Chlorella pyrenoidosa mutant strain J-1 with high biomass and high photosynthetic efficiency as well as construction method and application of chlorella pyrenoidosa mutant strain J-1

Abstract

The invention relates to the technical field of microalgae biology, in particular to a high-biomass and high-photosynthetic-efficiency chlorella pyrenoidosa mutant strain J-1 which is obtained by mutagenizing wild type chlorella pyrenoidosa through an atmospheric pressure room temperature plasma (ARTP) mutagenesis technology and combining an efficient screening strategy. When the strain is cultured for 4 days under a standard autotrophic condition, the biomass reaches 1.23 mg / mL, the OD750 reaches 6.27, and when the strain is cultured for 5 days under a high light stress condition, the OD750 reaches 16.70; according to the invention, the growth performance is obviously enhanced: the biomass of J-1 is higher than that of WT under autotrophic, mixotrophic or heterotrophic conditions, and the biomass of a J-1 mutant strain reaches 1.23 mg / mL and is increased by 53.8% compared with that of a wild type (0.80 mg / mL) after the J-1 mutant strain is cultured for 4 days under a standard autotrophic condition (50 [mu] mol * m <-2 > * s <-1 >); the OD750 reaches 6.27 and is higher than 5.75 of a wild type, the OD750 reaches 2.18 mg / mL (WT is 1.75 mg / mL) on the fourth day of mixotrophic culture, the OD750 reaches 1.39 mg / mL (WT is 1.05 mg / mL) on the fourth day of heterotrophic culture, and it is indicated that biomass can be efficiently accumulated in different nutritional modes.

Description

Technical Field

[0001] This invention relates to the field of microalgae biotechnology, specifically to a high-biomass, high-photosynthetic-efficiency, and high-nutritional-value Chlorella mutant strain J-1 obtained through atmospheric pressure room temperature plasma (ARTP) mutagenesis, and its application in the fields of food, health products, and feed additives. Background Technology

[0002] Auxenochlorella pyrenoidosa, rich in proteins, pigments, unsaturated fatty acids, and other bioactive substances, has broad application prospects in food, feed, biofuel, and pharmaceutical fields. However, there is still room for improvement in wild-type Auxenochlorella pyrenoidosa in terms of growth rate, biomass accumulation, and the content of specific components to meet the needs of industrial production.

[0003] Traditional microalgae breeding methods, such as ultraviolet mutagenesis and chemical mutagenesis, suffer from low mutagenesis efficiency, narrow mutation spectrum, and poor mutation stability. In recent years, atmospheric pressure room temperature plasma (ARTP) mutagenesis technology has shown great potential in the field of microbial breeding due to its advantages such as high efficiency, safety, and ease of operation.

[0004] While existing technologies have explored ways to improve microalgal growth or the content of specific components, few microalgal mutant strains have been found that comprehensively enhance growth, biomass, photosynthetic efficiency, and various nutrient components while also exhibiting desirable flavor characteristics. For example, some strains may increase biomass but decrease pigment or fatty acid content; others may have high photosynthetic efficiency but poor adaptability to culture conditions (such as multitrophic or heterotrophic). Therefore, there is an urgent need to develop a microalgal mutant strain with excellent overall performance, suitability for multiple culture modes, and good sensory characteristics. Summary of the Invention

[0005] This invention aims to provide a mutant strain J-1 of Chlorella proteoglycans obtained through ARTP technology. This mutant strain is significantly superior to the wild type in terms of growth rate (especially under high light conditions), biomass, content of major pigments, fatty acid content, and content of some amino acids. It also improves sensory characteristics and reveals its regulatory mechanisms in photosynthesis, nitrogen metabolism, and energy metabolism. This overcomes the shortcomings of existing technologies in microalgae, such as single performance and low overall benefits, and provides excellent germplasm resources for the industrial application of microalgae, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high biomass, high photosynthetic efficiency Chlorella proteoglycans mutant strain J-1, which is obtained by mutagenizing wild-type Chlorella proteoglycans using atmospheric pressure room temperature plasma (ARTP) mutagenesis technology, combined with an efficient screening strategy;

[0007] After 4 days of culture under standard autotrophic conditions, the biomass reached 1.23 mg / mL, and the OD... 750 The OD reached 6.27 after culturing for 5 days under high light stress. 750 It reached 16.70.

[0008] Furthermore, in the aforementioned standard autotrophic conditions, the light intensity is 50 μmol*m. -2 *s -1 Furthermore, under high light stress conditions, the light intensity is 200 μmol*m. -2 *s -1 .

[0009] A method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 includes the following steps:

[0010] Step 1, Raw material preparation: Store wild-type Chlorella proteoglycans in a refrigerator at 4°C. Before use, streak the algae on a solid plate for 3-5 days to activate and culture them. Then, use a mutagenesis device to induce mutagenesis.

[0011] Step 2, Activation Culture: Wild-type Chlorella proteoglycans were streaked onto BG11 solid medium and placed in a constant temperature culture system at 25±0.5 ℃ and a light intensity of 50 μmol*m. -2 *s -1 After culturing for 3-5 days, select a single algal colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of BG11 liquid medium supplemented with 5 g / L glucose. Culture in a shaker in the dark at 150 rpm and 25 ± 0.5 ℃. Subculture 3-5 times, each time for 4-5 days.

[0012] Step 3, Mutagenesis treatment: Adjust the concentration of the algal solution in the logarithmic growth phase to 2×10⁻⁶. 8 cells / mL, diluted with glycerol;

[0013] Step 4: Screening and cultivation to select single clones that are dark green and grow rapidly;

[0014] Step 5, Stability verification and preservation: The selected single clones were cultured for 10 consecutive generations. The growth performance and content of key nutrients were tested in each generation to ensure the genetic stability of the traits. The stable mutants were inoculated on slant culture medium and stored in a refrigerator at 4°C.

[0015] Furthermore, the mutagenesis device is the Tianmu Bio ARTP-M type ARTP mutagenesis breeding instrument, which is preheated and calibrated before use, and the mutagenesis gas is high-purity helium.

[0016] Furthermore, in step three, 200 μL of algal solution and 200 μL of 10% (v / v) glycerol are thoroughly mixed in a sterilized laminar flow hood; 10 μL of the mixed algal solution is evenly spread on the surface of a sterile slide, and the mutagenesis program is started.

[0017] Furthermore, in step four, the mutagenized slide is placed in a 10 mL centrifuge tube containing 300 μL of sterile water, 100 μL of resuspended liquid is taken and spread on BG11 solid plate medium supplemented with 5 g / L glucose, and placed in a constant temperature culture system (25±0.5 ℃) for dark induction culture.

[0018] Furthermore, it covers OD 750 Detection and analysis of biomass, pigments, fatty acids, amino acids, proteins, gene expression levels, simulated taste, and simulated smell.

[0019] Furthermore, the OD 750 The detection method was as follows: using a UV-Vis spectrophotometer, with BG11 medium as a blank control, the absorbance of the algal solution was measured at a wavelength of 750 nm. Each sample was measured 3 times and the average value was taken.

[0020] Biomass detection was performed as follows: a certain volume of algal solution was taken, and algal cells were collected on pre-dried and weighed filter paper using a diaphragm pump-linked filtration device. The filter paper containing the sample was washed 2-3 times with pure water, and then dehydrated and dried in a 105 ℃ oven. The sample was weighed using an analytical balance, and the biomass per unit volume of algal solution was calculated.

[0021] Furthermore, the pigment content was detected as follows: using the Folch method, a certain amount of algal cells were taken, and a mixed solution of chloroform and methanol (volume ratio 2:1) was added. The mixture was ground and extracted in the dark, and water was removed by 0.75% NaCl solution. The lower organic phase was collected by centrifugation, dried under nitrogen, and then reconstituted with acetone. The contents of lutein, zeaxanthin, β-carotene, chlorophyll a, chlorophyll b, and total carotenoids were detected by high performance liquid chromatography and calculated according to the standard curve.

[0022] Fatty acid content was detected using gas chromatography-mass spectrometry. The sample was reconstituted after extraction with organic reagents, then subjected to methyl esterification, and then injected for analysis. C17:0 was used as an internal standard. The types of palmitic acid, palmitoleic acid, hexadecanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were determined based on the retention time and mass spectrum. The content was calculated based on the peak area.

[0023] Furthermore, the amino acid content is detected by liquid chromatography-tandem mass spectrometry. After acid hydrolysis of algal cells, the mixture is filtered to remove impurities, and the filtrate is injected for analysis. The content of common amino acids such as tryptophan and alanine, and uncommon amino acids such as glucosamine, L-citrulline, and methionine are calculated according to the standard curve.

[0024] Protein content was determined by: breaking down algal cells, hydrolyzing them with alkaline solution, and then measuring the protein content using a BCA kit.

[0025] Gene expression level detection involved using high-throughput sequencing technology to obtain all mRNA sequences and abundance information transcribed from algal cells, and then using bioinformatics analysis to determine gene function and expression differences.

[0026] The present invention has at least the following beneficial effects:

[0027] 1. This invention significantly enhances growth performance: J-1 exhibits higher biomass than WT under autotrophic, multitrophic, and heterotrophic conditions, and even higher biomass under standard autotrophic conditions (50 μmol*m). -2 *s -1 After 4 days of culture, the biomass of the J-1 mutant reached 1.23 mg / mL, an increase of 53.8% compared to the wild type (0.80 mg / mL); OD 750 The concentration reached 6.27, higher than the wild type's 5.75. On the fourth day of mixed culture, it reached 2.18 mg / mL (WT was 1.75 mg / mL), and on the fourth day of heterotrophic culture, it reached 1.39 mg / mL (WT was 1.05 mg / mL), indicating that it can efficiently accumulate biomass under different nutritional modes.

[0028] 2. This invention exhibits excellent high light adaptability: under high light stress conditions (200 μmol*m -2 *s -1 After 5 days of cultivation, the OD of J-1 750 The value reached 16.70, which is 14.1% higher than that of the wild type (14.64), indicating stronger light tolerance and photosynthetic efficiency.

[0029] 3. This invention exhibits significant enrichment of high-value pigments: the lutein content of J-1 reaches 3.74 mg / g, which is 43.3% higher than that of the wild type (2.61 mg / g); the β-carotene content reaches 0.61 mg / g, which is 15.1% higher than that of the wild type (0.53 mg / g); the total carotenoid content reaches 4.97 mg / g; and the chlorophyll a and b content reach 24.79 mg / g and 6.76 mg / g, respectively, both significantly higher than that of the wild type.

[0030] 4. The present invention simultaneously enhances the content of functional fatty acids: The content of various fatty acids such as palmitic acid, palmitoleic acid, hexadecanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid in J-1 is slightly higher than that in WT, with contents of 17.23, 2.64, 9.88, 3.09, 10.63, 31.81, and 12.89 mg / g, respectively, which improves its oil quality and application value;

[0031] 5. The present invention improves the content of certain amino acids and enhances flavor characteristics: J-1 has higher contents of glucosamine, L-citrulline, methionine, methionine sulfoxide, L-2-aminobutyric acid, L-glutamic acid, and L-glutamine than WT, and electronic tongue detection shows that it has slightly less astringency than WT, electronic nose detection shows that it has slightly more volatile aromatic compounds than WT, and less putrid odor such as sulfur compounds than WT, indicating that it has better palatability and flavor in food and feed applications;

[0032] 6. Significantly enhanced photosynthetic capacity, nitrogen metabolism, and energy metabolism pathways: Gene expression analysis confirmed that J-1 showed significant upregulation of genes related to supplemental light protein, photosystem I, photosystem II, and photosynthetic electron transport; significant upregulation of genes related to nitrogen metabolism such as NRT nitrate transport and glutamate synthesis; and upregulation of key enzyme genes for glycolysis / gluconeogenesis such as glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and glyceraldehyde triphosphate dehydrogenase, as well as key enzyme genes for the TCA cycle such as citrate synthase, succinate dehydrogenase, and malate dehydrogenase. These findings explain its excellent growth and material accumulation capabilities at the molecular level. Attached Figure Description

[0033] Figure 1 This is a photograph of wild-type Chlorella proteoglycans on a solid plate, as shown in Example 1 of this invention.

[0034] Figure 2 This is a photograph of the J-1 mutant strain from Example 2 of the present invention, streaked on a solid plate.

[0035] Figure 3 In Example 3 of this invention, mutant strains J-1 and WT were cultured for 4 days under autotrophic, multitrophic, and heterotrophic conditions, respectively, to obtain OD. 750 Compared with biomass;

[0036] Figure 4 For the mutant strains J-1 and WT in Example 3 of this invention, at 200 μmol*m -2 *s -1 OD cultured for 4 days under high light intensity conditions 750 contrast;

[0037] Figure 5 This is a comparison of pigment content between mutant strain J-1 and WT in Example 4 of the present invention;

[0038] Figure 6 This is a comparison of the fatty acid content of mutant strain J-1 and WT in Example 5 of the present invention;

[0039] Figure 7 This is a comparison of the amino acid content of mutant strain J-1 and WT in Example 6 of the present invention;

[0040] Figure 8This is a comparison of the protein content of mutant strain J-1 and WT in Example 6 of the present invention;

[0041] Figure 9 This is a comparison of the transcriptional levels of photosynthesis-related genes in mutant strain J-1 of Example 7 of the present invention compared to WT;

[0042] Figure 10 This is a comparison of the transcriptional levels of nitrogen metabolism-related genes in mutant strain J-1 of Example 7 of the present invention compared with those in WT.

[0043] Figure 11 This is a comparison of the transcriptional levels of glycolysis / gluconeogenesis-related genes in mutant strain J-1 of Example 7 of the present invention compared with WT;

[0044] Figure 12 This is a comparison of the transcriptional levels of TCA cycle-related genes in mutant strain J-1 of Example 7 of the present invention compared with those in WT.

[0045] Figure 13 This is a comparison of the electronic tongue data of mutant strain J-1 in Example 8 of the present invention with that of WT;

[0046] Figure 14 This is a comparison of mutant strain J-1 from Example 8 of the present invention with WT electronic nose data. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] A method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1

[0049] A. Raw material preparation: Wild-type Chlorella proteoglycans were purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, with the code FACHB-9.

[0050] The mutagenesis device was the Tianmu BioARTP-M type ARTP mutagenesis breeding instrument, and the mutagenesis gas was high-purity helium.

[0051] B. Core preparation steps (in order):

[0052] After streaking wild-type Chlorella proteoglycans onto a solid culture medium, single algae were picked and cultured in shake flasks under dark conditions. Multiple passages were performed to ensure their stability and viability.

[0053] The algal solution in the logarithmic growth phase was diluted with glycerol and subjected to ARTP mutagenesis. After treatment, it was spread on a sugar-containing solid medium for culture.

[0054] Rapidly growing, dark green monoclonal clones were selected and named J-1. They were then subjected to 10 consecutive generations of subculturing to ensure the genetic stability of their traits, and the resources were preserved on slant culture medium.

[0055] C. Key process parameters:

[0056] Before ARTP mutagenesis, the cell concentration of algae in the logarithmic growth phase was adjusted to 2 × 10⁻⁶. 8 cells / mL.

[0057] The ARTP mutagenesis instrument parameters were set as follows: RF power 120 W, helium flow rate 10 L / min, and plasma nozzle-sample distance 2 mm.

[0058] In a sterilized laminar flow hood, thoroughly mix 200 μL of algal solution with 200 μL of 10% (v / v) glycerol. Spread 10 μL of the mixed algal solution evenly onto a sterile slide and initiate the mutagenesis program.

[0059] The mutagenized slides were transferred to 10 mL centrifuge tubes containing 300 μL of sterile water. Then, 100 μL of the resuspended liquid was aspirated and spread onto BG11 solid agar plates containing glucose. The plates were then placed in a constant temperature culture system (25±0.5 ℃) for dark induction culture.

[0060] The initial pH of BG11 medium was 7.1 ± 0.1, and its components and concentrations are shown in Table 1.

[0061] Table 1. Composition and concentration of BG11 culture medium

[0062]

[0063] The culture medium used for heterotrophic culture is normal BG11 medium with 5 g / L glucose added, and the solid culture medium is liquid medium with 10 g / L agar powder added.

[0064] Materials characterization (key evidence)

[0065] OD 750 Absorbance of algal solution at a wavelength of 750 nm;

[0066] Biomass: The dry matter mass of a given volume of algal cells;

[0067] Pigment content: including lutein, azadirachtin, β-carotene, chlorophyll a, chlorophyll b and total carotenoids;

[0068] Fatty acid content: including palmitic acid, palmitoleic acid, hexadecanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid;

[0069] Amino acid content: Includes common amino acids such as tryptophan and alanine, and less common amino acids such as glucosamine, L-citrulline, and methionine;

[0070] Protein content: The protein contained in a given biomass of algal cells;

[0071] Gene expression level: The expression of a specific genome in a sample compared to a control;

[0072] Simulated taste analysis: Detecting soluble flavor substances in algal liquid using an electronic tongue device;

[0073] Simulated olfactory analysis: using an electronic nose device to detect volatile substances in algal solutions.

[0074] Example 1: Activation and culture of Chlorella proteoglycans

[0075] The purchased wild-type Chlorella proteoglycans were streaked onto solid plates. Single algae were picked from the solid plates and transferred to liquid culture medium for activation. Using 250 mL Erlenmeyer flasks and BG11 liquid medium supplemented with 5 g / L glucose, heterotrophic subculturing was carried out at 25 ± 0.5 °C until the viability stabilized. Algal solutions in the logarithmic growth phase were selected as materials for subsequent mutagenesis.

[0076] Example 2: ARTP mutagenesis screening for high-growth, high-pigmentation mutants

[0077] Chlorella proteoglycans in the logarithmic growth phase were diluted 1:1 with 10% (v / v) glycerol and spread onto sterile slides. Mutagenesis was induced in a pure helium atmosphere. After mutagenesis, the algal solution was spread on BG11 solid medium supplemented with 5 g / L glucose and placed in a constant temperature culture system (25±0.5℃) for dark induction culture. Rapidly growing and dark green algal colonies were selected and passaged multiple times to determine their phenotypic stability.

[0078] Example 3: Comparison of growth and biomass of J-1 mutant strain

[0079] J-1 and WT were cultured for 4 days under autotrophic, multitrophic, and heterotrophic conditions, respectively, and OD was measured daily. 750 The growth rate and biomass accumulation of J-1 and WT were compared under different culture conditions. Additionally, J-1 and WT were cultured at 200 μmol*m... -2 *s -1 Cultured under high light intensity for 5 days, OD was measured daily. 750 .

[0080] OD 750 Measurement: The absorbance of the algal solution at the characteristic wavelength of 750 nm was directly measured using a UV-Vis spectrophotometer.

[0081] Biomass determination: 5 mL of algal solution was taken and algal cells were collected on pre-dried and weighed filter paper using a diaphragm pump-assisted filtration device. The cells were washed with pure water and then dehydrated and dried in a 105 °C oven. The filter paper containing the sample was then weighed in an analytical balance.

[0082] J-1 OD on the fourth day of autotrophy 750 The OD value reached 6.27, slightly exceeding the WT value of 5.75, but in both multitrophic and heterotrophic environments... 750 All were inferior to WT. In terms of biomass, J-1 was superior to WT under all three culture conditions: 1.23 mg / mL on day 4 in autotrophic culture, 2.18 mg / mL in mixed-trophic culture, and 1.39 mg / mL in heterotrophic culture; while WT's biomass on day 4 was 0.80 mg / mL in autotrophic culture, 1.75 mg / mL in mixed-trophic culture, and 1.05 mg / mL in heterotrophic culture. (At 200 μmol*m...) -2 *s -1 Under high light intensity cultivation, J-1 showed growth consistent with WT for the first three days, but surpassed WT from the third day onwards, reaching its fifth OD. 750 It reached 16.70, while the WT was only 14.64.

[0083] Example 4: Comparison of pigment content in J-1 mutant strains

[0084] Pigment content determination: Pigments were extracted using the Folch method, redissolved in acetone, filtered through a 0.22 μm organic phase filter, and qualitative and quantitative analysis was performed using high performance liquid chromatography (HPLC).

[0085] On the fourth day of autotrophic conditions, the levels of lutein, β-carotene, chlorophyll b, chlorophyll a, and total carotenoids in J-1 were 3.74, 0.61, 6.76, 24.79, and 4.97 mg / g, respectively, all of which were higher than the values ​​in WT (2.61, 0.53, 5.27, 21.53, and 4.35 mg / g).

[0086] Example 5: Comparison of fatty acid content in J-1 mutant strains

[0087] Determination of fatty acid content: Lipids were extracted using the Folch method, and after fatty acid methyl esterification, C17:0 was used as an internal standard. Qualitative and quantitative analysis was performed using gas chromatography-mass spectrometry (GC-MS).

[0088] Differences in fatty acid content between J-1 and WT on the fourth day of autotrophic diet: J-1 has slightly higher levels of palmitic acid, palmitoleic acid, hexadecanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid than WT, with levels of 17.23, 2.64, 9.88, 3.09, 10.63, 31.81, and 12.89 mg / g, respectively; while WT's levels are 15.89, 2.07, 9.17, 2.72, 8.80, 30.54, and 11.68 mg / g, respectively.

[0089] Example 6: Comparison of amino acid and protein content of J-1 mutant strain

[0090] Determination of amino acid content: Algal cells were ground and broken to extract amino acids. The samples were analyzed by LC-MS / MS using a ThermoQ-Exactive ultra-high resolution mass spectrometer combined with a Vanquish H ultra-high performance liquid chromatograph.

[0091] Protein content determination: After the algal cells were broken down, they were hydrolyzed with alkali, and then the protein content was determined using a BCA kit.

[0092] The difference in amino acid content between J-1 and WT on day 4 of autotrophic growth. J-1 had higher levels of glucosamine, L-citrulline, methionine, methionine sulfoxide, L-2-aminobutyric acid, L-glutamic acid, and L-glutamine (0.75, 6.80, 8.33, 10.27, 4.03, 163.71, and 43.77 ng / mg, respectively) than WT (0.25, 2.21, 7.42, 6.86, 2.73, 72.25, and 38.33). However, J-1 had lower levels of L-tryptophan, γ-aminobutyric acid, alanine, L-aspartic acid, L-phenylalanine, L-proline, L-serine, and L-threonine than WT.

[0093] J-1 has a protein content of 51.50%, slightly lower than the WT value of 54.68%.

[0094] Example 7: Gene expression analysis of the J-1 mutant strain

[0095] Eukaryotic RNA-seq: Obtaining transcriptome and gene expression information of algal cells using a next-generation sequencing platform.

[0096] Photosynthesis-related genes: J-1 showed significant upregulation of supplemental light protein, photosystem I, photosystem II and related genes for photosynthetic electron transport (e.g., LHCA5, LHCB2, psbS, psaN, petE, etc.), indicating that it has better photosynthetic capacity and light energy utilization efficiency than WT.

[0097] Nitrogen metabolism-related genes: J-1 significantly upregulated genes related to nitrogen metabolism pathways such as NRT nitrate transport and glutamate synthesis (e.g., NRT2, NR, CYP55, glnA, gdhA, etc.) compared to WT, indicating that its ability to absorb and assimilate nitrogen is enhanced.

[0098] Genes related to energy metabolism:

[0099] Glycolysis / gluconeogenesis: Compared with WT, J-1 showed upregulation of key enzyme genes related to glycolysis / gluconeogenesis, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glyceraldehyde triphosphate dehydrogenase (gpmB).

[0100] TCA cycle: Compared to WT, J-1 showed upregulated genes of key enzymes related to the energy metabolism TCA cycle, such as citrate synthase (gltA) and succinate dehydrogenase (SDH2). These results indicate that J-1 has higher efficiency in carbon metabolism and energy production.

[0101] Example 8: Sensory characteristics analysis of J-1 mutant and wild type under heterotrophic conditions

[0102] Electronic tongue detection: using Japanese INSENT model SA402B; Electronic nose detection: using German Airsense model PEN3.

[0103] J-1 has slightly less astringency than WT, but the other flavors are basically the same as WT. J-1 has slightly more volatile aromatic compounds than WT, but less putrid smells such as sulfur compounds.

[0104] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0105] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1, characterized in that: This mutant strain was obtained by mutagenesis of wild-type Chlorella proteoglycans using atmospheric pressure room temperature plasma (ARTP) mutagenesis technology, combined with an efficient screening strategy. After 4 days of culture under standard autotrophic conditions, the biomass reached 1.23 mg / mL, and the OD... 750 The OD reached 6.27 after culturing for 5 days under high light stress. 750 It reached 16.

70.

2. The high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 1, characterized in that: Under the specified standard autotrophic conditions, the light intensity is 50 μmol*m. -2 *s -1 Furthermore, under high light stress conditions, the light intensity is 200 μmol*m. -2 *s -1 .

3. The method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella mutant strain J-1 according to claim 1, characterized in that, Includes the following steps: Step 1, Raw material preparation: Store wild-type Chlorella proteoglycans in a refrigerator at 4°C. Before use, streak the algae on a solid plate for 3-5 days to activate and culture them. Then, use a mutagenesis device to induce mutagenesis. Step 2, Activation Culture: Wild-type Chlorella proteoglycans were streaked onto BG11 solid medium and placed in a constant temperature culture system at 25±0.5℃ and a light intensity of 50 μmol*m. -2 *s -1 After culturing for 3-5 days, select a single algal colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of BG11 liquid medium supplemented with 5 g / L glucose. Culture in a shaker in the dark at 150 rpm and 25±0.5 ℃. Subculture 3-5 times, each time for 4-5 days. Step 3, Mutagenesis treatment: Adjust the concentration of the algal solution in the logarithmic growth phase to 2×10⁻⁶. 8 cells / mL, diluted with glycerol; Step 4: Screening and cultivation to select single clones that are dark green and grow rapidly; Step 5, Stability verification and preservation: The selected single clones were cultured for 10 consecutive generations. The growth performance and content of key nutrients were tested in each generation to ensure the genetic stability of the traits. The stable mutants were inoculated on slant culture medium and stored in a refrigerator at 4 ℃.

4. The method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella mutant strain J-1 according to claim 3, characterized in that: The mutagenesis device is the Tianmu BioARTP-M type ARTP mutagenesis breeding instrument, which is preheated and calibrated before use, and the mutagenesis gas is high-purity helium.

5. The method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 3, characterized in that: In step three, 200 μL of algal solution and 200 μL of 10% (v / v) glycerol are thoroughly mixed in a sterilized clean bench; 10 μL of the mixed algal solution is evenly spread on the surface of a sterile slide, and the mutagenesis program is started.

6. The method for preparing a high-biomass, high-photosynthetic-efficiency Chlorella mutant strain J-1 according to claim 3, characterized in that: In step four, the mutagenized slide is placed in a 10 mL centrifuge tube containing 300 μL of sterile water, 100 μL of resuspended liquid is taken and spread on BG11 solid plate medium supplemented with 5 g / L glucose, and placed in a constant temperature culture system (25±0.5 ℃) for dark induction culture.

7. The material characterization method for a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 1, characterized in that: Covering OD 750 Detection and analysis of biomass, pigments, fatty acids, amino acids, proteins, gene expression levels, simulated taste, and simulated smell.

8. The material characterization method for a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 7, characterized in that: The OD 750 The detection method was as follows: using a UV-Vis spectrophotometer, with BG11 medium as a blank control, the absorbance of the algal solution was measured at a wavelength of 750 nm. Each sample was measured 3 times and the average value was taken. Biomass detection was performed as follows: a certain volume of algal solution was taken, and algal cells were collected on pre-dried and weighed filter paper using a diaphragm pump-linked filtration device. The filter paper containing the sample was washed 2-3 times with pure water, and then dehydrated and dried in a 105 ℃ oven. The sample was weighed using an analytical balance, and the biomass per unit volume of algal solution was calculated.

9. The material characterization method for a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 8, characterized in that: The pigment content was determined by the following method: an organic solvent extraction method was used. A certain amount of algal cells were taken and a mixed solution of chloroform and methanol (volume ratio 2:1) was added. The mixture was ground and extracted in the dark. Water was removed by 0.75% NaCl solution (0.75 times the volume of methanol). The lower organic phase was centrifuged, dried under nitrogen, and then reconstituted with acetone. The mixture was filtered through a 0.22 μm organic phase filter. The contents of lutein, zeaxanthin, β-carotene, chlorophyll a, chlorophyll b, and total carotenoids were determined by high performance liquid chromatography (HPLC) and calculated based on the standard curve. Fatty acid content was detected using gas chromatography-mass spectrometry. The sample was reconstituted after extraction with organic reagents, then subjected to methyl esterification, and then injected for analysis. C17:0 was used as an internal standard. The types of palmitic acid, palmitoleic acid, hexadecanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were determined based on the retention time and mass spectrum. The content was calculated based on the peak area.

10. The material characterization method for a high-biomass, high-photosynthetic-efficiency Chlorella proteoglycan mutant strain J-1 according to claim 9, characterized in that: The amino acid content was detected by liquid chromatography-tandem mass spectrometry. After acid hydrolysis of algal cells, the mixture was filtered to remove impurities, and the filtrate was injected for analysis. The contents of common amino acids such as tryptophan and alanine, and uncommon amino acids such as glucosamine, L-citrulline, and methionine were calculated according to the standard curve. Protein content was determined by: breaking down algal cells, hydrolyzing them with alkaline solution, and then measuring the protein content using a BCA kit. Gene expression level detection involved using high-throughput sequencing technology to obtain all mRNA sequences and abundance information transcribed from algal cells, and then using bioinformatics analysis to determine gene function and expression differences.

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