Non-green Chlamydomonas reinhardtii and its application

Abstract

The invention discloses non-green Chlamydomonas reinhardtii and its application. There are three strains of non-green Chlamydomonas reinhardtii. The yellow Chlamydomonas reinhardtii is named CHL-1yellow, with a deposit number of CCTCC M 2024746; the red Chlamydomonas reinhardtii is named CHL-2red, with a deposit number of CCTCC M 2024747; and the white Chlamydomonas reinhardtii is named CHL-3white, with a deposit number of CCTCC M 2024748. The Chlamydomonas reinhardtii of the invention has various properties, different colors and rich tastes. Compared with the Chlamydomonas reinhardtii before mutagenesis, the protein content has no obvious change after detection, and the chlorophyll content is reduced, thereby reducing its algae smell, having rich colors, and is a very excellent alternative protein product. In addition, the algae strain obtained by the mutagenesis method of the invention is confirmed to have high stability after more than three generations of subculture, and the operation method is simple and easy, without toxic consumables, and is safe and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to non-green Chlamydomonas reinhardtii and applications thereof. Background Art

[0002] Chlamydomonas reinhardtii is a unicellular eukaryotic green algae, which is a good material for studying photosynthetic system, material metabolism, and ciliary regulation. Chlamydomonas reinhardtii lives in fresh water, is easy to culture and reproduce, has a short cell cycle, and has excellent environmental stress tolerance.

[0003] The protein content of Chlamydomonas reinhardtii is higher than that of ordinary green algae, and the crude polysaccharide content is ≥10%. It is listed as a "generally recognized as safe" substance in the United States and can be used as a source of dietary protein. In my country, it was approved by the National Health Commission in 2022 and can be used as a new food ingredient.

[0004] Algae mainly fix carbon dioxide and solar energy through photosynthesis. At present, the traditional algae cultivation methods are mainly open and sealed. The open ones are mainly runway pools, shallow pools, etc., but due to their simple equipment, they are easy to cause pollution. The sealed photobioreactor is expensive and small in scale, and is not suitable for large-scale production. In addition, the fishy smell of algae in the existing technology needs to be optimized, and the meat taste is average.

[0005] Therefore, it is necessary to develop an algae with more diverse sensory colors and a richer meaty taste, which does not rely on limiting factors such as light for algae growth, and can be clean and sterile using closed fermentation in fermentation tanks, and has the advantages of a mature production equipment system, low pollution, and controllable costs. Summary of the invention

[0006] In order to overcome the above-mentioned problems of the prior art, the present invention provides a non-green Chlamydomonas reinhardtii, which has less algae smell and excellent color sensory perception, and a richer meat taste. The algae does not rely on limiting factors of algae growth such as light, and can achieve cleanliness and sterility by closed fermentation in a fermentation tank. It has the advantages of a mature production equipment system, low pollution, and controllable cost, and has great production prospects.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions:

[0008] In a first aspect of the present invention, a non-green Chlamydomonas reinhardtii strain is provided, wherein the non-green Chlamydomonas reinhardtii strain comprises at least one of the following three strains:

[0009] The yellow Chlamydomonas reinhardtii was named Chlamydomonas reinhardtii CHL-1yellow, with the deposit number of CCTCC M 2024746 and the deposit date of April 22, 2024;

[0010] The red Chlamydomonas reinhardtii is named Chlamydomonas reinhardtii CHL-2red, with the deposit number CCTCCM 2024747 and the deposit date April 22, 2024;

[0011] The white Chlamydomonas reinhardtii was named Chlamydomonas reinhardtii CHL-3white, the deposit number was CCTCC M 2024748, and the deposit date was April 22, 2024.

[0012] In a second aspect of the present invention, a non-green Chlamydomonas reinhardtii fermentation agent is provided, wherein the fermentation agent comprises:

[0013] Fermenting at least one of the non-green Chlamydomonas reinhardtii to obtain a fermentation liquid;

[0014] Alternatively, the fermentation liquid is spray-dried to obtain Chlamydomonas reinhardtii freeze-dried powder.

[0015] Furthermore, the fermentation liquid obtained by fermenting at least one strain of the non-green Chlamydomonas reinhardtii comprises:

[0016] (1) Obtaining Chlamydomonas reinhardtii seeds: Inoculate the dominant algae strain into liquid TAP medium and culture in shake flasks;

[0017] (2) Preparation of fermentation medium: The fermentation medium initially uses TAP medium, the acid tank uses 20% glacial acetic acid solution, the alkali tank uses sodium hydroxide solution, and the defoaming agent is prepared by using corn oil: triglycerol = 7:3, and sterilized at high temperature;

[0018] (3) Fermentation of mutant algae strains: Inoculate the mutant algae seeds cultured in shake flasks into a fermenter, start the fermentation program, set the fermentation parameters to a culture temperature of 22°C-30°C (preferably 24°C), pH 6.8-7.2 (preferably pH 7.0), a rotation speed of 100-200 rpm (preferably 150 rpm), and a dissolved oxygen content greater than 10%. Start feeding at an appropriate time and link the acid-base regulation system. After the fermentation is completed, obtain the fermentation liquid.

[0019] Furthermore, the fermentation liquid is spray-dried to obtain Chlamydomonas reinhardtii freeze-dried powder, comprising:

[0020] The fermentation liquid is centrifuged, and then the algae cells are collected, spread on a metal dish, and freeze-dried under low temperature vacuum to obtain the Chlamydomonas reinhardtii freeze-dried powder.

[0021] The freeze-dried Chlamydomonas reinhardtii powder obtained by fermentation is tested to be rich in protein and crude polysaccharide, which can supplement various nutrients required by the human body, and the secondary metabolites produced by fermentation have rich flavor substances.

[0022] In the third aspect of the present invention, provided is the use of the non-green Chlamydomonas reinhardtii or the fermentation agent of the non-green Chlamydomonas reinhardtii in the preparation of cosmetics.

[0023] Furthermore, the cosmetics include at least one of a facial mask, a facial cream, a hand cream and an eye cream.

[0024] In a fourth aspect of the present invention, provided is the use of the non-green Chlamydomonas reinhardtii or the fermentation agent of the non-green Chlamydomonas reinhardtii in the preparation of food for replacing protein products.

[0025] Furthermore, the food includes at least one of hamburger plant meat, sausage casings, pet food, pasta and rice food.

[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0027] The present invention provides non-green Chlamydomonas reinhardtii, which increases the diversity of food sources: most of the existing algae products are health foods - algae capsules or compressed particles, and the existing products of Chlamydomonas reinhardtii only have green Chlamydomonas reinhardtii algae powder on the market. The present invention uses mutagenesis technology to obtain non-green Chlamydomonas reinhardtii strains, which have a variety of colors and a richer meat taste compared with Chlamydomonas reinhardtii before mutagenesis, and are very good substitute protein products. It has been identified that the protein content, crude polysaccharide content and other indicators are the same as those of the wild-type algae strain, and the chlorophyll content is reduced, thereby reducing the fishy smell of algae. The mutant algae strains with different colors enrich the types of existing algae products and can be used as substitute protein raw materials to make algae protein hamburgers, cream and other foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a flow chart of the screening method of the present invention;

[0029] Figure 2 These are examples of liquid culture of wild type and various mutants. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0031] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0033] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0034] The original background algae strain used in the present invention is a Chlamydomonas reinhardtii, Latin name: Chlamydomonas reinhardtii, purchased from the Chlamydomonas Resource Center of the University of Minnesota, USA, numbered CC-1690 wild type mt+, also known as 21gr, which belongs to the Chlorophyta, Chlorophyceae, Volvoxales, Chlamydomonasaceae, Chlamydomonas genus.

[0035] The inventors of the present application obtained three mutants of Chlamydomonas reinhardtii through screening steps such as sample culture, sample preparation, atmospheric and room temperature plasma (ARTP) mutagenesis, coating culture, darkroom screening, and trait confirmation. The screening step uses light-shielding stress to screen and obtain chlorophyll synthesis defective algae strains, and then transfers them to fresh culture medium to obtain dominant algae strains (non-green Chlamydomonas reinhardtii) with stable mutant traits and rapid growth in light-shielding conditions under repeated stress environments.

[0036] According to existing literature reports, Chlamydomonas reinhardtii can be induced by ultraviolet light or by inserting gene mutations, and yellow and white algae species can be obtained by screening under specific light-proof conditions. However, this technology is different from the above technical scheme. It uses atmospheric and room temperature plasma technology (ARTP) for mutagenesis, and does not require the preparation of DNA fragments, enzyme cutting, purification and other steps to produce algae strains with stable genetic traits, and does not introduce exogenous DNA. It is more environmentally friendly, simpler and faster to operate, and more efficient in obtaining mutant algae strains. The mutant algae strains (non-green Chlamydomonas reinhardtii) screened by the present invention do not rely on limiting factors such as light for algae growth, and can be clean and sterile by using closed fermentation production in a fermenter. It has the advantages of a mature production equipment system, low pollution, and controllable cost, and has great production prospects.

[0037] More importantly, the present invention screened and obtained three non-green Chlamydomonas reinhardtii strains that have different characteristics from previous Chlamydomonas reinhardtii (i.e., unexpected technical effects): the Chlamydomonas reinhardtii obtained by the present invention through ARTP technology mutation has diverse traits, different colors and rich tastes. After testing, there is no significant difference in various nutritional elements compared with the Chlamydomonas reinhardtii before mutation, and it has a richer meat taste. The chlorophyll content is reduced, thereby reducing the fishy smell of algae, and it is a very good alternative protein product. In addition, the algae strain obtained by the mutagenesis method of the present invention has been found to have high stability after more than three generations of subculture, and the operation method is simple and easy, without toxic consumables, safe and environmentally friendly.

[0038] In a specific embodiment, the screening method of non-green Chlamydomonas reinhardtii of the present invention comprises the following steps:

[0039] S1: Sample culture: transfer the wild-type Chlamydomonas reinhardtii CC-1690 into liquid TAP medium for activation and proliferation;

[0040] In S1, the experimental conditions for activation and proliferation are: constant temperature (24°C), light (light: dark = 14:10), and aeration culture for 72 hours;

[0041] S2: ARTP technology mutagenesis treatment: take the activated algae cells and spread them on the sterilized slide, put them into the mutagenesis chamber, use high-purity helium as the plasma working gas for mutagenesis, set the instrument parameters: working voltage 120W, working gas flow 10slpm, mutagenesis time 40s, collect the cells into the centrifuge tube filled with liquid TAP culture medium after mutagenesis, shake them in a shaker, and recover them in the dark;

[0042] In S2, the speed of the shaking table is 50-150 rpm, preferably 100 rpm, and the time must be fixed at 8 hours, because cells will proliferate if the time is too long, which is not conducive to the selection of monoclonal algae strains.

[0043] S3: Spreading culture: The recovered algal cells were collected by low-speed centrifugation, resuspended in a small amount of liquid TAP medium, and spread on solid TAP medium;

[0044] S4: Darkroom screening: Invert the culture away from light and observe the cell budding;

[0045] S5: Confirmation of traits and preservation of algae: Select non-green algae strains, spread them on solid TAP culture medium, repeatedly perform light-protection stress, screen out the best growing and distinctly colored yellow, white, and red monoclonal algae colonies, and use liquid nitrogen to preserve the dominant algae strains.

[0046] The TAP medium can be purchased commercially. As a specific embodiment, the configuration method of the TAP medium used in the present invention is as follows, taking 1L as an example:

[0047] (1) Prepare a 1 mol / L tris solution by weighing 12.1 g of tris powder, diluting to 100 mL, and stirring evenly;

[0048] (2) Prepare phosphate buffer by dissolving 10.8 g of dipotassium hydrogen phosphate and 5.6 g of potassium dihydrogen phosphate in a small amount of water, making the volume to 100 mL and stirring evenly;

[0049] (3) Prepare buffer A by dissolving 20 g of ammonium chloride, 5 g of magnesium sulfate heptahydrate, and 2.5 g of calcium chloride dihydrate in a small amount of water in order, making the volume to 500 mL and stirring evenly;

[0050] (4) Prepare trace element stock solution: dissolve 22 g zinc sulfate heptahydrate, 11.4 g boric acid, 5.06 g manganese chloride tetrahydrate, 1.61 g cobalt chloride hexahydrate, 1.57 g copper sulfate pentahydrate, 1.1 g sodium molybdate tetrahydrate, 4.99 g ferrous sulfate heptahydrate, 50 g disodium ethylenediaminetetraacetic acid, and 20 g potassium hydroxide in a small amount of water, and make up to 1 L;

[0051] (5) Add the following solutions in the following order: 20 mL of 1 M tris solution, 1 mL of phosphate buffer, 10 mL of buffer A, 1 mL of trace element stock solution, and 1 mL of glacial acetic acid. Adjust the pH to 7.0 and finally make up to 1 L. For solid culture medium, add 15 g of agar powder. Sterilize at high temperature and high pressure, then cool for later use.

[0052] The mutant algae strains obtained by the ARTP technology mutagenesis screening of the present invention are different from ultraviolet and insertion gene mutations, do not introduce exogenous DNA, and have stable genetic traits, and algae strains of different colors are obtained through directional screening. And the mutation efficiency is high: a large number of mutated algae strains can be obtained in a short time by using simple operations, without using more reagents and consumables. The uniform plasma jet in the ARTP mutagenizer can act on the algae cells to be treated, and the chemically active ions rich in the jet act on the cell genetic material, which can induce gene mutations on the DNA, and finally obtain a large number of mutants. High biological safety: compared with molecular breeding methods, the present invention does not use toxic and harmful reagents or consumables, a large number of mutant algae strains can be obtained by simple operations, and no exogenous DNA is introduced, and no ecological pollution is caused to the external environment.

[0053] In other embodiments, the present invention provides a fermentation agent of non-green Chlamydomonas reinhardtii, wherein the fermentation agent comprises:

[0054] Fermenting at least one of the non-green Chlamydomonas reinhardtii to obtain a fermentation liquid;

[0055] Alternatively, the fermentation liquid is spray-dried to obtain Chlamydomonas reinhardtii freeze-dried powder.

[0056] Testing has shown that non-green Chlamydomonas reinhardtii and its fermentation agents not only contain multiple nutritional elements such as protein, crude polysaccharides, and multiple amino acids similar to ordinary algae strains, but also have various colors. The secondary metabolites produced through fermentation have rich flavor substances and can be used in food, cosmetics, biomedicine and other fields.

[0057] The following will describe in detail a non-green Chlamydomonas reinhardtii strain and its application in combination with examples and experimental data.

[0058] Example 1. Obtaining non-green Chlamydomonas reinhardtii

[0059] 1. Screening of mutant algae strains

[0060] 1.1 Sample culture: The wild-type Chlamydomonas reinhardtii CC-1690 preserved on solid culture medium was transferred to liquid TAP culture medium for activation and proliferation, and cultured at a constant temperature (24°C) with illumination (light:dark=14:10) and aeration for 72 hours.

[0061] 1.2 Sample preparation: Collect the activated algae cells by centrifugation at 2500 rpm, add fresh liquid TAP medium and resuspend to 5×10 6 Pieces / mL.

[0062] 1.3 Plasma mutagenesis at room temperature and pressure: Take 10uL of algae solution and evenly spread it on the surface of the slide, put it into the mutagenesis chamber, use high-purity helium as the working gas of plasma for mutagenesis, and then set the instrument parameters: working voltage 120W, working gas flow 10slpm (liters per minute), mutagenesis time 40s, after mutagenesis, collect the cells into a 50mL centrifuge tube containing 9mL of culture medium and shake at 100rpm in the dark for 8h.

[0063] 1.4 Spreading culture: Centrifuge the recovered algal cells at 2500 rpm, collect the lower layer of cell sediment, resuspend it with a small amount of liquid culture medium, and spread it evenly on the solid TAP culture medium.

[0064] 1.5 Darkroom screening: The solid culture plate was inverted and cultured in the dark for 10 days, and the cell budding was observed in the dark every 12 hours.

[0065] 1.6 Confirmation of traits and preservation of algae: The germinated monoclonal algae strains were re-inoculated onto fresh solid culture medium and cultured again in the dark, as described in 1.5 above, and repeated 7 times until the best growing, distinctly colored yellow, white, and red monoclonal algae colonies were screened, and the dominant algae strains were preserved in liquid nitrogen. The yellow, white, and red monoclonal algae colonies were also preserved.

[0066] The preservation date of the yellow reinhardtii of the present invention is April 22, 2024, and the preservation number is CCTCC M2024746. Its classification name is yellow reinhardtii (Chlamydomonas reinhardtii), and the preservation unit name is China Center for Type Culture Collection, and the address is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072.

[0067] The deposit date of the red Chlamydomonas reinhardtii of the present invention is April 22, 2024, and the deposit number is CCTCC M2024747. Its classification name is red Chlamydomonas reinhardtii, and the depository name is China Center for Type Culture Collection, and the address is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072.

[0068] The preservation date of the white reinhardtii of the present invention is April 22, 2024, and the preservation number is CCTCC M2024748. Its classification name is white reinhardtii (Chlamydomonas reinhardtii), and the preservation unit name is China Center for Type Culture Collection, and the address is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072.

[0069] Example 2: Preparation of Yellow Chlamydomonas reinhardtii Freeze-dried Powder

[0070] The mutant algae strain (yellow algae strain CHL-A7S80) with stable genetics was used for high-density culture in a 5L fermenter. The fermentation process was as follows:

[0071] 1. Obtaining Chlamydomonas reinhardtii seeds: Inoculate the dominant algae strain into liquid TAP medium and culture at 100 rpm in a shake flask until the concentration reaches 10 6 Pieces / mL.

[0072] 2. Preparation of fermentation medium: liquid TAP medium, 20% glacial acetic acid solution, 0.5 mol / L sodium hydroxide solution, defoaming agent (corn oil: triglycerol = 7:3) and feed medium are 50×TAP concentrate without Tirs in the fermenter. In addition, sodium acetate with a final concentration of 0.5 mol / L and acetic acid with a volume fraction of 20% are added and sterilized for use.

[0073] 3. Fermentation of mutant algae strains: Inoculate the mutant algae seeds cultured in shake flasks into the fermenter at a ratio of 0.5% of the culture medium volume over a flame, start the fermentation program, set the fermentation parameters to a culture temperature of 24°C, pH 7.0, a rotation speed of 150rpm, and dissolved oxygen greater than 10%. After 2 days when the dissolved oxygen is lower than 60%, start the feeding program, initially set at 2mL / h, double the feeding every 12h, observe the foaming through the window, and add 0.05% of the total volume of defoamer each time.

[0074] 4. Obtaining algae powder: After 6 days of fermentation, extract algae liquid every 6 hours, and stop the fermentation when the wet weight reaches 100g / L. Collect the fermented algae liquid, centrifuge at 4500rpm to obtain the cell precipitate, spread it on a metal dish, and freeze-dry it at low temperature and vacuum for more than 8 hours to obtain Chlamydomonas reinhardtii freeze-dried powder. Figure 2 shown.

[0075] Example 3: Preparation of white Chlamydomonas reinhardtii freeze-dried powder

[0076] Compared with Example 2, the difference is that in the process of preparing the freeze-dried Chlamydomonas reinhardtii powder, the white mutant algae strain preserved in Example 1 is used, and the other steps are the same as Example 2.

[0077] Example 4: Preparation of red Chlamydomonas reinhardtii freeze-dried powder

[0078] Compared with Example 2, the difference is that in the process of preparing the freeze-dried Chlamydomonas reinhardtii powder, the red mutant algae strain preserved in Example 1 is used, and the other steps are the same as in Example 2.

[0079] Comparative Example 1: Preparation of freeze-dried powder of wild-type Chlamydomonas reinhardtii

[0080] Compared with Example 2, the difference is that the wild-type Chlamydomonas reinhardtii species CC-1690 is directly used to prepare the Chlamydomonas reinhardtii freeze-dried powder.

[0081] Comparative Example 2: Freeze-dried powder of other algae strains obtained by the same screening method as in Example 1

[0082] Comparative Example 2 is the freeze-dried powder of the A5S22 algae strain. The screening method is the same as that of Example 1 of the present invention. The other red monoclonal algae colonies selected are named A5S22 algae strains and the freeze-dried powder is prepared by the method of Example 2.

[0083] Experimental Example 1: Determination of Nutrient Content and Sensory Evaluation

[0084] 1. The nutrient content of the freeze-dried Chlamydomonas reinhardtii powder prepared in the above examples and comparative examples was measured. The results are shown in Table 1.

[0085] Table 1 Carotene content of Chlamydomonas reinhardtii powder

[0086]

[0087]

[0088] From Table 1, we can see that

[0089] Comparative Example 1 is a freeze-dried powder prepared from wild-type Chlamydomonas reinhardtii, and its protein and chlorophyll contents are 51.2 g / 100 g and 20.5 mg / g, respectively.

[0090] In Comparative Example 1, the freeze-dried powders prepared in yellow, red and white colors respectively had protein and chlorophyll contents of 45.2 g / 100 g, 48.5 g / 100 g, 46.5 g / 100 g, 0.139 mg / g, 0.163 mg / g and 0.102 mg / g, respectively.

[0091] Compared with Comparative Example 1-Comparative Example 2, the chlorophyll content of Example 2-Example 4 is reduced.

[0092] 2. The freeze-dried Chlamydomonas reinhardtii powders prepared in the above examples and comparative examples were subjected to sensory evaluation. The results are shown in Table 2.

[0093] Sensory evaluation method: 20 tasters with normal sense of taste and smell formed a sensory evaluation panel. The fishy smell of the freeze-dried powder in comparative example 1 was recorded as 10, and the distilled water without fishy smell was recorded as 0. Each group of freeze-dried powder was subjected to a separate sensory evaluation test. Each person measured each sample three times and took the average value of each person, and then took the overall average value. The results are as follows:

[0094] Table 2

[0095]

[0096] As shown in Table 2, compared with Comparative Examples 1 and 2, the algae smell of Examples 2 to 4 is reduced. Application Example 1: Application to noodle and rice foods

[0097] Weigh the Chlamydomonas reinhardtii powder prepared in Example 2, Example 3 or Example 4, add 60g of Chlamydomonas reinhardtii powder to 1kg of wheat flour and mix them evenly for later use; control the water temperature at 30°C, add water to the prepared materials in the first step in proportion according to the standard of 1kg of wheat flour to 0.3L of water, mix them thoroughly for 10 minutes with a dough mixer to make dough; put the dough into a static maturation machine for 10 minutes. The prepared Chlamydomonas reinhardtii dough is used as a raw material, and other raw materials are added (according to conventional methods) to further process into noodles, pancakes, dumplings, buns, steamed buns, fried dough sticks and bread.

[0098] Application Example 2: Dietary Supplements

[0099] Weigh the Chlamydomonas reinhardtii powder prepared in Example 2, Example 3 or Example 4, and add it to food and drinks, such as soup, juice and salad, to increase the taste and nutritional value.

[0100] 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.

[0101] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0102] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A non-green Chlamydomonas reinhardtii, characterized in that: The non-green Chlamydomonas reinhardtii is any one of the following three strains: Yellow Chlamydomonas reinhardtii, named Chlamydomonas reinhardtii CHL-1yellow, with the deposit number CCTCCM 2024746 and the deposit date April 22, 2024; Red Chlamydomonas reinhardtii, named Chlamydomonas reinhardtii CHL-2red, with the deposit number of CCTCC M2024747 and the deposit date of April 22, 2024; White Chlamydomonas reinhardtii, named Chlamydomonas reinhardtii CHL-3white, with a deposit number of CCTCC M2024748 and a deposit date of April 22, 2024.

2. A non-green Chlamydomonas reinhardtii fermentation agent, characterized in that: The fermentation agent comprises: Fermentation broth obtained by fermenting at least one of the non-green Chlamydomonas reinhardtii strains according to claim 1; Alternatively, the fermentation liquid is spray-dried to obtain Chlamydomonas reinhardtii freeze-dried powder.

3. The fermentation agent of non-green Chlamydomonas reinhardtii according to claim 2, characterized in that: The method for preparing the fermentation liquid obtained by fermenting at least one strain of the non-green Chlamydomonas reinhardtii comprises: (1) Obtaining Chlamydomonas reinhardtii seeds: Inoculate the dominant algae strain into liquid TAP medium and culture in shake flasks; (2) Preparation of fermentation medium: The fermentation medium initially uses TAP medium, the acid tank uses 20% glacial acetic acid solution, the alkali tank uses sodium hydroxide solution, and the defoaming agent is prepared by using corn oil: triglycerol = 7:3, and sterilized at high temperature; (3) Fermentation of mutant algae strains: The mutant algae seeds cultured in shake flasks were inoculated into the fermentation tank, and the fermentation program was started. The fermentation parameters were set to a culture temperature of 22°C-30°C, a pH of 6.8-7.8, a rotation speed of 100-200 rpm, and a dissolved oxygen of more than 10%. Feeding was started at an appropriate time, and the acid-base adjustment system was linked. After the fermentation was completed, the fermentation liquid was obtained.

4. The fermentation agent of non-green Chlamydomonas reinhardtii according to claim 2, characterized in that: The method for preparing Chlamydomonas reinhardtii freeze-dried powder by spray-drying the fermentation liquid comprises: The fermentation liquid is centrifuged, and then the algae cells are collected, spread on a metal dish, and freeze-dried under low temperature vacuum to obtain the Chlamydomonas reinhardtii freeze-dried powder.

5. Use of the non-green Chlamydomonas reinhardtii according to claim 1 or the fermentation agent of the non-green Chlamydomonas reinhardtii according to claims 3-4 in the preparation of cosmetics.

6. The use according to claim 5, characterized in that the cosmetics include at least one of a facial mask, a facial cream, a hand cream and an eye cream.

7. Use of the non-green Chlamydomonas reinhardtii according to claim 1 or the fermentation agent of the non-green Chlamydomonas reinhardtii according to claims 3-4 in the preparation of food for replacing protein products.

8. The use according to claim 7, characterized in that the food comprises at least one of hamburger plant meat, sausage casings, pet food, pasta and rice food.

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