Novel microalgae

Abstract

To provide novel microalgae, and a microalgae product using the microalgae.SOLUTION: In one aspect, the present disclosure provides microalgae having an ability of highly producing at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, γ aminobutyric acid, hydroxyproline, calcium, and chlorophyll and / or property of exerting a high amino acid score. In one embodiment, the microalgae of the present disclosure an ability of producing fucoxanthin of 0.01 g or more, 0.02 g or more, 0.05 g or more, 0.07 g or more, 0.1 g or more, 0.2 g or more, 0.5 g or more, 0.6 g or more, 0.7 g or more, 0.8 g or more, 0.9 g or more, 1 g or more, 1.5 g or more, 2 g or more, 2.5 g or more, 3 g or more, 4 g or more, 5 g or more, 6 g or more, 7 g or more, 8 g or more, 9 g or more, or 10 g or more per 100 g of dry cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to useful ingredient-containing microalgae and microalgae products thereof. In particular, the present disclosure relates to microalgae that highly produce fucoxanthin and microalgae products thereof.

Background Art

[0002] Microalgae such as Chlorella and Euglena have attracted attention for their nutritional components such as contained vitamins and minerals, and are used as health foods and food materials (Patent Document 1 = JP 2018-70568 A).

[0003] In addition, microalgae can contain various components including useful components and harmful components. For example, chlorophyll widely contained in microalgae is known to be converted into pheophorbide and cause phototoxicity.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

[0007] In this disclosure, the one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary. [Effects of the Invention]

[0008] This disclosure enables the provision of microalgae or microalgae products (e.g., food) with novel functionalities. [Brief explanation of the drawing]

[0009] [Figure 1] This image shows the OPMS30543 strain in culture. The scale bar represents 50 μm. [Figure 2] This shows the spray-dried microalgae product of strain OPMS30543. [Figure 3] An exemplary photobioreactor is shown. The left image is a photograph of the bioreactor, where the elongated tube is a transparent component with an outer diameter of 60 mm. The right image is a schematic diagram of the bioreactor. [Modes for carrying out the invention]

[0010] The present disclosure is described below in best form. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0011] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0012] (definition, etc.) In this specification, "microalgae" refers to microscopic microorganisms (e.g., 0.1 μm to 1 mm) containing chloroplasts, which generally inhabit water. Microalgae include organisms of the prokaryotes Cyanobacteria, as well as eukaryotes Glaucophyta, Rhodophyta, Chlorophyta, Cryptophyta, Haptophyta, Heterokontophyta, Dinophyta, Euglenida, and Chlorarachniophyta.

[0013] The division Haptophyta includes the class Haptophyceae, which in turn includes the subclass Pavlovophycidae and the subclass rymnesiophycidae. The subclass Pavlovophycidae includes the order Pavlovales, which in turn includes the family Pavlovaceae, which includes Diacronema, Exanthemachrysis, Pavlova, and Rebecca. The genus Pavlova includes P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, and P. salina. Haptophytes are phytoplankton with a cell diameter of approximately 5-50 μm and are photosynthetic autotrophic organisms. Most haptophytes inhabit the ocean, but some species are also found in freshwater and salt lakes. The biomass of haptophytes in the open ocean is large, making them important as primary producers in the marine environment.

[0014] In this specification, “microalgae products” refers to products containing the thallus of microalgae or components of microalgae. Typically, microalgae products are dried products, products further processed from dried products, or concentrated extracts of specific components of microalgae (e.g., fucoxanthin).

[0015] In this specification, "food" means an article intended for consumption by animals (e.g., humans), and includes not only commonly used foods and beverages, but also food additives, functional foods (e.g., Foods for Specified Health Uses, Foods with Function Claims, Foods with Nutrient Function Claims, etc.) and supplements.

[0016] In this specification, “cosmetics” means any product intended to be worn, applied to the body by rubbing, spraying, or other similar methods for the purpose of cleansing, beautifying, enhancing attractiveness, altering appearance, or maintaining the health of the skin or hair of an animal (e.g., a human). In this specification, “cosmetics” is not limited to “cosmetics” as defined in the Pharmaceuticals and Medical Devices Act (formerly the Pharmaceutical Affairs Act), but may include, for example, quasi-drugs, pharmaceuticals, or general merchandise. In this specification, “quasi-drugs” is a classification between pharmaceuticals and cosmetics as defined in Japan’s “Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc.,” and includes products with mild effects on the human body, as well as machines and devices with mild effects on the human body. Examples of quasi-drugs include, but are not limited to, medicated cosmetics (including medicated soaps and medicated toothpastes), bath additives, quasi-drugs for pest control (such as insecticides), and designated quasi-drugs (such as drinks, mouthwashes, and some gastrointestinal medicines). In this specification, "pharmaceuticals" refers to drugs administered for the diagnosis, treatment, or prevention of diseases in humans or animals, and includes substances listed in the Japanese Pharmacopoeia, substances intended for use in the diagnosis, treatment, or prevention of diseases in humans or animals that are not medical devices, dental materials, medical supplies, or sanitary products (excluding quasi-drugs), and substances intended to affect the structure or function of the human or animal body that are not medical devices, dental materials, medical supplies, or sanitary products (excluding quasi-drugs and cosmetics).

[0017] In this specification, "chlorophyll" is used in the common sense of the art and is a substance often used to absorb light energy in the light-dependent reactions of photosynthesis. Microalgae that have chloroplasts may contain chlorophyll.

[0018] In this specification, "pheophorbite" is used in the ordinary sense in the art and is a substance often produced in microalgae by the decomposition of chlorophyll. Pheophorbite can be produced by the action of chlorophyllase on chlorophyll. Because it may cause health hazards such as skin damage, its content is regulated in processed chlorella products, etc. (May 8, 1981) (Environmental Food Safety Notification No. 99) (Notification from the Director of the Environmental Health Bureau of the Ministry of Health and Welfare to the Governors of each prefecture, the Mayors of each designated city, and the Heads of each special ward)).

[0019] In this specification, "fucoxanthin" is used in the ordinary sense in the art, and the following structure [ka] It is a substance that possesses [a certain characteristic]. Fucoxanthin is known to be easily decomposed by heating, light irradiation, and oxidation.

[0020] In this specification, "eicosapentaenoic acid (EPA)" is used in the ordinary sense of the art, and the following structure [ka] This refers to a substance that possesses [a certain characteristic].

[0021] In this specification, "hydroxyproline" is used in the ordinary sense of the art, and the following structure [ka] This refers to a substance that possesses [a certain characteristic].

[0022] In this specification, "dietary fiber" is used in the ordinary sense in the art and refers to the general term for indigestible components contained in foods that are not digested by human digestive enzymes.

[0023] In this specification, "amino acid score" is an index based on the 1985 WHO / FAO / UNU Joint Expert Committee report. The amino acid score is calculated as the number of milligrams of each essential amino acid per gram of nitrogen that makes up the protein, and is calculated as a ratio to the amino acid scoring pattern established as a standard by the Joint Committee of FAO / WHO, etc. On average, 1 gram of nitrogen that makes up a protein is equivalent to 6.25 grams of protein. The ratio of essential amino acids in the sample protein is compared with the standard essential amino acid pattern, and the value of the amino acid with the lowest value (first limiting amino acid) becomes the evaluation value.

[0024] In this specification, "culture" is used in the ordinary sense of the art and refers to the operation of maintaining cells in or on a culture medium in a viable state, the number of cells may increase, decrease, or remain constant during culture.

[0025] This disclosure also provides microalgae products containing purified components of microalgae. In this specification, “purified” substance (e.g., fucoxanthin) means a substance from which at least some of its naturally associated factors have been removed. Therefore, the purity of a purified substance is usually higher (i.e., more concentrated) than the substance in its normal state. As used herein, “purified” means, for example, the presence of an isomorphic substance at a concentration of 10–100%, preferably at least 75% by weight, more preferably at least 85% by weight, and even more preferably at least 95% by weight.

[0026] As used herein, “inducible strain,” “similar strain,” or “mutant strain” preferably contains a gene that includes a region substantially homologous to the DNA of the microalga in question, and such strain has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical when aligned with the whole genome sequence of the original strain using a computer homology program known in the art. This means a microalga modified by gene mutation, substitution, deletion, and / or addition, in which the induced strain still exhibits the biological functions of the original microalga, though not necessarily to the same degree. For example, gene mutations can be introduced using any known mutagen, UV, plasma, etc. In one embodiment, the “inducible strain,” “similar strain,” or “mutant strain” is a strain of the same genus and / or species as the original strain. For example, the biological functions of such microalga can be investigated by appropriate and available in vitro assays described herein or known in the art.

[0027] In this specification, "homology" of genes refers to the degree of identity between two or more gene sequences, and generally, having "homology" means having a high degree of identity or similarity. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether two types of genes have homology can be investigated by direct comparison of sequences, or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, genes have homology if their DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0028] Amino acids may be referred to herein by either their generally known three-letter code or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally recognized one-letter codes. In this specification, comparisons of similarity, identity, and homology of amino acid sequences and base sequences are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.7.1 (published October 19, 2017). The value of "identity" in this specification usually refers to the value obtained when aligning using BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value will be used as the identity value. If identity is evaluated in multiple regions, the highest value among them will be used as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

[0029] In one embodiment of this disclosure, the numerical value of identity, etc., "70% or more" may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more, and may be within the range of any two of the starting numerical values. The above "identity" is calculated by determining the proportion of homologous amino acids in two or more amino acid sequences according to the known method described above. Specifically, before calculating the proportion, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and gaps are introduced in parts of the amino acid sequences if necessary to maximize the proportion of identical amino acids. Methods for alignment, methods for calculating the proportion, methods for comparison, and computer programs related thereto are conventionally well known in the art (e.g., BLAST as described above). In this specification, "identity" and "similarity" may be expressed as values ​​measured by NCBI's BLAST unless otherwise specified. When comparing amino acid sequences with BLAST, the Blastp algorithm can be used by default. The measurement results are quantified as Positives or Identities.

[0030] In this specification, “biological function” refers to a specific function that a particular microalga may possess, which may include, but is not limited to, the ability to produce specific components such as fucoxanthin. In this disclosure, for example, the ability to produce fucoxanthin at high levels may be mentioned, but is not limited to these. In this specification, a biological function may be exerted by a corresponding “biological activity.” In this specification, “biological activity” refers to the activity that a particular microalga may possess in a particular environment, and includes activities that exert various functions (e.g., fucoxanthin production). Such biological activity can be measured by techniques well known in the art. Accordingly, “activity” refers to various measurable indicators that influence a response (i.e., have a measurable effect in response to some exposure or stimulus), which may include, for example, the amount of upstream or downstream proteins or other similar functions of the microalgae in this disclosure after some stimulus or event.

[0031] As used herein, the “amount” of an analyte in a bodily fluid sample generally refers to an absolute value that reflects the mass of the analyte detectable in the volume of the sample. However, the amount may also refer to a relative amount compared to another analyte. For example, the amount of an analyte in a sample may be greater than the control level or normal level of the analyte normally present in the sample.

[0032] In this specification, the term "about" means plus or minus 10% of the indicated value unless otherwise explicitly stated.

[0033] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure should not be limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination.

[0034] (New edible microalgae) In one aspect, the present disclosure provides microalgae having the ability to produce at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll in high concentrations and / or exhibiting a high amino acid score. The production capacity of various products (such as fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll) in the microalgae of the present disclosure is shown per 100g of dry cells when grown to 2g / L or higher.

[0035] In one embodiment, the microalgae of the present disclosure have the ability to produce 0.01g or more, 0.02g or more, 0.05g or more, 0.07g or more, 0.1g or more, 0.2g or more, 0.5g or more, 0.6g or more, 0.7g or more, 0.8g or more, 0.9g or more, 1g or more, 1.5g or more, 2g or more, 2.5g or more, 3g or more, 4g or more, 5g or more, 6g or more, 7g or more, 8g or more, 9g or more, or 10g or more of fucoxanthin per 100g of dry cells. Since fucoxanthin is known to have effects such as anti-obesity, anti-diabetic, antioxidant, anti-cancer, and angiogenesis inhibition, the microalgae of the present disclosure and its microalgae products that produce high levels of fucoxanthin are expected to exhibit these effects. The microalgae of the present disclosure can also contain high levels of eicosapentaenoic acid, which can reduce the degradation of fucoxanthin.

[0036] In one embodiment, the microalgae of the present disclosure have the ability to produce 0.01g or more, 0.02g or more, 0.05g or more, 0.07g or more, 0.1g or more, 0.2g or more, 0.5g or more, 0.6g or more, 0.7g or more, 0.8g or more, 0.9g or more, 1g or more, 1.5g or more, 2g or more, 2.5g or more, 3g or more, 3.5g or more, 4g or more, 4.5g or more, 5g or more, 6g or more, 7g or more, 8g or more, 9g or more, or 10g or more of eicosapentaenoic acid (EPA) per 100g of dry cells. Since EPA is known to have effects such as lowering triglycerides, inhibiting platelet aggregation, anti-inflammatory, anti-allergic, infection prevention, and mental stabilization, the microalgae of the present disclosure and their products that produce high levels of EPA are expected to exhibit these effects.

[0037] In one embodiment, the microalgae of the present disclosure have the ability to produce 0.1 mg or more, 0.2 mg or more, 0.5 mg or more, 0.7 mg or more, 1 mg or more, 2 mg or more, 5 mg or more, 7 mg or more, 10 mg or more, 20 mg or more, 50 mg or more, 60 mg or more, 70 mg or more, 80 mg or more, 90 mg or more, 100 mg or more, 150 mg or more, 200 mg or more, 250 mg or more, 300 mg or more, 400 mg or more, 500 mg or more, 700 mg or more, or 1000 mg or more of γ-aminobutyric acid per 100 g of dry cells. Since γ-aminobutyric acid is known to have effects such as lowering blood pressure and reducing stress, the microalgae of the present disclosure that produce high levels of γ-aminobutyric acid and their microalgae products are expected to exert these effects.

[0038] In one embodiment, the microalgae of the present disclosure have the ability to produce 0.005g or more, 0.01g or more, 0.02g or more, 0.05g or more, 0.07g or more, 0.1g or more, 0.15g or more, 0.2g or more, 0.25g or more, 0.3g or more, 0.4g or more, 0.5g or more, 0.6g or more, 0.7g or more, 0.8g or more, 0.9g or more, 1g or more, 1.5g or more, 2g or more, 3g or more, 4g or more, 5g or more, 7g or more, or 10g or more of hydroxyproline per 100g of dry cells. Hydroxyproline is a major component of collagen and, together with proline, is responsible for the stability of collagen. Therefore, the microalgae of the present disclosure and its microalgae products that produce high levels of hydroxyproline are expected to have the effect of maintaining skin firmness and removing wrinkles.

[0039] In one embodiment, the microalgae of the present disclosure have the ability to produce 0.1g or more, 0.2g or more, 0.5g or more, 0.7g or more, 1g or more, 1.5g or more, 2g or more, 2.5g or more, 3g or more, 3.5g or more, 4g or more, 4.5g or more, 5g or more, 6g or more, 7g or more, 8g or more, 9g or more, 10g or more, 15g or more, or 20g or more of calcium per 100g of dry cells. Since calcium is known to have effects such as bone strengthening, the microalgae of the present disclosure and its microalgae products that produce high levels of calcium are expected to exert such effects. Furthermore, by combining them with fucoxanthin, which has been reported to have an inhibitory effect on osteoclast differentiation, effects such as prevention of osteoporosis are expected.

[0040] In one embodiment, the microalgae of this disclosure have the ability to produce 0.5g or more, 0.7g or more, 1g or more, 1.5g or more, 2g or more, 5g or more, 7g or more, 8g or more, 9g or more, 10g or more, 12g or more, 15g or more, 17g or more, 20g or more, 22g or more, 25g or more, 27g or more, or 30g or more of dietary fiber per 100g of dry cells. Since dietary fiber is known to have effects such as preventing obesity, suppressing cholesterol increase, suppressing blood glucose increase, hydrogen gas production, antioxidant, and promoting bowel movements, the microalgae of this disclosure and its products that produce high levels of dietary fiber are expected to exert these effects. The dietary fiber produced by the microalgae of this disclosure may include fucoidan and the like.

[0041] In one embodiment, the microalgae of this disclosure are capable of producing 0.1 to 20 g of chlorophyll per 100 g of dry cells, for example, about 0.1 g, about 0.2 g, about 0.5 g, about 0.7 g, about 1 g, about 1.5 g, about 2 g, about 2.5 g, about 3 g, about 3.5 g, about 4 g, about 4.5 g, about 5 g, about 6 g, about 7 g, about 8 g, about 9 g, about 10 g, about 12 g, about 15 g, about 17 g, or about 20 g. Chlorophyll is a compound that can be converted to pheophorbite, and the microalgae of this disclosure can produce a relatively large amount of chlorophyll, but can be processed in a state where pheophorbite is suppressed, as described below.

[0042] In one embodiment, the microalgae of the Disclosure have the characteristic of exhibiting an amino acid score of 5 or higher, 10 or higher, 20 or higher, 30 or higher, 40 or higher, 50 or higher, 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, 95 or higher, or about 100. The microalgae of the Disclosure and their products having high amino acid scores can provide well-balanced amino acids for humans. In another embodiment, the microalgae of the Disclosure have the ability to produce about 1 to 70 g of protein per 100 g of dry cells, for example, about 1 g, about 2 g, about 5 g, about 7 g, about 10 g, about 12 g, about 15 g, about 17 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 50 g, about 60 g, or about 70 g. The microalgae and their products described herein, which have high protein production and high amino acid scores, can provide sufficient amino acids for humans in small quantities.

[0043] The microalgae of this disclosure may have any combination of the above-mentioned fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll productivity, and amino acid score, but in one embodiment, the microalgae of this disclosure may have the above-mentioned fucoxanthin productivity and at least one characteristic selected from the above-mentioned dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll productivity, and amino acid score. In one embodiment, the microalgae of this disclosure may have the above-mentioned fucoxanthin productivity and the above-mentioned gamma-aminobutyric acid or hydroxyproline productivity, and may optionally further have at least one characteristic selected from the above-mentioned dietary fiber, eicosapentaenoic acid, calcium, and chlorophyll productivity, and amino acid score.

[0044] In one embodiment, the microalgae of this disclosure exhibit low adhesion. For example, adhesion can be evaluated by measuring the weight of microalgae attached to a substrate made of a specific material when it is immersed in a culture solution for a specific time.

[0045] In one embodiment, the microalgae of the Disclosure may be algae of the family Pavlovaceae and may belong to the genera Diacronema, Exanthemachrysis, Pavlova, or Rebecca. The genus Diacronema includes D. ennorea, D. lutheri, D. noctivaga, D. virescens, D. viridis, and D. vlkianum. The genus Exanthemachrysis includes E. gayraliae. The genus Pavlova includes P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, and P. salina. The genus Rebecca includes R. helicata and R. salina. In a particular embodiment, the microalgae of the Disclosure are microalgae of the genus Pavlova. In a more particular embodiment, the microalgae of the Disclosure are P. granifera or P. gyrans. The Discloser has identified the newly discovered microalgae as P. granifera and P. gyrans and deposited them. The NBRC accession number is NBRC 114066. In one embodiment, the microalgae of the Disclosure are Pavlova OPMS30543 strain (the algal strain identified by accession number NBRC 114066) or Pavlova OPMS30543X strain, or derivatives thereof.

[0046] In one embodiment, the microorganism of the Disclosure is a derivative of strain OPMS30543 (the algal strain identified by accession number NBRC 114066) or strain OPMS30543X. Here, a derivative does not need to be a strain derived from these algal strains, but refers to a microalga that exhibits the biological functions of these algal strains, though not necessarily to the same degree. In one embodiment, the microalga that is a derivative of the Disclosure exhibits biological functions selected from the group consisting of the ability to produce at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll, as described above, and the characteristic of exhibiting an amino acid score, similar to strain OPMS30543 or strain OPMS30543X, but the degree of its biological function may differ from that of strain OPMS30543 or strain OPMS30543X. In one embodiment, the microorganism that is the derivative strain of the present disclosure is a microalga of the genus Pavlova, and more specifically, P. granifera or P. gyrans.

[0047] (Uses and functionality) As described above, the microalgae of this disclosure have the ability to produce at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll in high quantities and / or have a high amino acid score, and therefore can be suitably used as various microalgae products. Furthermore, since the microalgae of this disclosure can be suppressed in pheophorbite by treatment, they can be safely used, for example, in food. In one embodiment, the microalgae product of this disclosure may be in a dry state and may have a moisture content of 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 10% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, 0.2% by weight or less, 0.1% by weight or less, or 0.05% by weight or less. In one embodiment, the microalgae product of the present disclosure may be provided by immersing dried microalgae or a purified product thereof in oil and / or encapsulating them in capsules (e.g., soft capsules), which can be stabilized by being in these forms.

[0048] In one embodiment, the microalgae product of the Disclosure is a food product, and the food product may contain any amount of the microalgae of the Disclosure, for example, by weight, 0.01 to 100%, about 0.01%, about 0.02%, about 0.05%, about 0.07%, about 0.1%, about 0.2%, about 0.5%, about 0.7%, about 1%, about 2%, about 5%, about 7%, about 10%, about 20%, about 50%, about 70%, or about 100%. The microalgae of the Disclosure may have the characteristics of being soft and lacking a cell wall, so as not to give an unpleasant texture when ingested. If the taste or flavor of the microalgae of the Disclosure is a concern, it may be used in combination with any suitable flavoring agent, odoring agent, or masking agent, or the taste or flavor of the microalgae may be masked by means of coating or encapsulation. The microalgae described herein are rich in the beneficial components mentioned above and can exert their effects even with small amounts of intake, making them suitable for use as supplements and / or food additives in food products.

[0049] In one embodiment, the microalgae product of the present disclosure is a refined product containing a concentration of at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, hydroxyproline, calcium, and chlorophyll. Such a refined product may contain the desired component(s) in any amount, for example, by weight, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% or less, and / or about 10% or less, about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, about 95% or less, or about 100% or less. Such refined products may be used as food (e.g., supplements) or added to cosmetics, pharmaceuticals, and quasi-drugs. Depending on the concentrated component (e.g., fucoxanthin), the refined products of this disclosure may be unstable; in such cases, they may be combined with stabilizers (e.g., immersion in oil to avoid contact with air, or addition of antioxidants such as vitamin E and vitamin C).

[0050] In one specific embodiment, the microalgae product of the present disclosure is a purified product of fucoxanthin. In one embodiment, such a fucoxanthin product may include a purified product obtained by concentrating fucoxanthin from the microalgae of the Disclosure to a purity of 1 to 100% (for example, within any range defined by lower and / or upper limits of about 10%, about 2%, about 5%, about 7%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%, and / or less than or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 99%, or about 100%, e.g., 5 to 70%).

[0051] (Breeding method) The microalgae of this disclosure can be obtained by breeding methods, and in one aspect, this disclosure also provides such breeding methods. In one embodiment, the breeding method of this disclosure is provided under the following conditions • Seawater concentration: 50% seawater • Culture medium concentration: 2x IMK medium concentration ·Culture solution volume: 800mL • Light source: Side: Fluorescent lamp (100-150 μmol) • Light-dark cycle: 12 hours each for light and dark. ·pH: pH approximately 7.4 at the start of each culture • Aeration: Implemented • Mechanical stirring: None • Culture period: Approximately 10 days. ·Culture temperature: 25℃~28℃ The process includes a step of repeatedly subculturing a cell line of microalgae of the Pavlovales order under conditions that satisfy at least one of the following. In one embodiment, the step of repeatedly subculturing may be carried out under conditions that satisfy at least one or more of the following: seawater concentration: 50% seawater, culture medium concentration: IMK medium at twice the concentration, and culture period: approximately 10 days.

[0052] In one embodiment, the method of the present disclosure is a step of harvesting a cell line of Pavlovales microalgae cultured by the above method or the like when the cell density exceeds 2 g / L, and measuring fucoxanthin, eicosapentaenoic acid, and γ-aminobutyric acid of the cell line, and measuring at least one selected from the group consisting of hydroxyproline, calcium, dietary fiber, chlorophyll, and amino acid score, (i) Produces 1 g or more of fucoxanthin per 100 g of dried cells, (ii) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells, (iii) Produces 50 mg or more of gamma-aminobutyric acid per 100 g of dried cells, (iv) Produces 100 mg or more of hydroxyproline per 100 g of dry cells. (v) Produces 500 mg or more of calcium per 100 g of dry cells. (vi) Produces 5g or more of dietary fiber per 100g of dried cells. (vii) produce approximately 2 g or more of chlorophyll per 100 g of dried cells, (viii) The amino acid score is 85 or higher. The process may include selecting a cell line as a breeding line that satisfies at least one of the following conditions. The selection conditions vary depending on the performance of the target cell. (i) The amount of fucoxanthin produced per 100g of dried cells may be 1g or more, 1.5g or more, 2g or more, or 3g or more. (ii) The amount of eicosapentaenoic acid produced per 100g of dried cells may be 4g or more, 5g or more, 6g or more, or 8g or more. (iii) The amount of gamma-aminobutyric acid produced per 100g of dried cells may be 50mg or more, 70mg or more, 100mg or more, 150mg or more, or 200mg or more. (iv) The amount of hydroxyproline produced per 100g of dried cells may be 100mg or more, 150mg or more, 200mg or more, or 300mg or more. (v) The amount of calcium produced per 100g of dried cells may be 500mg or more, 700mg or more, 1g or more, or 1.5g or more. (vi) The amount of dietary fiber produced per 100g of dried cells may be 5g or more, 7g or more, 10g or more, or 15g or more. (vii) The amount of chlorophyll produced per 100g of dried cells may be approximately 2g or more, 3g or more, 4g or more, or 5g or more. (viii) The amino acid score may be 85 or higher, 90 or higher, 95 or higher, or approximately 100. The conditions for at least one arbitrary combination of the following may be satisfied.

[0053] In one embodiment, aeration may be performed by aeration stirring, and compressed air may be used. In one embodiment, the breeding method of the present disclosure is a method for breeding microalgae of the Pavlovales order. In one embodiment, for each subculturing, cells suspended from the supernatant of the culture are selectively used, thereby concentrating only the cell lines that survive well under the culture conditions. In one embodiment, the microalgae of the present disclosure are microalgae obtained by such a breeding method.

[0054] (Cultivation of microalgae) In one embodiment, the microalgae of the Disclosure can be cultured under any preferred conditions. In one embodiment, the culture can be subdivided into seed culture and main culture, etc. In one embodiment, seed culture may include several culture stages (for example, any combination of a test tube culture stage (about 100 mL), a PET bottle, flask or medium bottle culture stage (about 1 L or less), a photobioreactor of the Disclosure (about 5 L), a culture stage of 10 to 20 photobioreactors with a capacity of about 5 L or 2 to 4 photobioreactors with a capacity of about 25 L (about 50 to 100 L), and a larger photobioreactor culture stage (about 1000 L or more)). Unless otherwise specified, the culture conditions described below can be applied to any type of culture. The conditions in the process of culturing the microalgae (e.g., temperature, pH, stirring conditions, light irradiation conditions, and culture medium composition) can each be set to suit their own preferences. In one embodiment, the cultivation of microalgae may include multiple stages (e.g., seed culture and main culture, indoor contamination-free culture and outdoor rapid growth culture, acclimatization culture and main culture, etc.).

[0055] In one embodiment, microalgae can be cultured at a temperature of approximately 0°C to 80°C, more specifically, approximately 20°C to 30°C. Suitable upper temperature ranges include 80°C, 70°C, 60°C, 50°C, 40°C, 30°C, and 20°C, while lower temperature ranges include 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C. Any combination of these can be adopted as a suitable temperature range, as long as there is no contradiction. Any culture temperature can be used as long as the microalgae do not die. The culture temperature does not need to be constant, and strict temperature control is not necessary, especially when the culture tank is installed outdoors. It is preferable to expose the microalgae to a temperature at which they can suitably survive and grow for at least a portion of the culture period. If the temperature rises too high due to direct sunlight or other reasons, the temperature can be lowered by any cooling means (e.g., water cooling). For example, if the microalgae are haptophytes, they can suitably grow at a temperature of approximately 25 to 30°C.

[0056] In one embodiment, microalgae can be cultured at a pH of approximately 2 to 13. Suitable upper limits for pH include pH 13, pH 12, pH 11, pH 10, pH 9, pH 8.5, pH 8, pH 7.5, pH 7, and pH 6, while lower limits include pH 2, pH 3, pH 4, pH 5, pH 6, pH 6.5, pH 7, pH 7.5, and pH 8. Any combination of these can be adopted as a suitable pH range, as long as there is no contradiction. Any pH can be used as long as the microalgae do not die. The suitable pH may differ depending on the type of microalgae, but those skilled in the art can easily determine the suitable pH for the microalgae being used. It is preferable to avoid rapid pH changes during cultivation, and pH changes can be controlled using any suitable buffer (e.g., carbon dioxide, amine compounds, etc.). For example, if the microalgae are haptophytes, they can grow suitably in a slightly alkaline environment with a pH of approximately 8.

[0057] In one embodiment, microalgae may or may not be subjected to stirring conditions during cultivation. Means of stirring include, but are not limited to, aeration stirring, mechanical stirring (such as paddle stirring), running water stirring (e.g., using a pump), and shaking of the culture vessel. Depending on the stirring method, microalgae may be damaged, and since Euglena and haptophytes, which do not have cell walls, are relatively soft, it is preferable to avoid vigorous stirring that may destroy cells during cultivation.

[0058] In one embodiment, the microalgae can be cultured under light irradiation during at least a part of the culture period. Although it depends on the type of microalgae, the growth rate of the microalgae can be improved as long as the amount of light irradiated is high within a range where the microalgae are not damaged. For some microalgae, non-constant light irradiation may be preferable. Specific wavelength regions may be selectively irradiated. When culturing microalgae outdoors, it may be advantageous to utilize natural light. Even when culturing microalgae outdoors and using only natural light as a light source, the amount of light per microalgae cell can be controlled by adjusting the depth of the culture tank or the diameter of the photobioreactor. In particular, when growing haptophytes with a large amount of photosynthetic pigments, it may be advantageous to irradiate a high amount of light such as natural light. The amount of light energy that can be used can be, for example, about 30 μmol m -1 s -1 ~ about 3000 μmol m -2 s -1 , or about 30 μmol m -2 s -1 ~ about 1500 μmol m -2 s -1 and can be, preferably about 50 μmol m -2 s -1 ~ about 300 μmol m -2 s -1 . For example, when the microalgae are haptophytes, they can grow preferably with a light energy amount of about 100 μmol m -2 s -1 ~ about 150 μmol m -2 s -1 .

[0059] The composition of the culture medium used for culturing microalgae can be any suitable composition depending on the type of microalgae. Typical components that may be included in the medium include inorganic salts (e.g., potassium salts, sodium salts, calcium salts, magnesium salts), sugars (e.g., glucose), organic salts, nitrogen sources (nitrates, ammonium salts, etc.), phosphorus sources (inorganic phosphorus, phosphates, etc.), but other components may also be included. Nitrogen sources and phosphorus sources can be consumed as the microalgae grow, so they can be added as appropriate. In addition, if a carbon source (e.g., carbon dioxide) is added, it can be utilized by the microalgae. For example, when culturing haptophytes, since most haptophytes inhabit seawater to brackish water, a culture medium with a composition similar to that of seawater to brackish water (e.g., a culture medium containing about 50-75% of the salts found in seawater) or a culture medium with an osmotic pressure similar to that of seawater to brackish water can be suitably used.

[0060] In the cultivation step of the manufacturing method of this disclosure, it is preferable to increase the density of microalgae for the efficiency of cultivation. For example, microalgae can be cultivated to densities of at least 0.01 g / L, at least 0.02 g / L, at least 0.05 g / L, at least 0.07 g / L, at least 0.1 g / L, at least 0.2 g / L, at least 0.5 g / L, at least 0.7 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, at least 10 g / L, at least 20 g / L, at least 50 g / L, or at least 100 g / L in terms of dry weight of microalgae. The cultivation period may be continued until the desired microalgae density is achieved, a predetermined cultivation period may be set, or it may be continued indefinitely, such as in maintenance cultivation.

[0061] The microalgae of this disclosure can be cultured in any state, such as attached to a carrier, with each cell floating independently in a culture medium, with cells aggregated and floating in a culture medium, or in a mixture of these states.

[0062] In one embodiment, the microalgae of the Disclosure may be cultured under light irradiation conditions and then cultured under reduced light irradiation conditions. Since the microalgae of the Disclosure can grow under light irradiation conditions and increase in fucoxanthin content under reduced light irradiation conditions, controlling the light irradiation conditions in this way may be useful for producing microalgae products rich in fucoxanthin. In such culture conditions where the light irradiation conditions are controlled, the amount of light irradiation is approximately 30 μmol m³. -2 s -1 ~about 3000 μmol m -2 s -1 For example, approximately 3000 μmol m -2 s -1 , about 2000 μmol m -2 s -1 , approximately 1500 μmol m -2 s -1 , approximately 1000 μmol m -2 s -1 , approximately 700 μmol m -2 s -1 , approximately 500 μmol m -2 s -1 , about 200 μmol m -2 s -1 , approximately 150 μmol m -2 s -1 , approximately 100 μmol m -2 s -1 , approximately 50 μmol m -2 s -1 , approximately 30 μmol m -2 s -1 The light irradiation dose under the light irradiation reduction conditions is approximately 100 μmol m -2 s -1 For example, approximately 100 μmol m -2 s -1 , approximately 70 μmol m -2 s -1 , approximately 50 μmol m -2 s -1 , about 20 μmol m -2 s -1 , approximately 10 μmol m -2 s -1 , approximately 5 μmol m -2s -1 , about 2 μmol m -2 s -1 , approximately 1 μmol m -2 s -1 , or less, and any combination of these light irradiation conditions and light irradiation reduction conditions can be used. As the diameter of the reactor increases, the amount of light irradiated per microalgae cell may decrease. For example, if the weather is sunny before harvesting the microalgae, fucoxanthin may increase by shading them for 1-2 days before harvesting with a shading net (e.g., 50% shading).

[0063] (Manufacturing of microalgae products) A microalgae product can be produced from a culture containing the microalgae of this disclosure cultured according to the culture method described above. In one embodiment, the method for producing the microalgae product of this disclosure includes a step of subjecting the culture to a chlorophyllase inactivation treatment. By inactivating chlorophyllase, the production of pheophorbide can be suppressed. Examples of chlorophyllase inactivation treatments include, but are not limited to, heat treatment, any known protein denaturation treatment (temperature loading (low, high), chemical treatment (alcohol, strong acid, strong base, other denaturants), radiation irradiation (ultraviolet light, gamma rays, etc.)), etc.). The chlorophyllase inactivation treatment (e.g., heat treatment) can be carried out under any suitable conditions (means, time, etc.) for chlorophyllase inactivation, but conditions that do not destroy the microalgae and / or destroy the useful components of the microalgae are preferably applied. For example, since haptophytes can produce fucoxanthin, it is preferable to treat them under conditions where the degradation of fucoxanthin is minimal, for example, where the decrease in fucoxanthin when comparing before and after treatment is less than 0.01%, less than 0.02%, less than 0.05%, less than 0.07%, less than 0.1%, less than 0.2%, less than 0.5%, less than 0.7%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 60%, less than 70%, or less than 80%.

[0064] The chlorophyllase inactivation treatment is preferably carried out under conditions that control the amount of stress (for example, the amount of stimulation accumulated by any operation that increases the amount of pheophorbide produced in microalgae), and is preferably performed on microalgae that have not been subjected to a large amount of stress before the treatment. If chlorophyllase inactivation is performed on microalgae that have been subjected to a large amount of stress, a large amount of pheophorbide may have already been produced, and the pheophorbide inhibitory effect by chlorophyllase inactivation may not be sufficiently obtained. Controlling the amount of stress for pheophorbide inhibition can be achieved, for example, by maintaining a low density of microalgae and / or not concentrating the microalgae too much.

[0065] In one embodiment, after cultivation and before treatment to deactivate chlorophyllase, the microalgae of the present disclosure are not concentrated by 1000 times or more, 900 times or more, 800 times or more, 700 times or more, 600 times or more, 500 times or more, 400 times or more, 300 times or more, 200 times or more, 150 times or more, 100 times or more, 90 times or more, 80 times or more, 70 times or more, 60 times or more, 50 times or more, 40 times or more, 30 times or more, 20 times or more, 15 times or more, 10 times or more, 9 times or more, 8 times or more, 7 times or more, 6 times or more, 5 times or more, 4 times or more, 3 times or more, 2 times or more, or 1.5 times or more, or are not subjected to such a concentration operation.

[0066] In one embodiment, the process of inactivating chlorophylase is a heat treatment, which may be a heat treatment at approximately 50°C to 200°C, for example, approximately 50°C, 60°C, 70°C, 80°C, 85°C, 90°C, 95°C, 97°C, 100°C, 102°C, 105°C, 107°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, or 200°C. The heating time can range from approximately 10 seconds to 20 hours, for example, approximately 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7 hours, 10 hours, 20 hours, etc.

[0067] In one embodiment, the density of microalgae during the chlorophyllase inactivation treatment is approximately 0.01 to 100 g / L by dry weight, for example, less than or equal to approximately 100 g / L, less than or equal to approximately 70 g / L, less than or equal to approximately 50 g / L, less than or equal to approximately 40 g / L, less than or equal to approximately 30 g / L, less than or equal to approximately 20 g / L, less than or equal to approximately 15 g / L, less than or equal to approximately 10 g / L, less than or equal to approximately 7 g / L, less than or equal to approximately 5 g / L, less than or equal to approximately 4 g / L, less than or equal to approximately 3 g / L. The concentrations may be approximately 2 g / L or less, approximately 1 g / L or less, approximately 0.5 g / L or less, approximately 0.1 g / L or less, approximately 0.01 g / L or more, approximately 0.05 g / L or more, approximately 0.1 g / L or more, approximately 0.2 g / L or more, approximately 0.5 g / L or more, approximately 0.7 g / L or more, approximately 1 g / L or more, approximately 2 g / L or more, approximately 3 g / L or more, approximately 4 g / L or more, approximately 5 g / L or more, approximately 7 g / L or more, or approximately 10 g / L or more. If the density of microalgae exceeds, for example, 10 g / L, the overall inactivation of chlorophyllase may be insufficient. In one embodiment, the microalgae are not concentrated to the above concentrations between the time of cultivation and the treatment to inactivate chlorophyllase. In one embodiment, the microalgae are not diluted between the time of cultivation and the treatment to inactivate chlorophyllase.

[0068] In one embodiment, the microalgae are not subjected to advanced centrifugation before and / or during the chlorophyllase inactivation treatment, for example, 50G or more, 100G or more, 200G or more, 500G or more, 700G or more, 1000G or more, 1500G or more, 2000G or more, 2500G or more, 3000G or more, 3500G or more, 4000G or more, 4500G or more, 5000G or more, 6000G or more, 7000G or more, 8000G or more, 9000G or more, and Furthermore, the product is not exposed to gravitational acceleration of 10,000 G or more, and is not subjected to centrifugal processing for a period of time of approximately 10 seconds or more, approximately 30 seconds or more, approximately 1 minute or more, approximately 2 minutes or more, approximately 5 minutes or more, approximately 7 minutes or more, approximately 10 minutes or more, approximately 15 minutes or more, approximately 20 minutes or more, approximately 25 minutes or more, approximately 30 minutes or more, approximately 40 minutes or more, approximately 50 minutes or more, approximately 1 hour or more, approximately 1.5 hours or more, approximately 2 hours or more, approximately 2.5 hours or more, approximately 3 hours or more, approximately 4 hours or more, approximately 5 hours or more, approximately 7 hours or more, approximately 10 hours or more, or approximately 20 hours or more.

[0069] In one embodiment, the method for producing the microalgae product of the present disclosure includes a step of concentrating the microalgae. Any suitable means known in the art can be used to concentrate the microalgae, including, but not limited to, centrifugation, filtering, medium removal (such as evaporation), and the use of flocculants or precipitating agents. The concentration operation may increase the stress level of the microalgae. In particular, Euglena and Pavlovales, which lack cell walls, are relatively soft, and the concentration operation may increase pheophorbide production. It should be noted that the problem of pheophorbide increasing due to cell concentration treatment in Euglena and Pavlovales, which lack cell walls, is a problem that has been identified for the first time in this disclosure. For example, the amount of chlorophyll a+b in Chlorella chlamydomonas is often around 25 mg per gram of dried algae, although this varies depending on the culture conditions and timing. However, the amount of pavlova used in the example was 35.3 mg per gram of dried algae, which was unexpectedly high. Therefore, this disclosure addresses a problem that was not anticipated in conventional methods involving the concentration of microalgae, and furthermore, provides a means to solve it.

[0070] In one embodiment, the step of concentrating the microalgae before the chlorophylase inactivation treatment is omitted. When a culture medium containing unconcentrated microalgae is subjected to the chlorophylase inactivation treatment, more reagents and energy may be required compared to the case of concentration, potentially resulting in a higher environmental burden. However, the inventors have found a culture method that allows for the growth of microalgae (e.g., haptophytes) at a high density of 2 g / L or more (for example, a method using the culture apparatus of this disclosure described in detail below), so the environmental burden can be minimized even when the microalgae are subjected to the chlorophylase inactivation treatment without concentration.

[0071] In one embodiment, the process of treating microalgae includes killing the microalgae and / or other microorganisms. When providing microalgae products as food or food additives, the absence of living organisms may make the product easier to handle. Examples of such killing processes include, but are not limited to, heat treatment and irradiation.

[0072] In one embodiment, the method for producing the microalgae product of the present disclosure includes a step of drying the microalgae. The microalgae can be dried to the moisture content of the microalgae product of the present disclosure described above.

[0073] In one embodiment, the method for producing the microalgae product of this disclosure includes a step of separating the components of the microalgae. While the microalgae themselves may be useful, certain components may also be useful. Therefore, specific components contained in the microalgae may be separated from other microalgae components to increase the concentration of those specific components. In another embodiment, specific components (such as harmful components) may be separated and removed from the microalgae. For example, the inventors have found that the haptophyte Pavlova contains a large amount of fucoxanthin, so fucoxanthin may be separated and purified to produce the microalgae product of this disclosure.

[0074] In one embodiment, the present disclosure may provide a purified product of fucoxanthin. Fucoxanthin can be extracted from microalgae by using one or more organic solvents, including but not limited to alcohols such as methyl acetate, ethyl acetate, acetone, chloroform, toluene-methanol, ethanol, propanol, isopropanol, and n-butanol; ketones such as methyl ethyl ketone and acetone; esters such as methyl acetate and ethyl acetate; organochlorine hydrocarbons such as chloroform; aliphatic hydrocarbons such as hexane; and aromatic hydrocarbons such as benzene and toluene, either alone or in combination of two or more. In one embodiment, the fucoxanthin can be purified from the obtained extract using any known purification method, such as chromatography, resin adsorption, or crystallization.

[0075] (General technology) The molecular biological, biochemical, and microbiological methods used herein may be well-known and commonly used in the field.

[0076] (Note) In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else". In this specification, when it is specified that "within the range" of "two values", that range includes the two values ​​themselves.

[0077] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as they are specifically described herein.

[0078] The present disclosure has been described above with reference to preferred embodiments for ease of understanding. The present disclosure will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples]

[0079] Examples are described below. The reagents used are specifically those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakalai, R&D Systems, USCN Life Science INC, etc.) can be substituted.

[0080] (Example 1: Discovery of a novel microalga) The inventors created and tested various microalgae and discovered strain OPMS30543 (Pavlova granifera), a species of Pavlovaceae collected from the sea off Okinawa, as a microalgae that produces high levels of fatty acids. This strain was deposited (accession number NBRC 114066). Further investigation of the characteristics of this strain revealed that it produces high levels of fucoxanthin, suggesting that it is a microalgae with useful properties not found in conventional microalgae.

[0081] Two primers for 18S rDNA and two primers for 28S rDNA were designed for strain OPMS30543, and sequence analysis was performed, identifying the following nucleotide sequence. The results suggest that strain OPMS30543 is P. granifera. OPMS30543, 18S rDNA ·OPMS30543、28S rDNA

[0082] Since microalgae of the Pavlovaceae family were predicted to possess useful properties, we obtained and bred other microalgae of the Pavlovaceae family, including P. gyrans (strain OPMS30543X), P. pinguis, and P. lutheri, and investigated the properties of these species as well. As a result, it was confirmed that all of these species produce fucoxanthin. Here, strain OPMS30543X is a strain based on strain NBRC102809 (Pavlova gyrans).

[0083] Breeding was carried out as follows. The basic culture conditions are shown in the table below. [Table 1] *IMK medium (Daigo IMK medium, Nippon Pharmaceutical Co., Ltd., Tokyo) was prepared by dissolving it in MilliQ water to a 200-fold concentrated solution (dissolving one packet in 500 mL of water), then sterilizing it by filtration (0.22 μm) and storing it in a refrigerator in a light-shielded place.

[0084] Subculturing was performed after 10 to 12 days of culture. This period was sometimes adjusted according to the growth rate. During subculturing, approximately 5% to 20% of the culture immediately before subculturing was transferred to a new medium, adjusting as needed depending on the growth rate. During subculturing, aeration and other stirring were stopped, and the culture was allowed to stand for a while before selectively collecting the supernatant portion (where there were many swimming cells).

[0085] In cases where the condition of the culture deteriorated drastically, surviving cells were selected by micromanipulation or other methods as needed, and re-culture was performed in an environment with a culture scale reduced to a minimum of 2 mL. In cases where the proliferation of contaminating bacteria became severe, appropriate amounts of ampicillin or hypochlorite were added.

[0086] (Example 2: Cultivation of novel microalgae) OPMS30543 strain and OPMS30543X strain were cultured in a culture medium prepared by dissolving Marine Art SF-1 (Tomita Pharmaceutical, Tokushima) in water to a 50% seawater concentration, and then adding Daigo IMK medium (Nippon Pharmaceutical, Osaka) components to twice the concentration specified in the instructions for use. The culture was maintained at approximately 8 pH by adding CO2. An acrylic bioreactor, as shown in Figure 3, was used as the culture tank, and the culture was carried out outdoors. Strains OPMS30543 and OPMS30543X exhibited low adhesion to the culture tank walls and to each other, allowing for stable cultivation.

[0087] (Example 3: Component analysis of novel microalgae) The microalgae cultured in Example 2 were harvested when their density exceeded 2 g / L, and then heated, concentrated, and freeze-dried to prepare samples of strains OPMS30543 and OPMS30543X. These samples were sent to the Japan Food Research Laboratories (Tokyo) for component analysis of strains OPMS30543 and OPMS30543X (presumably Pavlova gyrans). The following representative components were identified: [Table 2] [Table 3]

[0088] (Example 4: Microalgae that produce high levels of fucoxanthin) In some cases, outdoor cultivation of the Pavlova strain using the method described in Example 2 resulted in high fucoxanthin productivity of 23.22 mg / g per dry weight of microalgae.

[0089] (Example 5: Treatment for microalgae products) As described above, strain OPMS30543 contains approximately 2250 mg / 100g (dry weight) of chlorophyll, and Pavlova, a haptophyte, can contain more chlorophyll than typical microalgae such as Chlorella. It is known that chlorophyll is metabolized in microalgae to pheophorbide, and pheophorbide is known to cause photosensitivity, so it is desirable to limit its intake by animals. Pheophorbide can be investigated as follows.

[0090] • Existing methods for quantifying pheophorbide The amount of chlorophyll breakdown products transferred from the ether extract solution of the pigment to 17% hydrochloric acid is converted to pheophorbide a (mg%). Weigh 100 mg of dried microalgae into a mortar, add approximately 0.5 g of sea sand and 20 ml of 85% (V / V) acetone, and quickly grind. Transfer the supernatant to a centrifuge tube. Add another 10 ml of acetone to the residue and repeat the procedure, then transfer the supernatant to a centrifuge tube and repeat this procedure once more. Next, centrifuge (3000 rpm, 5 minutes) and transfer the supernatant to a separatory funnel containing 30 ml of ethyl ether. Next, add 50 ml of 5% sodium sulfate solution to this ether-acetone mixture, shake gently, and discard the sodium sulfate layer. Repeat this washing procedure three more times, then add anhydrous sodium sulfate to dehydrate, remove the ether layer, and dilute the total volume to 50 ml with ethyl ether to obtain the dye stock solution. 20 ml of this dye stock solution was extracted by shaking with 20 ml of 17% hydrochloric acid, followed by 10 ml of the same hydrochloric acid. The hydrochloric acid layer was then transferred to a separatory funnel containing 150 ml of saturated sodium sulfate solution and 20 ml of ethyl ether. This was extracted by shaking, and the ether layer was separated. Ethyl ether was added to this to make a total volume of 20 ml, which was used as the decomposition product extract. This decomposition product extract was diluted with ethyl ether to the exact required concentration, and the absorbance at 667 nm was measured. The amount of chlorophyll decomposition products was calculated from the absorbance of the standard pheophorbide a and expressed as the amount of existing pheophorbide (mg%). For the absorbance of the standard pheophorbide a, the specific extinction coefficient at 667 nm of pheophorbide a, 70.2 (absorbance shown at 1 cm in a 0.1% solution), as described by SR Brown (J. Fish Res. Bd. Canada 25, 523-540. 1968), was used.

[0091] • Quantitative method for chlorophylase activity Incubate in aqueous acetone and convert the increase in chlorophyll degradation product production to the amount of pheophorbide a (mg%). 100 mg of dried microalgae is accurately weighed, and 10 ml of a mixture of cold M / 15 phosphate buffer (pH 8.0) and acetone (7:3) is added. The mixture is incubated at 37°C for 3 hours. The solution is then made weakly acidic with 10% hydrochloric acid, and the amount of pheophorbide is measured using the same method as for the quantification of existing pheophorbide. The increase is determined by subtracting the existing pheophorbide amount from the measured value, and this increase is defined as the chlorophyllase activity.

[0092] A 10 L (0.516 g / L) culture of the above Pavlova strain was centrifuged and concentrated 100-fold. The concentrate was then heated in an autoclave (100°C, 1 minute). Subsequently, the existing pheophorbide and chlorophylase activity were measured. Total pheophorbide amount = existing pheophorbide amount + chlorophylase activity. [Table 4]

[0093] In the unconcentrated culture medium, both the pre-existing pheophorbide and total pheophorbide levels were low. However, after centrifugation, the amount of pre-existing pheophorbide increased. Since heating suppressed chlorophylase activity, the total pheophorbide amount was equivalent to the amount of pre-existing pheophorbide.

[0094] Furthermore, an increase in the amount of existing pheophorbide was observed when the culture of the above-mentioned Pavlova strain was concentrated 100-fold using an MF membrane (for approximately 12 hours or more), and also when the culture of the above-mentioned Pavlova strain was passed through a cascade pump.

[0095] On the other hand, since chlorophylase, which catalyzes the production of pheophorbide, was thought to be inactivated by heating, we tested whether heating before concentration would suppress pheophorbide production.

[0096] The culture of the above Pavlova strain (0.592 g / L) was passed through a tube at a constant rate (10, 20, 40, or 80 mL / min) to an oil heater (105°C), and the heating time was adjusted. The heated liquid delivered from the oil heater was collected in a bottle on ice. The heat treatment times under each condition were approximately 8 minutes, 4 minutes, 2 minutes, and 1 minute. Each collected sample was centrifuged, and the existing pheophorbide, total pheophorbide, and chlorophylase activity were measured. As a result, it was found that sufficient preheating prevented an increase in the total pheophorbide amount even after subsequent centrifugation.

[0097] (Example 6: Extraction of fucoxanthin) The concentrated culture solution or dried algae of the OPMS30543 strain were extracted using 100% or 80% ethanol, and then filtered to obtain an extract. Impurities were removed from this extract using several types of resins to obtain fucoxanthin with a purity of 65% or higher. When the amount of fucoxanthin in the concentrated culture solution or dried algae is set to 100%, more than 50% of the fucoxanthin can be recovered.

[0098] (Example 7: Further acquisition of Pavlova strains) Related strains or derived strains can be obtained in the following manner. Microalgae are obtained from brackish water deposits or seawater. The characteristics of the obtained microalgae strains, such as fatty acid productivity and fucoxanthin productivity, are investigated. If necessary, the obtained microalgae strains are bred (for example, by the breeding method shown in Example 1) to obtain new strains. The breeding method (screening method for new strains) is as follows: The following conditions • Seawater concentration: 50% seawater • Culture medium concentration: 2x IMK medium concentration ·Culture solution volume: 800mL • Light source: Side: Fluorescent lamp (100-150 μmol) • Light-dark cycle: 12 hours each for light and dark. ·pH: pH approximately 7.4 at the start of each culture • Aeration: Implemented • Mechanical stirring: None • Culture period: Approximately 10 days. ·Culture temperature: 25℃~28℃ Then, by repeatedly subculturing cell lines of microalgae of the Pavlovales order, During each subculturing, cells suspended in the supernatant of the culture are selectively used, thereby concentrating only the cell lines that survive well under the aforementioned culture conditions. Then, the process of repeating the above culture subculturing is performed at least • Seawater concentration: 50% seawater • Culture medium concentration: 2x IMK medium concentration • Culture period: Approximately 10 days. The procedure will be carried out under the conditions that satisfy the following requirements. Subsequently, when the cell density exceeds 2 g / L, the cell line is collected, and fucoxanthin, eicosapentaenoic acid, and γ-aminobutyric acid are measured in the candidate cell line, while hydroxyproline, calcium, dietary fiber, chlorophyll, and amino acid scores are measured according to the above-described examples. The candidate cell lines that were measured (i) Produces 1 g or more of fucoxanthin per 100 g of dried cells, (ii) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells, (iii) Produces 50 mg or more of gamma-aminobutyric acid per 100 g of dried cells, (iv) Produces 100 mg or more of hydroxyproline per 100 g of dry cells. (v) Produces 500 mg or more of calcium per 100 g of dry cells. (vi) Produces 5g or more of dietary fiber per 100g of dried cells. (vii) produce approximately 2 g or more of chlorophyll per 100 g of dried cells, (viii) The amino acid score is 85 or higher. Cell lines that satisfy at least one of the following conditions are selected as further novel induced cell lines. This method can sometimes yield several plants in a single breeding attempt.

[0099] (Note) As described above, while the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of this disclosure should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0100] This disclosure provides novel microalgae and microalgae products using the same, which produce various useful components and are suitable for consumption, and can therefore be usefully used in various products, in particular as food. [Accession Number]

[0101] OPMS30543 strain (NBRC 114066)

Claims

1. This microalga is strain OPMS30543, identified by accession number NBRC 114066.

2. A food product comprising the microalgae described in claim 1.

3. The food according to claim 2, wherein the amount of pheophorbite per 100 g of the microalgae is 100 mg or less.

4. The following: (i) Produces 1 g or more of fucoxanthin per 100 g of dried cells, (ii) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells. (iii) Produces 50 mg or more of gamma-aminobutyric acid per 100 g of dried cells. (iv) Produces 100 mg or more of hydroxyproline per 100 g of dry cells. (v) Produces 500 mg or more of calcium per 100 g of dried cells, (vi) Produces 5g or more of dietary fiber per 100g of dried cells. (vii) Produces approximately 2 g or more of chlorophyll per 100 g of dried cells, (viiii) The amino acid score is 85 or higher. A method for breeding cell lines of microalgae of the genus Pavlova that satisfy at least one of the following conditions: • Seawater concentration: 50% seawater • Culture medium concentration: 2x IMK medium concentration ・Culture solution volume: 800mL Light source: Side: Fluorescent lamp (100-150 μmol) • Light-dark cycle: 12 hours of light and 12 hours of dark. ・pH: pH approximately 7.4 at the start of each culture Aeration: Implemented Mechanical stirring: None • Culture period: Approximately 10 days. ・Culture temperature: 25°C to 28°C A method comprising repeatedly subculturing the OPMS30543 strain, which is identified by accession number NBRC 114066, under conditions that satisfy the following, wherein for each subculturing, cells suspended from the supernatant of the culture are selectively used, thereby concentrating only the cell lines that survive well under the aforementioned culture conditions.

5. The process of repeating the culture subculturing is performed at least • Seawater concentration: 50% seawater • Culture medium concentration: 2x IMK medium concentration • Culture period: Approximately 10 days. The method according to claim 4, carried out under conditions that satisfy the following conditions.

6. The process involves collecting the cell line when the cell density exceeds 2 g / L and measuring the fucoxanthin, eicosapentaenoic acid, and γ-aminobutyric acid of the cell line, and at least one selected from the group consisting of hydroxyproline, calcium, dietary fiber, chlorophyll, and amino acid score, (i) Produces 1 g or more of fucoxanthin per 100 g of dried cells, (ii) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells. (iii) Produces 50 mg or more of gamma-aminobutyric acid per 100 g of dried cells. (iv) Produces 100 mg or more of hydroxyproline per 100 g of dry cells. (v) Produces 500 mg or more of calcium per 100 g of dried cells, (vi) Produces 5g or more of dietary fiber per 100g of dried cells. (vii) Produces approximately 2 g or more of chlorophyll per 100 g of dried cells, (viiii) The amino acid score is 85 or higher. The method according to claim 4 or 5, comprising the step of selecting a cell line as a breeding strain that satisfies at least one of the following conditions.

7. A microalga of the genus Pavlova prepared by the method described in any one of claims 4 to 6.

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