroxyl groups in the structure, can react with singlet oxygen, hydroxyl radicals and other free radicals to reduce the damage of oxidative stress to cells, and has excellent antioxidant biological activity.

[0003] At present, the methods for preparing lutein mainly include extraction, chemical synthesis and microbial synthesis. Among them, in the natural plant extraction method, biological materials rich in lutein are directly used as raw materials for extraction, which has the disadvantages of being limited by raw materials and solvent residues. In the chemical synthesis method, the conjugated double bond structure of lutein is constructed by organic chemical reaction to synthesize lutein, which has the problems of multiple isomers and serious pollution. In the microbial synthesis method, lutein is synthesized by the metabolic activity of microalgae, fungi, bacteria and other microorganisms, which can well adapt to the needs of sustainable development, and is a hot direction in the preparation process of lutein in recent years.

[0004] Chlorella is one of the commonly used materials in the natural plant extraction method, and is also an important advantage algal species of microalgae protein. It can produce lutein through different nutritional modes, has the advantages of easy scale-up culture and easy comprehensive utilization of biomass, and is one of the commonly used algae species in the production of lutein by microbial synthesis method. However, Chlorella is limited by intracellular metabolism such as key enzyme activity, precursor competition and metabolic diversion, improper control of fermentation conditions and oxidative degradation, and has the problems of low lutein yield, low protein content and a large amount of chlorophyll and other impurities in the obtained product, which need complex purification operation, etc., which has great limitations. SUMMARY

[0005] The first object of the present application is to solve the problems of low lutein yield, low protein content and a large amount of chlorophyll in the application of Chlorella in the microbial synthesis of lutein, and to provide Chlorella sorokiniana M6. The Chlorella sorokiniana M6 has excellent ability to co-produce lutein and protein, and almost does not synthesize chlorophyll, which has very important economic value and practical significance for realizing the commercial application of microalgae protein and lutein production.

[0006] A second objective of this invention is to provide the application of the above-mentioned Chlorella sorokiniana M6 in the preparation of lutein.

[0007] A third objective of this invention is to provide the application of the above-mentioned Chlorella sorokiniana M6 in the preparation of proteins.

[0008] A fourth objective of this invention is to provide a composition for the simultaneous synthesis of lutein and protein.

[0009] The fifth objective of this invention is to provide a method for the simultaneous synthesis of lutein and protein.

[0010] Specifically, the Chlorella sorokiniana M6 provided by this invention has the accession number GDMCC NO.66205.

[0011] This invention also provides the application of the above-mentioned Chlorella sorokiniana M6 in the preparation of lutein.

[0012] This invention also provides the application of the above-mentioned Chlorella sorokiniana M6 in protein preparation.

[0013] The composition for the simultaneous synthesis of lutein and protein provided by this invention comprises the above-mentioned Chlorella sorokiniana M6, a basal fermentation medium, a fed fermentation medium, and an induction fed medium; wherein, the basal fermentation medium comprises 10 g / L to 50 g / L glucose, 1 g / L to 2 g / L urea, 0.01 g / L to 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L to 0.6 g / L magnesium sulfate, 0.1 g / L to 0.2 g / L calcium chloride, 0.001 g / L to 0.01 g / L ferrous sulfate, 0.05 g / L to 0.1 g / L disodium EDTA, 0.001 g / L to 0.005 g / L manganese chloride, 0.6 mg / L to 1 mg / L zinc sulfate, 0.003 mg / L to 0.008 mg / L copper sulfate, and 10 mg... The fermentation feed medium consists of 600 g / L to 800 g / L glucose, 12 g / L to 18 g / L urea, 10 g / L to 15 g / L dipotassium hydrogen phosphate, 10 g / L to 15 g / L magnesium sulfate, and 1 g / L to 4 g / L trisodium citrate; the induction feed medium consists of 300 g / L to 320 g / L glucose, 360 g / L to 380 g / L urea, 20 g / L to 30 g / L dipotassium hydrogen phosphate, 20 g / L to 30 g / L magnesium sulfate, 3 g / L to 5 g / L trisodium citrate, and 15 μg / L to 25 μg / L vitamin B12.

[0014] The method for simultaneous synthesis of lutein and protein provided by this invention uses the above-mentioned composition for simultaneous synthesis of lutein and protein to synthesize lutein and protein, and includes the following steps: Chlorella sorokiniana M6 is inoculated into a fermentation basal medium and cultured in the dark; during the dark fermentation process, when the glucose concentration in the fermentation system is first consumed to no more than 5 g / L, the fermentation feed medium is used for feed treatment; when the glucose concentration in the fermentation system is consumed again to no more than 5 g / L, induction feed medium is added for induced expression.

[0015] Furthermore, the inoculum amount of Chlorella sorokiniana M6 is 8% (v / v) to 12% (v / v).

[0016] Furthermore, the temperature for the light-protected fermentation culture is 28℃~35℃, the dissolved oxygen content of the fermentation broth is 18%~25%, the stirring speed is 100rpm~800rpm, and the pH is 6~7.

[0017] Furthermore, in the fed-batch treatment, the amount of the fed-batch fermentation medium added is 30% (v / v) to 35% (v / v) based on the volume of the basal fermentation medium.

[0018] Furthermore, in the induced expression, the amount of the induction feed medium added is 30% (v / v) to 35% (v / v) based on the volume of the fermentation basal medium.

[0019] Furthermore, the induction time is 80h to 90h.

[0020] Biological Preservation

[0021] The Chlorella sorokiniana M6 provided by this invention was deposited on April 23, 2025, with accession number GDMCC NO.66205. The depository is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0022] Figure 1 The figure shows the experimental results of biomass testing of mutant algal strains C1 to C39 obtained by one EMS mutagenesis in Example 1 of this invention;

[0023] Figure 2 The figure shows the experimental results of testing the lutein content of mutant algal strains C1 to C39 obtained by EMS mutagenesis in Example 1 of this invention.

[0024] Figure 3 The figure shows the experimental results of biomass testing of mutant algal strains M1 to M17 obtained by secondary EMS mutagenesis in Example 1 of this invention.

[0025] Figure 4 The figure shows the experimental results of lutein content testing of mutant algal strains M1 to M17 obtained by secondary EMS mutagenesis in Example 1 of this invention.

[0026] Figure 5 The growth curves of the original algal strain 348, the mutant algal strain M2, and the mutant algal strain M6 obtained by secondary EMS mutagenesis in Example 1 of this invention are shown.

[0027] Figure 6 The images show the colony morphology of Chlorella sorokiniana 348 and M6 provided in Example 2 of this invention.

[0028] Figure 7The figure shows the experimental results of testing the lutein and protein content of Chlorella sorokiniana 348 and M6 provided in Example 2 of this invention. Detailed Implementation

[0029] Based on the existing problems of low lutein yield, low protein content, and large amounts of chlorophyll and other impurities in the products obtained from Chlorella used in lutein synthesis, the inventors of this invention, using Chlorella sorokiniana 348 (preserved in the Microalgae Germplasm Resource Bank of the Third Institute of Oceanography, Ministry of Natural Resources) as the original algal strain, conducted multiple mutagenesis treatments on the original algal strain using EMS chemical mutagenesis. Using algal colony color as the initial judgment criterion and biomass and lutein content as screening indicators, they creatively obtained a new Chlorella sorokiniana strain, M6. This M6 Chlorella sorokiniana has a deep yellow algal colony color, synthesizes almost no chlorophyll, and possesses excellent co-production capabilities of lutein and protein, showing promising application prospects in the large-scale production of microalgal proteins and lutein. Based on this, the technical solution of this invention was obtained.

[0030] This invention provides a Chlorella sorokiniana M6, which has the accession number GDMCC NO.66205.

[0031] This invention provides the application of the above-mentioned Chlorella sorokiniana M6 in the preparation of lutein.

[0032] This invention provides the application of the above-mentioned Chlorella sorokiniana M6 in protein preparation.

[0033] The present invention provides a composition for the simultaneous synthesis of lutein and protein, the composition specifically comprising the above-mentioned Chlorella sorokiniana M6, a fermentation basal medium, a fermentation fed medium, and an induction fed medium.

[0034] In this invention, the fermentation basal culture medium serves as the basic nutrient supply for supporting the growth, reproduction, and metabolic activities of *Chlorella sorokinina* M6. Specifically, it includes: 10 g / L to 50 g / L of glucose, specifically 10 g / L, 12 g / L, 18 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, or any value between these values; and 1 g / L to 2 g / L of urea, specifically 1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or 1.8 g / L. 0.01 g / L to 0.2 g / L of dipotassium hydrogen phosphate, specifically 0.01 g / L, 0.05 g / L, 0.08 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, or any value between them; 0.2 g / L to 0.6 g / L of magnesium sulfate, specifically 0.2 g / L, 0.28 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, or any value between them; 0.1 g / L to 0.2 g / L of calcium chloride, specifically 0.1 g / L, 0.13 g / L, 0.15 g / L, 0.18 g / L, 0.2 ...8 g / L, 0.13 g / L, 0.15 g / L, 0.18 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.28 g / L, 0.13 g / L, 0.15 g / L, 0.18 g / L, 0.28 g / L, 0.28 g / L, 0.13 g / L, 0.15 g / L, 0.2 g / L or any value between them; ferrous sulfate from 0.001 g / L to 0.01 g / L, specifically 0.001 g / L, 0.003 g / L, 0.005 g / L, 0.008 g / L, 0.01 g / L or any value between them; disodium ethylenediaminetetraacetate from 0.05 g / L to 0.1 g / L, specifically 0.05 g / L, 0.06 g / L, 0.075 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L or any value between them; manganese chloride from 0.001 g / L to 0.005 g / L, specifically 0.001 g / L, 0.002 g / L... 0.003 g / L, 0.0035 g / L, 0.004 g / L, 0.005 g / L, or any value between them; zinc sulfate at concentrations of 0.6 mg / L to 1 mg / L, specifically 0.6 mg / L, 0.65 mg / L, 0.7 mg / L, 0.78 mg / L, 0.8 mg / L, 0.825 mg / L, 0.9 mg / L, 1 mg / L, or any value between them; copper sulfate at concentrations of 0.003 mg / L to 0.008 mg / L, specifically 0.003 mg / L, 0.004 mg / L, 0.005 mg / L, 0.006 mg / L, 0.007 mg / L, 0.003 mg / L, 0.0035 g / L, 0.004 g / L, 0.005 g / L, 0.005 g / L, or any value between them.0.008 mg / L or any value between them; boric acid at concentrations of 10 mg / L to 20 mg / L, specifically 10 mg / L, 12 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 18 mg / L, 20 mg / L or any value between them; and cobalt nitrate at concentrations of 0.01 mg / L to 0.02 mg / L, specifically 0.01 mg / L, 0.015 mg / L, 0.0175 mg / L, 0.02 mg / L or any value between them; and a pH of 6 to 7, specifically 6, 6.2, 6.4, 6.5, 6.7, 6.9, 7 or any value between them.

[0035] In this invention, the fermentation feed medium is used to dynamically regulate the fermentation process to achieve stable cell growth through fermentation culture. Specifically, it includes: 600 g / L to 800 g / L of glucose, specifically 600 g / L, 610 g / L, 625 g / L, 650 g / L, 670 g / L, 700 g / L, 750 g / L, 800 g / L, or any value between these values; 12 g / L to 18 g / L of urea, specifically 12 g / L, 13 g / L, 14 g / L, 15 g / L, 15.75 g / L, 16 g / L, 17 g / L, 18 g / L, or any value between these values; and 10 g / L to 15 g / L of urea. The concentrations of dipotassium hydrogen phosphate (DHP) can be 10 g / L, 11 g / L, 12 g / L, 12.5 g / L, 13 g / L, 14 g / L, 15 g / L, or any value between them; magnesium sulfate (10 g / L to 15 g / L) can be 10 g / L, 11 g / L, 12.5 g / L, 13 g / L, 14 g / L, 15 g / L, or any value between them; and trisodium citrate (1 g / L to 4 g / L) can be 1 g / L, 1.5 g / L, 1.8 g / L, 2 g / L, 2.1 g / L, 2.3 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, or any value between them.

[0036] In this invention, the induction feeding medium is used for targeted supplementation of inducing agents and regulation of the nutrient environment to activate *Chlorella sorokinense*. Specific metabolic pathways related to lutein synthesis within *Sorbus stokiniana* M6 cells enhance the efficiency and yield of lutein and protein synthesis. These pathways include: 300 g / L to 320 g / L of glucose (specifically 300 g / L, 305 g / L, 310 g / L, 313 g / L, 318 g / L, 320 g / L, or any value between these values); 360 g / L to 380 g / L of urea (specifically 360 g / L, 363 g / L, 368 g / L, 370 g / L, 373 g / L, 375 g / L, 379 g / L, 380 g / L, or any value between these values); and 20 g / L to 30 g / L of dipotassium hydrogen phosphate (specifically 20 g / L, 21 g / L, 23 g / L, or any value between these values). L, 25g / L, 28g / L, 29g / L, 30g / L or any value between them; 20g / L to 30g / L magnesium sulfate, specifically 20g / L, 22g / L, 25g / L, 27g / L, 29g / L, 30g / L or any value between them; 3g / L to 5g / L trisodium citrate, specifically 3g / L, 3.2g / L, 3.5g / L, 3.8g / L, 4g / L, 4.2g / L, 4.5g / L, 4.9g / L, 5g / L or any value between them; 15μg / L to 25μg / L vitamin B12, specifically 15μg / L, 18μg / L, 20μg / L, 23μg / L, 25μg / L or any value between them.

[0037] This invention provides a method for the simultaneous synthesis of lutein and protein. The method uses the above-mentioned composition for the simultaneous synthesis of lutein and protein to synthesize lutein and protein, and specifically includes the following steps: taking the above-mentioned Chlorella sorokiniana M6 and inoculating it into a fermentation basal medium for light-protected fermentation culture.

[0038] In this invention, during the light-protected fermentation process, when the glucose concentration in the fermentation system is initially consumed to a level not exceeding 5 g / L, the fermentation feed medium is used for feeding. When the glucose concentration in the fermentation system is again consumed to a level not exceeding 5 g / L, induction feed medium is added to induce expression. This segmented light-protected fermentation mode allows the growth of the algal strain and the synthesis of lutein and protein to be independent, optimizing the growth state of the algal cells and increasing the yield per unit cell. This is of great significance for realizing the large-scale industrial co-production of lutein and protein.

[0039] In this invention, the inoculum amount of Chlorella sorokiniana M6 is preferably 8% (v / v) to 12% (v / v), specifically 8% (v / v), 8.2% (v / v), 8.5% (v / v), 9% (v / v), 9.5% (v / v), 10% (v / v), 11% (v / v), 12% (v / v) or any value between them.

[0040] In this invention, the conditions for the light-protected fermentation culture specifically include: the temperature is preferably 28℃~35℃, specifically 28℃, 29℃, 30℃, 31℃, 32℃, 34℃, 35℃ or any value between them; the dissolved oxygen content of the fermentation broth is preferably 18%~25%, specifically 18%, 19%, 20%, 21%, 23%, 25% or any value between them; the stirring speed is preferably 100rpm~800rpm, specifically 100rpm, 120rpm, 150rpm, 200rpm, 230rpm, 250rpm, 300rpm, 450rpm, 500rpm, 600rpm, 700rpm, 800rpm or any value between them; the pH is preferably 6~7, specifically 6, 6.3, 6.5, 6.8, 7 or any value between them.

[0041] In this invention, during the feeding process, the amount of the feeding medium added is preferably 30% (v / v) to 35% (v / v) based on the volume of the fermentation basal medium, specifically 30% (v / v), 31% (v / v), 32.5% (v / v), 33% (v / v), 34% (v / v), 35% (v / v) or any value between them.

[0042] In this invention, during the induced expression, the amount of the induction feed medium added is preferably 30% (v / v) to 35% (v / v) based on the volume of the fermentation basal medium, specifically 30% (v / v), 31.3% (v / v), 32% (v / v), 33.8% (v / v), 34% (v / v), 35% (v / v), or any value between them.

[0043] In this invention, the induction time is preferably 80h to 90h, specifically 80h, 81h, 83h, 84h, 86h, 88h, 90h or any value between them.

[0044] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0045] The algal strains and reagents involved in the following examples specifically include:

[0046] (1) Chlorella sorokiniana 348: Deposited at the Microalgae Germplasm Resource Bank of the Third Institute of Oceanography, Ministry of Natural Resources.

[0047] (2) MM liquid culture medium: including 10 g / L glucose, 0.6183 g / L urea, 0.1 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.15 g / L calcium chloride, 0.005 g / L ferrous sulfate, 0.075 g / L disodium ethylenediaminetetraacetate, 0.0035 g / L manganese chloride, 0.825 mg / L zinc sulfate, 0.005 mg / L copper sulfate, 15 mg / L boric acid and 0.0175 mg / L cobalt nitrate, with a pH of 6.5 ± 0.5.

[0048] (3) MM solid medium: The culture is basically the same as that of MM liquid medium, except that it also includes 2.0 wt% agar.

[0049] (4) Fermentation basal medium: 30 g / L glucose, 1.5 g / L urea, 0.1 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.15 g / L calcium chloride, 0.005 g / L ferrous sulfate, 0.075 g / L disodium ethylenediaminetetraacetate, 0.0035 g / L manganese chloride, 0.825 mg / L zinc sulfate, 0.005 mg / L copper sulfate, 15 mg / L boric acid and 0.0175 mg / L cobalt nitrate, pH 6.5 ± 0.5.

[0050] (5) Fermentation feed medium: including 750 g / L glucose, 15.75 g / L urea, 12.5 g / L dipotassium hydrogen phosphate, 12.5 g / L magnesium sulfate and 2.1 g / L trisodium citrate.

[0051] (6) Induction feeding medium: including 313 g / L glucose, 373 g / L urea, 25 g / L dipotassium hydrogen phosphate, 25 g / L magnesium sulfate, 4.2 g / L trisodium citrate and 20 μg / L vitamin B12.

[0052] Example 1

[0053] This embodiment uses Chlorella sorokiniana 348 as the original strain and constructs Chlorella sorokiniana M6 using EMS chemical mutagenesis, specifically including:

[0054] 1. Activation of the original strain: Take 1.2 mL of Chlorella sorokiniana 348 (cell density 1×10⁻⁸) 7 The algal colonies (cells / mL) were inoculated into MM liquid medium and mixed evenly to obtain algal solution; 100 μL of algal solution was spread onto MM solid medium and cultured upside down in the dark at 27℃ for 7 days. Then, a single algal colony was picked and inoculated into 100 mL of liquid medium and mixed evenly. The colonies were cultured in the dark at 160 rpm and 30℃ for 7 days to obtain the original seed algal solution.

[0055] 2. First EMS mutagenesis: (1) Take 20 mL of the original seed algal solution and centrifuge at 6500 rpm for 10 min. Remove the supernatant, collect the algal cells, and wash the algal cells 3 times with sterile water. Then, take the algal cells and mix them evenly with 10 mL of 1.2 M ethyl methanesulfonate solution. After mutagenesis treatment in the dark for 30 min, add 10 mL of 1 wt% sodium thiosulfate solution and mix evenly. Centrifuge at 6500 rpm for 10 min, remove the supernatant, collect the algal cells, and wash the algal cells 3 times with sterile water to obtain the first mutagenized algal cells.

[0056] (2) Mix 20 mL of MM liquid culture medium with the first-stage mutant algal cells, let stand at room temperature for 3 days, and then dilute 10, 10, and so on. 2 10 3 10 4 10 5 and 10 6 After dilution, 100 μL of the diluted algal solution was spread onto MM solid medium and cultured at 37°C in the dark until single algal colonies appeared. 39 algal colonies with lightened green color (light green, golden yellow, dark yellow, etc.) were obtained and named mutant algal strains C1 to C39.

[0057] (3) Mutant strains C1 to C39 were inoculated into 100 mL of MM liquid medium at an inoculation rate of 10 mL. After culturing in the dark at 160 rpm and 30 °C for 7 days, the cultures were centrifuged at 6500 rpm for 10 min, the supernatant was discarded, and the algal cells were collected. The algal cells were then vacuum-dried to constant weight to obtain the biomass (in g / L) of each mutant algal strain. The results are as follows: Figure 1 As shown.

[0058] (4) Take the vacuum-dried algal powder and add it to a 1:2 mixture of methanol and dichloromethane. Sonicate in an ice bath for 15 min, centrifuge at 6500 rpm for 5 min, collect the supernatant, and obtain the test solution. Perform high-performance liquid chromatography (HPLC) on the test solution to obtain the lutein content (mg / g) of each mutant algae. The results are as follows: Figure 2 As shown.

[0059] Depend on Figure 1 and 2 It can be seen that, compared with the original algal strain Chlorella sorokiniana 348, the biomass of mutant strains C2, C4, C9, C13, C14 and C23 was significantly increased, with the biomass increase being more than 1.2 times; while the lutein content of mutant strains C13, C20, C21 and C23 was significantly increased. Therefore, mutant strains C13, C20, C21 and C23 were selected for secondary EMS mutagenesis.

[0060] 3. Secondary EMS mutagenesis: (1) Following the method provided in “1. Activation of the original strain”, the mutant algal strains C13, C20, C21 and C23 were activated to obtain seed algal solution.

[0061] (2) Following the method provided in step (1) of “2. EMS mutagenesis”, the seed algal solution was subjected to mutagenesis treatment. Mutant algal strain C13 was mutated to produce 5 algal colonies with yellow color and larger algal colony diameter, which were named mutant algal strains M1 to M5. Mutant algal strain C20 was mutated to produce 9 algal colonies with yellow color and larger algal colony diameter, which were named mutant algal strains M6 to M14. Mutant algal strain C21 was mutated to produce 3 algal colonies with yellow color and larger algal colony diameter, which were named M15 to M17. Mutant algal strain C23 did not produce any algal colonies after mutagenesis.

[0062] (3) The biomass and lutein content of the obtained mutant algal strains M1-M17 were tested according to the methods provided in "2. Steps (3) and (4) of the first EMS mutagenesis". The results are as follows: Figures 3-5 As shown.

[0063] Depend on Figures 3-5 The results show that the biomass of the mutant algal strains M2 and M6 were 3.54 g / L and 3.49 g / L, respectively, and the lutein contents were 12.45 g / L and 13.02 g / L, respectively. Furthermore, the mutant algal strain M6 exhibited a faster growth rate, reaching a cell density of 2.97 × 10⁻⁶ cells / mL when it reached the stationary growth phase. 8 The cells / mL was 1.43 times that of the mutant strain M2, and Chlorella sorokiniana M6 was obtained through screening.

[0064] 4. Biological Preservation: Chlorella sorokiniana M6 was sent to the Guangdong Provincial Microbial Culture Collection Center on April 23, 2025 for preservation and confirmed to be viable. The preservation number is GDMCC NO.66205.

[0065] Example 2

[0066] This embodiment illustrates the application of Chlorella sorokiniana M6 provided in Example 1 in the simultaneous synthesis of lutein and protein, with the original Chlorella sorokiniana 348 as a control. Specifically, it includes:

[0067] 1. Preparation of seed algal solution: (1) Take 1.2 mL of Chlorella sorokiniana 348 and M6 (cell density of 1×10⁻⁶) respectively. 7 The algae (cells / mL) were inoculated into MM liquid medium and mixed thoroughly to obtain an algal solution. 100 μL of the algal solution was spread onto MM solid medium and incubated upside down in the dark at 27°C for 7 days. The algal colony morphology was observed, and the results are as follows: Figure 6 As shown.

[0068] Depend on Figure 6 The results show that, compared to the original Chlorella sorokiniana 348, the colony color of Chlorella sorokiniana M6 is dark yellow.

[0069] (2) Select a single algal colony and inoculate it into 100 mL of liquid culture medium. Mix well and culture in the dark at 160 rpm and 30 ℃ for 7 days to obtain seed algal solution.

[0070] 2. Fermentation culture of seed algae: Take 10% (v / v) of seed algae and inoculate it into 1.5L of fermentation basal medium. Fermentation culture was carried out in the dark under the conditions of dissolved oxygen of 20%, stirring speed of 200rpm, temperature of 30℃ and pH of 6.5±0.5.

[0071] During the light-protected fermentation process, 507 mL of fermentation feed medium was added when glucose was first consumed to 5 g / L; when glucose in the fermentation system was consumed again to below 5 g / L, 516 mL of induction feed medium was added and expression was induced for 84 h.

[0072] 3. Determination of lutein and protein content: (1) The lutein content of Chlorella sorokiniana 348 and M6 was tested according to the method provided in "2. Step (1) of the first EMS mutagenesis" in Example 1. The results are as follows: Figure 7 As shown.

[0073] (2) The protein content of Chlorella sorokiniana 348 and M6 was determined by the Kjeldahl method, and the results are as follows: Figure 7 As shown.

[0074] Depend on Figure 7 The results show that, compared with the original Chlorella sorokiniana 348, the Chlorella sorokiniana M6 provided by this invention has increased lutein and protein content, with the lutein content increasing by 1.91 times. This Chlorella sorokiniana M6 has excellent ability to synthesize lutein and protein simultaneously, and has broad prospects for industrial application.

[0075] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A Chlorella sorokiniana M6, characterized in that, The preservation number of Chlorella sorokiniana M6 is GDMCC NO.66205.

2. The application of Chlorella sorokiniana M6 as described in claim 1 in the preparation of lutein.

3. The use of Chlorella sorokiniana M6 as described in claim 1 in the preparation of proteins.

4. A composition for the simultaneous synthesis of lutein and protein, characterized in that, The composition comprises Chlorella sorokiniana M6 as described in claim 1, a fermentation basal medium, a fermentation fed medium, and an induction fed medium; The fermentation basal culture medium comprises 10 g / L to 50 g / L glucose, 1 g / L to 2 g / L urea, 0.01 g / L to 0.2 g / L dipotassium hydrogen phosphate, 0.2 g / L to 0.6 g / L magnesium sulfate, 0.1 g / L to 0.2 g / L calcium chloride, 0.001 g / L to 0.01 g / L ferrous sulfate, 0.05 g / L to 0.1 g / L disodium ethylenediaminetetraacetate, 0.001 g / L to 0.005 g / L manganese chloride, 0.6 mg / L to 1 mg / L zinc sulfate, 0.003 mg / L to 0.008 mg / L copper sulfate, 10 mg / L to 20 mg / L boric acid, and 0.01 mg / L to 0.02 mg / L cobalt nitrate, with a pH of 6 to 7. The fermentation feed medium includes 600 g / L to 800 g / L of glucose, 12 g / L to 18 g / L of urea, 10 g / L to 15 g / L of dipotassium hydrogen phosphate, 10 g / L to 15 g / L of magnesium sulfate and 1 g / L to 4 g / L of trisodium citrate. The induction feeding medium comprises 300 g / L to 320 g / L glucose, 360 g / L to 380 g / L urea, 20 g / L to 30 g / L dipotassium hydrogen phosphate, 20 g / L to 30 g / L magnesium sulfate, 3 g / L to 5 g / L trisodium citrate, and 15 μg / L to 25 μg / L vitamin B12.

5. A method for simultaneously synthesizing lutein and protein, characterized in that, The method uses the composition for simultaneous synthesis of lutein and protein as described in claim 4 to synthesize lutein and protein, and includes the following steps: taking the Chlorella sorokiniana M6 and inoculating it into a fermentation basal medium for light-protected fermentation culture; During the light-protected fermentation process, when the glucose concentration in the fermentation system is consumed to a level not exceeding 5 g / L for the first time, the fermentation feed medium is used for feeding treatment; when the glucose concentration in the fermentation system is consumed to a level not exceeding 5 g / L again, induction feed medium is added for induced expression.

6. The method for simultaneous synthesis of lutein and protein according to claim 5, characterized in that, The inoculum size of Chlorella sorokiniana M6 was 8% (v / v) to 12% (v / v).

7. The method for simultaneous synthesis of lutein and protein according to claim 5, characterized in that, The light-protected fermentation culture was conducted at a temperature of 28℃ to 35℃, with a dissolved oxygen content of 18% to 25% in the fermentation broth, a stirring speed of 100 rpm to 800 rpm, and a pH of 6 to 7.

8. The method for simultaneous synthesis of lutein and protein according to claim 7, characterized in that, In the fed-batch treatment, the amount of the fed-batch culture medium added is 30% (v / v) to 35% (v / v) based on the volume of the basal fermentation medium.

9. The method for simultaneous synthesis of lutein and protein according to claim 7, characterized in that, In the induced expression, the amount of the induction feed medium added is 30% (v / v) to 35% (v / v) based on the volume of the fermentation basal medium.

10. The method for simultaneous synthesis of lutein and protein according to claim 7, characterized in that, The induction time is 80h to 90h.

Images