EMS-ARTP compound mutation chlorella pyrenoidosa mutant strain and application thereof

Abstract

The invention discloses an EMS-ARTP compound mutation chlorella pyrenoidosa mutant strain and an application thereof. The preparation method comprises the following steps: mutagenizing a chlorella pyrenoidosa mutant strain CX41 by adopting an ethyl methanesulfonate (EMS) and normal-pressure and room-temperature plasma (ARTP) composite technology, screening and verifying to obtain a golden yellow chlorella pyrenoidosa mutant strain, naming the golden yellow chlorella pyrenoidosa mutant strain as chlorella pyrenoidosa Y11, and screening and verifying the golden yellow chlorella pyrenoidosa Y11 to obtain the chlorella pyrenoidosa Y11. The strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on the fifth floor of the building 59, No. 100 courtyard, Xianlie Middle Road, Guangzhou on October 23, 2024, and the preservation number is GDMCC No: 65324. The chlorella pyrenoidosa mutant strain Y11 has the characteristics of high yield of protein and rich carotenoids, and can be used for fermentation production of microalgae protein and carotenoids.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering, and particularly relates to an EMS-ARTP composite mutagenesis protein nucleus Chlorella mutant strain and application thereof. Background Art

[0002] Food protein is the most important nutrient for humans and the main carrier of life activities. The Food and Agriculture Organization of the United Nations (FAO) predicts that the world population will reach 9 billion in 2050, and the demand for food protein will increase by 30% to 50%, reaching 265 million tons. Animal protein from animal husbandry has been used as conventional food protein in the past and is still the main component of food protein. However, animal husbandry production occupies 30% of the land area and 70% of all agricultural land, causing greenhouse gas emissions, water pollution and land degradation, and bringing great pressure to the ecological environment. On the other hand, traditional animal husbandry may overuse hormones and antibiotics, leading to health risks of zoonotic virus transmission, parasites and pathogenic bacteria infection.

[0003] In order to alleviate the environmental and resource pressure faced by traditional protein production and meet consumers' growing demand for food safety and health, alternative proteins have attracted widespread attention in the food industry. At present, alternative proteins mainly come from four sources: plant protein, fungal protein, algae protein and insect protein. Among them, microalgae protein is an important source of alternative protein. They absorb carbon dioxide, grow rapidly, do not need to occupy arable land, and are nutritionally comprehensive, high in protein content, rich in various essential amino acids for the human body, and have extremely low allergenicity. They are a good sustainable source of future food protein.

[0004] As one of the microalgae currently produced on a large scale in my country, Chlorella vulgaris has an intracellular protein content of 50% to 65%. It is a single-cell protein source and has been recognized by the Food and Agriculture Organization of the United Nations as a "green and healthy food". It can be photosynthetically autotrophic or fermented at high cell density, and has great production potential. Compared with autotrophic culture, high-density fermentation can solve the problem of not relying on light, with high biomass, precise and controllable production parameters, and relatively low production costs. However, the intracellular protein content of algae cells is lower than that of photoautotrophy, while the lipid content is higher, which greatly reduces its commercial applicability as an alternative protein source. In addition, the Chlorella algae is dark green, and its fat-derived volatiles and sulfur-containing compounds will produce a strong fishy smell, and its use in food is less accepted by consumers.

[0005] The Chinese patent (patent number: ZL 2023105723078, title: A chlorophyll synthesis defective mutant of pyrenoid chlorella and its application) provides two chlorophyll synthesis defective mutants of pyrenoid chlorella CX41 and MC8, the algae of these two mutants are golden yellow, the chlorophyll a content is reduced by more than 98% and does not contain chlorophyll b, and the protein yield is 4.03g / L and 4.07g / L respectively. Although the above mutants solve the bottleneck of color and odor of wild-type pyrenoid chlorella, the dry weight of biomass is still low, resulting in low protein yield. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a mutant strain of Chlorella pyrenoidosa, which has high biomass, high protein content, and is rich in carotenoids, can be cultured by heterotrophic fermentation, and is suitable for large-scale industrial production.

[0007] Another object of the present invention is to provide an application of the Chlorella pyrenoidosa mutant.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A mutant strain of Auxenochlorella pyrenoidosa, named Auxenochlorella pyrenoidosa Y11, with a deposit number of GDMCC No: 65324, was deposited at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on October 23, 2024, on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The pyrenoid chlorella Y11 has the characteristic structure of typical Chlorophyta, Chlorophyceae, Chlorococcales, Oocystaceae, and Chlorella. When cultured in a sterile Basal medium at 28 to 30° C., the algae is spherical and golden yellow, the nutrient cell size is about 5 to 10 μm, and the nutrition mode is heterotrophic.

[0011] The 18S rRNA sequence of the Chlorella pyrenoidosa Y11 is shown in SEQ ID NO.1. The Chlorella pyrenoidosa mutant strain CX41 was subjected to composite mutagenesis using ethyl mesylate (EMS) and atmospheric and room temperature plasma (ARTP), and was obtained through high-throughput screening, shake flask culture and system evaluation verification.

[0012] A method for culturing the Chlorella pyrenoidosa mutant strain comprises the following specific steps: inoculating the Chlorella pyrenoidosa mutant strain into a culture medium and culturing the culture medium at 28-30°C.

[0013] The inoculation density of the pyrenoid Chlorella mutant when cultured in liquid culture medium is 1×10 6 ~1×10 7 cells / mL, preferably 5×10 6 ~1×10 7 cells / mL.

[0014] The culture medium is a Basal culture medium containing a carbon source and a nitrogen source, and its formula is as follows: glucose 10-30 g / L, NaNO3 1.25-3.75 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 1.0 g / L, ethylenediaminetetraacetic acid (EDTA) 0.5 g / L, H3BO3 0.1142 g / L, CaCl2·2H2O 0.111 g / L, FeSO4·7H2O 0.0498 g / L, ZnSO4·7H2O 0.0882 g / L, MoO3 0.0071 g / L, MnCl·4H2O 0.0142 g / L, CuSO4·5H2O 0.0157 g / L, CoNO3·6H2O 0.0061 g / L, and the pH is adjusted to 6.1. Basal solid culture medium is prepared by adding agar with a final concentration of 25 to 30 g / L to Basal culture medium.

[0015] The application of the pyrenoid Chlorella mutant in producing proteins and / or carotenoids.

[0016] The application of the pyrenoid Chlorella mutant in the production of protein and / or carotenoids is specifically as follows: the activated pyrenoid Chlorella mutant is inoculated into a Basal liquid culture medium containing a carbon source and a nitrogen source, fermented and cultured at 28-30°C in the dark, the algae liquid is collected by centrifugation, washed, and freeze-dried to obtain algae powder rich in protein and carotenoids.

[0017] The carotenoids include carotene and xanthophyll substances.

[0018] The carotene includes at least one of beta-carotene, alpha-carotene, phytoene, epsilon-carotene, gamma-carotene and lycopene.

[0019] The xanthophyll substances include at least one of lutein, antheraxanthin, zeaxanthin, violaxanthin, neoxanthin, β-cryptoxanthin and α-cryptoxanthin.

[0020] The inoculation density of the pyrenoid Chlorella mutant was 1×10 6 ~1×10 7 cells / mL; preferably 5×10 6 ~1×10 7cells / mL.

[0021] The formula of the Basal liquid culture medium containing carbon source and nitrogen source is as follows: glucose 10-30 g / L, NaNO3 1.25-3.75 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 1.0 g / L, ethylenediaminetetraacetic acid (EDTA) 0.5 g / L, H3BO3 0.1142 g / L, CaCl2·2H2O 0.111 g / L, FeSO4·7H2O 0.0498 g / L, ZnSO4·7H2O 0.0882 g / L, MoO3 0.0071 g / L, MnCl·4H2O 0.0142 g / L, CuSO4·5H2O 0.0157 g / L, CoNO3·6H2O 0.0061 g / L, and the pH is adjusted to 6.1.

[0022] The fermentation culture can be carried out in conventional containers in the art, such as triangular flasks (shake flasks) commonly used in laboratories.

[0023] The fermentation culture conditions are: 150-180r / min, 28-30°C in the dark for more than 24h; preferably: 160r / min, 30°C in the dark for more than 96h.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] (1) The present invention is based on EMS-ARTP compound mutagenesis, and after a series of screening, a high-protein-producing pyrenoid Chlorella mutant strain Y11 is finally obtained, and the algae is golden yellow. Compared with a single mutagenesis method, the use of a compound mutagenesis method is conducive to breaking the limitation of low mutation rate, obtaining more positive mutant algae strains, increasing gene mutation types, and expanding the mutation library.

[0026] (2) The mutant strain Y11 of the present invention was cultured heterotrophically under dark conditions, and its protein yield reached 5.04g / L; the biomass, protein content and yield reached 9.05g / L, 55.54%DW and 1.26g / L / d, respectively, which were significantly increased by 21.15%, 25.60% and 51.81% compared with the starting algae strain CX41; the amino acid score reached 94.41, and the total amount of branched-chain amino acids reached 6.96%DW; the total carotenoids, lutein and carotene substances reached 690.44μg / g, 564.65μg / g and 62.9μg / g, respectively, which were increased by 41.69%, 34.83% and 83.65% compared with the starting algae strain CX41. Therefore, the mutant strain Y11 of the present invention has the characteristics of high protein yield and rich carotenoids as the fermentation algae strain, and provides a high-quality algae species for the fermentation production of high-quality microalgae protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the algal state of the Chlorella pyrenoidosa mutant strain Y11.

[0028] Figure 2 This is the EMS-induced lethality curve of the starting strain CX41 of Chlorella pyrenoidosa in Example 1.

[0029] Figure 3 This is a graph showing the ARTP-induced lethality curve of the starting strain CX41 of Chlorella pyrenoidosa in Example 2.

[0030] Figure 4 It is a growth curve diagram of the pyrenoid Chlorella mutant strain in Example 4 during the expansion and passage process of 100 microdroplets in MMC.

[0031] Figure 5 This is a production performance diagram of 7 mutant strains and the starting algae strain CX41 in shake flask culture in Example 4; wherein a is the cell density; and b is the biomass.

[0032] Figure 6 It is a statistical graph of protein content, yield and yield of the 7 mutant strains in Example 5 and the starting algae strain CX41. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0034] The pyrenoid chlorella CX41 used in the present invention is a chlorophyll synthesis-deficient mutant strain CX41 of pyrenoid chlorella (Auxenochlorellapyrenoidosa), which has been disclosed in a Chinese patent (patent number: ZL 2023 105723078, entitled: A pyrenoid chlorella chlorophyll synthesis-deficient mutant strain and its application), and the deposit number is GDMCC No: 63357. The biomass concentration was determined by referring to the detection method in the patent document, the cell density was determined and the specific growth rate was calculated, the protein content was determined and the protein yield was calculated, and the amino acid analysis was performed; wherein,

[0035] The specific growth rate is calculated as follows:

[0036]

[0037] Where: C t1 , C t2Represent the cell density at time points t1 and t2, respectively, in cells / mL.

[0038] The protein yield was calculated as follows:

[0039]

[0040] Where: P represents protein content, the unit is %DW; W1 and W2 represent the biomass concentration at time points t1 and t2, respectively, the unit is g / L.

[0041] In the present invention, liquid chromatography-tandem mass spectrometry is used to determine the content of carotenoids, and the specific steps are as follows:

[0042] ① Drawing of standard curve

[0043] Prepare carotenoid standard solutions with concentrations of 0.001μg / mL, 0.005μg / mL, 0.01μg / mL, 0.05μg / mL, 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, 250μg / mL, and 400μg / mL, obtain the mass spectrometry peak intensity data of the corresponding quantitative signals of the standards at each concentration, and draw a standard curve of carotenoid concentration (μg / L, y) versus standard peak area (x). Where,

[0044] The liquid phase conditions mainly include:

[0045] a) Chromatographic column: YMC C30 (3 μm, 100 mm × 2.0 mm id);

[0046] b) Mobile phase: Phase A, methanol / acetonitrile (1:3, v / v) with 0.01% (w / v) 2,6-di-tert-butyl-p-cresol (BHT) and 0.1% (v / v) formic acid added; Phase B, methyl tert-butyl ether with 0.01% (w / v) BHT added;

[0047] c) Gradient elution program: 0 min A / B is 100:0 (V / V), 3 min is 100:0 (V / V), 5 min is 30:70 (V / V), 9 min is 5:95 (V / V), 10 min is 100:0 (V / V), 11 min is 100:0 (V / V);

[0048] d) Flow rate: 0.8 mL / min; column temperature: 28°C; injection volume: 2 μL.

[0049] Mass spectrometry conditions mainly include:

[0050] The atmospheric pressure chemical ionization source (APCI) temperature was 350°C and the curtain gas (CUR) was 25 psi. In the Q-Trap 6500+, each ion pair was scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0051] ② Determination of carotenoid content

[0052] Weigh 50 mg of algae powder sample and extract it with 0.5 mL of a mixed solution of n-hexane / acetone / ethanol (1:1:1, v / v / v) containing 0.01% BHT (g / mL); vortex at room temperature for 20 min, centrifuge at 4°C, 12000 r / min for 5 min, take the supernatant, repeat the extraction once, and then centrifuge to combine the supernatants; concentrate the obtained extract, re-dissolve it with 100 μL of a mixed solution of methanol / methyl tert-butyl ether (1:1, v / v), filter it through a 0.22 μm filter membrane, and store it in a brown injection bottle for LC-MS / MS analysis. The detection method is the same as step ① above.

[0053] In the embodiment of the present invention, Excel 2021 and Microsoft Origin 2021 were used to process and analyze the experimental data, and SPSS Statistics 27 was used to perform one-way analysis of variance and significance test on the data.

[0054] Example 1

[0055] The ethyl methanesulfonate mutagenesis method of Chlorella pyrenoidosa CX41 specifically comprises the following steps:

[0056] (1) Activation of CX41 strain and preparation of seed solution

[0057] Pick CX41 (deposit number GDMCC No: 63357) algae from the Basal slant culture medium stored at 4°C, streak continuously on the Basal solid plate, culture in the dark at 30°C for 3-4 rounds, pick a single algae colony and inoculate it into 20mL sterile Basal liquid culture medium containing 10g / L glucose, place a 50mL shake flask in a constant temperature shaker, and culture in the dark at 30°C and 160r / min for 96h to prepare the seed solution.

[0058] The Basal solid culture medium has the following formula (g / L): glucose 10, NaNO3 1.25, KH2PO4 1.25, MgSO4·7H2O 1.0, ethylenediaminetetraacetic acid (EDTA) 0.5, H3BO3 0.1142, CaCl2·2H2O 0.111, FeSO4·7H2O0.0498, ZnSO4·7H2O 0.0882, MoO3 0.0071, MnCl·4H2O 0.0142, CuSO4·5H2O 0.0157, CoNO3·6H2O 0.0061, agar 25-30, and pH adjusted to 6.1. The formula of the Basal liquid culture medium is obtained by removing the agar from the Basal solid culture medium. The plate culture temperature is 28-30°C.

[0059] (2) Preparation of the starting algal suspension of CX41 strain

[0060] Take 1 mL of CX41 seed solution in a 2 mL sterile centrifuge tube, centrifuge at 8000 r / min for 5 min to collect algal cells, add 1 mL of sterile water to the algal mud sediment, mix well, centrifuge and wash 3 times, and dilute to 10 with 10 mL of sterile water. 6 ~10 7 cells / mL to make CX41 algae suspension.

[0061] (3) Ethyl methanesulfonate mutagenesis

[0062] Take 6 groups of 1mL CX41 algae suspension in 2mL sterile centrifuge tubes, centrifuge at 8000r / min for 5min, and remove the supernatant. Prepare different concentrations of ethyl methanesulfonate (EMS) solution using sterile PBS buffer (pH 7.0), filter and sterilize with a 0.22μm microfiltration membrane, and add 1mL to 2mL centrifuge tubes containing CX41 algae cell pellets, so that the final concentration of EMS is 0, 100, 200, 300, 400, and 500mM, respectively. Each group has 3 parallel samples, tightly cover, and place on an oscillator for 30min at 30℃ and 500r / min in the dark. Immediately add 1mL of 5% (w / v) sodium thiosulfate solution to each tube to terminate the reaction, centrifuge to remove the mutagenic solution, add 1mL of sterile PBS buffer to rinse the algae cells, centrifuge, discard the supernatant, and wash the pellet again. After washing, the algal cell pellet was mixed with 1 mL of sterile water. The algal suspension was diluted in a gradient manner, and 100 μL was applied to Basal solid culture medium and placed in a 30°C incubator in the dark until a single algal colony appeared. The algal liquid without EMS was used as a control. The algal colonies of each mutagenesis group were counted and the lethality was calculated according to formula (a). The lethality curve was drawn. The results are shown in Figure 2As shown. The cell lethality increases with the increase of EMS concentration, reaching 100% lethality at 500mM EMS. Usually, a lethality of 90% to 95% is selected, and the positive mutation rate of the algae strain is higher. When the EMS concentration is 400mM, the lethality is 94.71%, so 400mM is selected as the optimal dose of EMS mutagenesis.

[0063]

[0064] Example 2

[0065] The atmospheric pressure and room temperature plasma mutagenesis method of Chlorella pyrenoidosa CX41 specifically comprises the following steps:

[0066] (1) Activation of CX41 strain and preparation of seed solution

[0067] The method is the same as in Example 1.

[0068] (2) Preparation of the starting algal suspension of CX41 strain

[0069] The method is the same as in Example 1.

[0070] (3) Atmospheric pressure room temperature plasma mutagenesis

[0071] Take 8 groups of 1mL CX41 algae suspension, mix them with equal volumes of 10% (v / v) sterile glycerol solution to form a new algae suspension, and make the final concentration of glycerol 5% (v / v) as a mutagenic protective agent. Take 10μL of the above new CX41 algae suspension, drop it in the center of a sterile metal slide, and place it in an ARTP mutagenizer for mutagenesis. Under the conditions of high-purity helium as the ARTP working gas source, power supply power of 120W, gas flow rate of 10SLM, and irradiation distance of 2mm, the algae cells on the 8 slides were induced to undergo mutagenesis. The treatment time was 0, 10, 15, 20, 22, 25, 30, and 35s, respectively, with 3 parallel samples for each time. After treatment, the sterile metal slide containing the algae suspension was gradiently diluted and spread on Basal solid culture medium, placed in a 30℃ incubator and cultured until a single algae colony appeared. The algae solution that had not been induced was used as a control. The algae colonies of each mutagenesis group were counted, and the lethality was calculated according to formula (a), and a lethality curve was drawn. The results are shown in Figure 3 As shown in the figure, the cell lethality increased with the extension of ARTP treatment time, reaching 100% lethality at 35s, and the cell lethality was 92.94% at 20s. Therefore, 20s was selected as the ARTP mutagenesis treatment time.

[0072] Example 3

[0073] The composite mutation breeding method of Chlorella pyrenoidosa specifically comprises the following steps:

[0074] (1) Activation of the starting algae strain CX41 and preparation of seed solution

[0075] The method is the same as in Example 1.

[0076] (2) Preparation of the starting algal suspension of CX41 strain

[0077] The method is the same as in Example 1.

[0078] (3) Ethyl methanesulfonate-normal pressure room temperature plasma mutagenesis

[0079] Take 1mL of activated CX41 seed solution in a 2mL sterile centrifuge tube, centrifuge at 8000r / min for 5min, and remove the supernatant. Take 1mL of 400mM sterile EMS solution (sterilized by 0.22μm microfiltration membrane filtration) and add it to the 2mL centrifuge tube containing the CX41 algae cell precipitate, cover it tightly, place it on an oscillator at 30℃, 500r / min in the dark for 30min, and immediately add 1mL of 5% (w / v) sodium thiosulfate solution to terminate the reaction, and centrifuge to remove the mutagenic solution. Add 1mL of sterile PBS buffer to rinse the algae cells, centrifuge, discard the supernatant, and wash the precipitate again. Add 1mL of sterile water to the washed algae cell precipitate to mix, and mix with an equal volume of 10% (v / v) sterile glycerol solution to form a new algae suspension. Take 10μL of the above new algae suspension, drop it in the center of a sterile metal slide, and place it in an ARTP mutagenizer for mutagenesis. The treatment was carried out for 20 seconds under the conditions of high-purity helium as the ARTP working gas source, power supply of 120W, gas flow rate of 10SLM, and irradiation distance of 2mm. The treated metal slide was eluted in 1mL of sterile water, inoculated into 20mL of Basal liquid culture medium containing 10g / L glucose, and cultured in the dark in a constant temperature shaker at 30℃ and 160r / min for 96h.

[0080] Example 4

[0081] The high-throughput screening of Chlorella pyrenoidosa mutants specifically comprises the following steps:

[0082] (1) Fully automated high-throughput micro-droplet high-throughput culture

[0083] The logarithmic phase composite mutagenesis culture solution obtained in Example 3 was diluted with sterile water to an OD of 450 When the concentration of OD is about 0.6 to 0.8, 10 mL of the dilution solution is transferred to the microbial droplet culture instrument (MMC). Before the initialization of the droplet microfluidic device, ultraviolet irradiation is performed for 30 minutes inside and outside the culture room. The microfluidic chip is initialized and the parameters are set: temperature 30°C, OD detection wavelength 450nm, detection cycle 0h (i.e. continuous detection), and the number of initial droplets generated is 100. 450 As a cell growth indicator, after three rounds of expansion and one round of subculture, OD 450 The highest droplets are harvested, and the results are as follows Figure 4 The results show that the OD of 100 microdroplets 450 The value increased with the extension of culture time. The droplets were expanded 3 times in total during the whole process, followed by 1 manual subculture. Finally, the microdroplet with the highest OD450 before and after subculture (number: droplet 23) was selected and collected.

[0084] (2) Screening on solid plate culture

[0085] The microdroplets collected by MMC were diluted and spread, and placed in a 30°C constant temperature incubator for dark culture for 10 days, and single algae colonies with fast growth, rounded and full surface, and large diameter were selected for plate streaking verification. Then, single clones of mutant strains with fast growth and large algae colonies were selected from the transfer plate and named Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, and Y22.

[0086] (3) 48-well plate liquid culture initial screening

[0087] The above 22 mutant monoclonal clones were inoculated into 48-well plates containing 800 μL of sterile Basal medium at a density of 5×10 6 ~1×10 7 cells / mL, 4 parallel wells per group. The covered microplate was placed on a microplate oscillator and cultured in the dark at 500r / min in a constant temperature incubator at 30°C for 4 days. The algae liquid on day 0 and day 4 was taken to determine the cell density and biomass. The average specific growth rate and biomass of the cells were used as indicators for evaluation, and mutants with growth advantages were screened. The results are shown in Table 1. As can be seen from Table 1, the average specific growth rate and cell density of Y1, Y5, Y6, Y7, Y9, Y11 and Y22 were significantly higher than those of the starting algae strain CX41 (p<0.05), among which the mutant strain Y9 had the highest final cell density, reaching 2.86×10 8 cells / mL; Y7 had the highest final biomass (4.54 g / L), which was 17.21% and 8.87% higher than CX41, respectively, while the biomass of other mutants had no significant difference from CX41. Based on the three indicators, the top 7 dominant mutants were selected from the 22 mutants, namely Y1, Y5, Y6, Y7, Y9, Y11, and Y22.

[0088] Table 1. Comparison of 22 mutant strains and the original algae strain CX41 in microplate culture

[0089] Average specific growth rate / ( / d) <![CDATA[Cell density / (×10 8 cells / mL)]]> Biomass / (g / L) CX41 0.97±0.00 2.44±0.02 4.17±0.05 Y1 0.99±0.00 2.62±0.00 4.31±0.02 Y2 0.96±0.01 2.35±0.06 4.11±0.11 Y3 0.96±0.01 2.29±0.10 4.04±0.18 Y4 0.97±0.00 2.45±0.02 3.57±0.23 Y5 1.00±0.00 2.68±0.00 4.29±0.00 Y6 0.99±0.01 2.62±0.13 4.25±0.05 Y7 1.00±0.00 2.75±0.02 4.54±0.18 Y8 0.93±0.01 2.06±0.04 3.68±0.03 Y9 1.01±0.00 2.86±0.02 4.35±0.08 Y10 0.96±0.01 2.36±0.08 4.1±0.10 Y11 1.01±0.00 2.84±0.00 4.38±0.11 Y12 0.95±0.00 2.27±0.02 3.93±0.07 Y13 0.96±0.00 2.28±0.02 4.13±0.02 Y14 0.90±0.00 1.82±0.00 3.93±0.00 Y15 0.96±0.00 2.31±0.04 3.92±0.01 Y16 0.97±0.00 2.4±0.02 3.61±0.04 Y17 0.88±0.00 1.68±0.02 3.64±0.07 Y18 0.82±0.00 1.36±0.02 3.13±0.00 Y19 0.83±0.00 1.4±0.00 3.81±0.15 Y20 0.96±0.00 2.33±0.02 3.93±0.20 Y21 0.94±0.00 2.15±0.04 3.22±0.08 Y22 1.00±0.01 2.77±0.14 4.2±0.01

[0090] (4) Shake flask culture rescreening

[0091] The mutant strains with growth advantages obtained by screening were prepared into seed solution according to the method in Example 1, and then inoculated into 100 mL of sterile Basal medium containing 30 g / L glucose and 3.75 g / L NaNO3 in a 250 mL shake flask at an inoculation density of 5×10 6 ~1×10 7 cells / mL, and cultured in a constant temperature shaker at 30°C and 160 r / min in the dark for 96 h. Samples were taken every 24 h to measure the cell density and biomass. The results are as follows Figure 5 As shown. Figure 5 It can be seen that the cell density of Y7 and Y11 (1.46 and 1.44×10 8 cells / mL) and biomass (9.4 and 9.05 g / L) were significantly higher than those of the starting algae strain CX41 (p<0.01). However, there was no positive correlation between the biomass and cell density of Y5, Y9, Y6, and Y22, which may be due to the difference in the content and cell size of the mutants. After the culture, the algae liquid of the seven mutants was collected, centrifuged, washed, and the algae powder was collected after freeze-drying and stored at -20℃ in the dark.

[0092] Example 5

[0093] The systematic evaluation of Chlorella pyrenoidosa mutants includes the following steps:

[0094] (1) Comparison of protein production performance of mutant strains

[0095] Protein content, yield and productivity of the seven mutants in dark culture in shake flasks Figure 6 As shown. The protein content of Y9 and Y11 was significantly higher than that of the starting algae strain CX41 (p<0.01); the three mutant strains with the highest protein production were Y11, Y7 and Y5. It can be seen that the mutant strain Y11 has the best protein production capacity, with protein content, yield and yield reaching 55.54% DW, 5.02g / L and 1.26g / L / d, respectively, which are significantly higher than other mutant strains and starting algae strains (p<0.01), and compared with the starting algae strain CX41, they increased by 25.60%, 52.12% and 51.81%, respectively.

[0096] (2) Amino acid analysis of mutant strain Y11 and starting strain CX41

[0097] Further amino acid analysis was performed on the mutant strain Y11 with the best protein production performance and the starting strain CX41. The results of the amino acid composition comparison with the starting strain CX41 under the same culture conditions are shown in Table 2. The total amino acid content of the optimal mutant strain Y11 reached 39.52% DW, which was 30.21% higher than that of the starting strain CX41; the contents of 18 amino acids were all increased, among which the content of arginine (Arg) increased the most, increasing by 51.75% compared with the starting strain CX41. Further analysis showed that the total amount of essential amino acids (EAA) was 16% DW, accounting for 40% of the total amino acids; branched-chain amino acids (BCAA), as a class of amino acids that are essential for muscle synthesis, reached 6.96% dry weight, which was 27.35% higher than that of CX41. In addition, the total amount of non-essential amino acids (NEAA) was 23.52% DW.

[0098] Table 2. Comparison of amino acid composition between mutant strain Y11 and starting strain CX41

[0099]

[0100]

[0101] The amino acid score AAS (AAS = amino acid content per gram of protein in algae powder (mg) / amino acid content per gram of ideal model or reference protein (mg) × 100) was calculated based on the essential amino acid composition. The reference protein can adopt the FAO / WHO essential amino acid model for human beings. The results are shown in Table 3. It can be seen that the amino acid score of Y11 reached 94.41, the limiting amino acids were valine, isoleucine and leucine, and the remaining amino acids were higher than the nutritional model standards recommended by FAO / WHO. Compared with the starting algae strain CX41, although Y11 had a slight decrease in the scores of methionine and cysteine, with specific values ​​of 35.65±0.40, its overall amino acid score was improved and the amino acid composition was better. In view of the comprehensive advantages of Y11, this mutant strain has good application potential in the field of microalgae fermentation to produce alternative proteins, and can be used as an efficient and sustainable source of high-quality protein in the future food industry.

[0102] Table 3. Comparison of amino acid scores between mutant strain Y11 and starting strain CX41

[0103]

[0104]

[0105] (3) Analysis of carotenoid content in mutant strain Y11 and starting strain CX41

[0106] Further analysis of the carotenoid content of mutant strain Y11 and starting strain CX41 revealed 6 carotenes (mainly β-carotene, α-carotene, phytoene) and 22 xanthophylls (mainly antheraxanthin, zeaxanthin, neoxanthin, lutein, β-cryptoxanthin, α-cryptoxanthin) (see Table 4). Among them, lutein was the highest in content, with the lutein content of mutant strain Y11 reaching 368.91 μg / g, an increase of 33.16% compared with starting strain CX41. Its carotene, lutein and total carotenoid contents were 83.65%, 34.83% and 41.69% higher than those of CX41, respectively.

[0107] Table 4. Carotenoid content of mutant strain Y11 and starting strain CX41

[0108]

[0109]

[0110] The algal state of Chlorella pyrenoidosa Y11 is as follows Figure 1 The 18S rRNA gene of mutant strain Y11 was further sequenced and analyzed (Guangdong Microbiological Analysis and Testing Center), and the gene sequence is shown in SEQ ID NO.1. The homology of 18S rRNA of mutant strain Y11 and the published pyrenoid Chlorella strains is 100%.

[0111] 18S rRNA gene sequence (SEQ ID NO.1):

[0112]

[0113] Based on the colony morphological characteristics and molecular identification results of the mutant strain Y11, the above mutant strain Y11 was named Auxenochlorella pyrenoidosa Y11, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on October 23, 2024, on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC 65324.

[0114] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A mutant strain of Chlorella pyrenoidosa, characterized in that: The name is Auxenochlorella pyrenoidosa Y11, the preservation number is GDMCC No: 65324, and it was deposited on October 23, 2024 at the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for culturing the mutant strain of Chlorella pyrenoidosa according to claim 1, characterized in that: The specific steps are as follows: inoculating the mutant strain of Chlorella vulgaris into the culture medium and culturing it at 28-30°C.

3. The method according to claim 2, characterized in that: The culture medium is a Basal culture medium containing a carbon source and a nitrogen source, and its formula is as follows: glucose 10-30 g / L, NaNO3 1.25-3.75 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 1.0 g / L, ethylenediaminetetraacetic acid EDTA 0.5 g / L, H3BO3 0.1142 g / L, CaCl2·2H2O 0.111 g / L, FeSO4·7H2O 0.0498 g / L, ZnSO4·7H2O 0.0882 g / L, MoO3 0.0071 g / L, MnCl·4H2O 0.0142 g / L, CuSO4·5H2O 0.0157 g / L, CoNO3·6H2O 0.0061 g / L, and the pH is adjusted to 6.

1.

4. Use of the Chlorella pyrenoidosa mutant according to claim 1 in producing proteins and / or carotenoids.

5. The use according to claim 4, characterized in that: The activated protein-nucleated Chlorella mutant strain is inoculated into a Basal liquid culture medium containing a carbon source and a nitrogen source, and fermented and cultured at 28-30° C. in the dark. The algae liquid is collected by centrifugation, washed, and freeze-dried to obtain algae powder rich in protein and carotenoids.

6. The use according to claim 5, characterized in that: The carotenoids include carotene and xanthophyll substances; The carotene includes at least one of β-carotene, α-carotene, phytoene, ε-carotene, γ-carotene and lycopene; The xanthophyll substances include at least one of lutein, antheraxanthin, zeaxanthin, violaxanthin, neoxanthin, β-cryptoxanthin and α-cryptoxanthin.

7. The use according to claim 5, characterized in that: The formula of the Basal liquid culture medium containing carbon source and nitrogen source is as follows: glucose 10-30 g / L, NaNO3 1.25-3.75 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 1.0 g / L, ethylenediaminetetraacetic acid EDTA 0.5 g / L, H3BO3 0.1142 g / L, CaCl2·2H2O 0.111 g / L, FeSO4·7H2O 0.0498 g / L, ZnSO4·7H2O 0.0882 g / L, MoO3 0.0071 g / L, MnCl·4H2O 0.0142 g / L, CuSO4·5H2O 0.0157 g / L, CoNO3·6H2O 0.0061 g / L, and the pH is adjusted to 6.

1.

8. The use according to claim 5, characterized in that: The inoculation density of the pyrenoid Chlorella mutant was 1×10 6 ~1×10 7 cells / mL; The fermentation culture conditions are: 150-180r / min, 28-30°C, and dark culture for more than 24 hours.

9. The use according to claim 8, characterized in that: The inoculation density of the pyrenoid Chlorella mutant was 5×10 6 ~1×10 7 cells / mL; The fermentation culture conditions are: 160r / min, 30°C, dark culture for more than 96h.

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