Chlorella pyrenoidosa mutant strain and application thereof

Abstract

The invention relates to the technical field of microalgae biology, in particular to a chlorella pyrenoidosa mutant strain and application thereof. According to the invention, a mutagenesis screening mode is adopted to obtain a chlorella pyrenoidosa mutant strain which is named as RMCP012, the chlorella pyrenoidosa mutant strain can still grow quickly after being continuously cultured for one year and is stable in character, and the protein content of the chlorella pyrenoidosa mutant strain is equivalent to that of a wild type chlorella pyrenoidosa mutant strain. Meanwhile, the algal strain of the mutant strain is light green, the decoloring process of industrial production of algal source protein is greatly reduced, the industrial cost is reduced, and the mutant strain is a high-quality algal strain capable of being used for industrial production of protein. The invention further relates to a heterotrophic culture mode of the chlorella pyrenoidosa, so that the chlorella pyrenoidosa can grow quickly, and protein with a relatively high level can be accumulated.

Description

Technical Field

[0001] The invention relates to the field of microalgae biotechnology, and in particular to a pyrenoid chlorella mutant and an application thereof. Background Art

[0002] With the growth of the global population, the improvement of people's health awareness and the rise of plant-based diets, especially the healthy lifestyle, the increasing trend of sports and fitness, and the demand for high-protein diets, the protein market is continuously expanding. Traditional animal proteins such as meat, dairy products and fish currently occupy a major part of the market, but the traditional protein market requires a long growth cycle to provide protein, and it is difficult to balance high quality, hormone-free and cost-effectiveness. More and more consumers choose to be vegetarian or reduce meat consumption, which has driven the market demand for plant protein and alternative protein. Today, the plant protein market is growing rapidly. Among them, algae-derived protein has the characteristics of short growth cycle and high protein quality. It is an excellent alternative protein option. These proteins are not only rich in nutrition, but also can adapt to the future low-carbon development direction and sustainable development needs.

[0003] Chlorella pyrenoidosa is a green single-cell freshwater algae, which is widely used in food, health care products and cosmetic additives due to its rich nutrients and efficient growth ability. Chlorella pyrenoidosa cells contain significant pyrenoids, which makes its protein content as high as 40-60%, exceeding high-protein foods such as beef and soybeans, and its protein components are rich in essential amino acids, with extremely high nutritional value. At the same time, it is rich in vitamins (such as vitamin B group, vitamin C, vitamin E) and minerals (such as iron, calcium, magnesium), which play an important role in human health.

[0004] As a new resource food, the regulations on the production, use and sale of algae-derived proteins in various countries are gradually becoming clearer. Among them, the US FDA regards chlorella products as GRAS (generally recognized as safe); China's new resource food clearly stipulates that protein-core chlorella meets the basic requirements of food and is non-toxic and harmless to the human body; Japan, Canada, Australia and New Zealand and other countries have stipulated the safety and use specifications of chlorella products as food ingredients and dietary supplements. Algae-derived proteins have shown great potential in the protein market. They not only provide high-quality nutrition, but also have significant environmental and sustainability advantages.

[0005] However, chlorella produces chlorophyll during its growth, which causes chlorella protein powder to appear green, which will affect consumer and market acceptance. At present, the decolorization technology of chlorella mainly includes physical separation, solvent extraction, chemical treatment, etc. The above treatments may cause nutrient loss. At the same time, the complex operation steps will increase the time, money, manpower, material and other costs in the production process, and there may also be potential safety issues. Therefore, there is an urgent need for an algae strain that is easy to decolorize and rich in protein. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a mutant strain of Chlorella pyrenoidosa, which has the advantages of stable properties, high protein content and easy decolorization, and can be used for industrial protein production.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In the first aspect, the present invention provides a mutant strain of Auxenochlorella pyrenoidosa, named RMCP012, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65141, a deposit date of September 14, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The present invention uses wild-type pyrenoid Chlorella as a starting strain, performs ultraviolet mutagenesis, and obtains a pyrenoid Chlorella mutant strain through a series of screening, which is named RMCP012. The pyrenoid Chlorella mutant strain RMCP012 grows rapidly, has a chlorophyll content significantly lower than that of the wild-type pyrenoid Chlorella, and has a high protein content, and is an excellent algae strain for producing alternative proteins. The pyrenoid Chlorella mutant strain RMCP012 is prepared into algae powder, and the chlorophyll and lutein content are much lower than the wild-type, showing a light green color with a white tint, which solves the problem of pigment decolorization in the industrial production process, reduces the processing steps, and greatly reduces the production cost of pyrenoid Chlorella.

[0010] In a second aspect, the present invention provides the use of the Auxenochlorella pyrenoidosa mutant in food, health products and dietary additives.

[0011] In a third aspect, the present invention provides a method for obtaining algae-derived protein, wherein the protein is produced by culturing the Auxenochlorella pyrenoidosa mutant.

[0012] Preferably, the Auxenochlorella pyrenoidosa mutant is cultured under dark conditions.

[0013] Preferably, the culture method includes at least one of autotrophic culture, heterotrophic culture and fermentation culture.

[0014] More preferably, the culture method is heterotrophic culture.

[0015] Preferably, the heterotrophic culture conditions are: culture temperature 20-35°C, pH value 6-8.

[0016] The method for heterotrophically culturing a mutant strain of Chlorella vulgaris provided by the present invention has simple culture conditions. When used for industrial fermentation of algae, the fermentation process is not affected by the natural environment such as temperature and light. During the fermentation process, all nutrients are absorbed by the algae, which greatly ensures the stability of algae production, thereby ensuring the rapid and stable acquisition of high-quality protein, and providing more possibilities for the entire protein market.

[0017] The beneficial effects of the present invention are:

[0018] The present invention uses wild-type pyrenoid Chlorella as a starting strain, performs ultraviolet mutagenesis, and obtains a pyrenoid Chlorella mutant strain through a series of screening, which is named RMCP012. The mutant strain produces almost no chlorophyll during dark culture, and the color of the algae strain is light green, and this trait can still be maintained after multiple subcultures.

[0019] The mutant strain of pyrenoid Chlorella of the present invention has the following characteristics: ① its growth is continuously monitored, and the material growth rate of the mutant strain is higher than that of the wild-type pyrenoid Chlorella; ② the chlorophyll and lutein content in the cells of the mutant strain are significantly lower than those of the wild-type pyrenoid Chlorella; ③ the protein accumulation content of the cells of the mutant strain is slightly higher than that of the wild-type pyrenoid Chlorella.

[0020] In the mutant strain of Chlorella vulgaris obtained by mutagenesis screening, the chlorophyll content is reduced or missing, resulting in a lighter color, which can simplify the processing steps, ensure the nutritional integrity, and can be used for industrial production of protein. The mutant strain of Chlorella vulgaris described in the present invention has the advantages of stable properties, rapid growth, and high protein content, and is an excellent high-quality algae strain for producing alternative proteins, and has broad application prospects in food additives, functional beverages, and nutritional supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the appearance of wild-type Chlorella vulgaris.

[0022] Figure 2 This is the appearance picture of the Chlorella pyrenoidosa mutant strain RMCP012.

[0023] Figure 3 Comparison results of the growth curves of wild-type Chlorella pyrenoidosa and mutant strain RMCP012.

[0024] Figure 4 This is the algal pollen picture of the Chlorella pyrenoidosa mutant strain RMCP012.

[0025] Figure 5 This is a comparison of the protein content of wild-type Chlorella vulgaris and mutant strain RMCP012 when they grew to the stationary phase.

[0026] Figure 6 This is a comparison of the chlorophyll content of wild-type Chlorella and mutant strain RMCP012 when they grew to the stationary phase.

[0027] Figure 7 This is a comparison of the lutein content of wild-type Chlorella and mutant strain RMCP012 when they grew to the stationary phase. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] The present invention provides a mutant strain of Auxenochlorella pyrenoidosa, named as RMCP012, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65141, a deposit date of September 14, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0030] Embodiment 1:

[0031] The invention uses wild-type Chlorella pyrenoidosa as a starting strain, performs ultraviolet mutagenesis, and obtains a Chlorella pyrenoidosa mutant strain named RMCP012 through a series of screening.

[0032] The specific process is as follows:

[0033] 1. Preparation of culture medium for Chlorella pyrenoidosa (heterotrophic culture)

[0034] Liquid culture medium formula: weigh raw materials to prepare a culture medium for Chlorella vulgaris, the formula of which is as follows (based on a total volume of 1 L): 10-20 g of glucose, 5-10 g of urea, 0.005-0.01 g of citric acid, 0.005-0.01 g of ammonium ferric citrate, 0.005-0.01 g of K2HPO4·3H2O, 0.05-0.1 g of MgSO4·7H2O, 0.05-0.1 g of CaCl2·2H2O, 0.03-0.05 g of Na2HCO3, and the balance of water; mix well and sterilize at a high temperature and high pressure of 105-125°C to obtain a culture medium for Chlorella vulgaris.

[0035] Preparation of culture medium for solid plate culture: Solid culture medium is based on liquid culture medium and 0.8%-2% agar powder is added.

[0036] 2. Prepare the mutagenesis working solution of Chlorella pyrenoidosa

[0037] Wild-type Chlorella pyrenoidosa (naturally isolated from Hubei waters) was cultured on plates (eg Figure 1 As shown in the figure, under sterile conditions, pick a single clone and add it to the prepared pyrenoid chlorella liquid culture medium (50mL) above. The culture conditions are: temperature 20-32℃, pH value 6-8, and dark culture. Culture for 3-5 days, at which time the pyrenoid chlorella is in the logarithmic growth phase. Take a small amount of algae liquid to observe the cell morphology under a microscope and count the cells at the same time. Then, dilute the algae liquid with sterile water to different gradient cell densities: 10 5 -10 6 / mL as the mutagenesis working solution.

[0038] 3. Mutation of Chlorella pyrenoidosa

[0039] Prepare the UV operating equipment, set the UV lamp power to 40-60w, keep the working solution 30-50cm away from the UV lamp, and treat for different time periods of 10-40min to determine the time when the UV lethality of the cells in the working solution is 85-95%. Then treat for this time period (23min), evenly dilute the treated algae solution and apply it to the solid culture medium of Chlorella pyrenoidosa, place it in a dark culture at 20-32°C, and observe the results after 7-10 days of culture.

[0040] 4. Screening of mutant strains of Chlorella pyrenoidosa

[0041] Through 2-3 times of ultraviolet mutagenesis and screening of strains with reduced pigments. After repeated mutagenesis and screening, the color of the strains became lighter and lighter, and finally several treated protein nucleus Chlorella plates were screened. The initial screening was carried out by picking the algae strains that appeared to be lighter or yellow in color in the plate and culturing them in new heterotrophic plates. The present invention found that in the early stage of mutagenesis, the cells were genetically unstable, the cells themselves would repair the damaged DNA, the phenotype would be unstable, and the cells might turn green again. Therefore, multiple subcultures are required to screen for strains with stable phenotypes.

[0042] After continuous cultivation for more than 8 generations, the present invention finally obtained a mutant strain of Chlorella pyrenoidosa, named RMCP012. Figure 2 The algae strain was deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 65141, the deposit date was September 14, 2024, and the deposit address was 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0043] Embodiment 2:

[0044] The growth status of the Chlorella pyrenoidosa mutant strain RMCP012 was analyzed, and the strain was continued to be subcultured.

[0045] Determine the growth characteristics of mutant RMCP012: Pick wild-type pyrenoid Chlorella (control) and mutant RMCP012 obtained through repeated screening and culture in 15mL heterotrophic liquid culture medium, then inoculate 10-20% of the culture medium into 100mL heterotrophic liquid culture medium for expansion, and culture in the dark at room temperature for 7-10 days. From the first day after inoculation into the 100mL system, the absorbance values ​​of wild-type pyrenoid Chlorella and mutant RMCP012 were continuously measured (the absorbance value was positively correlated with the biomass) to draw a growth curve, and the measurement was continued for 7 days. The results are as follows: Figure 3 shown.

[0046] In the early stage of culture, the growth of wild-type pyrenoid Chlorella was faster than that of mutant RMCP012, but when the mutant RMCP012 entered the logarithmic growth phase (3-5 days of culture), its growth rate was significantly higher than that of wild-type pyrenoid Chlorella. After the growth of mutant RMCP012 entered the plateau phase (after 5 days of culture), the absorbance value of mutant RMCP012 was higher than that of wild-type, indicating that the biomass of mutant RMCP012 was higher than that of wild-type pyrenoid Chlorella.

[0047] Embodiment 3:

[0048] The protein, chlorophyll and xanthophyll content of the Chlorella pyrenoidosa mutant strain RMCP012 were determined.

[0049] The wild type and mutant strain RMCP012 of Chlorella pyrenoidosa grown to the stationary phase in the dark for 7-10 days were collected, the strains were collected by centrifugation, and the pre-frozen strains were directly placed in a freeze dryer for freeze drying for 2-4 days to prepare algal powder ( Figure 4 ) for subsequent testing.

[0050] 1. Protein content determination

[0051] Detection method: Determine the protein concentration by BCA method, prepare BCA working solution, dilute the protein standard, then add the diluted sample to the ELISA plate for incubation, and finally measure the absorbance at 562nm by ELISA. Draw a standard curve based on the absorbance of the standard, and then calculate the concentration of the sample protein through the standard curve. The result is as follows: Figure 5 shown.

[0052] The protein concentration of the wild type obtained by extracting 0.1g of algae powder was 57.15mg / mL, and the protein concentration of the mutant strain RMCP012 of Chlorella pyrenoidosa was 58.88mg / mL. The results showed that the protein content of the mutant strain RMCP012 of Chlorella pyrenoidosa was slightly higher than that of the wild type of Chlorella pyrenoidosa.

[0053] 2. Determination of chlorophyll content

[0054] Detection method: Chlorophyll was extracted by organic solvent extraction and its content was determined. The sample was collected by centrifugation and the residual culture medium was washed with deionized water. It was then resuspended in acetone and kept in the dark overnight to extract chlorophyll, and then the volume was adjusted to 10 mL as the test solution. The maximum absorption peaks of chlorophyll in acetone are 663nm and 645nm. The absorbance values ​​were determined, and then the chlorophyll a (Chl a) content and chlorophyll b (Chl b) content were calculated according to formulas (1) and (2).

[0055] Chlorophyll a content (mg / L) = 12.7 × OD 663 nm -2.69×OD 645 nm ——Formula (1)

[0056] Chlorophyll b content (mg / L) = 22.9 × OD 645 nm -2.69×OD 663 nm ——Formula (2)

[0057] The results are as follows Figure 6As shown, 0.1 g of algal powder was collected to detect its chlorophyll content. The chlorophyll a and chlorophyll b contents of the Chlorella vulgaris mutant strain RMCP012 were 0.59 mg / L and 0.37 mg / L, respectively, while the chlorophyll a and chlorophyll b contents of the wild type were 7.21 mg / L and 15.96 mg / L, respectively.

[0058] The contents of chlorophyll a and chlorophyll b in the mutant strain RMCP012 of Chlorella pyrenoidosa were significantly lower than those in the wild-type Chlorella pyrenoidosa.

[0059] 3. Determination of lutein content

[0060] Detection method: The lutein content is determined by high performance liquid chromatography (HPLC). The sample is placed in a solvent (acetone, ethanol, methanol or DMSO) and ultrasonically crushed until the sample is colorless. The supernatant is collected and filtered through a 0.22 μm microporous filter to obtain a clear extract. The extract is injected into a high performance liquid chromatograph, and a reverse phase column (such as a C18 column) is used to separate the lutein by gradient elution (such as a solvent system such as methanol-water, acetonitrile-water, etc.). The lutein peak is detected by a UV-visible detector (usually at 445 nm), and the lutein content in the sample is analyzed by comparing the retention time and peak area of ​​the standard.

[0061] The results are as follows Figure 7 As shown, the lutein content of the wild-type mutant strain RMCP012 of Chlorella pyrenoidosa is 2.61 mg / L, and the lutein content of the wild-type Chlorella pyrenoidosa is 14.88 mg / L.

[0062] The lutein content of the mutant strain RMCP012 of Chlorella pyrenoidosa was significantly lower than that of the wild-type Chlorella pyrenoidosa.

[0063] Example 4: Analysis of pigment synthesis stability of mutant strain RMCP012

[0064] After nearly a year of subculture, the pyrenoid Chlorella mutant strain RMCP012 can still maintain its color stability, showing a light green color, almost white, which is significantly different from the dark green pyrenoid Chlorella wild type, indicating that the mutant strain RMCP012 is a pyrenoid Chlorella mutant strain with stable expression of chlorophyll synthesis defects.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A mutant strain of Auxenochlorella pyrenoidosa, characterized in that: It is named RMCP012, deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC NO: 65141, the deposit date is September 14, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Auxenochlorella pyrenoidosa mutant according to claim 1 in food, health products and dietary additives.

3. A method for obtaining algae-derived protein, characterized in that: The protein is produced by culturing the Auxenochlorella pyrenoidosa mutant according to claim 1.

4. The method for obtaining algae-derived protein according to claim 3, characterized in that: The Auxenochlorella pyrenoidosa mutant strain is cultured under dark conditions.

5. The method for obtaining algae-derived protein according to claim 4, characterized in that: The culture method includes at least one of autotrophic culture, heterotrophic culture and fermentation culture.

6. The method for obtaining algae-derived protein according to claim 5, characterized in that: The culture method is heterotrophic culture.

7. The method for obtaining algae-derived protein according to claim 6, characterized in that: The conditions of the heterotrophic culture are: culture temperature 20-35° C., pH value 6-8.

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