Method for screening and cultivating yellow chlorella pyrenoidosa mutant strain capable of rapidly growing
Through random mutagenesis treatment of plasma at normal pressure room temperature, Chlorella yellowin protein Chlorella mutant strain was screened, which solved the decolorization problem of Chlorella when used in food, achieved rapid growth and high biomass, reduced heterotrophic costs, and broadened the application market.
Patent Information
- Application Number
- CN202411701106.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-02-25
AI Technical Summary
When used in food, the existing Chlorella dry powder is mostly green or dark green, resulting in poor appearance and low consumer acceptance. At the same time, the existing decolorization methods are complicated to operate or the use of organic solvents lead to environmental pollution and high costs.
Through random mutagenesis treatment of plasma at normal pressure room temperature, chlorophyll-deficient Chlorella mutant strains were screened and cultivated, and their rapid growth and high biomass under heterotrophic conditions were used to solve the decolorization problem and optimize the carbon source concentration to improve growth efficiency.
The chlorophyll content of Chlorella ributycete mutant strain obtained was significantly lower than that of wild type, with fast growth rate, high biomass, and stable color traits, which solved the problem of decolorization of Chlorella in industrial applications, reduced heterotrophic costs, and broadened the Chlorella application market.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of microalgae breeding, and in particular to a method for screening and cultivating a fast-growing yellow protein-core Chlorella mutant. Background Art
[0002] In recent years, the world's population has continued to grow, and limited natural resources can hardly meet the global demand for food and feed. About 57% of the world's protein supply comes from plant sources (almost all from terrestrial plants); the remaining 43% comes from livestock and poultry. As the global greenhouse effect intensifies and environmental pollution leads to the depletion of land resources, traditional crops and meat products will find it difficult to maintain the food gap. Based on the fact that environmental protection, food demand and healthy eating concepts are deeply rooted in people's minds, it is urgent to establish a big food concept and develop a diversified food supply system that combines animals, plants and microorganisms, which has also promoted the entry of new proteins into the food market.
[0003] Chlorella has attracted much attention as a new type of green, healthy and sustainable food. Compared with traditional crops, it has a short growth cycle, a protein content of 50-60%, and is rich in a variety of active substances. It was approved as a new resource food by the state as early as 2012. It is also recognized as a "green and healthy food" by the Food and Agriculture Organization of the United Nations. At present, the market share of microalgae is also climbing year by year. The latest report released by Transparency Market Research shows that between 2020 and 2030, the global microalgae market is expected to grow at a healthy annual compound growth rate of about 5%, and the market value will reach US$3.2 billion by the end of 2030. Despite the above advantages, chlorella powder is only used as a colorant or added to food for marketing purposes, rather than as a food to replace traditional food. This is attributed to the fact that chlorella powder is mostly green or dark green, and adding it to food will result in poor appearance and low consumer acceptance. However, the existing decolorization methods, resin and activated carbon adsorption methods, are cumbersome in operation; however, the organic solvents used in the organic solvent extraction method are easy to pollute the environment, and the treatment of sewage further increases the cost, resulting in a significant increase in the decolorization cost in the industry, and a large amount of nutrients in the algae will be lost after treatment. Several methods have been developed for breeding new mutant algae strains, among which random mutagenesis is widely used as a non-genetic mutation method to mutate new algae species. Studies have shown that chemical reagents can also effectively reduce the chlorophyll content in algae and obtain chlorophyll-deficient Chlorella, but mutagens have caused environmental pollution and pose a certain threat to human health. The use of atmospheric pressure room temperature plasma technology can effectively induce strains to form mutants, while being simple to operate and environmentally friendly. It can be used as an important tool for breeding new algae species in the laboratory and efficiently and safely screen out target algae species.
[0004] Therefore, it is necessary to use normal pressure room temperature plasma for random mutagenesis treatment in order to obtain a new chlorophyll-deficient Chlorella mutant to solve the decolorization problem in algae powder processing, which is of great significance for the cultivation of new algae species and the application of Chlorella industry. Summary of the invention
[0005] The main purpose of the present invention is to provide a method for screening and cultivating fast-growing yellow protein-core Chlorella, the chlorophyll content of the yellow protein-core Chlorella is significantly lower than that of the wild-type protein-core Chlorella, and the biomass is significantly higher than that of the wild-type, which can solve the technical problem of decolorization of Chlorella in industrial applications, realize the cultivation of new algae species, and is more suitable for heterotrophic conditions than the original algae strain, and is more suitable for industrial fermentation.
[0006] To achieve the above object, the present invention provides the following technical solution: a method for screening and cultivating a mutant strain of Chlorella vulgaris capable of rapid growth, comprising the following steps:
[0007] Step 1: Algae strain activation: After streaking the wild-type algae strain in solid culture medium, pick a single algae colony for shake flask culture in the dark, culture it to the logarithmic growth phase and perform multiple subcultures to maintain its cell activity;
[0008] Step 2: Dilute the algae strain in the logarithmic growth phase with glycerol, perform mutagenesis treatment using an atmospheric pressure room temperature plasma mutagenizer (ARTP), and apply the treated algae suspension to a solid culture medium;
[0009] Step 3: Using color as the screening target, pick the yellow mutant with a sterile toothpick and place it in a fresh solid culture medium for multiple passages until the color phenotype is stable. After streaking the yellow mutant, pick a single algae colony and place it in a shake bottle for dark culture to obtain a purified yellow protein-core Chlorella mutant strain.
[0010] Step 4: Inoculate the yellow mutant algae strain into liquid culture medium with different glucose concentrations (5-50 g / L), culture in the dark at 25-30°C and 140 rpm on a shaker for 4-6 days;
[0011] Step 5: Collect the algae liquid, centrifuge, wash and dry, measure the biomass and glucose consumption, and screen for the optimal carbon source concentration;
[0012] Step 6: Cultivate Chlorella vulgaris at the optimal carbon source concentration, take samples every day to measure the biomass, and determine its growth curve and pigment content.
[0013] Furthermore, the culture medium used was BG-11 basal culture medium, and 10 g / L glucose was added to the BG-11 culture medium as a carbon source to heterotrophically culture the wild-type Chlorella pyrenoidosa until the logarithmic growth phase.
[0014] Furthermore, the second step is specifically performed as follows: dilute the wild-type protein-nucleated Chlorella in the logarithmic growth phase with an equal volume of 10% glycerol to an OD680 between 0.6 and 0.8 to obtain an algae suspension, aspirate 10uL of the algae suspension and evenly apply it to a sterile slide, place it in an atmospheric pressure room temperature plasma mutagenizer (ARTP) for treatment, place the slide treated with ARTP in 1mL90% physiological saline, and evenly apply the algae solution of each treatment group to a fresh solid culture medium of BG-11 containing 10g / L glucose at 28°C for heterotrophy.
[0015] Furthermore, the pyrenoid Chlorella in the first step is the wild type W, and the yellow pyrenoid Chlorella screened in the third step is defined as the mutant A19.
[0016] Furthermore, in the fourth step, 5 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L of glucose were added to the culture medium for heterotrophy.
[0017] Furthermore, the specific operation of the fifth step is as follows: Biomass determination: take the algae liquid into a weighed centrifuge tube and centrifuge it, wash it and re-centrifuge it 2-3 times, then place the algae precipitate in a 60°C vacuum drying oven and dry it for 12 hours to constant weight, and then weigh it to determine the biomass;
[0018] Glucose residue: Take 1 mL of the supernatant after centrifugation and add it to a colorimetric tube. Add 2 mL of DNS reagent and boil for 2 min. After cooling, dilute with pure water to 15 mL scale and measure the absorbance at 490 nm.
[0019] Furthermore, the method for determining the chlorophyll content is as follows: collect the cultured algae liquid, wash it with ddH2O after centrifugation, repeat the centrifugation twice, collect the algae precipitate and freeze-dry it to obtain algae powder, weigh a certain amount of freeze-dried algae powder, grind it sufficiently under the protection of liquid nitrogen, extract it with methanol, and extract it at 4°C in the dark until the algae residue turns white, collect the supernatant, and carry out the entire operation under light-proof conditions. Use a spectrophotometer to measure the absorbance at 665.2nm, 652.4nm and 470nm.
[0020] Furthermore, a method for obtaining and detecting lutein is provided: freeze-dried algae powder is weighed, low-temperature grinding is performed, anhydrous ethanol is added, and the mixture is shaken and extracted. The supernatant is collected by centrifugation, and anhydrous ethanol is added to the precipitate again and shaken and extracted until the algae powder turns white. The extract is collected, and the extract is centrifuged, the supernatant is taken and dried with nitrogen, and anhydrous ethanol is added to dissolve the pigment. The pigment is passed through a membrane and analyzed by high performance liquid chromatography. The whole process is carried out in the dark.
[0021] Furthermore, Chlorella vulgaris must be cultured in a BG-11 medium containing 5 g / L glucose under dark conditions for 4 days.
[0022] The present invention has at least the following beneficial effects:
[0023] 1. The present invention uses wild-type protein-nucleated Chlorella as the starting algae strain, uses a normal pressure room temperature plasma mutagenizer to treat it, and obtains yellow chlorophyll-deficient protein-nucleated Chlorella A19 through color screening. The mutant cell color is yellow, and the color trait is stable. Under heterotrophic conditions, the chlorophyll content is significantly lower than that of the wild type, but the growth rate is fast, the biomass is higher than that of the wild type, and its color, chlorophyll content and biomass have not changed significantly after multiple subcultures and inoculations. The successful cultivation of this mutant strain not only solves the technical problem of decolorization in algae powder production applications, but also broadens the application market of Chlorella and reduces the cost of heterotrophically obtaining high-density biomass. At the same time, other microalgae species can also use this method to mutate algae species, providing a new direction for laboratory cultivation of new algae species;
[0024] 2. The invention mainly includes the process steps of purification and activation culture of wild-type pyrenoid Chlorella, normal pressure and room temperature plasma mutagenesis treatment of algal cells, screening and purification culture of pyrenoid Chlorella color mutants, optimization of the optimal carbon source, and biomass and chlorophyll detection. The obtained pyrenoid Chlorella mutants have higher production and application value. Secondly, compared with the traditional Chlorella dry powder decolorized and used in food processing, it is safer and more efficient. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a photo of the wild-type Chlorella pyrenoidosa plate purification in Example 1;
[0026] Figure 2 This is a plate purification culture diagram of the chlorophyll-deficient proteoglenoid Chlorella A19 obtained by screening after atmospheric pressure and room temperature plasma mutagenesis in Example 2;
[0027] Figure 3 Example 3 is the residual glucose content of the mutant strain of Chlorella pyrenoidosa at different glucose concentrations under heterotrophic conditions at 27°C on the fourth day of cultivation;
[0028] Figure 4 Example 4 is a comparison of the growth curves of wild-type Chlorella pyrenoidosa and chlorophyll-deficient mutant strains under heterotrophic conditions at 27°C;
[0029] Figure 5 Example 5 is a comparison of chlorophyll a and chlorophyll b contents between wild-type Chlorella pyrenoidosa and chlorophyll-deficient mutant strains under heterotrophic conditions at 27°C;
[0030] Figure 6 Example 6 is a comparison of lutein content between wild-type Chlorella pyrenoidosa and chlorophyll-deficient mutant strains under heterotrophic conditions at 27°C;
[0031] Figure 7Example 7 is a comparison of protein content between wild-type Chlorella pyrenoidosa and chlorophyll-deficient mutant strains under heterotrophic conditions at 27°C;
[0032] Figure 8 Example 8 is a comparison of the amino acid composition and content of wild-type Chlorella pyrenoidosa and the chlorophyll-deficient mutant. DETAILED DESCRIPTION
[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0034] Example 1: Purification and activation culture of wild-type Chlorella pyrenoidosa;
[0035] The obtained wild-type Chlorella was streaked on a plate, and a single colony of the wild-type protein-core Chlorella was picked and expanded on a solid plate. The single colony of the wild-type protein-core Chlorella that can grow stably was inoculated into a 100 mL shaking bottle containing 40 mL of liquid culture medium, and heterotrophically cultured at 27°C for multiple generations to stabilize its growth activity. When it grows to the logarithmic phase, it is inoculated into a newly prepared culture medium at a 10% (v / v) inoculation amount for shaking heterotrophic culture as a seed solution for ARTP mutagenesis experiment.
[0036] The basic components of the heterotrophic culture medium include: NaNO3, K2HPO4·2H2O, MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, H3BO3, MnSO4·H2O, ZnSO4·7H2O, CuSO4·5H2O and (NH4)6MO7O24·4H2O, 5-10 g / L of glucose. After high temperature and high pressure sterilization, the heterotrophic culture medium suitable for protein nucleus Chlorella is obtained.
[0037] Example 2: Random mutagenesis and screening of chlorophyll-deficient Chlorella pyrenoidosa mutants;
[0038] The wild-type protein-nucleated Chlorella algae liquid grown to the logarithmic phase is diluted with 10% glycerol (v / v) to an OD680 between 0.6 and 0.8, the diluted algae suspension is evenly applied to a sterile slide, and treated under the protection of ultrapure helium for 0 to 25 seconds, the treated slide is placed in sterile physiological saline, the algae cells are vortexed, and then evenly applied to a BG-11 solid culture medium containing 10 g / L glucose, and cultured at 27° C. in the dark for 10 to 15 days until the algae colonies are completely grown, and different protein-nucleated Chlorella monoclonal mutants can be obtained.
[0039] After the algae colonies have fully grown, the color of the single algae colonies is used as the screening target for the first screening. Compared with the single algae colonies of the wild-type pyrenoid Chlorella, the algae colonies with light green, yellow or white visible to the naked eye are selected as mutants, which are picked with toothpicks and stored in BG-11 solid culture medium. The yellow pyrenoid Chlorella mutants obtained are passaged multiple times in BG-11 solid culture medium to determine the stability of their color traits.
[0040] Example 3: The mutant strain of Chlorella vulgaris was cultured under different glucose concentration conditions;
[0041] The yellow protein nucleus Chlorella colony cultured on the BG-11 plate was picked up into a shaking bottle for heterotrophic activation culture, and the culture medium was consistent with Example 1. In order to determine the optimal carbon source concentration, a 250mL conical flask was used as a culture container, 100mL of BG-11 culture medium was transferred, and the activated yellow protein nucleus Chlorella was inoculated at 10% (v / v) into the newly prepared culture medium for heterotrophy, and the glucose concentration gradient in the culture medium was set to 5g / L, 10g / L, 20g / L, 30g / L, and 40g / L. The conical flasks were placed in a constant temperature shaker, the speed was set to 140rpm, the temperature was set to 27°C, and the culture time was 4 days. On the fourth day, the algae solution was collected, the supernatant was centrifuged to measure the glucose content in the supernatant, and its glucose consumption was determined.
[0042] Glucose content determination: take 2mL of algae liquid, centrifuge at 5000rpm for 5min, take 1mL of supernatant and add it to 25mL colorimetric tubes, add 2mL of DNS reagent to each colorimetric tube, place in a boiling water bath for 2min, cool in running water, dilute to 15mL mark with ddH2O, use a spectrophotometer to measure the absorbance at 490nm, and calculate the glucose content in the supernatant.
[0043] Example 3 Effect Analysis: The mutants of Chlorella vulgaris were grown heterotrophically under different glucose concentrations. The glucose content in the culture solution of each treatment group on the 4th day of culture was compared. Figure 3 As shown: The optimal carbon source for the yellow protein nucleus Chlorella mutant is 5 g / L. When cultured in BG-11 heterotrophic medium containing 5 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L glucose for 4 days, the glucose content in the culture medium was 1.13 g / L, 6.02 g / L, 17.42 g / L, 27.72 g / L, and 37.09 g / L, respectively.
[0044] Example 4: Comparison of growth curves of wild-type Chlorella pyrenoidosa and yellow Chlorella pyrenoidosa mutants under heterotrophic conditions;
[0045] Wild-type pyrenoid Chlorella and yellow pyrenoid Chlorella mutants in the exponential growth phase were inoculated into BG-11 liquid culture medium, and the culture conditions were the same as those in Example 3, except that 5 g / L glucose was added as a heterotrophic carbon source. Algal liquid was collected every day and the biomass was measured.
[0046] Biomass determination: 2 mL of algae solution was taken into a pre-weighed centrifuge tube, centrifuged at 5000 rpm for 5 min, the supernatant was taken, washed with ddH2O and centrifuged, repeated twice, and then dried in a 60°C vacuum oven to constant weight, and the biomass was calculated.
[0047] Example 4 Effect Analysis: The final biomass of wild-type Chlorella pyrenoidosa and yellow Chlorella pyrenoidosa mutant under heterotrophic conditions. In the heterotrophic medium with 5 g / L glucose, the biomass of the yellow Chlorella pyrenoidosa mutant was 2.12 g / L on the second day of culture, and the highest could reach 2.14 g / L, which was 1.17 times the biomass of the wild-type Chlorella pyrenoidosa.
[0048] Example 5: Comparison of chlorophyll content between wild-type Chlorella pyrenoidosa and yellow Chlorella pyrenoidosa mutants;
[0049] Example 5 The culture conditions were the same as those of Example 4. The wild type and yellow protein nucleus Chlorella were cultured heterotrophically for 4 days, the algae solution was collected, centrifuged at 5000 rpm, washed with ddH2O and centrifuged twice, and the algae precipitate was collected and freeze-dried to obtain algae powder.
[0050] Chlorophyll content determination: accurately weigh 10 mg of freeze-dried algae powder, fully grind under liquid nitrogen protection, extract with 10 mL of methanol, extract at 4°C in the dark for 12 hours until the algae residue turns white, centrifuge at 8000 rpm for 10 minutes at 4°C to collect the supernatant, and perform the whole operation in dark conditions. Measure the absorbance at 665.2 nm, 652.4 nm and 470 nm with a spectrophotometer to calculate the chlorophyll content.
[0051] Example 5 Effect Analysis: The color of Chlorella is mainly due to the presence of chlorophyll and other substances in the algae, which makes the algae appear strong green. The chlorophyll content of the yellow protein-core Chlorella mutant and the wild-type protein-core Chlorella was measured and it was found that the chlorophyll content in the yellow protein-core Chlorella mutant was significantly reduced, with chlorophyll a of 3.23 mg / g and chlorophyll b of 4.04 mg / g, while the chlorophyll a and chlorophyll b contents in the wild-type protein-core Chlorella were 5.41 times and 1.73 times that of the yellow protein-core Chlorella, respectively. Figure 5 shown.
[0052] Example 6: Comparison of lutein content between wild-type Chlorella pyrenoidosa and mutant strains of Chlorella pyrenoidosa;
[0053] The culture conditions of Example 6 were the same as those of Example 5. The wild type and the yellow pyrenoid Chlorella mutant were cultured in the dark with 5 g / L glucose as the carbon source, and the algae liquid was collected and centrifuged and freeze-dried to obtain algae powder.
[0054] Lutein content detection: Take 10 mg of freeze-dried algae powder, grind it thoroughly with a mortar under the protection of liquid nitrogen, extract it with chromatographic grade acetone, shake it thoroughly, centrifuge it at 8000rpm and 4℃ for 5min, collect the algae residue, extract it twice with chromatographic grade acetone until the algae residue becomes colorless, combine the three acetone extracts, blow dry with nitrogen, and fully dissolve the pigment in 1mL chromatographic grade acetone. After passing through a membrane, analyze it with high performance liquid chromatography (HPLC). The whole process is carried out under light-proof conditions.
[0055] HPLC analysis method: chromatographic column Agilent Zorbax 300XDB-C18 (150mm×4.6mm, 5μm); column temperature 25℃; detection wavelength 460nm; mobile phase: A: pure ethyl acetate; B: acetonitrile: methanol: ultrapure water = 84:2:14; C: methanol: ultrapure water = 90:10. All mobile phases were filtered with a 0.45mm pore size filter membrane and ultrasonically degassed for later use.
[0056] The mobile phase elution conditions were as follows:
[0057]
[0058] Example 6 Effect Analysis: Lutein, as a natural plant pigment with multiple biological activities, has physiological functions such as anti-oxidation and coloring. Figure 6 As shown: the lutein content of the yellow pyrenoid Chlorella mutant is 5.1 mg / g, which is 1.16 times that of the wild-type pyrenoid Chlorella. The yellow pyrenoid Chlorella obtained by ARTP random mutagenesis screening has improved color properties and can grow rapidly, which can further broaden the application range of Chlorella.
[0059] Example 7: Comparison of protein content between wild-type Chlorella pyrenoidosa and mutant strains of Chlorella xanthophylla;
[0060] Example 7 The culture conditions of Chlorella are consistent with those of Example 5 and Example 6. Protein content determination: Accurately aspirate 1 ml of algae solution, centrifuge at 5000 rpm for 5 min, remove the supernatant, wash with ddH2O and repeat the centrifugation twice. Add 100uL 2M NaOH solution to the algae precipitate, bathe in 80°C water for 10 min to fully extract the protein in the algae, add 0.9 mL ddH2O, centrifuge at 13000 rpm for 5 min, repeat twice to obtain a total of 1.9 mL supernatant. Add 20uL supernatant and 180uL BCA working solution to the ELISA plate, incubate at 37°C for 30 min, measure the absorbance at 562nm, and calculate the protein content.
[0061] Example 7 Effect Analysis: Figure 7 As shown: the protein content of the mutant strain of yellow pyrenoid Chlorella is 26%, which is not much different from the protein content of the wild-type pyrenoid Chlorella (30%).
[0062] Example 8: Comparison of amino acid composition and content between wild-type Chlorella pyrenoidosa and mutant strains of Chlorella xanthophylla;
[0063] Example 8 The culture conditions of Chlorella were the same as those of Example 5. On the 4th day of culture, the algae solution was collected, centrifuged at 5000 rpm for 5 min, washed with ddH2O, resuspended, and centrifuged twice, and the algae precipitate was freeze-dried to obtain algae powder.
[0064] Amino acid determination: accurately weigh 20 mg of freeze-dried algae powder, acid hydrolyze with 6 M hydrochloric acid at 110 ° C for 22 h, cool and dilute to 50 mL with 0.02 M hydrochloric acid solution, take 1 mL of the hydrolyzate and evaporate it to dryness with a rotary evaporator, add 2-3 drops of ultrapure water and continue rotary evaporation to remove the acid, dry it and dilute with 2 mL of 0.02 M hydrochloric acid, and use LC20AD-API TRAP 3200MD HPLC-MS / MS system (Shimadzu Corporation, Kyoto, Japan) to analyze the amino acid content in the hydrolyzate.
[0065] Example 8 Effect Analysis: Protein, as one of the important nutrients, plays an important role in maintaining normal physiological activities in organisms. Amino acids are the basic units of protein, and their composition and content largely determine the quality level of protein. Figure 8 As shown: 17 kinds of amino acids were detected in both the wild type and the mutant strains, and the essential amino acids were complete. The content of sulfur-containing amino acids cysteine (Cys) and methionine (Met) in the mutant strain A19 was nearly 4 times higher than that in the wild type Chlorella pyrenoidosa. The EAA / TAA value of the mutant strain A19 was 44.74%, which was higher than that of the wild type Chlorella pyrenoidosa (42.90%), and the EAA / NEAA ratio was 0.81, which basically met the protein reference model required by the United Nations and the Agricultural Organization (FAO / WHO). The cultivation of new algae strains further improved the quality of amino acids in proteins and can be used as a substitute for traditional foods to meet the diversified needs of the market and consumers.
[0066] The cultivation of new algae species in this embodiment refers in particular to a method for screening and cultivating yellow protein-core Chlorella mutants that can grow rapidly by using atmospheric pressure room temperature plasma (ARTP) random mutation. It includes the following process steps: activation culture of wild-type protein-core Chlorella, random mutagenesis induced by ARTP and screening of yellow mutants, screening of optimal glucose concentration, obtaining and detecting biomass, chlorophyll, and xanthophyll, and determining the composition and content of proteins and amino acids;
[0067] This example fully verifies the probability of using ARTP random mutation to screen out chlorophyll-deficient protein-core Chlorella, and the obtained yellow protein-core Chlorella A19 has higher biomass and excellent amino acid composition, significantly reduced chlorophyll content and bright yellow color, which solves the problem of Chlorella decolorization in industrial production, provides an excellent substitute source for traditional food, and provides a new direction for laboratory cultivation of new algae species. The method of random mutagenesis and screening of new monoclonal mutants proposed in the present invention can be applied to mutations of other microalgae species.
[0068] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for screening and cultivating a fast-growing mutant of Chlorella vulgaris, characterized in that: The following steps are involved: Step 1: Algae strain activation: After streaking the wild-type algae strain in solid culture medium, pick a single algae colony for shake flask culture in the dark, culture it to the logarithmic growth phase and perform multiple subcultures to maintain its cell activity; Step 2: Dilute the algae strain in the logarithmic growth phase with glycerol, perform mutagenesis treatment using an atmospheric pressure room temperature plasma mutagenizer (ARTP), and apply the treated algae suspension to a solid culture medium; Step 3: Using color as the screening target, pick the yellow mutant with a sterile toothpick and place it in a fresh solid culture medium for multiple passages until the color phenotype is stable. After streaking the yellow mutant, pick a single algae colony and place it in a shake bottle for dark culture to obtain a purified yellow protein-core Chlorella mutant strain. Step 4: Inoculate the yellow mutant algae strain into liquid culture medium with different glucose concentrations (5-50 g / L), culture in the dark at 25-30°C and 140 rpm on a shaker for 4-6 days; Step 5: Collect the algae liquid, centrifuge, wash and dry, measure the biomass and glucose consumption, and screen for the optimal carbon source concentration; Step 6: Cultivate Chlorella vulgaris at the optimal carbon source concentration, take samples every day to measure the biomass, and determine its growth curve and pigment content.
2. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 1, characterized in that: The culture medium used was BG-11 basal culture medium. 10 g / L glucose was added to the BG-11 culture medium as a carbon source to culture the wild-type Chlorella pyrenoidosa heterotrophically until the logarithmic growth phase.
3. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 2, characterized in that: The specific operation of the second step is as follows: the wild-type protein nucleus Chlorella in the logarithmic growth phase is diluted with an equal volume of 10% glycerol to an OD680 between 0.6 and 0.8 to obtain an algae suspension, 10uL of the algae suspension is evenly applied to a sterile slide, and the slide is placed in an atmospheric pressure room temperature plasma mutagenizer (ARTP) for treatment, and the slide treated with ARTP is placed in 1mL90% physiological saline, and the algae liquid of each treatment group is evenly applied to a fresh solid culture medium of BG-11 containing 10g / L glucose under heterotrophic conditions at 28°C.
4. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 2, characterized in that: The pyrenoid Chlorella in the first step is the wild type W, and the yellow pyrenoid Chlorella screened in the third step is defined as the mutant A19.
5. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 1, characterized in that: In the fourth step, 5 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L of glucose were added to the culture medium for heterotrophy.
6. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 5, characterized in that: The specific operation of the fifth step is as follows: Biomass determination: take the algae liquid into a weighed centrifuge tube and centrifuge it. After washing, re-centrifuge it 2-3 times and then place the algae precipitate in a 60℃ vacuum drying oven to dry for 12 hours to constant weight, and then weigh it to determine the biomass; Glucose residue: Take 1 mL of the supernatant after centrifugation and add it to a colorimetric tube. Add 2 mL of DNS reagent and boil for 2 min. After cooling, dilute with pure water to 15 mL scale and measure the absorbance at 490 nm.
7. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 6, characterized in that: Chlorophyll content determination method: collect the cultured algae liquid, wash with ddH2O after centrifugation, repeat centrifugation twice, collect the algae precipitate and freeze-dry it to obtain algae powder, weigh a certain amount of freeze-dried algae powder, grind it fully under the protection of liquid nitrogen, extract it with methanol, and extract it at 4°C in the dark until the algae residue turns white, collect the supernatant, and carry out the whole operation under light-proof conditions. Measure the absorbance at 665.2nm, 652.4nm and 470nm with a spectrophotometer.
8. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 5, characterized in that: The method for obtaining and detecting lutein is as follows: freeze-dried algae powder is weighed, low-temperature grinding is performed, anhydrous ethanol is added and shaken for extraction, the supernatant is collected by centrifugation, anhydrous ethanol is added to the precipitate again and shaken for extraction until the algae powder turns white, the extract is collected, the extract is centrifuged, the supernatant is taken and dried with nitrogen, anhydrous ethanol is added to dissolve the pigment, and the pigment is analyzed by high performance liquid chromatography after being passed through a membrane. The whole process is carried out under light-proof conditions.
9. The method for screening and cultivating a fast-growing mutant of Chlorella vulgaris according to claim 8, characterized in that: Chlorella vulgaris must be cultured in BG-11 medium containing 5 g / L glucose in the dark for 4 days.