White chlorella pyrenoidosa mutant strain and application thereof

Abstract

The invention relates to a white chlorella pyrenoidosa mutant strain and application thereof, and belongs to the technical field of microalgae biology. The chlorella pyrenoidosa mutant strain is named as YYZM024, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.40787, and the preservation date is September 19, 2023. The mutant strain is cultured in a glucose-containing heterotrophic culture medium, and after the mutant strain is cultured under the dark condition of 25-28 DEG C for 7-10 days, the content of chlorophyll in cells is still stably maintained at a relatively low level, and the color characterization is very stable. The chlorophyll content of the mutant strain is not obviously changed after passage inoculation for six months, and dry algae powder is always milky white, so that the genetic character of the mutant strain is relatively stable, and the mutant strain can be used as an algae species for large-scale production of algae source protein or an algae powder source of food additives.

Description

Technical Field

[0001] The present invention belongs to the field of microalgae biotechnology, and particularly relates to a white mutant strain of Chlorella pyrenoidosa and its application. Background Art

[0002] In recent years, the concepts of environmental protection, animal welfare, and healthy diet have become increasingly popular, promoting the market application of alternative proteins in human diets. The development of new alternative protein foods based on the concepts of sustainable production, nutrition, health, and environmental protection has witnessed rapid growth. Microalgae have attracted much attention due to their rich metabolites and unique physiological characteristics, and have a wide range of applications in renewable energy, biomedicine, food industry, and environmental monitoring. Microalgae are important raw materials for foods, feeds, drugs, cosmetics, bioenergy, etc.

[0003] Chlorella is a large group of single-celled green algae with a protein content as high as 50%-65% of the dry weight, rich in various bioactive substances, and is a sustainable food resource for high-quality protein. Chlorella can carry out photosynthetic autotrophic production or high-cell-density heterotrophic fermentation, with great production potential, and is regarded as an important new plant protein resource in the field of alternative proteins. Although the taxonomic naming of some Chlorella germplasms has changed in recent years, at least in Europe, more and more Chlorella species are applied in the food field and are not restricted by the new food resource regulations. However, due to the dark green color of the dry powder of wild-type Chlorella, its sensory properties are poor, and it is likely to cause confusion among consumers when used in foods or food additives, restricting its wide application in the food field.

[0004] To solve the color interference, decolorization is currently the main technical means. By optimizing extraction methods such as supercritical CO2 extraction and organic solvent extraction, although the color has been improved to a certain extent, the chlorophyll removal is incomplete, and it will cause high costs, environmental pollution, and loss of nutrients. In the industrial production of microalgae protein, the cost of the decolorization link accounts for about 10-15% of the total protein extraction cost. The decolorization process methods are mostly physical adsorption decolorization and chemical decolorization. Commonly used physical adsorption decolorizing agents include activated carbon, diatomaceous earth, etc.; chemical decolorization is mainly through the way of organic solvent extraction, mainly using ethanol, acetone, etc. The existing decolorization methods usually do not completely remove chlorophyll, the decolorization effect is not ideal, and other colors will be produced after the pigment is oxidized. Moreover, when decolorizing microalgae, whether using physical methods or chemical methods, it will cause losses to the active ingredients beneficial to the human body in microalgae, reduce the nutritional value of microalgae, and lower the yield of the target product. In addition, the organic solvents used in decolorization will also cause environmental pollution and produce polluted wastewater, and the treatment of such polluted wastewater will further increase the production cost, and the trace residues of organic solvents will also affect the product quality.

[0005] For the above reasons, the inventors expect to fundamentally solve the technical problems related to decolorization by screening out light-colored or white algal strains without chlorophyll or with extremely low chlorophyll content. Summary of the Invention

[0006] (1) Technical Problems to be Solved

[0007] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides a white mutant strain of Chlorella pyrenoidosa and its application. The chlorophyll content in the cells of this mutant strain of Chlorella pyrenoidosa is significantly lower than that of the wild type, making the dried algal powder show a distinct white (milky white) color, and it solves the technical problem that it is difficult to remove the color of the dried powder of Chlorella pyrenoidosa. This mutant strain of Chlorella pyrenoidosa is obtained by mutagenesis and screening of the wild-type Chlorella pyrenoidosa, and it can provide an excellent algal strain for the production of Chlorella pyrenoidosa protein.

[0008] (2) Technical Solutions

[0009] To achieve the above object, the main technical solutions adopted by the present invention include:

[0010] In the first aspect, the present invention provides a white mutant strain of Chlorella pyrenoidosa, named YYZM024, which is deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO.40787, the deposit date is September 19, 2023, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] In the second aspect, the present invention provides an application of a white mutant strain of Chlorella pyrenoidosa in the preparation of food or food additives.

[0012] Preferably, the dried powder of the above-mentioned mutant strain of Chlorella pyrenoidosa is prepared and used in foods or food additives other than infant foods.

[0013] In the third aspect, the present invention provides a method for obtaining algal-derived protein, which produces protein by culturing a mutant strain of Chlorella pyrenoidosa.

[0014] Optionally, the culturing method is heterotrophic culture, the culture medium is heterotrophic BG11 medium, and the carbon source in the culture medium is 15 - 30 g / L glucose.

[0015] Optionally, the culturing conditions are static culture in the dark at 25 - 28 °C, and the culturing time is 7 - 10 days.

[0016] (3) Beneficial Effects

[0017] The beneficial effects of the present invention are as follows: The wild-type strain of Chlorella pyrenoidosa is used as the starting strain in the present invention, and normal temperature and pressure plasma mutagenesis is carried out to obtain the Chlorella pyrenoidosa mutant strain YYZM024. The cells of the Chlorella pyrenoidosa mutant strain YYZM024 are brightly milky white, and the color does not change with the increase in the passage times of the algal strain. When the mutant strain is cultured heterotrophically in the dark, its chlorophyll content is significantly lower than that of the wild type, and its protein content is basically the same as that of the wild type. After six months of subculture inoculation, the chlorophyll content and protein content do not change significantly, and the genetic traits are stable. This mutant strain can be used as the algal strain for the production of Chlorella pyrenoidosa algal powder, broadening the application market for microalgae protein production.

[0018] The mutant algal strain of the present invention not only retains the high nutritional value and high production efficiency of Chlorella, but also solves the difficult problem of decolorization of traditional Chlorella, improves the sensory acceptance of consumers, and broadens the food application scenarios. Brief Description of the Drawings

[0019] Figure 1 It is a photograph of the wild-type strain of Chlorella pyrenoidosa;

[0020] Figure 2 It is a photograph of the chlorophyll synthesis-deficient strain YYZM024 obtained by mutagenesis screening;

[0021] Figure 3 It is a photograph of the color mutant strain of the wild-type strain of Chlorella pyrenoidosa;

[0022] Figure 4 It is a photograph of the white mutant strain of the wild-type strain of Chlorella pyrenoidosa after subculture;

[0023] Figure 5 It is a comparison of the growth conditions of the wild-type Chlorella pyrenoidosa and the mutant algal strain under 25°C in the dark;

[0024] Figure 6 It is a comparison of the chlorophyll fluorescence values of the wild-type Chlorella pyrenoidosa and the mutant algal strain under 25°C in the dark;

[0025] Figure 7 It is a comparison of the protein contents of the wild-type Chlorella pyrenoidosa and the mutant algal strain under 25°C in the dark.

[0026] Figure 8 It is a photograph of the dry algal powder of the Chlorella pyrenoidosa mutant strain YYZM024 screened in the present invention. Detailed Embodiments

[0027] In order to better explain the present invention for easy understanding, the present invention will be described in detail below with reference to the drawings through specific embodiments.

[0028] The present invention provides a white mutant strain of Chlorella pyrenoidosa, named YYZM024, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC NO. 40787, the deposit date is September 19, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This mutant strain uses the wild-type Chlorella pyrenoidosa as the material, and adopts the atmospheric and room temperature plasma mutagenesis (ARTP) technology to mutate Chlorella pyrenoidosa. Under the condition of using helium as the protective gas, 10 8 cells are treated for 35 - 40 seconds. Screening is carried out on the heterotrophic BG11 medium, and the obtained Chlorella pyrenoidosa strain with chlorophyll synthesis defect is further verified. Under dark conditions, after culturing this mutant strain in the heterotrophic BG11 medium for 7 - 10 days, the chlorophyll content of the algal cells is significantly lower than that of the wild type, and its protein content is 42.1%, which is basically the same as the wild-type protein content of 45.9%. The wild-type Chlorella pyrenoidosa is dark green (such as Figure 1 ), and the mutant strain screened by the mutation of the present invention is milky white with distinct color (such as Figure 2 ). The chlorophyll content and protein content of this mutant strain have not changed significantly after six months of subculture and inoculation. Therefore, this mutant strain can be used as the algal species source for large-scale production of white Chlorella pyrenoidosa powder.

[0029] 1. The process of obtaining the Chlorella pyrenoidosa mutant strain YYZM024 is as follows:

[0030] (1) Preparation of the Chlorella pyrenoidosa medium

[0031] The heterotrophic Chlorella pyrenoidosa medium contains KH2PO4 0.7 - 7 g / L, K2HPO4 0.3 - 3 g / L, MgSO4·7H2O 0.3 - 2 g / L, FeSO4·7H2O 0.003 - 0.02 g / L, vitamin B1 10 - 20 g / L, glucose 20 - 30 g / L. After high-temperature and high-pressure sterilization, the heterotrophic Chlorella pyrenoidosa BG11 medium can be obtained.

[0032] (2) Preparation of the Chlorella pyrenoidosa seed liquid

[0033] The wild-type Chlorella pyrenoidosa (collected from local wild waters in Yunnan and identified as wild-type Chlorella pyrenoidosa) is cultured in the BG11 medium. A single clone of the wild-type algal strain is picked from the BG11 medium slant and streaked on the BG11 solid plate medium containing 20 g / L glucose, and cultured under light at 25°C for 7 days to obtain an activated single clone. Subsequently, a single algal colony is picked and inoculated into 20 mL of sterile BG11 medium containing 20 g / L glucose, and continuously cultured for 5 days to prepare the seed liquid.

[0034] (3) Mutation and Screening of Chlorella pyrenoidosa

[0035] Take the seed liquid of Chlorella pyrenoidosa in the logarithmic growth phase and measure its cell density. Take 1 - 2×10 8 cells and use the atmospheric and room temperature plasma mutagenesis (ARTP) technology to mutate Chlorella pyrenoidosa. Under the condition of using helium as the protective gas, treat for 10 8 cells for 35 - 40 seconds to make the lethality rate about 95%. Resuspend the algal cells and coat them on the BG11 solid plate, and culture them in the dark at 25°C for 10 - 15 days to obtain monoclonal Chlorella pyrenoidosa with different mutations.

[0036] Determine the treatment conditions with a lethality rate higher than 95% as the optimal mutagenesis conditions. Select cells in the logarithmic growth period for multi - batch mutagenesis and then coat them on the BG11 solid plate for culture. Use the color of the single algal colonies grown as an index for primary screening. Compared with the wild - type dark - dark - green algal colonies, select the algal colonies with a visibly lighter color (light green, yellow, white, etc.) as the primary - screened mutant strains, collect and preserve them. Pick the white monoclonal onto a new BG11 solid plate and perform multiple passages to observe the color stability.

[0037] 2. Analysis and Testing. The test items are as follows:

[0038] (1) Analysis of the Stability of Pigment Synthesis in Mutants

[0039] Starting from the wild - type Chlorella pyrenoidosa strain, after multi - batch mutagenesis, 1 color - mutant strain was obtained through primary screening (such as Figure 3 ). Since the algal colonies presented milky white in Figure C and had the most stable traits during the continuous sub - screening process, finally, the algal strain in Figure C of Figure 3 was selected as the target mutant strain for screening and was passaged multiple times; Figure 3 The mutant strain in Figure C of Figure 4 was continuously sub - screened on the heterotrophic BG11 solid plate (as shown in Figure 4 , Figures C1, C2, and C3 in Figure 4 are mutant strains with gradually extended passage times). After 8 - passage culture, the color characteristics were stable (as shown in Figure C3 of

[0040] (2) Growth, Chlorophyll and Protein Content Determination

[0041] Take several sterile 250 mL vented flasks, add 100 mL of heterotrophic BG11 liquid medium on the ultra - clean workbench, use a sterile pipette tip to pick monoclonal algal colonies into the BG11 liquid medium, and culture them in the dark at 25°C for 7 days. After growing to the plateau phase, centrifuge to collect the algae for protein content determination. The specific process is as follows:

[0042] ① First, simultaneously place the chlorophyll-deficient mutant strains of Chlorella pyrenoidosa (the yellow chlorophyll-deficient strain of Chlorella pyrenoidosa and the white chlorophyll-deficient strain of Chlorella pyrenoidosa, among which the yellow chlorophyll-deficient strain of Chlorella pyrenoidosa has been deposited, and the deposit number is CGMCC NO.40458; the white chlorophyll-deficient strain of Chlorella pyrenoidosa is named YYZM024) and the wild-type Chlorella pyrenoidosa in 100 mL of heterotrophic medium, and continuously culture them for 8 days under dark conditions at 25 °C to make a growth curve and measure the chlorophyll fluorescence value. The results are as follows: As Figure 5 shown, under dark culture, the growth rates of the two mutant algal strains are slower compared to the wild-type algal strain and the selected strain, which is consistent with the growth prediction of the chlorophyll synthesis-deficient algal strains; during the experimental culture period, the growth trends of YYZM024, CGMCC NO.40458, and the wild-type Chlorella pyrenoidosa strain (WT) are the same; however, throughout the experimental cycle, the growth rate of the YYZM024 algal strain is slightly lower than that of the other two algal strains. Compared with CGMCC NO.40458 and the wild-type algal strain (WT), the chlorophyll fluorescence value of the selected mutant target algal strain YYZM024 is significantly reduced, and the chlorophyll fluorescence value of the target algal strain YYZM024 has been stably at a low level throughout the dark culture period (compared with the wild-type algal strain and CGMCC NO.40458); during the experimental culture period, the characteristics of CGMCC NO.40458 are stable and it has always maintained a yellow state, but the chlorophyll fluorescence value content still increased significantly in the later stage of the experimental culture, while the YYZM024 algal strain has always maintained a low level state, as Figure 6 shown.

[0043] ② Protein content determination

[0044] After continuously culturing the wild-type algal strain, the selected mutant target algal strain YYZM024 (hereinafter referred to as the target algal strain) and the selected strain (CGMCC NO.40458) in the dark, measure their protein content. It is found that the protein content of the target algal strain is basically the same as that of the wild-type algal strain. Because the chlorophyll fluorescence value of the selected strain increased in the later stage of the experimental culture, making its growth rate faster than that of the target algal strain YYZM024, so the protein content of the selected strain is higher than that of YYZM024, as Figure 7 shown.

[0045] Although the protein content and growth rate of YYZM024 are slightly lower than those of the screened strain, under comprehensive comparison, the screened strain cannot completely solve the technical problem of difficult microalgae decolorization in industrial production, and its trait stability is poor, unable to ensure the stability of color traits throughout the fermentation production process. Therefore, based on the objective of the present invention to provide an algal strain with the comprehensive advantages of high protein content and low chlorophyll content, while taking into account the stability of mutant traits, YYZM024 algal strain is finally selected as the target algal strain.

[0046] (3) Color of the dry powder of the Chlorella pyrenoidosa mutant strain YYZM024

[0047] The Chlorella pyrenoidosa mutant strain YYZM024 is made into dry powder through general processes such as centrifugation, washing, separation, and drying. The color of both the mutant strain and the prepared dry powder is distinct milky white (see Figure 8 ), which solves the technical problems such as poor sensory properties of algal powder color and the need for decolorization. At the same time, it can maintain a relatively high protein content, improve the protein yield from algae, and reduce the protein extraction cost, and is particularly suitable as a food raw material or food additive.

[0048] In the BG11 heterotrophic medium, when the mutant strain YYZM024 of the present invention is cultured under dark conditions, its chlorophyll content is significantly lower than that of the wild type and remains at a low level throughout the growth cycle, and its protein content is also comparable to that of the wild type. After six months of subculture inoculation, the protein content has not changed significantly, and the color remains distinct and stable milky white, indicating that the genetic traits of the mutant strain YYZM024 are stable. The mutant strain YYZM024 can be used as an algal species for the production of Chlorella pyrenoidosa white algal powder, broadening the application market for microalgae protein production.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A white mutant strain of Chlorella pyrenoidosa, named YYZM024, was deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO.40787, the deposit date was September 19, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the white mutant strain of Chlorella pyrenoidosa described in claim 1 in the preparation of food or food additives.

3. The application according to claim 2, characterized in that, Prepare the dry powder of the mutant strain of Chlorella pyrenoidosa described above for use in foods or food additives other than infant foods.

4. A method for obtaining algal protein, characterized in that, Produce protein by culturing the mutant strain of Chlorella pyrenoidosa described in claim 1.

5. The method according to claim 4, wherein The culturing method is heterotrophic culture, the culture medium is heterotrophic BG11 medium, and the carbon source in the culture medium is 15 - 30 g / L glucose.

6. The method according to claim 4, characterized in that The culturing conditions are static culture in the dark at 25 - 28 °C, and the culturing time is 7 - 10 days.

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