Method for producing protein using algae

Abstract

To provide a method for maintaining or increasing the content of rubisco protein in algal cells.SOLUTION: A method for producing a protein, comprising culturing an alga in a medium containing sulfate ions and / or thiosulfate ions under conditions that do not deplete the sulfate ions and / or thiosulfate ions, thereby maintaining or increasing the content of a rubisco protein in the algal body of the alga.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] According to United Nations population forecasts, 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 of growth can be achieved by adding a substance that inhibits cell growth, such as sulfate, to the culture medium. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2017-502680 Summary of the Invention [Problem to be solved by the invention]

[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 in 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. [Means for solving the problem]

[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 the 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. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0010] [Figure 1] This figure shows the growth of Chromochloris zofingiensis UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test culture media for UTEX32 strain. [Figure 2]FIG. 1 shows the change in protein content of the UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test culture media for the UTEX32 strain. [Figure 3] FIG. 1 shows changes in protein production by the UTEX32 strain when 1 mM of different sulfates or thiosulfates was added to five test culture media for the UTEX32 strain. [Figure 4] FIG. 1 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. [Figure 5] FIG. 1 shows the change in protein content in algal cells when the medium for the UTEX32 strain contained five times the amount of magnesium sulfate as a sulfur source compared to the control. [Figure 6] FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the UTEX32 strain containing five times the amount of magnesium sulfate as the control, together with the control. [Figure 7] FIG. 1 shows the change in protein production when the medium for the UTEX32 strain contained five times as much magnesium sulfate as the sulfur source as the control. [Figure 8] Figure 1000 shows the results of an investigation into whether the sulfur source-dependent rate-limiting effect on algal growth is observed among different species of microalgae. Figure 1001 shows the results for Chromochloris zofingiensis UTEX32, Figure 1002 shows the results for Chlamydomonas sp. JSC4, Figure 1003 shows the results for Synechocystis sp. PCC6803, Figure 1004 shows the results for Synechococcus elongatus PCC7942, and Figure 1005 shows the results for Chlorella sorokiniana UTEX1230. [Figure 9]FIG. 1 shows changes 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. DETAILED DESCRIPTION OF THE INVENTION

[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 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, "sulfate ions and / or thiosulfate ions" will also be referred to as "sulfate ions, etc.", and "sulfate and / or thiosulfate salts" will also be referred to as "sulfate salts, etc."

[0013] (1)Algae In this specification, the 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 cultivation, environmental tolerance, etc., it is preferable to use one or more species of algae selected from the group consisting of the genus Chromochloris, the genus Chlamydomonas, the genus Synechocystis, the genus Synechococcus, the genus Chlorococcum, the genus Arthrospira, and the genus 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 Chlorella genus include Chlorella sorokiniana, Chlorella pyrenoidosa, Chlorella ellipsoidea, Chlorella vulgaris, Chlorella regularis, and Chlorella salina.

[0017] (2) Algae cultivation In the method of the present application, a culture medium is added containing 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 selected appropriately 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 1.0 mM sulfate ions are added to the medium, the medium will contain 1.3 mM sulfate ions.

[0023] (ii) When preparing a new medium, the medium must 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 discovery that the protein that decreases in the algae cells in the later stage of cultivation is Rubisco protein and that this decrease is due to the depletion of sulfur in the culture medium was first discovered by the present invention. Furthermore, the discovery that the content of Rubisco protein 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 the growth of the algae cells can be maintained well were also first discovered by the present invention. Furthermore, in the method described in Patent Document 1, the growth of microalgae is suppressed by the addition of sulfate, but in the method of the present application, the growth of microalgae is not suppressed, as will be described in the Examples below. Therefore, the method of the present application can be said to be advantageous for protein and biomass production.

[0028] As shown in the Examples 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, 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 stage 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 then 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 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 sulfate ions, etc., to the culture medium at, for example, 0.75 to 2.0 mM, 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 culture systems 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] As for the culture conditions, in order to promote the growth of algae, the light intensity, temperature, CO2 concentration, etc. are set to, for example, light intensity: 100 μmol-photons / (m 2 ·s), temperature: 25°C, CO2 concentration: 2%.

[0040] (3) The content of Rubisco protein in algae The present invention relates to a method for maintaining or increasing the content of Rubisco protein in the algae. As described above, the present inventors discovered that the protein content in the algae decreases in the later stages of algal culture. Therefore, the present inventors investigated the types of proteins whose content decreases using SDS-PAGE. As a result, as shown in the Examples below, it was found that the content of Rubisco protein significantly decreases in the later stages of culture.

[0041] Rubisco protein (ribulose-1,5-bisphosphate carboxyltransferase / oxygenase) is an enzyme that plays a central role in CO2 fixation. Based on the SDS-PAGE results, the decrease in protein content in the algae and the decrease in algal biomass production during the later stages of cultivation were thought to be due to a decrease in the efficiency of CO2 fixation caused by a decrease in Rubisco protein content. Because various proteins exist within the algae, it is unclear whether the proteins whose content decreases during the later stages of cultivation are photosynthetic proteins or other proteins. It was previously unknown 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 maintaining or increasing the content compared to when the algae are cultured without adding sulfate 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% of when the algae are cultured without adding sulfate 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 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 produce 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 algal growth for a long period of time. In other words, by maintaining or increasing the content of the Rubisco protein, CO2 fixation is facilitated and photosynthesis is carried out efficiently. As a result, good growth 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 sulfate or the like to the medium.

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

[0049] [2. Methods for maintaining algae photosynthesis for a long period of time] The method for maintaining algal photosynthesis for a long period of time according to the present invention 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 for long-term maintenance of photosynthesis of the algae. 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 for maintaining photosynthesis of the algae 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 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 medium contains 0.75 to 2.0 mM of sulfate ions and / or thiosulfate ions. <1> A method for producing the protein described in . <3> The medium contains sulfate ions and / or thiosulfate ions at a concentration of 0.75 to 2.0 mM at the start of algae culture. <2> A method for producing the protein described in . <4> The algae is one or more species selected from the group consisting of Chromochloris, Chlamydomonas, Synechocystis, Synechococcus, and Chlorella. <1> from <3> A method for producing a protein according to any one of the above. <5> The algae is one or more species selected from the group consisting of Chromochloris zofingiensis, Chlamydomonas sp., Synechocystis sp., Synechococcus elongatus, and Chlorella sorokiniana; <1> from <4> A method for producing a protein according to any one of the above. <6> A method for maintaining algal photosynthesis for a long period of time, which includes 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. [Example]

[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 scoop of cells from Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were then illuminated under a daylight fluorescent light with 30-40 μmol-photons / (m 2 The resulting mixture was incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). Next, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). 2 The plants were pre-cultured under a light condition of 100 s for 5 days.

[0054] Sodium sulfate (NaSO), potassium sulfate (KSO), magnesium sulfate (MgSO), ammonium sulfate ((NHSO), and sodium thiosulfate (NaSO) were prepared as sulfur sources. 70 ml of BG-11 medium containing 1 mM 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 cultivation was initiated. As a control, 70 ml of standard BG-11 medium was used. Standard BG-11 medium contains 0.3 mM MgSO4. Therefore, in this example, the BG-11 medium contained 1.3 mM of sulfate, etc., which means that it contained 1.3 mM of sulfate ions.

[0056] The culture conditions were as follows: the medium was used, CO2 concentration: 2% CO2, light: 100 μmol-photons / (m 2 The conditions were: temperature: 25°C, agitation: 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] Algal growth: 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] (result) Figure 1 shows the growth of the UTEX32 strain when the type of sulfate and other salts added to the medium was varied. 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 sulfate or thiosulfate was added to the BG-11 medium, resulting in a total of 1.3 mM, in addition to the 0.3 mM sulfate originally contained in the medium. In other words, sulfate ions and the like were added to the medium at 1.3 mM. Regardless of the type of sulfate and the like, 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 slowed from day 9. This phenomenon is thought to be due to the sulfur component limiting the growth of the UTEX32 strain.

[0063] Figure 2 shows the change in protein content of the UTEX32 strain when the type of sulfate and other salts added to the culture medium was varied. 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 over time. When sulfate and other salts were added, the decrease in protein content was slower than in the control, remaining at approximately 50% even at the end of culture (day 14). On the other hand, the protein content of the control decreased more rapidly than when sulfate and other salts were added, dropping to approximately 20% by the end of culture. This phenomenon in the control is thought to be due to the sulfur component being rate-limiting for protein production by the UTEX32 strain.

[0064] Figure 3 shows the change in protein production by the UTEX32 strain when the type of sulfate salts added to the medium was varied. Regardless of the type of sulfate salts, the protein production was significantly higher than that of the control. However, 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 sources are not depleted> (method) A single scoop of cells from Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were then illuminated under a daylight fluorescent light with 30-40 μmol-photons / (m 2 The resulting mixture was incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). Next, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). 2 The plants were pre-cultured under a light condition of 100 s for 5 days.

[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 to an initial concentration of 0.1 optical density (OD) at 750 nm, and cultivation was initiated. 70 ml of standard BG-11 medium was used as a control. Standard BG-11 medium contains 0.3 mM MgSO4. Therefore, in this example, the BG-11 medium contained 1.5 mM sulfate, which means that it contained 1.5 mM 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 within the algal bodies. Furthermore, changes in soluble proteins within 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] (result) 4 shows the growth of the UTEX strain when the medium for the UTEX32 strain contained five times the amount of magnesium sulfate as a sulfur source compared to the control. As in Example 1, growth in the control strain was delayed from the ninth day of culture. This phenomenon is thought to be due to the sulfur component limiting the growth rate of the UTEX32 strain.

[0070] Figure 5 shows the change in protein content within 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 decrease in protein content indicates a change in the amount of protein within the algae. Therefore, SDS-PAGE was performed to identify the proteins whose amounts within the algae were changed.

[0072] Figure 6 shows the results of SDS-PAGE analysis 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 after the start of culture. On the other hand, changes in the composition were observed in the control on days 9 and 14 after the start of culture, when the growth of the algae had deteriorated and the protein content within the algae had changed significantly. Specifically, a significant decrease in 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, was confirmed in the control.

[0073] The results shown in Figure 6 confirm that when the sulfur source is rate-limiting for protein production, a decrease in the protein content of the algae is due to a decrease in the protein with a molecular weight of approximately 50 kDa. This protein with a molecular weight of approximately 50 kDa is presumed to be the Rubisco protein (52.5 kDa), an important protein responsible for CO2 fixation during photosynthesis.

[0074] When protein production by the sulfur source becomes rate-limiting, the growth of the algae also declines, as shown in Figure 4. This phenomenon is thought to be due to a decrease in the amount of CO2 fixed due to a decrease in Rubisco protein. In other words, the results shown in Figure 6 are 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 within the algae compared to the control, and the production amount (on the 14th day of culture) was approximately four times higher than the control. These results confirm that avoiding the 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 illuminated under a daylight fluorescent light with 30-40 μmol-photons / (m 2 The algal suspension was then incubated for 3 days under a light condition of 30-40 μmol-photons / (m 2 The plants were pre-cultured under a light condition of 100 s for 5 days.

[0077] The microalgae used were the freshwater eukaryotic algae Chromochloris zofingiensis strain UTEX32, the brackish water eukaryotic algae Chlamydomonas sp. strain JSC4, the freshwater prokaryotic algae Synechocystis sp. strain PCC6803, the freshwater prokaryotic algae Synechococcus elongatus strain PCC7942, and the freshwater eukaryotic algae Chlorella sorokiniana strain UTEX1230. 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 cultivation was initiated. 70 ml of standard BG-11 medium was used as a control. Standard BG-11 medium contains 0.3 mM MgSO4. Therefore, in this example, the BG-11 medium contained 1.5 mM sulfate salt, which means that it contained 1.5 mM sulfate ions.

[0079] The culture conditions were as follows: the medium was used, CO2 concentration: 2% CO2, light: 100 μmol-photons / (m 2 The conditions were: 1·s, temperature: 25°C, agitation: 100 rpm. The culture medium was sampled on the 2nd, 4th, 7th, 9th, 11th, and 14th days after the start of cultivation, and the absorbance of the culture medium 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] (result) The results are shown in Figure 8. 1001 represents the results for Chromochloris zofingiensis strain UTEX32, 1002 for Chlamydomonas sp. strain JSC4, 1003 for Synechocystis sp. strain PCC6803, 1004 for Synechococcus elongatus strain PCC7942, and 1005 for Chlorella sorokiniana strain UTEX1230.

[0081] All algae tested, except for Chlorella sorokiniana strain UTEX1230, showed similar growth trends. These results strongly suggest that the sulfur source affects the content of Rubisco, a key protein in CO2 fixation during photosynthesis, even among different algae. Furthermore, strain 1005 is expected to show similar trends as strains 1001-1004 if the culture period is extended.

[0082] Chlamydomonas sp. JSC4, which grows in brackish water, 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 over the course of the culture period, such as in culture, sulfur is rate-limiting for 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 marine.

[0084] Example 4 <Setting the sulfur source concentration range required for biomass production up to approximately 6 g / L> (method) A single scoop of cells from Chromochloris zofingiensis UTEX32 strain was picked up and suspended in BG-11 medium. The cells were then illuminated under a daylight fluorescent light with 30-40 μmol-photons / (m 2 The resulting mixture was incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). Next, a portion of the suspension containing the UTEX32 algae was placed in fresh BG-11 medium and incubated for 3 days under a light condition of 30-40 μmol-photons / (m s). 2 The plants were pre-cultured under a light condition of 100 s for 5 days.

[0085] Six 70 ml aliquots of N- and P-enriched BG-11 medium were prepared. The medium contained 0.3 mM sulfate. Magnesium sulfate was added as sulfate to each medium at concentrations of 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 sulfate (sulfate ions). The concentrations shown in the legend in Figure 9 represent the total sulfate (sulfate ions) concentration in the medium. The culture conditions were as follows: using the medium, CO2 concentration: 2% CO2, light: 100 μmol-photons / (m 2 ·s), 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 biomass production. Biomass production here 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 after day 8, and so data from that point onwards has been omitted to avoid complexity. The same is true for "0.45 mM" from day 10 onwards, "0.60 mM" from day 11 onwards, and "0.75 mM" 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 table for these data is also omitted.

[0089] As shown in Figure 9, it can be seen that 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 was sufficient to add sulfate ions and / or thiosulfate ions in the medium at a concentration of about 0.75 to 1.3 mM. [Industrial Applicability]

[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. 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. 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. 2. 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. 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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