Chlorella pyrenoidosa white mutant strain FZU65-W1 and application thereof

Abstract

The invention discloses a chlorella pyrenoidosa white mutant strain FZU65-W1 and application of the chlorella pyrenoidosa white mutant The classification name of the chlorella pyrenoidosa white mutant strain FZU65-W1 is chlorella pyrenoidosa, the chlorella pyrenoidosa white mutant strain is preserved in the China General Microbiological Culture Collection Center on September 11, 2024, the preservation number is CGMCC N0.46151, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Compared with an original strain FZU65, the chlorella pyrenoidosa white mutant strain FZU65-W1 has the advantages that the growth speed of algae cells under the heterotrophic condition is higher, and meanwhile, the cells are white due to the fact that chlorophyll and carotenoid are not synthesized in the cells. The chlorella pyrenoidosa white mutant strain FZU65-W1 can be used for producing high-quality protein through heterotrophic fermentation, so that not only can a later-stage protein extraction and preparation process be simplified, but also the sensory acceptance of algae powder can be improved, and a high-quality germplasm resource is provided for industrial production and application of microalgae protein.

Description

Technical Field

[0001] The invention relates to the field of mutant screening of dominant microalgae strains, and in particular to a white mutant strain of Chlorella pyrenoidosa FZU65-W1 and applications thereof. Background Art

[0002] As the global population continues to grow and people's demand for higher quality food continues to grow, the world's natural resources will inevitably face greater pressure. Protein is an essential nutrient in the human diet, providing most of the nitrogen required by humans. The current global annual protein demand is about 202 million tons, and it is estimated that the world's demand for animal-based protein will double by 2050, which has raised concerns about the sustainability of protein sources and food security.

[0003] Microalgae can synthesize all types of amino acids, and the protein content of most microalgae is comparable to or even higher than that of traditional protein source plants, making them ideal strategic protein sources. The unicellular green algae Chlorella pyrenoidosa was approved as a new resource food by my country in 2012. It is one of the few microalgae that are consumed in large quantities by humans, mainly due to its high protein content and balanced amino acid composition. However, the commercial production of Chlorella pyrenoidosa is mainly obtained by culturing in open runway pools in a photoautotrophic mode. Due to the low production efficiency and high production cost of this cultivation method, the widespread application of Chlorella pyrenoidosa is greatly limited. Heterotrophic cultivation, as a more economical and efficient industrial production model, is a potential way to reduce the production cost of Chlorella pyrenoidosa and increase production capacity. However, Chlorella pyrenoidosa can synthesize chlorophyll and carotenoids under heterotrophic or autotrophic conditions, which also makes it have a strong grassy flavor and green color. It is reported that the current application of microalgae in food still faces some challenges, mainly due to their sensory characteristics, including strong green and grassy flavors, so consumers generally have low sensory acceptance. In addition, in the reported extraction and preparation methods of microalgae proteins, additional steps such as decolorization and deodorization are often required, which is mainly caused by the excessively high pigment content in the algae powder. Therefore, in order to improve the sensory acceptance of microalgae-based foods and simplify the extraction and preparation process of microalgae proteins, it is necessary to further develop and screen microalgae germplasm that does not synthesize chlorophyll and carotenoids, but there are few related research reports. Since random mutagenesis does not introduce exogenous genes, it may be a more effective way to use this method to genetically improve the production algae species and fundamentally inhibit the synthesis of chlorophyll and carotenoids. Summary of the invention

[0004] The purpose of the present invention is to provide a white mutant strain of Chlorella pyrenoidosa FZU65-W1 and an application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a white mutant strain of Chlorella pyrenoidosa FZU65-W1, which is classified and named Chlorella pyrenoidosa ( Chlorella pyrenoidosa ), which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 11, 2024, with the deposit number CGMCC No.46151, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The white mutant strain of Chlorella pyrenoidosa FZU65-W1 is a multiple white mutant strain of Chlorella pyrenoidosa with pigment synthesis defect obtained by mutagenesis screening with the chemical reagent ethyl methanesulfonate on the basis of Chlorella pyrenoidosa FZU65. The chlorophyll and carotenoid contents of the white mutant strain FZU65-W1 of Chlorella vulgaris were almost undetectable under heterotrophic fermentation conditions, and the biomass concentration was about 1.09 times that of the original strain FZU65 of Chlorella vulgaris.

[0006] The second aspect of the present invention provides the use of the above-mentioned Chlorella pyrenoidosa white mutant strain FZU65-W1 in producing protein.

[0007] The third aspect of the present invention provides a method for producing protein, which is to produce protein by culturing the above-mentioned Chlorella pyrenoidosa white mutant strain FZU65-W1; The culture method is heterotrophic culture; The heterotrophic culture comprises the following steps: S1: Activation culture: a single clone of the white mutant strain FZU65-W1 of Chlorella vulgaris was inoculated into the Modified HA-SK liquid culture medium, and activated and cultured in a shaker at 25-35°C and 150 rpm until the logarithmic growth phase to obtain an activated culture solution; S2: Seed culture: dilute the activated culture medium 5 times with Modified HA-SK liquid medium, and culture in a shaker at 25-35°C and 150 rpm for 3 days to obtain seed culture medium; S3: Fermentation culture: Inoculate the seed culture medium into Modified HA-SK liquid medium, control the initial cell concentration to 0.90-0.15 g / L, and culture in a shaker at 25-35°C and 150 rpm until the carbon source is exhausted, then add 0.2-0.8 g / L of sodium nitrate and 0.2-0.8 g / L of trisodium citrate dihydrate to the culture medium, and continue to culture in a shaker at 25-35°C and 150 rpm for 1-3 days; The formula of Modified HA-SK liquid culture medium is as follows: glucose 15g / L, sodium nitrate 2.25g / L, magnesium sulfate heptahydrate 0.3g / L, calcium chloride dihydrate 0.15g / L, potassium dihydrogen phosphate 0.6g / L, Tris 0.5g / L, EDTA·2Na 0.115g / L, boric acid 8.82mg / L, ferrous sulfate heptahydrate 18mg / L, cobalt chloride hexahydrate 1.42mg / L, sodium molybdate dihydrate 1.705mg / L, zinc sulfate heptahydrate 24.1mg / L, cobalt nitrate hexahydrate 0.49mg / L, copper sulfate pentahydrate 1.57mg / L; The fourth aspect of the present invention provides the use of the above-mentioned Chlorella pyrenoidosa white mutant strain FZU65-W1 in the preparation of food or food additives.

[0008] The significant advantages of the present invention are: The present invention provides a white mutant strain of Chlorella pyrenoidosa FZU65-W1, which is a mutant strain with a fast growth rate and defective pigment synthesis, and the algal cells are white; under heterotrophic culture conditions, the white mutant strain of Chlorella pyrenoidosa FZU65-W1 can accumulate high-content protein, and its amino acid composition is very superior. The use of the white mutant strain of Chlorella pyrenoidosa FZU65-W1 of the present invention can not only simplify the extraction and preparation process of the later protein, but also improve the sensory acceptance of the algae powder, and can significantly improve the industrialization prospect of using microalgae to produce high-quality protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a photo of the solid plate culture of the white mutant strain of Chlorella pyrenoidosa.

[0010] Figure 2 These are photos of shake flask cultures of the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa.

[0011] Figure 3 Comparison of biomass concentration between the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa.

[0012] Figure 4 It is the pigment content of the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa.

[0013] Figure 5 The amino acid composition of the white mutant strain FZU65-W1 of Chlorella pyrenoidosa. DETAILED DESCRIPTION

[0014] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is based on the technical solution of the present invention and is experimentally performed, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0015] In the following examples, the formula of Modified BG-11 liquid culture medium is: glucose 10 g / L, sodium nitrate 1.5 g / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, potassium hydrogen phosphate 0.03 g / L, citric acid 0.006 g / L, Tris 0.5 g / L, EDTA·2Na 1 mg / L, ammonium ferric citrate 6 mg / L, sodium carbonate 0.02 g / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.81 mg / L, sodium molybdate dihydrate 0.39 mg / L, zinc sulfate heptahydrate 0.222 mg / L, cobalt nitrate hexahydrate 0.49 mg / L, copper sulfate pentahydrate 0.079 mg / L.

[0016] In the following examples, the formula of the Modified BG-11 solid plate is: Modified BG-11 liquid medium + 1.5 wt% agar.

[0017] In the following examples, the composition of the Modified HA-SK liquid culture medium is: glucose 15 g / L, sodium nitrate 2.25 g / L, magnesium sulfate heptahydrate 0.3 g / L, calcium chloride dihydrate 0.15 g / L, potassium dihydrogen phosphate 0.6 g / L, Tris 0.5 g / L, EDTA·2Na 0.115 g / L, boric acid 8.82 mg / L, ferrous sulfate heptahydrate 18 mg / L, cobalt chloride hexahydrate 1.42 mg / L, sodium molybdate dihydrate 1.705 mg / L, zinc sulfate heptahydrate 24.1 mg / L, cobalt nitrate hexahydrate 0.49 mg / L, and copper sulfate pentahydrate 1.57 mg / L.

[0018] Example 1: Mutagenesis screening of white mutants of Chlorella pyrenoidosa Scrape two rings of algae mud from the pyrenoid chlorella FZU65 plate with an inoculation loop, inoculate into a 250mL conical flask containing 100mL Modified BG-11 liquid medium, and culture in a light-proof shaker at 25°C and 150rpm until the logarithmic growth phase to obtain the pyrenoid chlorella FZU65 seed solution. The obtained pyrenoid chlorella FZU65 seed solution was transferred to a sterilized centrifuge tube, centrifuged at 8000rpm for 5min, discarded the supernatant, washed the precipitate twice with sterile water, and then added 10mL of sterile water and ethyl methanesulfonate (EMS) with a final concentration of 2.5wt% to the precipitate, placed in a shaker at 25°C and 150rpm for dark treatment for 1h, and then added 10mL of sodium thiosulfate to stop the EMS reaction, centrifuged, discarded the supernatant, washed the precipitate twice with sterile water, and then added 10mL of sterile water to the precipitate, shaken well, and obtained the mutagenic algae solution. The obtained mutagenic algae solution was transferred to a 250 mL conical flask containing 100 mL of Modified BG-11 liquid culture medium and cultured in a shaker at 25 °C and 150 rpm for 24 hours in the dark. After the culture was completed, 1 mL of the culture solution was diluted with sterile water in a gradient manner (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), 100 μL of the dilution was spread on a Modified BG-11 solid plate, and cultured at 25°C in the dark for 7 days. The pyrenoid Chlorella white mutant monoclonal FZU65-W1, FZU65-W2 and FZU65-W3 were separated from the solid plate by naked eye observation. The pyrenoid Chlorella white mutant monoclonal FZU65-W1, FZU65-W2 and FZU65-W3 were transferred to the Modified BG-11 solid plate, and cultured in a 25°C incubator in the dark for 5 days. Figure 1 shown.

[0019] Example 2: Comparison of growth ability and pigment production between the original strain and the white mutant strain of Chlorella vulgaris The original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa were scraped from the Modified BG-11 solid plate and inoculated into 250mL conical flasks containing 100mL Modified BG-11 liquid culture medium, respectively, and cultured in a light-proof shaker at 25℃ and 150rpm for 3 days. Then the culture solution was inoculated into a 250mL conical flask containing 150mL Modified BG-11 liquid culture medium, and the initial cell concentration was controlled to 0.1g / L, and cultured in a light-proof shaker at 25℃ and 150rpm for 6 days. After the culture was completed, samples were retained to determine the biomass concentration and pigment content. The biomass concentration was determined by the cell dry weight method, and the pigment content was determined by organic solvent extraction and colorimetry.

[0020] Figure 2 These are photos of shake flask cultures of the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa. It can be clearly seen that the culture fluid of the original strain of Chlorella pyrenoidosa FZU65 is green, while the culture fluids of the white mutant strains of Chlorella pyrenoidosa FZU65-W1, FZU65-W2 and FZU65-W3 are all white.

[0021] Figure 3 Figure 2 is the biomass concentration of the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa under shake flask culture conditions. It can be obviously seen that the biomass concentration of the white mutant strain of Chlorella pyrenoidosa FZU65-W1 is relatively high, which is 1.09, 1.07 and 1.02 times that of the original strain of Chlorella pyrenoidosa FZU65, the white mutant strain of Chlorella pyrenoidosa FZU65-W2 and the white mutant strain of Chlorella pyrenoidosa FZU65-W3 respectively.

[0022] Figure 4 Figure 2 shows the pigment content of the original strain of Chlorella pyrenoidosa and the white mutant strain of Chlorella pyrenoidosa under shake flask culture conditions. It can be obviously seen that the pigment content of the white mutant strain of Chlorella pyrenoidosa FZU65-W1 is significantly lower than that of the original strain of Chlorella pyrenoidosa FZU65, and its chlorophyll a, chlorophyll b and carotenoid contents are reduced by 97.08%, 84.64% and 98.33% respectively compared with the original strain of Chlorella pyrenoidosa FZU65.

[0023] Combination Figures 2 to 4 It can be seen that the growth rate of the white mutant strain FZU65-W1 of Chlorella pyrenoidosa is not only faster than that of the original strain FZU65 of Chlorella pyrenoidosa, but also its algal cells hardly synthesize chlorophyll and carotenoids, making the algal cells appear white.

[0024] The white mutant strain of Chlorella pyrenoidosa FZU65-W1 was sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation and was classified and named Chlorella pyrenoidosa ( Chlorella pyrenoidosa ), the deposit date is September 11, 2024, the deposit number is CGMCC No.46151, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Example 3: Heterotrophic protein production by the white mutant FZU65-W1 S1: Activation culture: Pick a single clone of the white mutant strain FZU65-W1 of Chlorella vulgaris, inoculate it into a 100 mL conical flask containing 20 mL of Modified HA-SK liquid culture medium, and culture it in a light-proof shaking incubator at 25°C and 150 rpm until the logarithmic growth phase to obtain an activated culture solution; S2: Seed culture: Transfer the activated culture medium of S1 to a 250 mL conical flask containing 80 mL of Modified HA-SK liquid medium, and culture in a shaker at 25°C and 150 rpm in the dark for 3 days to obtain the seed culture medium; S3: Fermentation culture: The seed culture of S2 was inoculated into a 250 mL conical flask containing 100 mL Modified HA-SK liquid culture medium, and the initial cell concentration was controlled to 0.90 g / L. The culture was incubated in a shaker at 25°C and 150 rpm in the dark until the carbon source was exhausted. Then, 0.2 g / L of sodium nitrate and 0.2 g / L of trisodium citrate dihydrate were added to the culture medium, and the culture was continued in a shaker at 25°C and 150 rpm in the dark for 2 days.

[0026] After the cultivation, the biomass concentration, protein content and amino acid composition were determined. The biomass concentration was determined by the cell dry weight method, the protein content was determined by a protein extraction and analysis kit, and the amino acid composition was determined by a fully automatic amino acid analyzer. The algal biomass was measured to be 4.88 g / L and the protein content was 35%. Figure 5 It can be seen that the amino acid composition of the white mutant strain FZU65-W1 of Chlorella pyrenoidosa is very superior, with an essential amino acid index of 1.12.

[0027] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A white mutant strain of Chlorella pyrenoidosa FZU65-W1, characterized in that: Chlorella pyrenoidosa Chlorella pyrenoidosa ), which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 11, 2024, with the deposit number CGMCC No.46151, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the white mutant strain FZU65-W1 of Chlorella vulgaris according to claim 1 in producing protein.

3. A method for producing protein, characterized in that: The protein is produced by culturing the white mutant strain FZU65-W1 of Chlorella vulgaris according to claim 1.

4. The method according to claim 3, characterized in that: The culture method is heterotrophic culture.

5. Use of the white mutant strain FZU65-W1 of Chlorella pyrenoidosa according to claim 1 in preparing food or food additives.

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