CN116555040B - Yellow chlorella pyrenoidosa mutant strain and application thereof - Google Patents
Yellow chlorella pyrenoidosa mutant strain and application thereof Download PDFInfo
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Abstract
The invention relates to a yellow chlorella pyrenoidosa mutant strain and application thereof, belonging to the technical field of microalgae biology. The chlorella pyrenoidosa mutant strain is named YYAM019 and is preserved in China general microbiological culture collection center (CGMCC) No.40458 and the preservation date is 2023, 4 and 27. The mutant strain is cultured in a heterotrophic culture medium containing glucose, and after 7-10 days of culture under the dark condition of 25-28 ℃, the chlorophyll content in cells is still stably maintained at a lower level, and the color characterization is stable. The chlorophyll content of the mutant strain is not changed obviously after six months of subculture, so that the genetic character of the mutant strain is deduced to be relatively stable, and the mutant strain can be used as an algae source for large-scale production of algae-derived proteins.
Description
Technical Field
The invention belongs to the technical field of microalgae biology, and particularly relates to a yellow chlorella pyrenoidosa mutant strain and application thereof.
Background
In recent years, environmental protection, animal welfare and healthy diet concepts are becoming deeper and deeper, market application of the substitute protein in human diets is being promoted, and development of novel substitute protein foods based on sustainable, nutritional, healthy and environment-friendly concepts is being developed at a high speed. Chlorella (Chlorella) is a large class of unicellular Chlorella, with protein content up to 50% -65% of dry weight, rich in various bioactive substances, and used as sustainable food resource for high-quality proteins. Chlorella can be used for photosynthesis autotrophic production and high cell density heterotrophic fermentation, has great production potential, and is regarded as an important new plant protein resource in the field of protein substitution. Although some of the chlorella germplasm designations have changed in taxonomy in recent years, there is an increasing use of chlorella species in the food field, at least in europe, without being limited by new resource food regulations. However, the dry powder of wild type chlorella is dark green, so that the dry powder has poor sense, is easy to cause confusion of consumers when being used for food or food additives, and limits the wide application in the food field. In order to solve the color interference, decolorization is the main technical means at present. In the industrial production of microalgae proteins, the cost of the decoloring link is about 10-15% of the total protein extraction cost. Most of the decolorization process methods are physical adsorption decolorization and chemical decolorization. The common physical adsorption decolorizer is activated carbon, diatomite and the like; the chemical decolorization is mainly carried out by means of organic solvent extraction, mainly using ethanol, acetone and the like. The prior decoloring method generally has incomplete removal of chlorophyll, and when the microalgae is decolored, no matter the physical method or the chemical method is used for decoloring, the active ingredients of the microalgae are negatively influenced, so that the nutritional value of the microalgae is lost, and the yield of products is reduced. In addition, the organic solvent used in the decolorization can produce environmental pollution, the treatment of such polluted wastewater can further increase the production cost, and trace residues of the organic solvent can also affect the product quality.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the invention provides a yellow chlorella pyrenoidosa mutant strain and application thereof, wherein the chlorophyll content in the chlorella pyrenoidosa mutant strain cells is significantly lower than that of a wild type, so that the dry algae powder is vividly yellow (brilliant yellow), and the technical problem that the color of the chlorella pyrenoidosa dry powder is difficult to remove is solved. The chlorella pyrenoidosa mutant strain is obtained by utilizing wild chlorella pyrenoidosa to carry out mutagenesis and screening, and can provide excellent algae strains for producing chlorella pyrenoidosa protein.
(II) technical scheme
In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:
In a first aspect, the invention provides a yellow chlorella pyrenoidosa Chlorella pyrenoidosa mutant, designated YYAM019, deposited in China general microbiological culture Collection center, having a deposit number of: CGMCC No.40458, the preservation date is 2023, 4 and 27, and the preservation address is North Star Xili No.1, 3 in the Chaoyang district of Beijing city.
In a second aspect, the invention provides an application of a yellow chlorella pyrenoidosa Chlorella pyrenoidosa mutant strain in preparing food or food additives.
Preferably, the chlorella pyrenoidosa Chlorellapyrenoidosa mutant dry powder is prepared and used in foods or food additives except infant foods.
In a third aspect, the present invention provides a method for obtaining an algae-derived protein by culturing a mutant chlorella pyrenoidosa Chlorellapyrenoidosa strain to produce the protein.
Alternatively, the culture method is heterotrophic culture, the culture medium is heterotrophic BG11 culture medium, and the carbon source in the culture medium is 15-30g/L glucose.
Alternatively, the culturing condition is at 25-28deg.C, and the culturing time is 7-10 days.
(III) beneficial effects
The beneficial effects of the invention are as follows: the invention takes a wild type chlorella pyrenoidosa strain as a starting strain to carry out plasma mutagenesis at normal temperature and normal pressure to obtain a chlorella pyrenoidosa mutant strain YYAM019. The chlorella pyrenoidosa mutant YYAM019 cells are vividly yellow, and the color is not changed along with the increasing of the passage times of the chlorella pyrenoidosa. The mutant has a chlorophyll content significantly lower than that of the wild type and a protein content higher than that of the wild type when the mutant is cultivated in the dark heterotrophy. The protein content is not changed obviously after six months of passage inoculation, and the genetic character is stable. The mutant strain can be used as an algae seed for producing the chlorella pyrenoidosa powder, and widens the application market for producing the microalgae protein.
The mutant chlorella strain not only maintains the high nutritive value and high production efficiency of the chlorella, but also solves the difficult problem of decoloration of the traditional chlorella, improves the sensory acceptability of consumers and widens the application scene of food.
Drawings
FIG. 1 is a photograph of a wild type Chlorella pyrenoidosa strain;
FIG. 2 is a photograph showing chlorophyll synthesis-deficient algae strain YYAM019 obtained by mutagenesis screening;
FIG. 3 is an SEM image of a color mutant strain of a wild type Chlorella pyrenoidosa strain;
FIG. 4 is an SEM image of a color mutant strain of wild type Chlorella pyrenoidosa after subculturing;
FIG. 5 is a comparison of growth of wild type Chlorella pyrenoidosa and mutant strain in the dark at 25 ℃;
FIG. 6 is a comparison of chlorophyll fluorescence values of wild type Chlorella pyrenoidosa and mutant strain in darkness at 25 ℃;
FIG. 7 shows the protein content comparison of wild type Chlorella pyrenoidosa and mutant strain of Chlorella pyrenoidosa in darkness at 25 ℃.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
The invention provides a yellow chlorella pyrenoidosa Chlorellapyrenoidosa mutant, named YYAM019, which is preserved in China general microbiological culture Collection center, and has the preservation number of: CGMCC No.40458, the preservation date is 2023, 4 and 27, and the preservation address is North Star Xili No. 1,3 in the Chaoyang district of Beijing city. The mutant strain uses the wild type of the chlorella pyrenoidosa as a material, adopts the normal temperature and pressure plasma mutagenesis (ARTP) technology to carry out the chlorella pyrenoidosa mutagenesis, and treats 10 8 cells under the condition of taking helium gas as a protective gas for 35-40 seconds. Screening on heterotrophic BG11 culture medium, and further verifying the obtained Chlorella pyrenoidosa strain with defective chlorophyll synthesis. The chlorophyll content of the algae cells is obviously reduced compared with that of the wild type after the mutant strain is cultured for 7-10 days by using a heterotrophic BG11 culture medium under the dark condition, the protein content is 53.7 percent and is 7.8 percent higher than that of the wild type protein. The wild type chlorella pyrenoidosa is dark greenish (shown in figure 1), and the mutant strain screened by the mutation of the invention is vividly yellow (shown in figure 2). The chlorophyll content and the protein content of the mutant strain are not changed obviously after six months of subculture, so that the mutant strain can be used as an algae source for producing the chlorella pyrenoidosa powder on a large scale.
1. The process for obtaining the chlorella pyrenoidosa mutant YYAM019 is as follows:
(1) Preparation of chlorella pyrenoidosa culture medium
The heterotrophic chlorella pyrenoidosa BG11 culture medium comprises KH2PO40.7-7g/L,K2HPO40.3-3g/L,MgSO4·7H2O0.3-2g/L,FeSO4·7H2O0.003-0.02g/L, vitamin B110-20g/L and glucose 20-30g/L, and can be obtained after high-temperature high-pressure sterilization.
(2) Preparation of chlorella pyrenoidosa seed liquid
Wild type chlorella pyrenoidosa (collected from local wild water areas in Yunnan and identified as wild type chlorella pyrenoidosa) is cultured in a BG11 culture medium, a wild type strain monoclonal is selected from the inclined plane of the BG11 culture medium, streaked on a BG11 solid flat-plate culture medium containing 20g/L glucose, and cultured for 7 days under the illumination of 25 ℃ to obtain an activated monoclonal. Then, a single algae is picked up, inoculated into 20mL of sterile BG11 medium containing 20g/L glucose, and continuously cultured for 5 days to prepare seed liquid.
(3) Mutagenesis and screening of Chlorella pyrenoidosa
Taking the chlorella pyrenoidosa seed liquid in logarithmic growth phase, and measuring the cell density. 1-2 multiplied by 10 3 cells are taken for carrying out the mutagenesis of the chlorella pyrenoidosa by adopting the normal temperature and pressure plasma mutagenesis (ARTP) technology, 10 8 cells are treated under the condition of taking helium gas as protective gas, the treatment time is 35-40 seconds, the lethality of the cells is about 95 percent, the chlorella pyrenoidosa cells are resuspended and coated on a BG11 solid plate, and the chlorella pyrenoidosa monoclonal with different mutations can be obtained after the reverse culture for 10-15 days in the dark at 25 ℃.
And determining the treatment condition with the mortality rate higher than 95% as the optimal mutagenesis condition, carrying out multi-batch mutagenesis on cells in the logarithmic growth period, then coating BG11 solid flat-plate culture, carrying out primary screening by taking the color of the grown single algae drop as an index, and selecting macroscopic algae drops with light green (light green, yellow, white and the like) as primary screening mutant strains compared with wild dark-black algae drops, and collecting and preserving. Yellow monoclonals were picked onto new BG11 solid plates for multiple passages and observed for color stability.
2. Analysis and testing, test items are as follows:
(1) Analysis of mutant pigment Synthesis stability
Starting from wild chlorella pyrenoidosa strains, performing multi-batch mutagenesis, and performing primary screening to obtain 3 color mutant strains (shown in figure 3), wherein the C is bright yellow algae, and the character is the most stable in the continuous passage screening process, so that the C in figure 3 is finally selected as a screened target mutant strain, and the target mutant strain is subjected to multiple passages; the mutant of panel C in fig. 3 was continuously passaged and screened on heterotrophic BG11 solid plates (as shown in fig. 4, panels a, B, and C in fig. 4 are mutants with sequentially prolonged passaging times), and after 8 times of subculture, the color characteristics were stable (as shown in panel C in fig. 4, clear and stable yellow color was exhibited).
(2) Growth conditions, chlorophyll and protein content determination
Taking a plurality of sterile 250mL aeration bottles, adding 100mL heterotrophic BG11 liquid culture medium on an ultra-clean workbench, picking up monoclonal algae by using a sterile middle gun head, placing the monoclonal algae into the BG11 liquid culture medium, culturing in darkness at 25 ℃ for 7 days, centrifuging to collect the algae after the algae grow to a platform period, and measuring the protein content. The specific process is as follows:
① Mutant strains (two mutant strains after passage screening are compared, wherein the mutant strain obtained by screening is named YYAM019, the other strain is the screening strain) and wild type chlorella pyrenoidosa are simultaneously placed in 100mL heterotrophic culture medium, and are continuously cultured for 5 days under the dark condition of 25 ℃ to be used as a growth curve and the chlorophyll fluorescence value is measured. The results are shown in the figure: as shown in fig. 5, in dark culture, the growth rate of two mutant algal strains was slower than that of the wild-type algal strains and the screened strains, which is consistent with the defect of chlorophyll synthesis of the mutant algal strains per se; the growth rates of YYAM019 and the screened strain were identical in the early stage of the test culture, but the screened strain appeared to be partially greened (mutant character was unstable) in the later stage of the test, so that the growth rate was faster than YYAM019. The chlorophyll fluorescence value of the target strain YYAM and the selected strain was significantly reduced compared to the wild-type strain, and the chlorophyll fluorescence value of the target strain YYAM019 was always stable at a low level throughout the dark culture period (compared to the wild-type strain and the selected strain), as shown in fig. 6.
② Protein content determination
After continuously culturing a wild-type strain, a target strain YYAM019 (hereinafter referred to as a target strain) and a selected strain, which are mutated, in the dark, the protein content of the selected strain is measured, and the protein content of the selected strain and the target strain is found to be higher than that of the wild-type strain, because the selected strain shows a green returning condition in the later stage of experimental culture, the growth speed is higher than YYAM019, and the protein content of the selected strain is higher than YYAM019, as shown in fig. 7.
Although YYAM019 has slightly lower protein content and growth speed compared with the screened strain, the screened strain cannot completely solve the technical problem of difficult decolorization of microalgae in process production and has poor character stability under comprehensive comparison, so the aim of the invention is to provide a comprehensive dominant algae strain with high protein content and low chlorophyll content, and meanwhile, the stability of mutation characters is considered, so that the YYAM019 algae strain is finally selected as a target algae strain.
(3) Chlorella pyrenoidosa mutant YYAM019 dry powder color
The chlorella pyrenoidosa mutant YYAM019 is prepared into dry powder by common processes such as centrifugation, washing, separation, drying and the like, and the mutant and the prepared dry powder are bright yellow in color, so that the technical problems of poor organoleptic properties of the dry powder, need of decolorization and the like are solved, and the chlorella pyrenoidosa mutant YYAM has higher protein content, can improve the yield of algae-derived protein and reduce the protein extraction cost.
The mutant YYAM019 of the present invention has a significantly lower chlorophyll content than the wild type and a higher protein content than the wild type when cultured in BG11 heterotrophic medium under dark conditions. The protein content is not changed obviously after six months of passage inoculation, the color is still bright and stable yellow, and the hereditary character of the mutant YYAM019 is stable. The mutant YYAM019 can be used as an algae seed produced by the chlorella pyrenoidosa algae powder, and widens the application market for the production of microalgae proteins.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (6)
1. A yellow chlorella pyrenoidosa (Chlorella pyrenoidosa) mutant, designated YYAM019, is deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.40458, the preservation date is 2023, 4 and 27, and the preservation address is North Star Xili No. 1, 3 in the Chaoyang area of Beijing city.
2. Use of the yellow chlorella pyrenoidosa mutant strain of claim 1 in the preparation of a food or food additive.
3. The use according to claim 2, wherein the chlorella pyrenoidosa mutant strain dry powder is prepared for use in foods other than infant foods or in food additives.
4. A method for obtaining an algae-derived protein, characterized by culturing the chlorella pyrenoidosa mutant strain of claim 1 to produce the protein.
5. The method according to claim 4, wherein the culture method is heterotrophic culture, the culture medium is heterotrophic BG11 culture medium, and the carbon source in the culture medium is 15-30g/L glucose.
6. The method according to claim 4, wherein the culturing is carried out in the dark at 25-28℃for 7-10 days.
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| JP2006101795A (en) * | 2004-10-07 | 2006-04-20 | Yakult Honsha Co Ltd | Chlorella having high chlorophyl and carotinoid contents and method for producing the same |
| CN112226452A (en) * | 2020-09-03 | 2021-01-15 | 广东海洋大学 | Chlorella pyrenoidosa chloroplast transgene system and application thereof |
| CN114854596A (en) * | 2022-05-26 | 2022-08-05 | 珠海元育生物科技有限公司 | Chlorella protothecoides mutant strain with high protein yield and application thereof |
| CN116024092A (en) * | 2022-08-17 | 2023-04-28 | 中国农业科学院都市农业研究所 | Chlorella pyrenoidosa improved variety resource with high protein yield and high genetic transformation efficiency and application thereof |
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| KR102276935B1 (en) * | 2020-02-06 | 2021-07-12 | 한양대학교 산학협력단 | Novel violaxanthin-overproducing strain of chlorella vulagaris and the method for producing violaxanthin using the same |
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| JP2006101795A (en) * | 2004-10-07 | 2006-04-20 | Yakult Honsha Co Ltd | Chlorella having high chlorophyl and carotinoid contents and method for producing the same |
| CN112226452A (en) * | 2020-09-03 | 2021-01-15 | 广东海洋大学 | Chlorella pyrenoidosa chloroplast transgene system and application thereof |
| CN114854596A (en) * | 2022-05-26 | 2022-08-05 | 珠海元育生物科技有限公司 | Chlorella protothecoides mutant strain with high protein yield and application thereof |
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