Antioxidant, method for removing oxygen radicals, and method for producing antioxidant

Abstract

The present disclosure provides a new antioxidant using cyanobacteria. An antioxidant according to the present disclosure contains a secretion product of cyanobacteria.

Description

Antioxidant, method for removing active oxygen, and method for producing antioxidant

[0001] The present disclosure relates to an antioxidant, a method for removing active oxygen, and a method for producing an antioxidant.

[0002] Conventionally, attempts have been made to obtain useful components using cyanobacteria and the like.

[0003] For example, Patent Document 1 describes a method for obtaining water-soluble compounds such as phycocyanin from cyanobacteria, in which cyanobacteria mixed with an aqueous solution are subjected to ultrasonic treatment under predetermined conditions to extract the water-soluble compounds.

[0004] Patent Document 2 describes polysaccharides released from Cyanoseis sp. CCY 0110 for use in pharmaceuticals, veterinary medicine, or cosmetics. Cyanoseis sp. CCY 0110 is a type of cyanobacterium and is a highly efficient producer of extracellular polymeric substances, releasing up to 75% of the polymer into the culture medium. The release kinetics of polysaccharides from Cyanoseis sp. CCY 0110 can be controlled by adding divalent cations.

[0005] Patent Document 3 describes a method for producing a mycosporine-like amino acid. This method includes the steps of culturing a microorganism that produces the mycosporine-like amino acid extracellularly, separating the microorganism from the extracellular culture medium, and recovering the mycosporine-like amino acid from the extracellular culture medium. The microorganism is Escherichia coli, yeast, actinomycete, microalgae, or a microorganism belonging to Labyrinthulea.

[0006] Non-Patent Document 1 describes a promoter that enables efficient production of heterologous proteins in cyanobacteria.

[0007] International Publication No. WO 2018 / 229364 International Publication No. WO 2018 / 042378 International Publication No. WO 2015 / 174427

[0008] Jie Zhou et al., “Discovery of a super-strong promoter enable efficient production of heterologous proteins in cyanobacteria”, Scientific Reports, Research, 2014, Vol.4, Article No. 4500 (2014)

[0009] The techniques described in the above documents need to be reconsidered from the viewpoint of obtaining antioxidants using cyanobacteria. Therefore, the present disclosure provides a novel antioxidant using cyanobacteria.

[0010] The present disclosure provides an antioxidant comprising a cyanobacterial secretion.

[0011] The antioxidant of the present disclosure is a novel antioxidant that can be obtained using cyanobacteria.

[0012] FIG. 1 is a flowchart illustrating an example of a method for producing an antioxidant according to the present embodiment. FIG. 2 is a diagram schematically illustrating an example of a gene sequence of a vector DNA used in the genetic modification of cyanobacteria. FIG. 3 is a diagram illustrating the results of electrophoresis of DNA fragments amplified from modified cyanobacteria according to the examples. FIG. 4 is a graph illustrating the results of measuring the activity of lipopolysaccharide (LPS) in culture supernatants according to the examples and comparative examples. FIG. 5 is a graph illustrating the change over time in absorbance differences in antioxidant capacity evaluation samples according to the examples and comparative examples. FIG. 6A is a graph illustrating the change over time in skin moisture content when a moisturizing evaluation sample according to the examples and pure water was used. FIG. 6B is a graph illustrating the change over time in skin moisture content when a moisturizing evaluation sample according to the comparative example and pure water was used. FIG. 7A is a graph illustrating the change over time in skin moisture content when a moisturizing evaluation sample according to the examples and pure water was used. FIG. 7B is a graph illustrating the change over time in skin moisture content when a moisturizing evaluation sample according to the comparative example and pure water was used. FIG. 8 is a graph illustrating the ultraviolet absorption spectra of culture supernatants according to the examples and comparative examples. 9A and 9B are graphs showing the relationship between the cotyledon expansion rate of pansies and the number of days elapsed since sowing of pansy seeds when irrigated with culture supernatant or pure water according to the examples, respectively.

[0013] (Findings Forming the Basis of the Present Disclosure) In cosmetics, quasi-drugs, pharmaceuticals, foods, beverages, and the like that claim to reduce deterioration of the skin or the body and improve the quality of the skin or the body, for example, antioxidants are expected to prevent skin aging and body aging, for example, antioxidants are expected to prevent skin problems such as age spots, wrinkles, and sagging.

[0014] It is becoming clear that the oxidation of biological tissues by reactive oxygen species is closely related to lifestyle-related diseases such as cancer, arteriosclerosis, and heart disease, as well as aging. Reactive oxygen species is a general term for highly reactive compounds containing oxygen. Examples of reactive oxygen species include hydroxyl radicals (.OH), alkoxyl radicals (LO.), peroxyl radicals (LOO.), hydroperoxyl radicals (HOO.), nitric oxide (NO.), nitrogen dioxide (NO2.), and superoxide anions (O2.). - These reactive oxygen species are highly reactive and oxidize biological substances such as proteins, lipids, and DNA, causing protein denaturation, lipid peroxidation, and genetic damage, and are thought to be involved in the onset of various diseases.

[0015] Reactive oxygen species can be removed by ascorbic acid, tocopherol, carotenoids, various polyphenols, etc. Therefore, it is expected that the use of antioxidants will be effective in preventing various diseases associated with reactive oxygen species.

[0016] Compositions formulated with synthetic chemicals to achieve desired effects and benefits are based on Western medical principles and aim to improve appearance or resolve problems in a short-term approach. On the other hand, organic antioxidants can be developed based on Eastern medical principles, which have complex plant-derived effects and benefits, and aim to enhance self-healing or barrier function in a long-term manner without impairing the body's natural functions. Compositions formulated with synthetic chemicals can irritate the skin or organism, leading to a trend toward organic products. In recent years, natural-derived products have continued to grow in markets for cosmetics, quasi-drugs, pharmaceuticals, food, and beverages, and interest in products that claim to be natural or organic has increased. However, creating organic antioxidants is not easy. Microorganisms often do not produce large quantities of the target compounds, making it difficult to obtain commercially meaningful quantities of these compounds using microorganisms.

[0017] Cyanobacteria have attracted the attention of many researchers and businesses in recent years. Cyanobacteria contain numerous compounds with high added value, and many researchers and businesses are interested in extracting compounds such as physcocyanin from cyanobacteria. However, to use compounds produced inside wild-cyanobacteria, the cyanobacteria must be disrupted to extract the compounds. The technology described in Patent Document 1 requires ultrasonic treatment of cyanobacteria mixed with an aqueous solution under specified conditions, making the process for obtaining the desired compounds complicated and likely to increase the cost of producing compositions containing the compounds. The technology described in Patent Document 2 produces polysaccharides released from Cyanoseis sp. CCY 0110, but it is unclear whether the resulting compounds have antioxidant properties. Furthermore, it is understood that the amount of polysaccharides released from Cyanoseis sp. CCY 0110 is limited. The technology described in Patent Document 3 produces mycosporine-like amino acids, but it is unclear whether the resulting compounds have antioxidant properties. The technology described in Non-Patent Document 1 is expected to produce desired proteins inside cyanobacterial cells. However, proteins produced inside cyanobacterial cells are difficult to secrete outside the cells, and in order to obtain these proteins, it is necessary to disrupt the cyanobacterial cells and extract the proteins produced inside the cells.

[0018] Therefore, the present inventors have conducted extensive research and have found that a specific method can be used to obtain a cyanobacterial secretion product that has antioxidant effects. Based on this new finding, the present inventors have completed the antioxidant of the present disclosure.

[0019] Cosmetics, quasi-drugs, pharmaceuticals, etc. that claim to reduce skin aging and improve skin quality are often required to prevent inflammation, darkening, dryness, and other skin-related effects caused by ultraviolet rays such as UV-A and UV-B. UV-A is a type of ultraviolet light that quickly darkens skin after exposure to sunlight. It can penetrate clouds and window glass to reach the dermis deep within the skin and cause skin photoaging, such as wrinkles and sagging. UV-B is a type of ultraviolet light that primarily acts strongly on the epidermis, causing redness and inflammation in the skin several hours after exposure to sunlight. UV-B increases melanin and can cause sunburn-related spots or freckles. Repeated sunburn also contributes to skin photoaging. In addition, when humans are exposed to ultraviolet rays contained in sunlight, reactive oxygen species are generated inside human cells, which can lead to skin oxidation and skin aging. Therefore, to protect the skin from UV rays, it is considered to use a topical skin composition that has at least one effect selected from the group consisting of antioxidant effect and UV absorption effect.

[0020] Compositions for topical skin applications include those formulated with chemically synthesized substances to achieve desired effects and efficacy. These compositions, formulated based on Western medical principles, aim to improve skin appearance or resolve skin problems in a short-term approach. On the other hand, organic topical skin compositions based on Eastern medical principles can also be developed, possessing multiple plant-derived effects and benefits, and aiming for long-term benefits such as enhancing self-healing ability or barrier function without impairing the skin's natural functions. Compositions formulated with chemically synthesized substances can cause skin irritation, leading to a trend toward organic skin care products. In recent years, the market for cosmetics, quasi-drugs, and pharmaceuticals for topical skin applications has seen a steady growth in naturally derived products, leading to growing interest in topical skin compositions that claim to be natural or organic. However, creating organic products for topical skin applications is not easy. Microorganisms often do not produce large quantities of target compounds, making it difficult to obtain commercially meaningful quantities of these compounds using microorganisms.

[0021] The technology described in Patent Document 1 requires ultrasonic treatment of cyanobacteria mixed with an aqueous solution under specified conditions, making the process for obtaining the desired compound complicated and likely to increase the production costs of compositions containing the compound. The technology described in Patent Document 2 obtains polysaccharides released from Cyanoseis sp. CCY 0110, but it is unclear whether the resulting composition has the desired effects, such as antioxidant and UV-absorbing effects. Furthermore, it is understood that the amount of polysaccharides released from Cyanoseis sp. CCY 0110 is limited. The technology described in Patent Document 3 obtains mycosporine-like amino acids, but it is unclear whether the resulting composition has the desired effects, such as antioxidant and UV-absorbing effects. The technology described in Non-Patent Document 1 is expected to produce desired proteins inside cyanobacterial cells. However, proteins produced inside cyanobacterial cells are difficult to excrete, and therefore, to obtain the proteins, it is necessary to disrupt the cyanobacterial cells and extract the proteins produced inside the cells.

[0022] Therefore, the present inventors have conducted extensive research and have found that a specific method can be used to obtain a cyanobacterial secretion that can have a desired effect. Based on this new finding, the present inventors have completed the topical skin composition of the present disclosure.

[0023] In cosmetics, quasi-drugs, pharmaceuticals, and the like that claim to alleviate skin aging and improve skin quality, moisturizers, for example, are important for preventing the decline of the skin's barrier function and maintaining healthy skin conditions. When the skin's barrier function declines, the stratum corneum cannot retain sufficient moisture, causing the skin to dry out and becoming more susceptible to external stimuli such as ultraviolet rays. Irritated skin is prone to various problems and can even become sensitive. For sensitive skin, moisturizing is important for preventing the decline of the skin's barrier function and maintaining healthy skin conditions.

[0024] In addition to moisturizing, it is also important to protect the skin from UV rays by, for example, absorbing them. Moisturizers are sometimes required to prevent inflammation, darkening, dryness, and other skin problems caused by UV rays such as UV-A and UV-B. UV-A is UV radiation that quickly darkens the skin after sun exposure. It can penetrate clouds and window glass and reach the dermis deep within the skin, causing skin photoaging such as wrinkles and sagging. UV-B is UV radiation that primarily acts strongly on the epidermis, causing redness and inflammation in the skin several hours after sun exposure. UV-B increases melanin and can cause sun spots or freckles. Repeated sunburns like this also lead to skin photoaging.

[0025] When humans are exposed to ultraviolet rays contained in sunlight, active oxygen is generated inside human cells, which can cause skin oxidation and skin aging. For this reason, in addition to moisturizing, antioxidants are also important. Antioxidants are important for preventing accelerated skin aging, which can cause skin problems such as age spots, wrinkles, and sagging.

[0026] Moisturizing compositions are often formulated with chemically synthesized substances to achieve the desired effects and benefits. These compositions, formulated based on Western medical principles, aim to improve appearance or resolve skin problems in a short-term approach. On the other hand, organic moisturizing agents, based on Eastern medical principles, can be developed with complex plant-derived effects and benefits, aiming for long-term benefits such as enhancing self-healing or barrier function without impairing the body's natural functions. Compositions formulated with chemically synthesized substances can cause skin irritation, leading to a trend toward organic products. In recent years, natural-derived products have continued to grow in the cosmetics, quasi-drug, and pharmaceutical markets, leading to growing interest in moisturizing agents that claim to be natural or organic. However, creating organic moisturizing agents is not easy. Microorganisms often do not produce large quantities of the target compounds, making it difficult to obtain commercially meaningful quantities of these compounds using microorganisms.

[0027] The technology described in Patent Document 1 requires ultrasonic treatment of cyanobacteria mixed with an aqueous solution under specified conditions, making the process for obtaining the desired compound complicated and likely to increase the production costs of compositions containing the compound. The technology described in Patent Document 2 obtains polysaccharides released from Cyanoseis sp. CCY 0110, but it is unclear whether the resulting components have moisturizing effects. It is also understood that the amount of polysaccharides released from Cyanoseis sp. CCY 0110 is limited. The technology described in Patent Document 3 obtains mycosporine-like amino acids, but it is unclear whether the resulting components have moisturizing effects. The technology described in Non-Patent Document 1 is expected to produce desired proteins inside cyanobacterial cells. However, proteins produced inside cyanobacterial cells are difficult to excrete, and therefore, to obtain the proteins, it is necessary to disrupt the cyanobacterial cells and extract the proteins produced inside the cells.

[0028] Therefore, the present inventors have conducted extensive research and have found that a specific method can be used to obtain a cyanobacterial secretion that has a moisturizing effect. Based on this new finding, the present inventors have completed the moisturizing agent of the present disclosure.

[0029] (Embodiments of the Present Disclosure) Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component arrangement positions, connection configurations, process conditions, steps, and step sequences shown in the following embodiments are merely examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, components that are not recited in the independent claims that represent the highest concepts are described as optional components. Note that each figure is a schematic diagram and is not necessarily an exact illustration. In each figure, substantially identical components are assigned the same reference numerals, and redundant explanations may be omitted or simplified. Furthermore, below, numerical ranges do not only represent strict meanings but also include substantially equivalent ranges. Furthermore, in this specification, each of the bacterial body and cell represents a single cyanobacterial individual.

[0030] (Embodiments) Hereinafter, embodiments will be specifically described with reference to FIGS. 1 and 2. In this specification, the homology between nucleotide sequences and amino acid sequences is calculated using the Basic Local Alignment Search Tool (BLAST) algorithm. Specifically, it is calculated by performing pairwise analysis using the BLAST program available on the website of the National Center for Biotechnology Information (NCBI). Information regarding the nucleotide sequences of genes and the amino acid sequences of proteins encoded by those genes is publicly available, for example, on the above-mentioned NCBI website, and can be referenced at any time.

[0031] (Antioxidant) The antioxidant according to this embodiment contains a secretion of cyanobacteria. In this specification, a secretion refers to a substance that is excreted outside the cyanobacteria by the action of the cyanobacteria themselves. Therefore, if a substance that is produced inside the cyanobacteria and remains inside the cyanobacteria is obtained by an artificial method such as crushing the cyanobacteria, the substance does not fall under the category of a secretion as defined in this specification. Having an antioxidant effect means, for example, that the degree of antioxidant effect is greater than when no antioxidant is used.

[0032] Cyanobacteria, also known as blue-green algae or cyanobacteria, are a group of prokaryotes that capture light energy using chlorophyll and use the energy to split water and generate oxygen during photosynthesis. Cyanobacteria are highly diverse, ranging from unicellular species such as Synechocystis sp. PCC6803 to multicellular, filamentous species such as Anabaena sp. PCC7120. They also vary in habitats, from thermophilic species such as Thermosynechococcus elongatus to marine species such as Synechococcus elongatus and freshwater species such as Synechocystis. Many species also possess unique characteristics, such as species that possess gas vesicles and produce toxins, such as Microcystis aeruginosa, and Gloeobacter violaceus, which lacks thylakoids but instead possesses proteins called phycobilisomes, which act as light-harvesting antennae, in the plasma membrane.

[0033] The antioxidant contains, for example, secretions secreted to the outside of the cyanobacteria, but does not contain the cyanobacteria themselves or components derived from the cyanobacteria themselves. In other words, the antioxidant contains, for example, only secretions secreted to the outside of the cyanobacteria as components derived from the cyanobacteria.

[0034] The antioxidant may further have at least one effect selected from the group consisting of a moisturizing effect and an ultraviolet absorbing effect. In this specification, having a moisturizing effect means, for example, that the moisturizing effect is greater than when no antioxidant is used. In this specification, having an ultraviolet absorbing effect means, for example, that the ultraviolet absorbing effect is greater than when no antioxidant is used.

[0035] When an antioxidant has an ultraviolet absorbing effect in addition to an antioxidant effect, the antioxidant can contribute to improving the high temperature tolerance and stress tolerance of a plant, and therefore can be used to improve the high temperature tolerance and stress tolerance of a plant.

[0036] The secreted product contained in the antioxidant is, for example, a secreted product of modified cyanobacteria. The modified cyanobacteria is, for example, genetically modified to be able to excrete the secreted product to the outside of the cyanobacteria. The modified cyanobacteria can, for example, excrete compounds that are difficult to excrete from wild-type cyanobacteria that have not been genetically modified. The modified cyanobacteria is, for example, genetically modified to be able to secrete components that have antioxidant effects.

[0037] Modified cyanobacteria are wild-type cyanobacteria whose genomic DNA has been artificially modified, resulting in mutated traits of the cells. Wild-type cyanobacteria are also called parent cyanobacteria. The method for modifying the genomic DNA of wild-type cyanobacteria is not limited to a specific method. Examples of such methods include genetic recombination, genome editing, and mutagenesis. For example, natural transformation-based genetic recombination (see Reference A below) or genome editing using the CRISPR-Cpf1 system (see Reference B below) can be used to modify the genomic DNA of cyanobacteria. Reference A: Hirose, Yu et al., Photosynthesis Research Methods, Low Temperature Science, Vol. 67, Institute of Low Temperature Science, Hokkaido University, 2008, pp. 9-15, ISSN: 1880-7593, http: / / hdl.handle.net / 2115 / 39084. Reference B: Ungerer et al., 2016, Scientific Reports, 6: 39681. In modified cyanobacteria, for example, the function of a protein involved in the binding between the outer membrane and the cell wall is suppressed or lost. As a result, the binding between the cell wall and the outer membrane is partially reduced in the modified cyanobacteria. In other words, the amount and strength of the binding between the cell wall and the outer membrane are reduced. This makes it easier for the outer membrane to partially detach from the cell wall. As a result, in the modified cyanobacteria, proteins and metabolic products produced within the bacterial cell are more likely to leak out of the bacterial cell. This makes it easier for proteins and metabolic products produced within the modified cyanobacterium to be secreted outside the cells, allowing the intracellularly produced substances to be collected as secretions without, for example, performing extraction processes for the intracellularly produced substances, such as disrupting the cells. This makes it less likely that the physiological activity and yield of the intracellularly produced substances secreted outside the cells will be reduced. Therefore, the physiological activity and yield of substances that can contribute to the provision of at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect, among the intracellularly produced substances of the modified cyanobacterium, are less likely to be reduced. As a result, the antioxidant can more advantageously provide at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0038] The genes modified in the modified cyanobacteria are not limited to specific genes. In the modified cyanobacteria, a gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate-modifying enzyme, which is involved in binding between the outer membrane and the cell wall, is modified. This tends to reduce the binding between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane of the modified cyanobacterium and the covalently bound glycans on the surface of the cell wall, making it easier for the outer membrane to partially detach from the cell wall, as described above. Reduced binding refers to a reduction in the amount and strength of binding.

[0039] The modified gene in the engineered cyanobacterium is, for example, slr1841, slr0688, or slr1908 found in Synechocystis sp. PCC 6803. Alternatively, the gene may be a gene equivalent to slr1841, slr0688, or slr1908 found in cyanobacteria of the genus Synechococcus, Anabaena, or Nostoc. slr1841 and slr1908 encode SLH domain-containing outer membrane proteins. These proteins consist of a C-terminal region embedded in the lipid membrane that forms the outer membrane of cyanobacteria and an N-terminal SLH domain protruding from the lipid membrane, and are widely distributed in cyanobacteria (see Reference C below). The region embedded in the lipid membrane forms a channel that allows hydrophilic substances to pass through the outer membrane. Meanwhile, the SLH domain functions to bind to the cell wall (see Reference D below). In order for the SLH domain to bind to the cell wall, the covalently bound glycans in the peptidoglycan must be modified with pyruvate (see Reference E below). The cell wall-pyruvate-modifying enzyme, which catalyzes the pyruvate modification of the covalently bound glycans in the peptidoglycan, has been identified in the Gram-positive bacterium Bacillus anthracis and named CsaB (see Reference F below). The cell wall-pyruvate-modifying enzyme can be encoded by slr0688. Reference C: Kojima et al., 2016, Biosci. Biotech. Biochem., 10: 1954-1959 Reference D: Kowata et al., 2017, J. Bacteriol., 199: e00371-17 Reference E: Kojima et al., 2016, J. Biol. Chem., 291: 20198-20209 Reference F: Mesnage et al., 2000, EMBO J., 19: 4473-4484 The above antioxidants can be produced, for example, by a method comprising secreting a secretion from cyanobacteria to obtain a component having antioxidant effect.

[0040] FIG. 1 is a flowchart showing an example of a method for producing an antioxidant. As shown in FIG. 1 , in step S1, cyanobacteria capable of secreting a secretion product contained in the antioxidant are prepared. In preparing the cyanobacteria, the state of the cyanobacteria is adjusted so that the secretion product can be secreted. For example, step S1 may include restoring bacterial cells from a freeze-dried form or glycerol stock of the cyanobacteria, or may include recovering cyanobacteria that have finished secreting the secretion product in step S2 described below. The prepared cyanobacteria may be the modified cyanobacteria described above. In this case, step S1 may include, for example, genetically modifying a parent cyanobacteria to produce the modified cyanobacteria, or may include restoring bacterial cells from a freeze-dried form or glycerol stock of the modified cyanobacteria. Step S1 may also include recovering the modified cyanobacteria that have finished secreting the secretion product in step S2 described below.

[0041] The modified cyanobacteria are genetically modified, for example, by vector DNA. The vector DNA is, for example, a plasmid or virus designed for genetic modification. The vector DNA contains, for example, a gene sequence of a homologous region of the target site of genetic modification, a gene sequence for suppressing expression of the target site of genetic modification, and a gene sequence that functions as a marker for genetic modification.

[0042] Figure 2 is a schematic diagram showing an example of the gene sequence of a vector DNA used for genetic modification of cyanobacteria. The target region for genetic modification using this vector DNA is the cyanobacterial slr0688 gene. The vector DNA contains homologous region 1 (see SEQ ID NO: 1), which is the sequence immediately upstream of the transcription start point of the slr0688 gene, and homologous region 2 (see SEQ ID NO: 2), which is the sequence immediately downstream of the transcription start point of the slr0688 gene. In the vector DNA, the sequence for suppressing expression of the slr0688 gene is the promoter sequence of the cyanobacterial petE gene (see SEQ ID NO: 3). This sequence suppresses expression of the slr0688 gene. The vector DNA contains a resistance gene for the antibiotic kanamycin (see SEQ ID NO: 4) as a genetic modification marker. This gene serves as an indicator for selecting modified cyanobacteria with newly introduced sequences via homologous recombination. The vector DNA also contains the E. coli mazF gene (see SEQ ID NO: 5) as a genetic modification marker. This gene acts as a lethal gene and serves as a marker for selecting modified cyanobacteria in which the existing sequence has been deleted by homologous recombination. The sequence for regulating this gene is the promoter sequence of the cyanobacterial nrsB gene (see SEQ ID NO: 6). This sequence allows for the expression of the lethal gene to be selectively controlled.

[0043] The method for constructing the above-mentioned vector DNA is not limited to a specific method. This vector DNA can be constructed, for example, by artificial gene synthesis. For example, genomic DNA is extracted from the cells of the parent cyanobacterium. A sequence corresponding to the homologous region of the target site of gene modification is amplified from the genomic DNA using the Polymerase Chain Reaction (PCR) method. This vector DNA can be constructed by introducing the resulting gene fragment into plasmid DNA. The source plasmid DNA is not limited to a specific DNA. The source plasmid DNA is preferably pUC19. pUC19 is easily introduced into E. coli and replicates within E. coli. The method for introducing the gene fragment into plasmid DNA is not limited to a specific method. For example, the method involves linearizing the plasmid DNA using any restriction enzyme and then ligating the linearized plasmid DNA using Takara Bio's DNA Ligation Kit or In-Fusion® HD Cloning Kit. This method allows the gene fragment to be easily introduced into the plasmid DNA.

[0044] The method for suppressing the expression of a target gene by genetic modification is not limited to a specific method, and includes, for example, introducing, immediately upstream of the target gene, a promoter sequence of a gene whose expression is weaker than that of the target gene, or a promoter sequence that can arbitrarily regulate the expression of the target gene.

[0045] A promoter is a regulatory sequence involved in the efficiency of the transcription initiation reaction of a gene. In a broad sense, a promoter can include both a regulatory sequence that exists relatively close to the transcription initiation site and a regulatory sequence that exists away from the transcription initiation site and that operates. Examples of promoters that can regulate the expression status in cyanobacteria are the promoters of the petE gene, the nirA gene, and the psbA2 gene of cyanobacteria (see document G below). For example, the promoter of the petE gene can be expressed when cells containing the promoter are Cu 2+This promoter is expressed only in a growth environment where ions are present (see Reference H below). Reference G: Heidorn et al., 2011, Methods in Enzymology, 497, 539-579 Reference H: Briggs et al., 1990, Plant Molecular Biology, 15, 633-642 The marker for genetic modification used in the genetic modification of cyanobacteria is not limited to a specific marker. As described above, an antibiotic resistance gene or a lethal gene is preferably used as the marker for genetic modification. By introducing a gene sequence containing an antibiotic resistance gene into a parent cyanobacterium, only modified cyanobacteria into which the gene sequence has been introduced can grow even in the presence of antibiotics. This makes it easy to select modified cyanobacteria. The antibiotic resistance gene is not limited to a specific resistance gene. Examples of antibiotic resistance genes include kanamycin, spectinomycin, and chloramphenicol resistance genes. By deleting a gene sequence containing a lethal gene from modified cyanobacteria, only modified cyanobacteria lacking the gene sequence can grow even under conditions in which the lethal gene is induced. This facilitates the selection of modified cyanobacteria. The lethal gene is not limited to a specific gene. Examples of lethal genes include the mazF gene of Escherichia coli and the sacB gene of Bacillus subtilis (see Reference I below). Reference I: Cheah et al., 2012, Biotechnol. Prog., 29(1), 23-30. As mentioned above, preferably, a promoter sequence capable of regulating expression in cyanobacteria is added upstream of the lethal gene. By regulating the expression of the lethal gene using this sequence, the lethal gene is not expressed before genetic modification, which can easily prevent interference with cyanobacterial growth.

[0046] As shown in FIG. 1 , in step S2, the cyanobacteria prepared in step S1 are allowed to secrete secretions. If the cyanobacteria is the modified cyanobacteria described above, proteins and metabolic products produced within the bacterial cell are likely to be secreted outside the outer membrane. These intracellularly produced substances may include substances that contribute to the antioxidant effect of the antioxidant. For example, in step S2, the modified cyanobacteria are cultured under specified conditions, thereby causing substances that contribute to the antioxidant effect of the antioxidant to be secreted outside the bacterial cell.

[0047] Cyanobacteria can generally be cultured using liquid culture or a modified method thereof using BG-11 medium. Therefore, modified cyanobacteria can also be cultured in a similar manner. The culture period for cyanobacteria to produce antioxidants is a period during which proteins and metabolic products can accumulate at high concentrations under conditions of sufficient bacterial growth, and may be, for example, 1 to 3 days or 4 to 7 days. Examples of culture methods include aeration and agitation culture and shaking culture. By culturing the modified cyanobacteria under these conditions, the modified cyanobacteria produce intracellularly produced substances, such as proteins and metabolic products, and secrete and excrete these intracellularly produced substances into the culture medium. These intracellularly produced substances include substances that contribute to the antioxidant effect of the antioxidant. When the secreted substances into the culture medium are recovered, the culture medium is filtered or centrifuged to remove solids such as cells, and the culture supernatant is recovered. According to this production method, substances that contribute to the antioxidant effect of the antioxidant are secreted extracellularly by the cyanobacteria. Therefore, there is no need to disrupt the cells to obtain the substances that contribute to the antioxidant effect of the antioxidant, and the cyanobacteria remaining after the secretion is collected can be used repeatedly to produce antioxidants.

[0048] The method for collecting the secretions secreted into the cyanobacterial culture solution is not limited to the above method. For example, the secretions in the culture solution may be collected while the cyanobacteria are being cultured. For example, a permeable membrane that allows the secretions to pass through may be used, and the secretions that have permeated the membrane may be collected. In this case, the secretions in the culture solution can be collected while the cyanobacteria are being cultured, eliminating the need for a process to remove the cyanobacterial cells from the culture solution. This allows for easier and more efficient production of antioxidants.

[0049] The antioxidant may be the cyanobacterial culture medium itself, the culture medium from which cyanobacteria have been removed, or a substance separated from the cyanobacterial culture medium by membrane separation or the like. The antioxidant may be in liquid, cream, or powder form. A powder antioxidant can be produced, for example, by drying a liquid containing the secretions by spray drying or the like.

[0050] As described above, the process of recovering and disrupting the cyanobacterial cells from the culture medium is no longer necessary, thereby reducing the damage and stress suffered by the cyanobacteria. This reduces the productivity of the secretion of cyanobacterial secretions, allowing the cyanobacteria to be used for a long period of time. Thus, for example, antioxidants can be obtained easily and efficiently by using modified cyanobacteria.

[0051] The secretions contained in the antioxidant are not limited to specific compounds. The secretions may contain, for example, at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids. This allows the antioxidant to have the desired effect. It is believed that the secretions containing phycocyanin tend to enhance the antioxidant's antioxidant ability. It is believed that the secretions containing mycosporine-like amino acids tend to enhance the antioxidant's ultraviolet absorption ability. It is believed that the secretions containing phycocyanin, polysaccharides, or amino acids tend to enhance the antioxidant's moisturizing properties. It is believed that the secretions containing nucleic acids or adenosine tend to enhance the antioxidant's whitening effect.

[0052] The antioxidant may contain components other than the secretions. The antioxidant may contain, for example, an aqueous component, an oily component, a surfactant, or a fragrance. Examples of aqueous components include water, glycerin, ethanol, BG, PEG, and sodium hyaluronate. Examples of oily components include squalane, mineral oil, petrolatum, and vegetable oil. Examples of surfactants include cationic surfactants, anionic surfactants, nonionic surfactants, and zwitterionic surfactants. The antioxidant may contain other components as needed. The antioxidant may contain, for example, sodium nitrate, magnesium sulfate, potassium chloride, or an organic acid having a carboxy group. The antioxidant may be contained in pharmaceuticals, quasi-drugs, cosmetics, foods, or beverages.

[0053] The above-mentioned antioxidant can provide a method for removing active oxygen. In this method, active oxygen is removed by the antioxidant. In this case, the antioxidant may be attached to the skin by a method such as application or spraying, or may be taken into the body orally or by injection. The antioxidant may also be used on plants.

[0054] (Composition for topical application to skin) The composition for topical application to skin according to this embodiment contains a secretion of cyanobacteria. The composition for topical application to skin has at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect. Having an antioxidant effect means, for example, that the degree of antioxidant effect is greater than when the topical application to skin is not used. Having an ultraviolet absorbing effect means, for example, that the degree of ultraviolet absorbing effect is greater than when the topical application to skin is not used.

[0055] The topical skin composition contains, for example, the secretions secreted to the outside of the cyanobacteria, but does not contain the cyanobacteria itself or any components derived from the cyanobacteria itself. In other words, the antioxidant topical skin composition contains, for example, only the secretions secreted to the outside of the cyanobacteria as the components derived from the cyanobacteria.

[0056] The topical skin composition may further have a moisturizing effect. Having a moisturizing effect means, for example, that the moisturizing effect is greater than when the topical skin composition is not used. The basic moisturizing effects can be classified into three categories: a "moisturizing effect" that replenishes moisture in the stratum corneum; a "softening effect" that provides and retains moisturizing components to give the skin flexibility; and an "evaporation-inhibiting effect" that confines moisture or moisturizing components in the stratum corneum. The level of moisturizing effect can be evaluated, for example, by measuring the change in skin moisture content over time after applying the topical skin composition to the skin.

[0057] The secretion contained in the topical skin composition is, for example, a secretion from a modified cyanobacterium. The modified cyanobacterium has been genetically modified, for example, to be able to excrete the secretion to the outside of the cyanobacterium. The modified cyanobacterium can, for example, excrete a compound that is difficult to excrete from wild-type cyanobacteria that have not been genetically modified. The modified cyanobacterium has been genetically modified, for example, to be able to secrete a component having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0058] The above explanations regarding cyanobacteria, cyanobacterial secretions, modified cyanobacteria, and modified cyanobacterial cultures in antioxidants also apply to compositions for topical application to the skin, unless technically inconsistent.

[0059] The topical composition for skin can be produced, for example, by a method comprising secreting a secretion from cyanobacteria to obtain a component having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect. The above explanation regarding the method for producing the antioxidant also applies to the method for producing the topical composition for skin, unless technically inconsistent.

[0060] For example, when the modified cyanobacteria are cultured, they produce proteins and metabolic products, which are then secreted into the culture medium. These intracellularly produced substances include substances that contribute to the topical skin composition having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect. When recovering the secretions secreted into the culture medium, the culture medium is filtered or centrifuged to remove solids such as cells (i.e., bacteria) from the culture medium, and the culture supernatant is recovered. According to this production method, the substances that contribute to the topical skin composition having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect are secreted extracellularly by the cyanobacteria. Therefore, there is no need to disrupt the cells to obtain the substances that contribute to the topical skin composition having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect. Therefore, the cyanobacteria remaining after the secretions are recovered can be repeatedly used to produce topical skin compositions.

[0061] The topical composition for skin application may be a cyanobacterial culture solution itself, a culture solution from which cyanobacteria have been removed, or a composition separated from a cyanobacterial culture solution by membrane separation or the like. The topical composition for skin application may be in the form of a liquid, cream, or powder. A powdered topical composition for skin application can be produced, for example, by drying a liquid containing secretions by spray drying or the like.

[0062] As described above, the need for recovering and disrupting the cyanobacterial cells from the culture medium is eliminated, reducing the damage and stress suffered by the cyanobacteria. This reduces the productivity of the secretion of cyanobacterial secretions, allowing the cyanobacteria to be used for a long period of time. Thus, for example, by using modified cyanobacteria, a topical skin composition can be easily and efficiently obtained.

[0063] The secretions contained in the topical skin composition are not limited to specific compounds. The secretions may contain, for example, at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids. This allows the topical skin composition to advantageously have at least one effect selected from the group consisting of antioxidant and ultraviolet absorbing effects. It is believed that the inclusion of phycocyanin in the secretions increases the antioxidant capacity of the topical skin composition. It is believed that the inclusion of mycosporine-like amino acids in the secretions increases the ultraviolet absorbing capacity of the topical skin composition. It is believed that the inclusion of phycocyanin, polysaccharides, or amino acids in the secretions increases the moisturizing properties of the topical skin composition. It is believed that the inclusion of nucleic acids or adenosine in the secretions increases the whitening effect of the topical skin composition.

[0064] The topical skin composition may contain components other than the above-mentioned secretions. The topical skin composition may contain, for example, an aqueous component, an oily component, a surfactant, or a fragrance. Examples of aqueous components include water, glycerin, ethanol, BG, PEG, and sodium hyaluronate. Examples of oily components include squalane, mineral oil, petrolatum, and vegetable oil. Examples of surfactants include cationic surfactants, anionic surfactants, nonionic surfactants, and zwitterionic surfactants. The topical skin composition may contain other components as needed. The topical skin composition may contain, for example, sodium nitrate, magnesium sulfate, potassium chloride, or an organic acid having a carboxy group.

[0065] The above-mentioned composition for external use on skin can provide a skin care method that includes applying the composition for external use on skin. The method for applying the composition for external use on skin to the skin may be application or spraying.

[0066] (Moisturizing Agent) The moisturizing agent according to the present embodiment contains a secretion of cyanobacteria. Having a moisturizing effect means, for example, that the moisturizing effect is greater than when the moisturizing agent is not used. The basic functions of moisturizing can be classified into three categories: a "moisturizing effect" that replenishes moisture in the stratum corneum; a "softening effect" that provides and retains moisturizing components to give flexibility to the skin; and an "evaporation suppression effect" that traps moisture or moisturizing components in the stratum corneum. The degree of moisturizing effect can be evaluated, for example, by measuring the change in skin moisture content over time after applying the moisturizing agent to the skin.

[0067] The moisturizing agent contains, for example, a secretion secreted to the outside of the cyanobacteria, but does not contain the cyanobacteria itself or any components derived from the cyanobacteria itself. In other words, the moisturizing agent contains, for example, only the secretion secreted to the outside of the cyanobacteria as a component derived from the cyanobacteria.

[0068] The moisturizer may further have at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect. In this specification, "having an antioxidant effect" means, for example, that the degree of antioxidant effect is greater than when no moisturizer is used. In this specification, "having an ultraviolet absorbing effect" means, for example, that the degree of ultraviolet absorbing effect is greater than when no moisturizer is used.

[0069] The secretion contained in the moisturizing agent is, for example, a secretion from a modified cyanobacterium. The modified cyanobacterium is, for example, genetically modified to be able to excrete the secretion to the outside of the cyanobacterium. The modified cyanobacterium can, for example, excrete a compound that is difficult to excrete from wild-type cyanobacteria that have not been genetically modified. The modified cyanobacterium is, for example, genetically modified to be able to secrete a component that has a moisturizing effect.

[0070] The above explanations regarding cyanobacteria, cyanobacterial secretions, modified cyanobacteria, and modified cyanobacterial cultures in antioxidants also apply to moisturizers, unless technically inconsistent.

[0071] The moisturizing agent can be produced by, for example, a method including secreting a secretion from cyanobacteria to obtain a component having moisturizing effect. The above explanation regarding the method for producing an antioxidant also applies to the method for producing a moisturizing agent, unless technically inconsistent.

[0072] For example, when the modified cyanobacteria are cultured, the modified cyanobacteria produce proteins and metabolic products, which are then secreted and excreted into the culture medium. These intracellularly produced substances include substances that contribute to the moisturizing effect of the moisturizer. When recovering the secretions secreted into the culture medium, the culture medium is filtered or centrifuged to remove solid matter such as cells (i.e., bacterial cells) from the culture medium, and the culture supernatant is recovered. According to this production method, the substances that contribute to the moisturizing effect of the moisturizer are secreted outside the cyanobacterial cells. Therefore, there is no need to disrupt the cells to obtain the substances that contribute to the moisturizing effect of the moisturizer. Therefore, the cyanobacteria remaining after recovery of the secretions can be repeatedly used to produce moisturizers.

[0073] The method for collecting the secretions secreted into the cyanobacterial culture solution is not limited to the above method. For example, the secretions in the culture solution may be collected while the cyanobacteria are being cultured. For example, a permeable membrane that allows the secretions to pass through may be used, and the secretions that have permeated the membrane may be collected. In this case, the secretions in the culture solution can be collected while the cyanobacteria are being cultured, eliminating the need for a process to remove the cyanobacterial cells from the culture solution. This allows for easier and more efficient production of moisturizing agents.

[0074] The moisturizing agent may be the cyanobacterial culture medium itself, the culture medium from which the cyanobacteria have been removed, or a substance separated from the cyanobacterial culture medium by membrane separation or the like. The moisturizing agent may be in liquid, cream, or powder form. A powder moisturizing agent can be produced, for example, by drying a liquid containing the secretions by spray drying or the like.

[0075] As described above, the process of recovering and disrupting the cyanobacterial cells from the culture medium is no longer necessary, thereby reducing the damage and stress suffered by the cyanobacteria. This reduces the productivity of the secretion of the cyanobacterial secretion product, allowing the cyanobacteria to be used for a long period of time. Thus, for example, by using the modified cyanobacteria, moisturizers can be obtained easily and efficiently.

[0076] The secretions contained in moisturizers are not limited to specific compounds. The secretions may contain, for example, at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids. This allows the moisturizer to have the desired effect. It is believed that the inclusion of phycocyanin in the secretions makes it easier for the moisturizer to have high antioxidant capacity. It is believed that the inclusion of mycosporine-like amino acids in the secretions makes it easier for the moisturizer to have high ultraviolet absorption capacity. It is believed that the inclusion of phycocyanin, polysaccharides, or amino acids in the secretions makes it easier for the moisturizer to have high moisturizing properties. It is believed that the inclusion of nucleic acids or adenosine in the secretions makes it easier for the moisturizer to have a whitening effect.

[0077] The moisturizer may contain components other than the above-mentioned secretions. The moisturizer may contain, for example, an aqueous component, an oily component, a surfactant, or a fragrance. Examples of aqueous components include water, glycerin, ethanol, BG, PEG, and sodium hyaluronate. Examples of oily components include squalane, mineral oil, petrolatum, and vegetable oil. Examples of surfactants include cationic surfactants, anionic surfactants, nonionic surfactants, and zwitterionic surfactants. The moisturizer may contain other components as needed. The moisturizer may be a moisturizer that moisturizes by directly providing moisture to the stratum corneum, or an emollient that suppresses moisture evaporation by forming a film on the stratum corneum surface by the moisturizer. The moisturizer may contain, for example, sodium nitrate, magnesium sulfate, potassium chloride, or an organic acid having a carboxy group.

[0078] The moisturizer described above can provide a skin care method that includes applying the moisturizer to the skin. The moisturizer can be applied to the skin by application or spraying.

[0079] (Additional Note) From the above description, the following techniques are disclosed.

[0080] (Technology 1) An antioxidant containing a secretion of cyanobacteria.

[0081] (Technology 2) The antioxidant according to Technology 1, which has an ultraviolet absorbing effect.

[0082] (Technology 3) The antioxidant according to Technology 1 or 2, wherein the secretion product is a secretion product of a modified cyanobacterium that has been genetically modified so as to be able to excrete the secretion product to the outside of the cyanobacterium.

[0083] (Technology 4) The antioxidant according to Technology 3, wherein the modified cyanobacterium has a suppressed or lost function of a protein involved in binding between the outer membrane and the cell wall.

[0084] (Technology 5) The antioxidant according to Technology 4, wherein the modified cyanobacterium has a gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate modifying enzyme involved in binding between the outer membrane and the cell wall modified.

[0085] (Technology 6) The antioxidant according to Technology 5, wherein the gene is a first gene which is slr1841, slr0688, or slr1908 possessed by Synechocystis sp. PCC 6803, or a second gene which is a gene corresponding to the first gene possessed by a cyanobacterium of the genus Synechococcus, the genus Anabaena, or the genus Nostoc.

[0086] (Technology 7) The antioxidant described in Technology 3, wherein the modified cyanobacterium is genetically modified using vector DNA, the vector DNA being a plasmid or virus designed for genetic modification, and the vector DNA includes a gene sequence of a homologous region of a site targeted for the genetic modification, a gene sequence for suppressing expression of the target of the genetic modification, and a gene sequence that functions as a marker for the genetic modification.

[0087] (Technology 8) The antioxidant according to any one of Techniques 1 to 7, wherein the secretory product contains at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids.

[0088] (Technology 9) A method for removing active oxygen, comprising removing active oxygen with an antioxidant containing a secretion of cyanobacteria.

[0089] (Technology 10) A method for producing an antioxidant, comprising causing cyanobacteria to secrete a secretion to obtain a component having a moisturizing effect.

[0090] (Technology 11) The production method according to Technology 10, wherein the cyanobacteria is genetically modified so as to be able to secrete the component.

[0091] (Technology 12) A composition for external application to the skin, comprising a secretion of a cyanobacterium, and having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0092] (Technology 13) The composition for external use on skin according to Technology 12, further having a moisturizing effect.

[0093] (Technology 14) The composition for external application to skin according to Technology 12 or 13, wherein the secretion is a secretion of a modified cyanobacterium that has been genetically modified so as to be able to excrete the secretion to the outside of the cyanobacterium.

[0094] (Technology 15) The composition for external application to skin according to Technology 14, wherein the modified cyanobacterium has a suppressed or lost function of a protein involved in binding between the outer membrane and the cell wall.

[0095] (Technology 16) The topical skin composition according to Technology 15, wherein the modified cyanobacterium has a gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate modifying enzyme involved in binding between the outer membrane and the cell wall modified.

[0096] (Technology 17) The topical composition for skin according to Technology 16, wherein the gene is a first gene which is slr1841, slr0688, or slr1908 possessed by Synechocystis sp. PCC 6803, or a second gene which is a gene corresponding to the first gene possessed by cyanobacteria of the genus Synechococcus, the genus Anabaena, or the genus Nostoc.

[0097] (Technology 18) The topical skin composition according to Technology 14, wherein the modified cyanobacteria is genetically modified using vector DNA, the vector DNA being a plasmid or virus designed for genetic modification, and the vector DNA includes a gene sequence of a homologous region of the target site of the genetic modification, a gene sequence for suppressing expression of the target of the genetic modification, and a gene sequence that functions as a marker for the genetic modification.

[0098] (Technology 19) The composition for external use on skin according to any one of techniques 12 to 18, wherein the secretion contains at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids.

[0099] (Technology 20) A skin care method comprising applying to the skin a composition for external use on the skin, the composition containing a secretion of cyanobacteria and having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0100] (Technology 21) A method for producing a composition for external use on skin, comprising causing cyanobacteria to secrete a secretion to obtain a component having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0101] (Technology 22) The method for producing a composition for external use on skin according to Technology 21, wherein the cyanobacteria has been genetically modified to be able to secrete the component.

[0102] (Technology 23) A moisturizer containing the secretions of cyanobacteria.

[0103] (Technology 24) The moisturizing agent according to Technology 23, further having at least one effect selected from the group consisting of an antioxidant effect and an ultraviolet absorbing effect.

[0104] (Technology 25) The moisturizing agent according to Technology 23 or 24, wherein the secretion is a secretion of a modified cyanobacterium that has been genetically modified so as to be able to excrete the secretion to the outside of the cyanobacterium.

[0105] (Technology 26) The moisturizing agent according to Technology 25, wherein the modified cyanobacterium has a suppressed or lost function of a protein involved in binding between the outer membrane and the cell wall.

[0106] (Technology 27) The moisturizing agent according to Technology 26, wherein the modified cyanobacterium has a gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate modifying enzyme involved in binding between the outer membrane and the cell wall modified.

[0107] (Technology 28) The moisturizing agent according to Technology 27, wherein the gene is a first gene which is slr1841, slr0688, or slr1908 possessed by Synechocystis sp. PCC 6803, or a second gene which is a gene corresponding to the first gene possessed by a cyanobacterium of the genus Synechococcus, the genus Anabaena, or the genus Nostoc.

[0108] (Technology 29) A moisturizing agent according to Technology 25, wherein the modified cyanobacteria is genetically modified using vector DNA, the vector DNA being a plasmid or virus designed for genetic modification, and the vector DNA includes a gene sequence of a homologous region of the target site of the genetic modification, a gene sequence for suppressing expression of the target of the genetic modification, and a gene sequence that functions as a marker for the genetic modification.

[0109] (Technology 30) The moisturizing agent according to any one of Techniques 23 to 29, wherein the secretion contains at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids.

[0110] (Technology 31) A skin care method comprising applying a moisturizer containing a cyanobacterial secretion to the skin.

[0111] (Technology 32) A method for producing a moisturizing agent, comprising causing cyanobacteria to secrete a secretion to obtain a component having a moisturizing effect.

[0112] (Technology 33) The production method according to Technology 32, wherein the cyanobacteria is genetically modified so as to be able to secrete the component.

[0113] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0114] (Preparation of cyanobacterial culture supernatant solution) Escherichia coli HST08 strain and wild-type cyanobacterium Synechocystis sp. PCC 6803 strain (parent cyanobacteria) were each cultured in 5 mL of LB medium for 16 hours. In the LB medium, the Tripton concentration was 1.0% by mass, the yeast extract concentration was 0.5% by mass, and the NaCl concentration was 1.0% by mass. Additionally, these were cultured in 10 mL of BG-11 medium for 120 hours. BG-11 medium was prepared as a 1 L aqueous solution containing the components shown in Table 1. Cells derived from 1 mL of fully grown culture medium were subjected to Promega's Wizard® Genomic DNA Purification Kit to extract cellular genomic DNA.

[0115]

[0116] Next, to construct the plasmid DNA used for gene modification, a total of seven DNA fragments (fragments 1 to 7) were amplified by PCR. Toyobo's KOD One® PCR Master Mix -Blue- was used for PCR. For fragment 1, the E. coli replication origin and the ampicillin antibiotic resistance gene were amplified using primer 1 (see SEQ ID NO:7) and primer 2 (see SEQ ID NO:8) with the pUC19 plasmid as a template. Next, for fragments 2 to 5, the promoter sequence of the petE gene was amplified using primer 3 (see SEQ ID NO:9) and primer 4 (see SEQ ID NO:10) with the cyanobacterium-derived genomic DNA as a template. For fragment 2, the promoter sequence of the nrsB gene was amplified using primer 5 (see SEQ ID NO:11) and primer 6 (see SEQ ID NO:12). For fragment 4, the homologous region 1 of the slr0688 gene was amplified using primer 7 (see SEQ ID NO:13) and primer 8 (see SEQ ID NO:14). For fragment 5, primer 9 (see SEQ ID NO: 15) and primer 10 (see SEQ ID NO: 16) were used to amplify homologous region 2 of the slr0688 gene. For fragment 6, primer 11 (see SEQ ID NO: 17) and primer 12 (see SEQ ID NO: 18) were used to amplify the mazF gene using the above-mentioned genomic DNA derived from E. coli as a template. For fragment 7, primer 13 (see SEQ ID NO: 19) and primer 14 (see SEQ ID NO: 20) were used to amplify the resistance gene for the antibiotic kanamycin using the pSL2680 plasmid as a template.

[0117] The seven fragments were ligated using Clontech's In-Fusion® Snap Assembly Master Mix. The resulting plasmid DNA was introduced into Takara Bio's E. coli HST08 Premium Competent Cells using the heat shock method. The resulting E. coli was cultured in the presence of 50 μg / mL of the antibiotic ampicillin, and cells into which the plasmid DNA had been introduced were selected. The replicated plasmid DNA in these cells was extracted using Promega's Wizard® Plus SV Minipreps DNA Purification System to obtain the pNRSmazF-petE0688 plasmid (see Figure 2) used for gene modification.

[0118] The parent cyanobacterial cells were cultured in 10 mL of BG-11 medium inside a 125 mL Erlenmeyer flask. The culture was incubated at 30°C for 120 hours with 100 μmol photons m -2 s -1 The transformation was performed in an artificial climate chamber under illumination with 1000 W of white LED light and shaking at 160 rotations per minute (rpm). A 0.5 mL culture of the fully grown parent cyanobacterium was mixed with 3 μg of the pNRSmazF-petE0688 plasmid, and homologous recombination was carried out by natural transformation to introduce the petE promoter sequence immediately upstream of the slr0688 gene. The transformed cells were grown on BG-11 agar medium for 2 days and then transferred to agar medium containing 20 μg / mL of the antibiotic kanamycin and grown for 7 days. Cells were transferred to another agar medium using an Immobilon-NC Triton-free MCE 0.45 μm 82 mm disc membrane filter (Merck Millipore).

[0119] The modified cyanobacterial colonies thus obtained were transferred twice to new agar plates and grown there, allowing homologous recombination to propagate throughout the entire genome of the cells. To confirm this, colony PCR was performed using the obtained colonies as templates. Promega's GoTaq® Green Master Mix was used for colony PCR. Figure 3 shows the results of electrophoresis of DNA fragments amplified by colony PCR. The left side of Figure 3 shows the results of colony PCR using primer 15 (see SEQ ID NO: 21) and primer 16 (see SEQ ID NO: 22). This colony PCR involved amplification of the genomic sequence located upstream of the site of homologous recombination and the genomic sequence located downstream of that site. The right side of Figure 3 shows the results of colony PCR using primer 16 and primer 17 (see SEQ ID NO: 23). This colony PCR involved amplification of the genomic sequence located downstream of the site of homologous recombination and the sequence in the kanamycin resistance gene where homologous recombination occurred. The center lane in Figure 3 shows the results of electrophoresis using Nippon Gene's Gene Ladder Wide 2 as a DNA size marker. Regarding the electrophoresis results for each DNA fragment in the left and right lanes, the numbers 1 to 5 indicate the colony numbers used as templates. In addition, "P" indicates the results of electrophoresis for DNA fragments amplified using the pNRSmazF-petE0688 plasmid as a control, and "N" indicates the results of electrophoresis for DNA fragments amplified using the genomic DNA derived from the parent cyanobacterium as a control. The results shown in Figure 3 confirm that homologous recombination was widespread throughout the genome, and it is understood that a modified cyanobacterium into which the promoter sequence of the petE gene was introduced was obtained.

[0120] The modified cyanobacterial cells were cultured in 1 L of BG-11 medium for 120 hours, similar to the parent cyanobacterial cells. As a control, parent cyanobacterial cells were also cultured simultaneously. The resulting culture solutions were then passed through a Merck Millipore Millex®-GS Syringe Filter Unit (0.22 μm) to remove the cells. This resulted in a culture supernatant solution containing the secretions of the modified cyanobacterium according to the example, and a culture supernatant solution of the parent cyanobacterial cells according to the comparative example.

[0121] (LPS Detection) The culture supernatant solutions of the Examples and Comparative Examples were each subjected to measurement of lipopolysaccharide (LPS), a characteristic component indicative of outer membrane detachment in cyanobacteria. Specifically, 0.2 mL of each culture supernatant solution was measured for LPS activity using a Seikagaku Corporation Endospecy ES-24S set and Pyrocolor diazo reagent. Figure 4 is a graph showing the results of LPS activity measurements for the culture supernatants of the Examples and Comparative Examples. As shown in Figure 4, the LPS activity of the culture supernatant solutions of the Examples was higher than that of the Comparative Example, suggesting that the outer membrane had partially detached in the modified cyanobacteria. In contrast, the LPS activity of the culture supernatant solution of the Comparative Example was lower, suggesting that the LPS was not secreted to the outside of the parent cyanobacteria.

[0122] (Measurement of antioxidant capacity) Diphenylpicrylhydrazyl (DPPH) is a stable radical. The antioxidant capacity of a test substance can be evaluated by measuring its active oxygen scavenging capacity, which is the degree to which the test substance removes the unpaired electron of DPPH. 150 μL of the test substance taken from the culture supernatant of the Example or the culture supernatant of the Comparative Example was mixed with 300 μL of DPPH methanol solution to obtain a sample for evaluating antioxidant capacity. The culture supernatant solution of the Example and the culture supernatant solution of the Comparative Example had turbidities of 0.4 and 1.9, respectively. The concentration of DPPH in the DPPH methanol solution was 4 mg / L. The absorbance A at a wavelength of 520 nm was 520nm From the absorbance A at a wavelength of 780 nm as a baseline 780nmThe time dependence of the difference obtained by subtracting the absorbance was measured. A JASCO V-650 UV-visible spectrophotometer was used to measure absorbance. Figure 5 is a graph showing the time change in the absorbance difference for samples for evaluating antioxidant capacity according to the Examples and Comparative Examples. For the sample for evaluating antioxidant capacity according to the Comparative Example, which used the culture supernatant solution as the test substance, the change in absorbance due to active oxygen disappeared after about 35 minutes from the start of the reaction, and the antioxidant capacity of the culture supernatant solution according to the Comparative Example was low. On the other hand, for the sample for evaluating antioxidant capacity according to the Examples, which used the culture supernatant solution as the test substance, the absorbance due to active oxygen continued to decrease even 60 minutes after the start of the reaction, indicating that the antioxidant capacity of the culture supernatant solution according to the Examples was very high, and that the culture supernatant solution according to the Examples contained secretions that contributed to its high antioxidant capacity.

[0123] (Measurement of Moisture Retention) After wiping off oil from the forearm of the subject, the sample for evaluating moisture retention according to the example and pure water were applied at 20 μL / cm 2A sample of each of the samples was dropped onto the forearm of a subject, and the change in skin moisture content over time after air drying was measured. The moisturizing evaluation sample according to the example was prepared by concentrating the culture supernatant solution according to the example ten times by freeze-drying. The turbidity of the culture supernatant solution according to the example was 0.4. This measurement was performed 10 times at the same location on the subject's forearm. Pure water was used as a reference. A Corneometer CM825 manufactured by Integral Corporation was used to measure skin moisture content. The measurement environment during the test was 56% relative humidity and 25°C temperature. FIG. 6A is a graph showing the change in skin moisture content over time when the moisturizing evaluation sample according to the example and pure water were used. The change in skin moisture content over time was measured in the same manner, except that the moisturizing evaluation sample according to the comparative example was used instead of the moisturizing evaluation sample according to the example. The moisturizing evaluation sample according to the comparative example was prepared by concentrating the culture supernatant solution according to the comparative example ten times by freeze-drying. The turbidity of the culture supernatant solution according to the comparative example was 1.9. 6B is a graph showing the change in skin moisture content over time when using a comparative example moisturizing evaluation sample and pure water. The dashed lines in FIGS. 6A and 6B represent the average values ​​measured 30 minutes after the sample and pure water were dropped onto the subject's forearm. Comparing FIGS. 6A and 6B, the moisturizing evaluation sample of the example exhibited higher moisturizing properties than the moisturizing evaluation sample of the comparative example. This suggests that the secretion of the modified cyanobacteria contained in the culture supernatant solution of the example contains components that may contribute to its moisturizing effect.

[0124] The change in skin moisture content over time was measured on another day using the moisture-retaining evaluation sample and pure water according to the example, and the moisture-retaining evaluation sample and pure water according to the comparative example, in the same manner as described above. The measurement environment during the test was 70% relative humidity and 26°C temperature. Figure 7A is a graph showing the change in skin moisture content over time when the moisture-retaining evaluation sample and pure water according to the example were used. Figure 7B is a graph showing the change in skin moisture content over time when the moisture-retaining evaluation sample and pure water according to the comparative example were used. The dashed lines in Figures 7A and 7B indicate the average values ​​measured 30 minutes after the sample and pure water were dropped onto the subject's forearm. Comparing Figures 7A and 7B reveals that the moisture-retaining evaluation sample according to the example exhibited higher moisture retention than the moisture-retaining evaluation sample according to the comparative example, even in a high-humidity environment.

[0125] (Evaluation of UV Absorption Ability) The absorbance of the culture supernatant solution according to the Example and the Comparative Example was measured at a wavelength of approximately 300 nm to evaluate the UV-B absorption ability. The turbidity of the culture supernatant solution according to the Example was 0.4, and the turbidity of the culture supernatant solution according to the Comparative Example was 1.9. Absorbance measurements were performed using a JASCO V-650 UV-Visible Spectrophotometer. To evaluate UV-B absorption ability, a correction was performed in the absorption spectrum, treating the line connecting wavelengths 260 nm and 340 nm as the background. Figure 8 is a graph showing the UV absorption spectra of the culture supernatant solutions according to the Example and the Comparative Example. This graph was obtained using the above correction. As shown in Figure 8, the culture supernatant solution according to the Example exhibited approximately twice the absorbance at a wavelength of 300 nm compared to the culture supernatant solution according to the Comparative Example. These results suggest that the secretions of the modified cyanobacteria contained in the culture supernatant solution according to the Example contain components that may contribute to the UV absorption effect.

[0126] (Evaluation of Plant High Temperature Tolerance and Stress Tolerance) Pansy seeds were sown in commercially available rock wool to prepare 12 rock wools sown with pansy seeds. Then, 1 mL of the culture supernatant solution according to the example was irrigated into six rock wools at the time of sowing. Additionally, 1 mL of pure water was irrigated into the remaining six rock wools at the time of sowing. Each rock wool sown with pansy seeds was placed in an environment of 40°C and 40,000 lux of illumination for six hours each day. After sowing, the presence or absence of cotyledon expansion of the pansy seeds was confirmed daily. The cotyledon expansion rate of pansies sown in rock wool irrigated with the culture supernatant solution according to the example and the cotyledon expansion rate of pansies sown in rock wool irrigated with pure water were calculated. The cotyledon expansion rate is the ratio of the number of rock wools in which cotyledon expansion was confirmed to the number of rock wools in which pansy seeds were sown. The results are shown in Figure 9A.

[0127] Spinach seeds (variety: Mirage) were sown into commercially available rock wool to prepare 12 rock wools seeded with spinach seeds. Then, 1 mL of the culture supernatant solution according to the example was irrigated into six rock wools at the time of sowing. Additionally, 1 mL of pure water was irrigated into the remaining six rock wools at the time of sowing. Each rock wool with the sown spinach seeds was placed in an environment of 40°C and 40,000 lux of illumination for six hours each day. After sowing, the presence or absence of cotyledon development was checked daily. The cotyledon development rate of spinach sown in rock wool irrigated with the culture supernatant solution according to the example and the cotyledon development rate of spinach sown in rock wool irrigated with pure water were calculated. The cotyledon development rate is the ratio of the number of rock wools with confirmed cotyledon development to the number of rock wools with spinach seeds sown. The results are shown in Figure 9B.

[0128] 9A and 9B, the cotyledon expansion rate in the rock wool irrigated with the culture supernatant solution according to the example was higher than that in the rock wool irrigated with pure water within 30 days after sowing the seeds. This suggests that the use of the culture supernatant solution according to the example improves the high-temperature tolerance and stress tolerance of plants such as pansies and spinach.

[0129] The antioxidants of the present disclosure can be used in applications such as improving the high temperature tolerance and stress tolerance of pharmaceuticals, quasi-medical products, cosmetics, foods, beverages, and plants, for example.

Claims

1. Antioxidants, including those secreted by cyanobacteria.

2. The antioxidant according to claim 1, which has an ultraviolet absorbing effect.

3. The antioxidant according to claim 1, wherein the secretion product is a secretion product of a modified cyanobacterium that has been genetically modified to be capable of excreting the secretion product to the outside of the cyanobacterium.

4. The antioxidant according to claim 3, wherein the function of a protein involved in binding between the outer membrane and the cell wall of the modified cyanobacterium is suppressed or lost.

5. The antioxidant described in claim 4, wherein the modified cyanobacterium has a modified gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate modifying enzyme involved in binding between the outer membrane and the cell wall.

6. The antioxidant according to claim 5, wherein the gene is a first gene which is slr1841, slr0688, or slr1908 possessed by Synechocystis sp. PCC 6803, or a second gene which is a gene corresponding to the first gene possessed by a cyanobacterium of the genus Synechococcus, the genus Anabaena, or the genus Nostoc.

7. The antioxidant described in claim 3, wherein the modified cyanobacterium is genetically modified by vector DNA, the vector DNA being a plasmid or virus designed for genetic modification, and the vector DNA containing a gene sequence of a homologous region of the target site of the genetic modification, a gene sequence for suppressing expression of the target of the genetic modification, and a gene sequence that functions as a marker for the genetic modification.

8. The antioxidant according to claim 1, wherein the secreted product contains at least one selected from the group consisting of nucleic acids, adenosine, polysaccharides, amino acids, phycocyanin, and mycosporine-like amino acids.

9. A method for removing active oxygen, comprising removing active oxygen with an antioxidant containing a secretion of cyanobacteria.

10. A method for producing an antioxidant, comprising causing cyanobacteria to secrete a secretion to obtain a component having an antioxidant effect.

11. The method of claim 10, wherein the cyanobacteria are genetically modified to be capable of secreting the component.

Images