Method for producing protein using alga

Abstract

The purpose of the present invention is to provide a method for maintaining or increasing the content of rubisco protein in an alga body. Provided is a method for producing a protein, which makes it possible to maintain or increase the content of rubisco protein in an alga body of an alga by adding sulfate ions and / or thiosulfate ions to a culture medium and culturing the alga under conditions where the sulfate ions and / or the thiosulfate ions are not depleted.

Description

Method for producing proteins using algae

[0001] The present invention relates to a method for producing a protein using algae.

[0002] According to United Nations population projections, the world population is expected to grow to 8.5 billion in 2030, 9.7 billion in 2050, and 10.8 billion in 2100, which means there is a possibility that protein supplies will be insufficient by 2050. As such, a protein crisis is predicted in the near future, but there are limits to how much can be done by simply increasing meat production. Therefore, there are high hopes for protein production using microorganisms, and microalgae are one such promising microorganism.

[0003] As a method for enriching microalgae proteins, Patent Document 1 discloses a method for retarding the growth of Chlorella using a fermentation medium depleted of nitrogen-free nutrients. Patent Document 1 also describes that this retardation can be achieved by adding a substance that inhibits cell growth, such as sulfate, to the culture medium.

[0004] Japanese Patent Application Publication No. 2017-502680

[0005] The present inventors have confirmed that poor growth of microalgae occurs in the later stages of cultivation, resulting in a decrease in the protein content within the algae and a decrease in biomass production. This phenomenon is fatal in protein production. According to the description in Patent Document 1, adding sulfate to algae that are already growing poorly is expected to further impair growth.

[0006] However, the present inventors have discovered that adding sulfate ions and / or thiosulfate ions to an algal culture medium can avoid this poor growth and maintain or increase the protein content within the algae. Furthermore, the present inventors have discovered that the protein that decreases in the later stage of culture and whose content is maintained or increased by the addition of sulfate ions and / or thiosulfate ions is Rubisco protein. The "later stage of culture" refers to the period when, when algae are cultured in a general culture medium, some of the nutrients in the medium begin to be depleted.

[0007] One aspect of the present invention aims to provide a method for maintaining or increasing the content of Rubisco protein in algae.

[0008] In order to solve the above problems, one embodiment of the present invention provides a protein production method that maintains or increases the content of Rubisco protein in algal cells by adding sulfate ions and / or thiosulfate ions to a culture medium and culturing algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions.

[0009] According to one aspect of the present invention, the content of Rubisco protein in algal cells can be maintained or increased, thereby enabling stable protein production by the algae even in the later stage of cultivation, and increasing biomass production.

[0010] 1 shows the growth status of the Chromochloris zofingiensis UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test medium sections of the UTEX32 strain. 2 shows the change in protein content of the UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test medium sections of the UTEX32 strain. 3 shows the change in protein production of the UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test medium sections of the UTEX32 strain. 4 shows the growth status of the UTEX32 strain when the medium for the UTEX32 strain contains five times the amount of magnesium sulfate as a sulfur source compared to the control. 5 shows the change in protein content in the algae when the medium for the UTEX32 strain contains five times the amount of magnesium sulfate as a sulfur source compared to the control. 1 shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the UTEX32 strain containing five times the amount of magnesium sulfate compared to the control, together with a control. 2 shows the change in protein production when the medium for the UTEX32 strain contains five times the amount of magnesium sulfate as a sulfur source compared to the control. 3 shows the results of an investigation using various microalgae to determine whether the rate-limiting effect of the sulfur source on algal growth is observed between different species of algae. 1001 shows the results using Chromochloris zofingiensis UTEX32 strain, 1002 Chlamydomonas sp. JSC4 strain, 1003 Synechocystis sp. PCC6803 strain, 1004 Synechococcus elongatus PCC7942 strain, and 1005 Chlorella sorokiniana UTEX1230 strain. This figure shows the change in biomass production when magnesium sulfate was added at various concentrations as a sulfur source to the medium for the UTEX32 strain and the strain was cultured for 14 days.

[0011] An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. In this specification, unless otherwise specified, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

[0012] 1. Protein Production Method A protein production method according to one embodiment of the present invention (hereinafter referred to as the method of the present application) is a method for maintaining or increasing the content of Rubisco protein in algal cells of algae by adding sulfate ions and / or thiosulfate ions to a culture medium and culturing algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions. Hereinafter, in this specification, "sulfate ions and / or thiosulfate ions" will also be referred to as "sulfate ions, etc.", and "sulfate and / or thiosulfate" will also be referred to as "sulfate, etc."

[0013] (1) Algae In this specification, algae may be macroalgae or microalgae. The method of the present application maintains or increases the content of Rubisco protein in the algal cells of the algae. Because both macroalgae and microalgae contain Rubisco protein, the method can be applied to either algae.

[0014] Macroalgae are multicellular algae, such as wakame seaweed, kelp, mozuku seaweed, and nori seaweed. Microalgae are primarily unicellular algae, such as diatoms, dinoflagellates, blue-green algae, green algae, and red algae. Microalgae are preferred as algae because they are easy to mass-cultivate and their proteins can be easily obtained. The following explanation will be given for the case where the algae is microalgae.

[0015] As the microalgae, from the viewpoint of excellent protein productivity, proliferation ability, ease of culturing, environmental tolerance, etc., it is preferable to use one or more types of algae selected from the group consisting of the genera Chromochloris, Chlamydomonas, Synechocystis, Synechococcus, Chlorococcum, Arthrospira, and Chlorella.

[0016] Examples of microalgae that can be used in the genus Chromochloris include Chromochloris zofingiensis. Examples of microalgae that can be used in the genus Chlamydomonas include Chlamydomonas sp. and Chlamydomonas reinhardtii. Examples of microalgae that can be used in the genus Synechocystis include Synechocystis sp. Examples of microalgae that can be used in the genus Synechococcus include Synechococcus elongatus. Examples of microalgae that can be used in the genus Chlorella include Chlorella sorokiniana, Chlorella pyrenoidosa, Chlorella ellipsoidea, Chlorella vulgaris, Chlorella regularis, and Chlorella salina.

[0017] (2) Cultivation of Algae In the method of the present application, a culture medium is added with sulfate ions and the algae are cultured under conditions that do not deplete the sulfate ions. The culture medium is not particularly limited and may be appropriately selected depending on the taxonomic group and culture characteristics of the algae. For example, for freshwater algae, terrestrial algae, hot spring algae, and saline algae, media such as BG-11, AAF-6, AF-6, AF-AC, BBM, C, CYT, HUT, M-11, MAF-6, MDM, MG, and MGM can be used. Furthermore, for marine algae and brackish water algae, media such as BESM, ESM, IMK, K, MF, and MKM can be used.

[0018] In the method of the present application, sulfate ions and the like are added to the medium based on the finding that the decrease in protein production and biomass production in the later stage of cultivation is due to the depletion of sulfur in the medium.

[0019] The method described in Patent Document 1 does not increase the total production amount of biomass because it includes a step of restricting the growth of microalgae. Patent Document 1 also describes that sulfate is added to inhibit the growth of microalgae.

[0020] On the other hand, the present inventors have found that the amount of protein produced and the amount of biomass produced by the microalgae decrease significantly in the later stage of the microalgae culture, and have conducted extensive research into the cause of this. As a result, as shown in the Examples below, it has been found that the decrease in protein production is caused by sulfur depletion due to the consumption of sulfur in the culture medium by the microalgae, and that the protein whose production decreases is mainly Rubisco protein.

[0021] Therefore, sulfate ions and the like were added to the medium, and algae were cultured under conditions that did not deplete the sulfate ions and / or thiosulfate ions. Note that, in this specification, "adding sulfate ions and the like to the medium" includes the following aspects.

[0022] (i) In addition to the sulfate ions, etc. originally contained in the medium, additional sulfate ions, etc. are added to the medium. For example, if a medium originally contains 0.3 mM sulfate ions and the medium is further supplemented with 1.0 mM sulfate ions, the medium will contain 1.3 mM sulfate ions.

[0023] (ii) When preparing a new medium, the medium should contain sulfate ions, etc.

[0024] When algae are cultured, sulfate ions and the like in the medium are consumed by the algae as the culture progresses. Therefore, if the concentration of sulfate ions and the like in the medium remains the same as the concentration originally contained in the medium, sulfate ions and the like will decrease, and eventually the "rate limitation by sulfate ions and the like" described below will occur. In this specification, "depletion of sulfate ions and the like" refers to a state in which sulfate ions and the like in the medium have decreased to the extent that the "rate limitation by sulfate ions and the like" occurs. In addition, in this specification, "conditions that do not deplete sulfate ions and the like" refers to a state in which sulfate ions and the like are contained in the medium to an extent that does not cause the "rate limitation by sulfate ions and the like."

[0025] The above conditions can be achieved by experimentally determining the amount of sulfate ions, etc. consumed by the algae before protein production within the algae becomes rate-limiting, and then adding sulfate ions, etc. that can replenish this amount to the culture medium at the start of cultivation or during cultivation.

[0026] As a result, it was revealed that regardless of the type of algae, the content of Rubisco protein in the algae bodies could be maintained or increased without decreasing, even in the later stages of cultivation, compared to before the later stages of cultivation, and that the growth conditions of the algae could be maintained in good condition.

[0027] Thus, the present invention was the first to discover that the protein that decreases in the algae in the later stage of cultivation is Rubisco protein, and that this decrease is due to the depletion of sulfur in the medium. Furthermore, the present invention was the first to discover that the Rubisco protein content can be maintained or increased even in the later stage of cultivation by culturing algae under conditions that do not deplete sulfate ions, etc., and that good algal growth can be maintained. Furthermore, in the method described in Patent Document 1, the addition of sulfate inhibits the growth of microalgae, but in the method of the present application, as described below in the Examples, the growth of microalgae is not inhibited, and therefore the method of the present application can be said to be advantageous for protein and biomass production.

[0028] As shown in the examples described below, the type of sulfate salt from which the sulfate ions are derived is not particularly limited, as it does not affect the content of Rubisco protein in the algae or the growth conditions of the algae. Examples of sulfate salts that can be used include sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, and sodium thiosulfate. These may be used alone or in combination. In the case of (i) above, the sulfate ions originally contained in the medium and the sulfate ions added to the medium may be derived from the same sulfate salt or different sulfate salts.

[0029] The method for adding sulfate ions and the like to the medium is not particularly limited. For example, sulfate ions and the like can be added to the medium by adding the sulfate salt and the like to the medium. The order of addition may be reversed. That is, the medium may be added to the sulfate salt and the like.

[0030] The medium usually contains about 0.3 mM of sulfate ions, etc., originally. However, if the culture is continued at this content, the protein content in the algae decreases when the rate-limiting effect of sulfate ions, etc. begins. Here, the "rate-limiting effect of sulfate ions, etc. begins" varies depending on the type of algae, but generally occurs after the fourth day from the start of the algae culture. In this specification, "rate-limiting effect of sulfate ions, etc." refers to the rate-limiting effect of protein production in the algae due to the depletion of sulfate ions, etc. The time when sulfate ions, etc. are added to the medium may be any time before the sulfate ions, etc. are depleted. Therefore, as long as the time is before the sulfate ions are depleted, the time may be any time before the start of the algae culture, at the start of the culture, or during the culture.

[0031] The sulfate ions and the like are preferably contained in the medium at a concentration of 0.75 to 2.0 mM. The relationship between the amount of sulfate ions and the like contained in the medium and the final protein content or biomass production amount in the algae varies somewhat depending on the type of algae. However, when the concentration of sulfate ions and the like contained in the medium is 0.75 to 2.0 mM, the protein content and biomass production amount can be effectively maintained or increased without decreasing even in the later stage of cultivation, generally regardless of the type of algae. Therefore, adding sulfate ions and the like to the medium at a concentration of 0.75 to 2.0 mM and culturing algae in the medium corresponds to an example of a condition that does not deplete the sulfate ions and the like. From this perspective, the concentration of sulfate ions and the like is more preferably 0.75 to 1.5 mM, and even more preferably 0.90 to 1.5 mM.

[0032] The concentration of sulfate ions and the like contained in the medium is preferably the concentration at the start of the culture (initial concentration). If the medium contains sulfate ions and the like at a concentration of 0.75 to 2.0 mM at the start of the culture, the concentration of sulfate ions and the like will be a concentration that will cover in advance the depletion of sulfate ions and the like that may occur in the later stages of the culture. Therefore, algae culture can be smoothly carried out without the need to take the time to check the concentration of sulfate ions and the like in the medium during the culture.

[0033] However, the concentration of sulfate ions, etc. contained in the medium may be the concentration during the culture. For example, algae culture may be started without adding sulfate ions, etc. to the medium, and sulfate ions, etc. may be added to the medium at a concentration of 0.75 to 2.0 mM before the sulfate ions, etc. are depleted.

[0034] The concentration of sulfate ions and the like contained in the culture medium is not necessarily limited to 0.75 to 2.0 mM. For example, even if the concentration is less than 0.75 mM, as long as sulfate ions and the like are not depleted, algae can be cultured under conditions that do not deplete sulfate ions and the like, thereby maintaining or increasing the content of Rubisco protein in the algae. The same applies when the concentration exceeds 2.0 mM.

[0035] Furthermore, when the concentration of sulfate ions, etc. contained in the culture medium is, for example, 0.75 to 2.0 mM, this concentration covers in advance the depletion of sulfate ions, etc. that may occur in the later stages of culture, and therefore it is not necessary to maintain this concentration at 0.75 to 2.0 mM until the end of culture. For example, in Example 4, algae are cultured using a medium containing sulfate ions, etc. at a concentration of 0.75 mM. In this case, it is expected that the concentration of sulfate ions, etc. in the medium will fall below 0.75 mM as the culture progresses, but the effects of the present invention can be achieved without performing any subsequent operations to maintain this concentration.

[0036] In addition to sulfate ions, the medium contains components necessary for algae growth, such as nitrogen, phosphorus, potassium, magnesium, and calcium. The inventors' investigations revealed that increasing the concentrations of these components other than sulfate ions in the medium alone did not increase the amount of protein and biomass produced within the algae. This finding revealed that the depletion of sulfate ions and other components due to their consumption was the primary cause of the decrease in protein content and biomass production.

[0037] Therefore, by adding, for example, 0.75 to 2.0 mM of sulfate ions or the like to the culture medium, the growth of the algae in the later stage of culture is maintained or enhanced, and as a result, the protein content and productivity in the algae can be maintained or increased.

[0038] The method for culturing the algae in the medium containing sulfate ions, etc. is not particularly limited, but it is preferable to use a closed culture system to avoid contamination by bacteria and to use a method that allows for mass culture in order to improve the efficiency of the culture. Examples of such a culture system include a bag-type culture device, a glass tube-type (horizontal) culture device, a glass tube-type (vertical) culture device, or a polyethylene tube-type culture device.

[0039] The culture conditions were determined based on the light intensity, temperature, CO ２ The concentration etc. is, for example, light intensity: 100 μmol-photons / (m ２ ・s), temperature: 25℃, CO ２ The concentration is preferably 2%.

[0040] (3) Rubisco Protein Content in Algal Bodies The present method is a method for maintaining or increasing the Rubisco protein content in the algal bodies of the algae. As described above, the present inventors have found that the protein content in the algal bodies decreases in the later stages of algal culture. Therefore, the present inventors used SDS-PAGE to investigate the types of proteins whose content decreases. As a result, as shown in the Examples below, it was found that the Rubisco protein content significantly decreases in the later stages of culture.

[0041] Rubisco protein (ribulose-1,5-bisphosphate carboxyltransferase / oxygenase) is a CO ２ From the results of SDS-PAGE, it was found that the decrease in the protein content in the algae and the decrease in the algal biomass production in the later stage of cultivation were due to the decrease in the content of Rubisco protein. ２ This was thought to be due to a decrease in fixation efficiency. Because various proteins exist within the algae, it is unclear whether the proteins whose content decreases in the algae during the later stages of cultivation are photosynthetic proteins or other proteins. It was not previously known that the proteins that decrease during the later stages of cultivation are Rubisco proteins.

[0042] The inventors then discovered that the Rubisco protein content did not recover when other nutrients were added to the medium, but that the content could be maintained or increased when sulfate ions, etc. were added to the medium. This surprising and previously unknown finding was that the Rubisco protein content in algae depends on the sulfur content in the medium, and that the content can be maintained or increased by supplementing the sulfate ions, etc. in the medium, which decrease as the culture progresses.

[0043] It is preferable to add sulfate ions and the like to the medium from the start of culture rather than adding them after the protein content in the algae begins to decrease, which allows for continuous, uninterrupted protein production and makes it easier to achieve the desired protein content.

[0044] "Maintaining or increasing the Rubisco protein content" means that the content is unchanged or increased compared to when the algae are cultured without adding the sulfate salt or the like to the medium, or compared to a reference point in time. "Not changing the content" means that the content is within ±5% compared to when the algae are cultured without adding the sulfate salt or the like to the medium, or compared to the Rubisco protein content at a reference point in time. The "reference point in time" refers to the start point of algal culture.

[0045] The maintenance or increase of the Rubisco protein content in the algae can be confirmed, for example, by recovering the algae after cultivation and performing the BCA method, which is a protein quantification method. The algae can also be recovered from the culture medium by, for example, centrifugation.

[0046] The resulting algal biomass containing high concentrations of protein can be used for processing purposes such as meat substitutes (block meat, minced meat, etc.), functional foods, health foods, functional materials such as bioplastics, liquid fuel, and animal feed. By subjecting the algal cells to the mass-culture method described above, the method of the present application can obtain large quantities of algal cells that maintain or increase the Rubisco protein content within the algal cells. Therefore, the method of the present application can be considered a groundbreaking method that can address the protein crisis described above.

[0047] The method of the present application not only maintains or increases the content of the Rubisco protein, but also maintains good growth of the algae for a long period of time. ２ This allows for smooth immobilization and efficient photosynthesis. As a result, good growth of the algae can be maintained for a long period of time. Therefore, the method of the present application can also increase biomass production compared to methods that do not include a step of adding sulfates or the like to the medium.

[0048] Such effects will also contribute to achieving, for example, Goal 2 of the Sustainable Development Goals (SDGs) advocated by the United Nations, "Zero Hunger."

[0049] [2. Method for maintaining algal photosynthesis for a long period of time] The method for maintaining algal photosynthesis for a long period of time of the present application includes adding sulfate ions and / or thiosulfate ions to a culture medium and culturing the algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby preventing a decrease in photosynthesis of the algae due to sulfur depletion. This allows photosynthesis of the algae to be maintained for a long period of time. As described in [1.] above, adding sulfate ions, etc. to a culture medium and culturing the algae under conditions that do not deplete the sulfate ions, etc., can maintain or increase the content of Rubisco protein in the algae. Therefore, as described in [1.] above, the cultured algae can efficiently perform photosynthesis. In other words, the method allows photosynthesis of the algae to be maintained in a good state for a long period of time compared to when the method is not used. The configuration of the method is as described in [1.] above.

[0050] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0051] [Summary] The present invention includes the following aspects. <1> A protein production method comprising adding sulfate ions and / or thiosulfate ions to a culture medium and culturing algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby maintaining or increasing the content of Rubisco protein in the algal cells of the algae. <2> The protein production method according to <1>, wherein the culture medium contains 0.75 to 2.0 mM of sulfate ions and / or thiosulfate ions. <3> The protein production method according to <2>, wherein the culture medium contains 0.75 to 2.0 mM of sulfate ions and / or thiosulfate ions at the start of algal culture. <4> The protein production method according to any one of <1> to <3>, wherein the algae are one or more species of algae selected from the group consisting of the genera Chromochloris, Chlamydomonas, Synechocystis, Synechococcus, and Chlorella. <5> The method for producing a protein according to any one of <1> to <4>, wherein the algae are one or more species selected from the group consisting of Chromochloris zofingiensis, Chlamydomonas sp., Synechocystis sp., Synechococcus elongatus, and Chlorella sorokiniana. <6> A method for maintaining algal photosynthesis for a long period of time by adding sulfate ions and / or thiosulfate ions to a medium and culturing the algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby preventing a decrease in photosynthesis of the algae due to sulfur depletion.

[0052] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0053] Example 1 Relationship between types of sulfates, etc., and algae growth, protein content, and protein production (Method) A single stroke of cells from Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were then incubated under a daylight fluorescent light of 30 to 40 μmol-photons / m ２ Then, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated under a light condition of 30 to 40 μmol-photons / (m ２ The plants were pre-cultured for 5 days under light conditions of 1000 x 1000 s.

[0054] Sodium sulfate (Na ２ SO ４ ), potassium sulfate (K ２ SO ４ ), magnesium sulfate (MgSO ４ ), ammonium sulfate ((NH ４ ) ２ SO ４ ), sodium thiosulfate (Na ２ S ２ O ３ ) was prepared as a sulfur source. 70 ml of BG-11 medium containing 1 mM of each of these was prepared.

[0055] The pre-cultured UTEX32 strain was added to each of these media so that the initial concentration reached an optical density (OD) at 750 nm of 0.1, and then the culture was initiated. As a control, 70 ml of standard BG-11 medium was used. Standard BG-11 medium contained no MgSO. ４ Therefore, in this example, the BG-11 medium contained 1.3 mM of sulfate salts, etc., and 1.3 mM of sulfate ions.

[0056] The culture conditions were as follows: ２ Concentration: 2% CO ２ , light: 100μmol-photons / (m ２The conditions were: temperature: 25°C, stirring: 100 rpm. The culture medium was sampled on the 2nd, 4th, 7th, 9th, 11th, and 14th days after the start of the culture and subjected to the following analyses.

[0057] Growth of algal bodies: The optical density (OD) of the culture medium at 750 nm was measured and used as an index of growth. The culture medium was centrifuged to collect algal cells, and the weight of the freeze-dried cells was measured to calculate the biomass production.

[0058] The protein content in the algal cells was calculated by centrifuging the culture medium to recover the algal cells, and quantifying the protein in the cells by the BCA method.

[0059] Protein production was calculated by assessing biomass production and protein content of algal cells.

[0060] (Results) The growth status of the UTEX32 strain when the type of sulfate and other salts added to the medium was changed is shown in Figure 1. The horizontal axis represents the number of days from the start of culture, with the start of culture being set as day 0. The vertical axis represents the absorbance at 750 nm.

[0061] In this example, 1 mM of sulfate or thiosulfate was added to the BG-11 medium, and combined with the 0.3 mM of sulfate originally contained in the medium, the medium contained 1.3 mM of sulfate ions, etc. In other words, the medium contained 1.3 mM of sulfate ions, etc. In this case, regardless of the type of sulfate, etc., the growth of the UTEX32 strain showed similar trends and was significantly better than the control.

[0062] In the control (normal BG-11 medium), growth retardation occurred from day 9 of culture. This phenomenon is thought to be due to the sulfur component limiting the growth rate of the UTEX32 strain.

[0063] Figure 2 shows the change in protein content of the UTEX32 strain when the type of sulfate, etc. added to the culture medium was changed. Measurement of the protein content within the algae confirmed that, under all conditions, the protein content reached a maximum on the seventh day of culture and then decreased with the passage of culture days. When sulfate, etc. was added, the decrease in protein content was slower than in the control, and it remained at about 50% even at the end of culture (day 14). On the other hand, the protein content of the control decreased at a greater rate than when sulfate, etc. was added, dropping to about 20% by the end of culture. This phenomenon in the control is thought to be due to the sulfur component limiting the rate of protein production by the UTEX32 strain.

[0064] The change in protein production of the UTEX32 strain when the type of sulfate salts added to the medium was varied is shown in Figure 3. Regardless of the type of sulfate salts, the protein production was significantly higher than that of the control. On the other hand, the protein production of the control was significantly lower than that of the medium containing sulfate salts.

[0065] These results confirm that the most important factor for protein production by microalgae is not the type of sulfur source (sulfate or thiosulfate), but rather ensuring that growth and protein content are not limited by sulfur source depletion.

[0066] Example 2 Analysis of Protein Production and Protein Species Under Conditions Where Sulfur Source is Not Depleted (Method) A single stroke of cells from Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were incubated under a daylight fluorescent light of 30 to 40 μmol-photons / (m ２ Then, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated under a light condition of 30 to 40 μmol-photons / (m ２ The plants were pre-cultured for 5 days under light conditions of 1000 x 1000 s.

[0067] 70 ml of BG-11 medium containing 1.2 mM magnesium sulfate as a sulfate salt was prepared. The pre-cultured UTEX32 strain was added to each of these media so that the initial concentration was an optical density (OD) at 750 nm of 0.1, and the culture was then initiated. 70 ml of regular BG-11 medium was used as a control. Regular BG-11 medium contained no MgSO 4 . ４ Therefore, in this example, the BG-11 medium contained 1.5 mM of sulfate, which means that the medium contained 1.5 mM of sulfate ions.

[0068] The culture conditions were the same as in Example 1. The culture medium was sampled on the second, fourth, ninth, and fourteenth days after the start of culture, and the optical density (OD) of the culture medium at 750 nm was measured and used as an index of growth. The culture medium was centrifuged to recover algal cells, and the protein content of the cells was quantified using the BCA method to calculate the protein content in the algal bodies. Furthermore, changes in soluble proteins in the algal bodies were analyzed using SDS-PAGE. Characteristic proteins were identified. Identification was performed by inferring from the molecular weights of known proteins.

[0069] (Results) Figure 4 shows the growth of the UTEX strain when the medium for the UTEX32 strain contained five times as much magnesium sulfate as the sulfur source as the control. As in Example 1, in the control, growth was delayed from the ninth day of culture. This phenomenon is thought to be due to the sulfur component limiting the growth of the UTEX32 strain.

[0070] 5 shows the change in protein content in the algae when the medium for the UTEX32 strain contained five times the amount of magnesium sulfate as a sulfur source compared to the control. No change in protein content was observed up to the fourth day after the start of cultivation. However, after the fourth day, the protein content decreased gradually when the sulfur source was added, whereas the protein content in the control decreased rapidly.

[0071] The phenomenon of a decrease in protein content indicates a change in the amount of protein within the algae. Therefore, SDS-PAGE was performed to identify proteins whose amounts within the algae were changed.

[0072] 6 shows the results of SDS-PAGE of the UTEX32 strain containing five times the amount of magnesium sulfate as the control, together with the control. No change in the composition of soluble proteins within the algae was observed up to day 4 from the start of culture. On the other hand, changes in the composition were observed in the control on days 9 and 14 from the start of culture, when the growth of the algae had deteriorated and the protein content within the algae had changed significantly. Specifically, it was confirmed that the amount of a protein with a molecular weight of approximately 50 kDa, which had been the major protein since the early stages of culture, had significantly decreased in the control.

[0073] From the results shown in Figure 6, it was confirmed that when the sulfur source is rate-limiting for protein production, a decrease in the protein with a molecular weight of approximately 50 kDa causes a decrease in the protein content in the algal cells. ２ It was speculated that this was the Rubisco protein (52.5 kDa), an important protein responsible for fixation.

[0074] When protein production is rate-limiting due to the sulfur source, the growth of the algae also declines, as shown in Figure 4. This phenomenon occurs when the amount of Rubisco protein decreases, resulting in CO ２ In other words, the results shown in Figure 6 were consistent with the growth test data shown in Figure 4.

[0075] Figure 7 shows the change in protein production when the medium for the UTEX32 strain contained five times the amount of magnesium sulfate as a sulfur source compared to the control. As shown in Figure 7, the addition of a sulfur source dramatically increased the protein content in the algae compared to the control, and the production amount (on the 14th day of culture) was about four times higher than the control. This result confirmed that avoiding a decrease in protein content due to sulfur source depletion is extremely effective in improving protein production by algae.

[0076] Example 3 Evaluation using different types of microalgae (Method) A single stroke of microalgae cells was picked up and suspended in BG-11 medium. The cells were then exposed to a daylight fluorescent light of 30 to 40 μmol-photons / (m ２ The algal cells were then incubated for 3 days under a light condition of 30-40 μmol-photons / (m ２ The plants were pre-cultured for 5 days under light conditions of 1000 x 1000 s.

[0077] The microalgae used were the freshwater eukaryotic algae Chromochloris zofingiensis UTEX32 strain, the brackish water eukaryotic algae Chlamydomonas sp. JSC4 strain, the freshwater prokaryotic algae Synechocystis sp. PCC6803 strain, the freshwater prokaryotic algae Synechococcus elongatus PCC7942 strain, and the freshwater eukaryotic algae Chlorella sorokiniana UTEX1230 strain. The pre-culture procedure was performed for each of these microalgae.

[0078] 70 ml of BG-11 medium containing 1.2 mM magnesium sulfate as a sulfate salt was prepared. The pre-cultured microalgae were added to the medium so that the initial concentration was an optical density (OD) at 750 nm of 0.1, and the culture was initiated. As a control, 70 ml of regular BG-11 medium was used. The regular BG-11 medium contained no MgSO 4 . ４Therefore, in this example, the BG-11 medium contained 1.5 mM of sulfate, which means that the medium contained 1.5 mM of sulfate ions.

[0079] The culture conditions were as follows: ２ Concentration: 2% CO ２ , light: 100μmol-photons / (m ２ The conditions were: temperature: 25°C, stirring: 100 rpm. The culture solution was sampled on the 2nd, 4th, 7th, 9th, 11th, and 14th days after the start of the culture, and the absorbance of the culture solution at 750 nm was measured. This allowed us to examine whether the rate-limiting effect of the sulfur source on algal growth was observed between different algae species.

[0080] (Results) The results are shown in Figure 8. 1001 represents the results obtained using Chromochloris zofingiensis UTEX32 strain, 1002 represents the results obtained using Chlamydomonas sp. JSC4 strain, 1003 represents the results obtained using Synechocystis sp. PCC6803 strain, 1004 represents the results obtained using Synechococcus elongatus PCC7942 strain, and 1005 represents the results obtained using Chlorella sorokiniana UTEX1230 strain.

[0081] All algae tested, except for Chlorella sorokiniana UTEX1230, showed similar growth trends. These results indicate that the sulfur source plays a key role in CO2 production during photosynthesis, even among different algae. ２ This strongly suggests that the amount of Rubisco protein, a key protein in the immobilization, is affected. It is expected that 1005 will show a similar trend to 1001-1004 if the culture period is extended.

[0082] Chlamydomonas sp. JSC4, which grows in brackish waters, can also grow in marine environments. Although algae grow in marine environments, the marine environment has a high concentration of sulfur sources, so sulfur is not thought to be a growth limiting factor as long as the algae grow in the marine environment.

[0083] However, in environments where sulfur may be depleted as the culture period progresses, such as in the case of culture, sulfur is rate-limiting for the growth of brackish water algae, just as it is for freshwater algae (see 1002). In other words, the sulfur source is rate-limiting for the synthesis of Rubisco protein in brackish water algae. Therefore, it is thought that the sulfur source is important for algal growth and protein production, regardless of whether the algae are freshwater or saltwater.

[0084] Example 4: Setting the concentration range of sulfur source necessary for biomass production up to about 6 g / L (Method) A single stroke of cells of Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were incubated under a daylight fluorescent light of 30 to 40 μmol-photons / (m ２ Then, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated under a light condition of 30 to 40 μmol-photons / (m ２ The plants were pre-cultured for 5 days under light conditions of 1000 x 1000 s.

[0085] Six 70 ml aliquots of BG-11 medium enriched with N and P were prepared. The medium contained 0.3 mM sulfate. Magnesium sulfate was added as sulfate to each medium at 0 mM, 0.15 mM, 0.3 mM, 0.45 mM, 0.6 mM, and 1.0 mM, and the medium was cultured for 14 days. In other words, the medium contained a total of 0.3 mM, 0.45 mM, 0.6 mM, 0.75 mM, 0.9 mM, and 1.3 mM of sulfate (sulfate ions). The concentrations shown in the legend in Figure 9 represent the total concentration of sulfate (sulfate ions) contained in the medium. The culture conditions were as follows: using the medium, CO ２ Concentration: 2% CO ２ , light: 100μmol-photons / (m２ The conditions were: temperature: 25°C, stirring: 100 rpm.

[0086] From the third day to the fourteenth day of the culture, the culture medium was sampled every day and the dry weight was measured to determine the amount of biomass produced.

[0087] The results are shown in Figure 9. The horizontal axis represents the number of days from the start of culture, with the start of culture being day 0. The vertical axis represents the amount of biomass production. Here, biomass production refers to the dry weight of algal cells contained in 1 L of culture medium. In Figure 9, for example, in the "0.30 mM" test group, data from day 8 onwards is not plotted. This is because biomass production did not increase from day 8 onwards, and so data from that point onwards has been omitted to avoid complexity. The same is true for the "0.45 mM" test group from day 10 onwards, the "0.60 mM" test group from day 11 onwards, and the "0.75 mM" test group from day 14 onwards.

[0088] For example, for the "0.45 mM" test group, data is plotted from the 5th day onwards, but the data for the 3rd and 4th days are substantially the same as those for the "0.30 mM" test group, and therefore are omitted from the table. Similarly, the data for the "0.60 mM" test group before the 6th day is substantially the same as those for the "0.45 mM" test group. Furthermore, the data for the "0.75 mM" test group before the 8th day is substantially the same as those for the "0.60 mM" test group. Furthermore, the data for the "0.90 mM" and "1.30 mM" test groups before the 9th day is substantially the same as those for the "0.75 mM" test group. Therefore, the data for these test groups are also omitted from the table.

[0089] As shown in Figure 9, the concentration of the sulfur source is rate-limiting for biomass production. To maintain the linearity of the graph, it was necessary to add a sulfur source to achieve the desired biomass production amount (final algal concentration).

[0090] From these results, it was confirmed that in order to obtain a biomass production amount of about 6 g / L, it is sufficient to add sulfate ions and / or thiosulfate ions in the medium at a concentration of about 0.75 to 1.3 mM.

[0091] The present invention can be used for the production of proteins using algae. Therefore, the algae based on the present invention can be used for processing purposes such as meat substitutes (block meat, minced meat, etc.), functional foods, health foods, functional materials such as bioplastics, liquid fuels, and animal feed.

Claims

1. A method for producing a protein, comprising adding sulfate ions and / or thiosulfate ions to a culture medium and culturing algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby maintaining or increasing the content of Rubisco protein in the algal cells of the algae.

2. The method for producing a protein according to claim 1, wherein the medium contains 0.75 to 2.0 mM of sulfate ions and / or thiosulfate ions.

3. The method for producing a protein according to claim 2, wherein the medium contains sulfate ions and / or thiosulfate ions at a concentration of 0.75 to 2.0 mM at the start of algae cultivation.

4. The method for producing a protein according to claim 1, wherein the algae is one or more species of algae selected from the group consisting of the genera Chromochloris, Chlamydomonas, Synechocystis, Synechococcus, and Chlorella.

5. The method for producing a protein according to any one of claims 1 to 4, wherein the algae is one or more species of algae selected from the group consisting of Chromochloris zofingiensis, Chlamydomonas sp., Synechocystis sp., Synechococcus elongatus, and Chlorella sorokiniana.

6. A method for maintaining algal photosynthesis for a long period of time, by adding sulfate ions and / or thiosulfate ions to a culture medium and culturing algae under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby avoiding a decrease in photosynthesis of the algae due to sulfur depletion.

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