JP2021013313A - Novel microalgae - Google Patents

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

The present disclosure relates to microalgae containing useful ingredients and microalgae products thereof. In particular, the present disclosure relates to microalgae that highly produce fucoxanthin and microalgae products thereof.

Microalgae such as chlorella and euglena are attracting attention for their nutritional components such as vitamins and minerals, and are used as health foods and food materials (Patent Document 1 = JP-A-2018-70568).

In addition, microalgae may contain various components including useful components and harmful components. For example, chlorophyll, which is widely contained in microalgae, is known to be converted to pheophorbite and cause phototoxicity.

JP 2018-70568

As a result of diligent research, the present inventors provide novel microalgae belonging to the Pavlova family that produce various useful components such as fucoxanthin. The present inventors have found that this microalgae can be processed in a state in which pheophorbite is suppressed.

Accordingly, the present disclosure provides:
(Item 1)
A microalga of the Pavlova family that produces 1 g or more of fucoxanthin per 100 g of dried cells.
(Item 2)
A microalga of the Pavlova family that produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells.
(Item 3)
A microalga of the Pavlova family that produces 50 mg or more of γ-aminobutyric acid per 100 g of dried cells.
(Item 4)
A microalga of the Pavlova family that produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(Item 5)
A microalga of the Pavlovaceae family that produces 1 g or more of fucoxanthin per 100 g of dried cells and 50 mg or more of γ-aminobutyric acid per 100 g of dried cells and / or 100 mg or more of hydroxyproline per 100 g of dried cells.
(Item 6)
Pavlova microalgae that produce 500 mg or more of calcium per 100 g of dried cells.
(Item 7)
Microalgae of the Pavlova family that produce 5 g or more of dietary fiber per 100 g of dried cells.
(Item 8)
Pavlova microalgae that produce about 2 g or more of chlorophyll per 100 g of dried cells.
(Item 9)
Microalgae of the Pavlova family with an amino acid score of 85 or higher.
(Item 10)
1 g or more of fucoxanthin is produced per 100 g of dried cells, and the following characteristics (a) to (g):
(A) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells.
(B) Produces 50 mg or more of γ-aminobutyric acid per 100 g of dried cells.
(C) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(D) Produces 500 mg or more of calcium per 100 g of dried cells.
(E) Produces 5 g or more of dietary fiber per 100 g of dried cells.
(F) Produces about 2 g or more of chlorophyll per 100 g of dried cells, and (g) has an amino acid score of 85 or more.
Pavlova microalgae having one or more of the properties of.
(Item 11)
The microalga according to any one of items 1 to 10, which belongs to the genus Pavlova.
(Item 12)
P. granifera or P.I. The microalga according to any one of items 1 to 10, which is a gyrans.
(Item 13)
Microalgae which is OPMS30543 strain (strain specified by accession number NBRC 114066).
(Item 14)
Any one of items 1 to 12, which is an inducible strain of OPMS30543 strain (strain specified by accession number NBRC 114066) and has the characteristics of the microalgae according to any one or more of items 1 to 10. The microalgae described in.
(Item 15)
A food containing the microalgae according to any one of items 1 to 14.
(Item 16)
The food according to item 15, wherein the amount of pheophorbite per 100 g of the microalgae is 100 mg or less.
(Item 17)
A microalgae product comprising the purified microalgae according to any one of items 1 to 14.
(Item 18)
A microalgae product comprising the fucoxanthin-purified microalgae according to any one of items 1 to 14.
(Item 19)
The microalgae product according to item 18, wherein the fucoxanthin purified product has a fucoxanthin purity of 5 to 70%.
(Item 20)
The microalgae product according to item 18, wherein the fucoxanthin purity of the purified fucoxanthin product is about 70% or more.
(Item 21)
The microalgae product according to any one of items 17 to 20, wherein the microalgae or a purified product thereof is immersed in oil.
(Item 22)
The microalgae product according to any one of items 17 to 21, wherein the microalgae or a purified product thereof is encapsulated.
(Item 23)
A method for breeding microalgae of the order Pavlova under the following conditions: ・ Seawater concentration: 50% seawater ・ Medium concentration: IMK medium double concentration ・ Culture solution volume: 800 mL
-Light source: Side: Fluorescent lamp (100 to 150 μmol)
・ Light-dark cycle: 12 hours each for light and dark ・ pH: pH about 7.4 at the start of each culture
・ Aeration: Implementation ・ Mechanical stirring: None ・ Culture period: Conducted for a period of about 10 days ・ Culture temperature: 25 ° C to 28 ° C
A step of repeating the culture passage of a Pavlova microalgae cell line under the condition of satisfying at least one of the above.
Here, for each subculture, cells floating from the supernatant portion of the culture are selectively used, thereby concentrating only cell lines that survive well under the culture conditions. how to.
(Item 24)
Repeat the steps of repeating the culture passage at least: -Seawater concentration: 50% seawater-Medium concentration: IMK medium double concentration-Culture period: Conditions that satisfy one or more of the implementations with a period of about 10 days. The method according to item 23, which is carried out in.
(Item 25)
When the cell density exceeds 2 g / L, the cell line is recovered and fucoxanthin, eicosapentaenoic acid, and γ-aminobutyric acid of the cell line are added to the group consisting of hydroxyproline, calcium, dietary fiber, chlorophyll, and amino acid score. The step of measuring at least one selected
(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 γ-aminobutyric acid per 100 g of dried cells.
(Iv) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(V) Produces 500 mg or more of calcium per 100 g of dried cells.
(Vi) Produces 5 g or more of dietary fiber per 100 g of dried cells,
(Vii) produces about 2 g or more of chlorophyll per 100 g of dried cells, and (viii) has an amino acid score of 85 or more.
The method according to item 23 or 24, which comprises the step of selecting a cell line satisfying at least one of the above conditions as a breeding strain.
(Item 26)
Microalgae of the order Pavlova produced by the method according to any one of items 23 to 25.

In the present disclosure, it is intended that the above one or more features may be provided in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.

The present disclosure makes it possible to provide microalgae having new functionality or microalgae products thereof (for example, food products).

The OPMS30543 strain in the cultured state is shown. The scale bar indicates 50 μm. A spray-dried OPMS 30543 strain microalgae product is shown. An exemplary photobioreactor is shown. On the left is a photograph of the bioreactor, the vertically long tube is a transparent member with an outer diameter of 60 mm. The right is a schematic diagram of the bioreactor.

Hereinafter, the present disclosure will be described with reference to the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of their plural, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate.

(Definition, etc.)
As used herein, the term "microalgae" refers to microorganisms having a microscopic size (for example, 0.1 μm to 1 mm) including chloroplasts, and generally inhabits water. Microalgae include the prokaryotic phylum Cyanobacteria, and the eukaryotic phylum Glaucophyta, phylum Red algae (Red algae), Chlorarachniophyta, and cryptophytes. Phylum (Cryptophyta), Hapt phylum (Haptophyta), Heterokontophyta, Whirlpool phylum (Dinophyta), Euglena (Euglenida) and Chlorarachnioides (Glaucophyta) Includes organisms of the phylum Chlorarachniophyta.

The phylum Haptophyta (Haptophyta) includes the class Haptophyceae, and the class Haptophycea includes the subclass Pavlovophycidae and the subclass primnesiophycea. The Pavlova subclass (Pavlovaphycidae) includes the order Pavlova, the order Pavlova (Pavlovales) includes the family Pavlovaceae, and the family Pavlova (Pavlovaceae) includes Diacronema, Excelca, and Xanthema. Is done. For the genus Pavlova, P. calceolate, P.M. granifera, P. et al. gyrans, P.M. lutheri, P.M. pinguis, P.I. Salina is included. Haptophytes are phytoplankton with a cell diameter of about 5 to 50 μm and are autotrophic organisms that perform photosynthesis. Most live in the ocean, but some species are also found in freshwater and salt lakes. The biomass of haptophytes in the open ocean is large, and it is important as a primary producer of the ocean.

As used herein, the term "microalgae product" refers to a microalgae body or a product containing some components of the microalgae. Typically, the microalgae product is a dried product, or a product further processed from the dried product, or an extract enriched with certain components of the microalgae (eg, fucoxanthin).

As used herein, the term "food" refers to an article intended to be ingested by an animal (for example, a human), and the food includes food additives and functionality in addition to commonly used foods and beverages. Foods (for example, foods for specified health use, foods with functional claims, foods with nutritional function, etc.), and supplements are included.

As used herein, "cosmetics" are worn and worn by an animal (eg, human) to cleanse, beautify, enhance its attractiveness, change its appearance, or keep its skin or hair healthy. Refers to any product intended to be used by rubbing, spraying or similar methods. In the present specification, the “cosmetics” is not limited to “cosmetics” under the so-called Pharmaceutical and Medical Devices Act (former Pharmaceutical Affairs Law), and may be, for example, quasi-drugs, pharmaceuticals, or miscellaneous goods. In this specification, "quasi-drugs" is an intermediate classification between pharmaceuticals and cosmetics as defined in Japan's "Act on Securing Quality, Effectiveness, and Safety of Pharmaceuticals, Medical Devices, etc." , Including those with a mild action on the human body, and also includes machinery and equipment with a gentle action on the human body. Examples of quasi-drugs include medicated cosmetics (including medicated soap, medicated toothpaste, etc.), bath salts, quasi-drugs for pest control (insecticides, etc.) and designated quasi-drugs (drinks, mouthwash, etc.) Quasi-drugs, etc.), but are not limited to these. In the present specification, the term "pharmaceutical product" refers to a drug given to diagnose, treat, or prevent a human or animal disease, and is contained in the Japanese Pharmacy, or a diagnosis or treatment of a human or animal disease. Or those intended for prophylactic use that are not machinery, dental materials, medical or sanitary products (excluding non-pharmaceutical products), and the structure or function of the human or animal body. Includes items that are intended to affect and are not machinery / equipment, dental materials, medical / sanitary items (excluding non-pharmaceutical products and cosmetics)

As used herein, "chlorophyll" is a substance used in the usual sense in the art and is often used to absorb light energy in the light reaction of photosynthesis. Microalgae with chloroplasts may contain chlorophyll.

As used herein, "pheophorbite" is used in the usual sense in the art and is often the substance produced by the decomposition of chlorophyll in microalgae. Pheophorbite can be produced by the action of chlorophyllase on chlorophyll. The content of processed chlorella products is regulated (May 8, 1981) (Kanshoku No. 99) (Kanshoku No. 99) because it may cause hygienic hazards that cause skin disorders. Notification to the governor of each prefecture, the mayor of each ordinance-designated city, and the mayor of each special ward).

As used herein, "fucoxanthin" is used in the usual sense in the art and has the following structure:

It is a substance having. Fucoxanthin is known to be easily decomposed by heating, light irradiation, oxidation and the like.

As used herein, "eicosapentaenoic acid (EPA)" is used in the usual sense in the art and has the following structure:

Refers to a substance that has.

As used herein, "hydroxyproline" is used in the usual sense in the art and has the following structure:

Refers to a substance that has.

As used herein, "dietary fiber" is used in the usual sense in the art and is a general term for indigestible components contained in foods that are not digested by human digestive enzymes.

In the present specification, the "amino acid score" is an index based on the report of the WHO / FAO / UNU Joint Expert Council in 1985, and the calculation method of the amino acid score is that each essential amino acid occupies 1 g of nitrogen constituting the protein. It is expressed by the number of mg of, and is calculated as a ratio to the amino acid scoring pattern based on the joint committee by FAO / WHO and the like. On average, 1 g of nitrogen constituting a protein corresponds to 6.25 g of protein. The evaluation value is the value of the lowest value amino acid (first limiting amino acid) by comparing the ratio of the essential amino acid in the protein of the sample with the reference essential amino acid pattern.

As used herein, "culture" is used in the conventional sense in the art and refers to an operation of keeping cells alive in or on a medium, even if the number of cells increases during the culture. It may be reduced, reduced, or maintained.

The present disclosure also provides microalgae products containing purified components of microalgae. As used herein, a "purified" substance (eg, fucoxanthin, etc.) is one from which at least some of the factors naturally associated with the substance have been removed. Therefore, the purity of the substance in the purified substance is usually higher (ie, concentrated) than in the state in which the substance is normally present. The term "purified" as used herein is of the same type, eg, 10-100%, preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight. It means that the substance is present.

The "inducible strain", "similar strain" or "mutant strain" used herein is preferably a region substantially homologous to the DNA of the microalgae of interest, although not intended to be limiting. Such strains contain at least 30%, 40, in various embodiments, when aligned by a computer homology program known in the art and compared to the sequence of the entire genome of the original strain. Has a whole genome sequence that is%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical. This is a microalga modified by mutation, substitution, deletion and / or addition of a gene, although the inducible strain may still exhibit the biological function of the original microalga to not necessarily the same degree. It means microalgae. For example, gene mutations can be introduced using any known mutagen, UV, plasma and the like. 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, suitable and available in vitro assays described herein or known in the art can be used to study the biological function of such microalgae.

In the present specification, "homology" of a gene means the degree of identity of two or more gene sequences to each other, and generally, having "homology" means a high degree of identity or similarity. Say. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes are homologous can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When two gene sequences are directly compared, the 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%, the genes are homologous.

Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code. In the present specification, comparison of amino acid sequence and base sequence similarity, identity and homology is calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.7.1 (issued 2017.10.19). The value of "identity" in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

In one embodiment of the present disclosure, "70% or more", which is a numerical value such as identity, is, 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 numerical values that are the starting points thereof. The above "identity" is calculated by calculating the ratio of the number of amino acids homologous in an amino acid sequence between two or a plurality of amino acids according to a known method as described above. Specifically, before calculating the proportions, align the amino acid sequences of the amino acid sequences to be compared and introduce a gap in part of the amino acid sequence if necessary to maximize the proportion of the same amino acid. To do. Methods for alignment, methods for calculating proportions, comparison methods, and related computer programs are well known in the art (eg, BLAST, described above). As used herein, "identity" and "similarity" can be expressed as values measured by NCBI BLAST unless otherwise specified. Blastp can be used with default settings for the algorithm when comparing amino acid sequences in BLAST. The measurement results are quantified as Positives or Identities.

As used herein, the term "biological function" refers to a specific function that a microalgae may have when referring to a microalgae, which includes, for example, the ability to produce a specific component such as fucoxanthin. Etc., but are not limited to them. In the present disclosure, for example, the ability to produce fucoxanthin at a high level can be mentioned, but the present invention is not limited thereto. As used herein, a biological function can be exerted by a corresponding "biological activity". As used herein, the term "biological activity" refers to an activity that a microalga can have in a certain environment, and includes an activity that exerts various functions (for example, production of fucoxanthin). Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that affect a response (ie, have a measurable effect in response to some exposure or stimulus), eg, some of the microalgae of the present disclosure. A measure of the amount of protein upstream or downstream after stimulation or event or other similar function may also be included.

As used herein, the "amount" of an analyte in a body fluid sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in the volume of the sample. However, the quantity also contemplates a relative quantity compared to another analyte quantity. For example, the amount of analyte in the sample may be greater than the control or normal level of the analyte normally present in the sample.

As used herein, the term "about" refers to the value shown plus or minus 10%, unless otherwise stated otherwise.

(Preferable embodiment)
The preferred embodiments of the present disclosure will be described below. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in consideration of the description in the present specification. It is also understood that the following embodiments of the present disclosure may be used alone or in combination.

(New edible microalgae)
In one aspect, the present disclosure exhibits the ability to produce at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll, and / or a high amino acid score. To provide microalgae with. The production capacity of various products (fucoxanthin, dietary fiber, eicosapentaenoic acid, γ-aminobutyric acid, hydroxyproline, calcium, and chlorophyll, etc.) in the microalgae of the present disclosure is when they grow to 2 g / L or more. It shows per 100 g of dried cells.

In one embodiment, the microalgae of the present disclosure are 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. 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 Has the ability to produce 7 g or more, 8 g or more, 9 g or more or 10 g or more of fucoxanthin. Since fucoxanthin is known to have anti-obesity, anti-diabetes, antioxidant, anti-cancer, and angiogenesis-suppressing effects, the microalgae of the present disclosure and its microalgae products that produce fucoxanthin at high levels. Is expected to produce these effects. The microalgae of the present disclosure can also contain a high amount of eicosapentaenoic acid, and the decomposition of fucoxanthin can be reduced.

In one embodiment, the microalgae of the present disclosure are 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. 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, It has the ability to produce 4.5 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 of ecosapentaenoic acid (EPA). Since EPA is known to have effects such as lowering triglyceride, suppressing platelet aggregation, anti-inflammatory, anti-allergy, prevention of infectious diseases, and mental stability, the microalgae of the present disclosure that produce high EPA and the microalgae thereof. Microalgae products are expected to exert these effects.

In one embodiment, the microalgae of the present disclosure are 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, per 100 g of dried cells. 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 γ-amino Has the ability to produce butyric acid. Since γ-aminobutyric acid is known to have effects such as lowering blood pressure and reducing stress, the microalgae of the present disclosure and its microalgae products that produce high γ-aminobutyric acid can exert these effects. Be expected.

In one embodiment, the microalgae of the present disclosure are 0.005 g or more, 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. 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 , 1.5 g or more, 2 g or more, 3 g or more, 4 g or more, 5 g or more, 7 g or more, or 10 g or more has the ability to produce hydroxyproline. Since hydroxyproline is a major component of collagen and is a substance that plays a role in collagen stability together with proline, the microalgae of the present disclosure and its microalgae products that produce high hydroxyproline maintain skin tension and wrinkles. It is expected to have the effect of removing.

In one embodiment, the microalgae of the present disclosure is 0.1 g or more, 0.2 g or more, 0.5 g or more, 0.7 g or more, 1 g or more, 1.5 g or more, 2 g or more, 2 g or more, per 100 g of dried cells. .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 ability to produce calcium Have. Since calcium is known to have effects such as bone strengthening, the microalgae of the present disclosure and its microalgae products, which produce high calcium, are expected to exert such effects, and further, they are broken. When combined with fucoxanthin, which has been reported to have an osteoclast differentiation inhibitory effect, it is expected to have effects such as prevention of osteoporosis.

In one embodiment, the microalgae of the present disclosure are 0.5 g or more, 0.7 g or more, 1 g or more, 1.5 g or more, 2 g or more, 5 g or more, 7 g or more, 8 g or more, 9 g or more per 100 g of dried cells. It has the ability to produce 10 g or more, 12 g or more, 15 g or more, 17 g or more, 20 g or more, 22 g or more, 25 g or more, 27 g or more or 30 g or more of dietary fiber. Since dietary fiber is known to have effects such as obesity prevention, cholesterol elevation suppression, blood glucose elevation suppression, hydrogen gas production, antioxidants, and promotion of defecation, the microalgae of the present disclosure that produce high dietary fiber. And its microalgae products are expected to exert these effects. The dietary fiber produced by the microalgae of the present disclosure may include fucoidan and the like.

In one embodiment, the microalgae of the present disclosure is 0.1 to 20 g per 100 g of dried cells, eg, about 0.1 g, about 0.2 g, about 0.5 g, about 0.7 g, about 1 g, about. 1.5g, about 2g, about 2.5g, about 3g, about 3.5g, about 4g, about 4.5g, about 5g, about 6g, about 7g, about 8g, about 9g, about 10g, about 12g, about It has the ability to produce 15 g, about 17 or about 20 g of chlorophyll. Chlorophyll is a compound that can be converted to pheophorbite, and the microalgae of the present disclosure can produce a relatively large amount of chlorophyll, but can be processed in a state in which pheoholbite is suppressed as described below.

In one embodiment, the microalgae of the present disclosure are 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85. It has the property of exhibiting an amino acid score of 90 or more, 95 or more, or about 100. The microalgae of the present disclosure having a high amino acid score and the microalgae products thereof can provide a well-balanced amino acid for humans. Also, in one embodiment, the microalgae of the present disclosure are about 1-70 g, eg, about 1 g, about 2 g, about 5 g, about 7 g, about 10 g, about 12 g, about 15 g, about 17 g per 100 g of dried cells. Has the ability to produce 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 of protein. The microalgae and their microalgae products of the present disclosure, which have high protein production and high amino acid score, may provide sufficient amino acids for humans in small amounts.

The microalgae of the present disclosure may have properties of any combination of fucoxanthin, dietary fiber, eicosapentaenoic acid, γaminobutyric acid, hydroxyproline, calcium, and chlorophyll productivity, as well as amino acid scores described above. In one embodiment, the microalgae of the present disclosure are selected from the fucoxanthin productivity described above and the dietary fiber, eicosapentaenoic acid, γaminobutyric acid, hydroxyproline, calcium, and chlorophyll productivity described above, as well as the amino acid score. Can have at least one characteristic. In one embodiment, the microalgae of the present disclosure may have the fucoxanthin productivity described above and the γ-aminobutyric acid or hydroxyproline productivity described above, optionally the dietary fiber, eicosapentaenoic acid, the above. It may further have at least one property selected from calcium and chlorophyll productivity, as well as an amino acid score.

In one embodiment, the microalgae of the present disclosure exhibit low adhesion. For example, the adhesiveness can be evaluated by measuring the weight of microalgae attached to the base material when the base material of a specific material is immersed in the culture solution for a specific time.

In one embodiment, the microalgae of the present disclosure can be Pavlova family algae and can belong to the genus Diacronema, Exanthemachrysis, Pavlova or Rebecca. For the genus Diacronema, D. ennorea, D.I. lutheri, D.M. noctivaga, D.M. virescens, D. viridis and D. vrkianum is included. In the genus Exanthemachysis, E.I. Gayraliae is included. For the genus Pavlova, P. calceolate, P.M. granifera, P. et al. gyrans, P.M. lutheri, P.M. pinguis and P. Salina is included. For the genus Rebecca, R. helicata and R. Salina is included. In certain embodiments, the microalgae of the present disclosure are microalgae of the genus Pavlova. In a more specific embodiment, the microalgae of the present disclosure are P.I. granifera or P.I. gyrans. The present discloser has described the newly discovered microalgae as P.I. granifera and P.I. It was identified as gyrans and deposited. The NBRC number, which is the accession number, is NBRC 114066. In one embodiment, the microalgae of the present disclosure is Pavlova OPMS30543 strain (algae strain identified by Accession No. NBRC 114066) or Pavlova OPMS30543X strain, or an inducible strain thereof.

In one embodiment, the microorganism of the present disclosure is an OPMS30543 strain (algae strain identified by Accession No. NBRC 114066) or an inducible strain of OPMS30543X strain. Here, the inducible strain does not have to be a strain obtained based on these algae strains, and the biological functions of these algae strains do not necessarily have to be the same degree, but microalgae. Point to. In one embodiment, the inducible microalgae of the present disclosure, like the OPMS30543 or OPMS30543X strains, are fucoxanthin, dietary fiber, eicosapentaenoic acid, γ-aminobutyric acid, hydroxyproline, calcium as described above. , And the ability to produce at least one of chlorophyll, as well as the biological function selected from the group consisting of properties exhibiting an amino acid score, but the degree of biological function differs from the OPMS30543 or OPMS30543X strains. May be. In one embodiment, the inducible strain of the present disclosure is a microalga of the genus Pavlova, more specifically Pavlova. granifera or P.I. It can be gyrans.

(Use and functionality)
The microalgae of the present disclosure are capable of producing at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, gamma-aminobutyric acid, hydroxyproline, calcium, and chlorophyll as described above and / or have a high amino acid score. Therefore, it can be suitably used as various microalgae products. In addition, since the microalgae of the present disclosure can suppress pheophorbite by treatment, they can be safely used in foods, for example. In one embodiment, the microalgae products of the present disclosure can be dry, eg, 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 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, 0.05% by weight It can be: In one embodiment, the microalgae products of the present disclosure may be provided by immersing the dried microalgae of the present disclosure or purified products thereof in oil and / or encapsulating them in capsules (eg, soft capsules). It can be stabilized by taking the form of.

In one embodiment, the microalgae product of the present disclosure is a food product, which may contain any amount of the microalgae of the present disclosure, eg, 0.01-100% by weight. 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 present disclosure do not have a cell wall and may have the property of being soft, so that they do not give an unpleasant texture when ingested. If the taste and flavor of the microalgae of the present disclosure are of concern, they may be used in combination with any suitable flavoring agent, odorant, masking agent, or finely formed by means such as coating or encapsulation. The taste and flavor of algae may be masked. Since the microalgae of the present disclosure are rich in the above-mentioned useful components and can exert their effects even when ingested in a small amount, they are also suitable for use as supplements and / or food additives in foods.

In one embodiment, the microalgae product of the present disclosure is a refined product comprising concentrated at least one of fucoxanthin, dietary fiber, eicosapentaenoic acid, hydroxyproline, calcium, and chlorophyll. Such refined products may contain any amount of the desired ingredient (s), but for example, by weight, about 5% or more, about 10% or more, about 20% or more, about 30%. 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%, and / or about 10% or less, about Includes 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 obtain. Such refined products may be used as foods (for example, supplements), or may be added to cosmetics, pharmaceuticals, quasi-drugs, and the like. Depending on the components to be concentrated (eg, fucoxanthin), the refined product of the present disclosure may be unstable, and in such cases, it may be combined with a stabilizer (eg, immersion in oil and contact with air). Avoid, add antioxidants such as vitamin E and vitamin C, etc.).

In one specific embodiment, the microalgae product of the present disclosure is a refined product of fucoxanthin. In one embodiment, such refined fucoxanthin products are 1-100% (eg, about 1% or more, about 2% or more, about 5% or more, about 7% or more) fucoxanthin from the microalgae of the present disclosure. , About 10% or more, about 15% 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%, 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 Percentage of any range defined by the lower and / or upper limits of% or less, about 90% or less, about 95% or less, about 97% or less, about 99% or less or about 100% or less, eg 5 to 70%). It may contain a purified product concentrated to be.

(Breeding method)
The microalgae of the present disclosure can be obtained by breeding methods, and in one aspect, the disclosure also provides such breeding methods. In one embodiment, the breeding method of the present disclosure has the following conditions: -Seawater concentration: 50% seawater-Medium concentration: IMK medium double concentration-Culture volume: 800 mL
-Light source: Side: Fluorescent lamp (100 to 150 μmol)
・ Light-dark cycle: 12 hours each for light and dark ・ pH: pH about 7.4 at the start of each culture
・ Aeration: Implementation ・ Mechanical stirring: None ・ Culture period: Conducted for a period of about 10 days ・ Culture temperature: 25 ° C to 28 ° C
The step of repeating the culture passage of the cell line of the Pavlova microalgae under the condition of satisfying at least one of the above is included. In one embodiment, at least one of the steps of repeating the culture passage is carried out with a target of seawater concentration: 50% seawater, medium concentration: IMK medium double concentration, and culture period: about 10 days. It can be performed under conditions that satisfy one or more.

In one embodiment, in the method of the present disclosure, a cell line of microalgae of the order Pavlova cultured by the above method or the like is recovered when the cell density exceeds 2 g / L, and the cell lines fucoxanthin and eikosapentaene are collected. A step of measuring acid, γ-aminobutyric acid, at least one selected from the group consisting of hydroxyproline, calcium, dietary fiber, chlorophyll and amino acid scores.
(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 γ-aminobutyric acid per 100 g of dried cells.
(Iv) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(V) Produces 500 mg or more of calcium per 100 g of dried cells.
(Vi) Produces 5 g or more of dietary fiber per 100 g of dried cells,
(Vii) produces about 2 g or more of chlorophyll per 100 g of dried cells, and (viii) has an amino acid score of 85 or more.
The process of selecting a cell line satisfying at least one of the above conditions as a breeding strain may be included. The selection conditions vary depending on the performance of the target cells.
(I) The amount of fucoxanthin produced per 100 g of dried cells may be 1 g or more, 1.5 g or more, 2 g or more, or 3 g or more.
(Ii) The amount of eicosapentaenoic acid produced per 100 g of dried cells may be 4 g or more, 5 g or more, 6 g or more, or 8 g or more.
(Iii) The amount of γ-aminobutyric acid produced per 100 g of dried cells may be 50 mg or more, 70 mg or more, 100 mg or more, 150 mg or more, or 200 mg or more.
(Iv) The amount of hydroxyproline produced per 100 g of dried cells may be 100 mg or more, 150 mg or more, 200 mg or more, or 300 mg or more.
(V) The amount of calcium produced per 100 g of dried cells may be 500 mg or more, 700 mg or more, 1 g or more, or 1.5 g or more.
(Vi) The amount of dietary fiber produced per 100 g of dried cells may be 5 g or more, 7 g or more, 10 g or more, or 15 g or more.
(Vii) The amount of chlorophyll produced per 100 g of dried cells may be about 2 g or more, 3 g or more, 4 g or more, or 5 g or more.
(Viii) The amino acid score may be 85 or higher, 90 or higher, 95 or higher, or about 100.
At least one of the conditions of any combination can be satisfied.

In one embodiment, the aeration may be aeration agitation, where compressed air may be used. In one embodiment, the breeding method of the present disclosure is a method of breeding microalgae of the order Pavlova. In one embodiment, for each subculture, cells floating from the supernatant portion of the culture are selectively used, thereby allowing the cell line to survive well under the culture conditions. Concentrate only. In one embodiment, the microalgae of the present disclosure are microalgae obtained by such a breeding method.

(Culture of microalgae)
In one embodiment, the microalgae of the present disclosure can be cultivated under any suitable conditions. In one embodiment, the culture can be subdivided into seed cultures, main cultures and the like. In one embodiment, the seed culture is performed in multiple culture stages (eg, in vitro culture stage (about 100 mL), PET bottle, flask or medium bottle culture stage (about 1 L or less), of the photobioreactor of the present disclosure. Culturing stage (about 5 L), culturing 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. (Any combination of culture steps (about 1000 L or more)) may be included. Unless otherwise specified, the culture conditions described below can be applied to any type of culture. Conditions (for example, temperature, pH, stirring conditions, light irradiation conditions, and medium composition) in the step of culturing microalgae can be preferably set. In one embodiment, the culture of microalgae may include multiple stages (eg, seed culture and main culture, indoor contamination-free culture and outdoor fast growth culture, acclimatization and main culture, etc.).

In one embodiment, the microalgae can be cultivated at a temperature of about 0 ° C to 80 ° C, more specifically about 20 ° C to 30 ° C. The upper limit of the appropriate temperature can be 80 ° C., 70 ° C., 60 ° C., 50 ° C., 40 ° C., 30 ° C., 20 ° C., and the lower limit can be 0 ° C., 5 ° C., 10 ° C., 15 ° C. , 20 ° C., 25 ° C., 30 ° C., etc., and any combination thereof can be adopted as an appropriate 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 have to be constant, and strict temperature control may not be required, especially when the culture tank is installed outdoors. For at least a part of the culture period, it is preferable to bring the microalgae to a temperature at which they can preferably survive and grow. When the temperature rises too much due to direct sunlight or the like, the temperature can be lowered by any cooling means (for example, water cooling). For example, when the microalgae are haptophytes, they can grow favorably at a temperature of about 25-30 ° C.

In one embodiment, the microalgae can be cultured at a pH of about 2-13. The upper limit of the appropriate pH can be pH13, pH12, pH11, pH10, pH9, pH8.5, pH8, pH7.5, pH7, pH6, etc., and the lower limit can be pH2, pH3, pH4, pH5, etc. pH 6, pH 6.5, pH 7, pH 7.5, pH 8, etc. can be mentioned, and any combination thereof can be adopted as an appropriate 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 set a suitable pH for the microalgae to be used. It is preferable not to cause a sudden pH change during culturing, and any suitable buffer (for example, carbon dioxide, amine compound, etc.) can be used to control the pH change. For example, when the microalgae are haptophytes, they can grow favorably in a weakly alkaline environment with a pH of about 8.

In one embodiment, the microalgae may or may not be subject to stirring conditions during culturing. Examples of the means for stirring include, but are not limited to, aeration stirring, mechanical stirring (paddle stirring, etc.), running water stirring (for example, using a pump), stirring by shaking the culture tank, and the like. Depending on the stirring means, microalgae may be damaged, and especially Euglena and haptophytes having no cell wall are relatively soft, so it may be preferable to avoid vigorous stirring that destroys cells in culture.

In one embodiment, the microalgae can be cultured under light irradiation for at least a portion of the culture period. Although it depends on the type of microalgae, the larger the amount of light irradiated in the range where the microalgae is not damaged, the higher the growth rate of the microalgae can be. Depending on the microalgae, non-constant light irradiation may be preferable. A specific wavelength region may be selectively irradiated. When culturing microalgae outdoors, it may be advantageous to utilize natural light. Even when microalgae are cultured outdoors and only natural light is used 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 having 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 ^-2 s ^-1 to about 3000 μmol m ^-2 s ^-1 , or about 30 μmol m ^-2 s ^-1 to about 1500 μmol m ^-2 s ^-1. 50μmol m ^-2 s ^-1 ~ about 300 [mu] mol m ^-2 s ^-1 may be preferred. For example, when the microalgae are Haptophyte it may suitably grown with light energy level of about 100μmol m ^-2 s ^-1 ~ about 150μmol m ^-2 s ^-1.

The composition of the medium used for culturing the microalgae can be any suitable one according to the type of the microalgae. Inorganic salts (eg, potassium salt, sodium salt, calcium salt, magnesium salt), sugars (eg, glucose), organic salts, nitrogen sources (nitrate, ammonium salt, etc.), phosphorus are typical components that can be contained in the medium. Sources (inorganic phosphorus, phosphates, etc.) may be mentioned, but other components may be included. Nitrogen sources, phosphorus sources, etc. can be added as appropriate because they can be consumed with the growth of microalgae. Moreover, when a carbon source (for example, carbon dioxide) is added, it can be utilized for microalgae. For example, when culturing haptophytes, most of the haptophytes live in seawater to brackish water, so a medium having a composition similar to that of seawater to brackish water (for example, a medium containing about 50 to 75% of salts in seawater) or seawater to A medium close to the osmotic pressure of brackish water can be preferably used.

In the culturing step in the production method of the present disclosure, it is preferable to increase the density of microalgae in order to improve the efficiency of culturing. For example, in terms of dry weight of microalgae, 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 1.5g / L, at least 2g / L, at least 2.5g / L, at least 3g / L, at least 3.5g / L, at least 4g / L, at least 4.5g / L, at least 5g / L, at least 5 Cultivate to a density of .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. Can be done. The culture period may be continued until the desired microalgae density is achieved, a predetermined culture period may be specified, or maintenance culture may be continued indefinitely.

The microalgae of the present disclosure include a state in which cells are attached to a carrier or the like, a state in which each cell floats independently in a medium, a state in which cells aggregate and float in a medium, and a state in which these states coexist. It can be cultured in any state.

In one embodiment, the microalgae of the present disclosure may be cultivated under light irradiation conditions and then under reduced light irradiation conditions. The microalgae of the present disclosure grow under light irradiation conditions, and the amount of fucoxanthin can be increased under conditions where light irradiation is reduced. Therefore, controlling the light irradiation conditions in this way contains a large amount of fucoxanthin. It can be useful in the production of algal products. Under the culture conditions for controlling such light irradiation conditions, the light irradiation amount under the light irradiation conditions is about 30 μmol m ^-2 s ^-1 to about 3000 μmol m ^-2 s ^-1 , for example, about 3000 μmol m ^-2 s ^-1. , about 2000μmol m ^-2 s ^-1, about 1500μmol m ^-2 s ^-1, about 1000 micro mol m ^-2 s ^-1, about 700μmol m ^-2 s ^-1, about 500 [mu] mol m ^-2 s ^-1, about 200 [mu] mol m ^{- 2} s ^-1, about 150μmol m ^-2 s ^-1, about 100μmol m ^-2 s ^-1, about 50μmol m ^-2 s ^-1, may be about 30μmol m ^-2 s ^-1, of the light irradiation reducing conditions light irradiation amount is about 100 [mu] mol m ^-2 s ^-1 or less, for example, about 100 [mu] mol m ^-2 s ^-1, about 70μmol m ^-2 s ^-1, about 50 [mu] mol m ^-2 s ^-1, about 20 [mu] mol m ^-2 s ^-1 , about 10 μmol m ^-2 s ^-1 , about 5 μmol m ^-2 s ^-1 , about 2 μmol m ^-2 s ^-1 , about 1 μmol m ^-2 s ^-1 , or less, any of these A combination of the light irradiation amount of the light irradiation condition and the light irradiation reduction condition of the above can be used. As the thickness of the reactor increases, the amount of light irradiation per cell of microalgae can decrease. For example, when the weather before collecting microalgae is sunny, fucoxanthin can be increased by shading with a shading net (for example, 50% shading) for 1 to 2 days before collecting.

(Manufacturing of microalgae products)
A microalgae product can be produced from a culture containing the microalgae of the present disclosure cultured according to the above culture method. In one embodiment, the method for producing a microalgae product of the present disclosure comprises a step of inactivating chlorophyllase. By inactivating chlorophyllase, the production of pheophorbide can be suppressed. Treatments that inactivate chlorophyllase include, for example, heat treatment, any known protein denaturation treatment (temperature load (low temperature, high temperature)), drug treatment (alcohol, strong acid, strong base, other denaturing agents), irradiation (ultraviolet rays). , Gamma rays, etc.)), but are not limited to these. Treatments that inactivate chlorophyllase (eg, heat treatment) can be performed under any suitable conditions (means, time, etc.) that inactivate chlorophyllase, but do not destroy microalgae and / or microscopic. Conditions that do not destroy the useful components of the algae can be preferably applied. For example, since haptophytes can produce fucoxanthin, the decomposition of fucoxanthin is small, for example, the reduction of fucoxanthin when compared before and after treatment is less than 0.01%, less than 0.02%, 0.05. Less than%, 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%, 5 Less than%, 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%.

The treatment for inactivating chlorophyllase is preferably carried out under conditions that control the amount of stress (for example, the amount of stimulation accumulated by an arbitrary operation that increases the amount of pheophorbide produced in microalgae). It is preferable to carry out for microalgae in which the amount of stress given before is not large. When a treatment for inactivating chlorophyllase is applied to microalgae subjected to a large amount of stress, it is possible that a large amount of pheophorbide has already been produced, and the effect of suppressing pheophorbide by inactivating chlorophyllase is sufficient. May not be obtained. Controlling the amount of stress for the suppression of pheophorbide can be achieved, for example, by maintaining a low density of microalgae and / or not concentrating the microalgae significantly.

In one embodiment, after culturing and before the treatment for inactivating chlorophyllase, the microalgae of the present disclosure are 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 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 It is not concentrated above or is not subjected to such enrichment operations.

In one embodiment, the treatment for inactivating chlorophyllase is a heat treatment, which is about 50 ° C to 200 ° C, eg, about 50 ° C, about 60 ° C, about 70 ° C, about 80 ° C, about 85 ° C, about. 90 ° C, about 95 ° C, about 97 ° C, about 100 ° C, about 102 ° C, about 105 ° C, about 107 ° C, about 110 ° C, about 120 ° C, about 130 ° C, about 140 ° C, about 150 ° C, about 160 ° C , About 170 ° C, about 180 ° C, about 190 ° C, about 200 ° C and the like. The heat treatment time is about 10 seconds to 20 hours, for example, about 10 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 5 minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20 minutes. , About 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about It can be 7 hours, about 10 hours, about 20 hours, and so on.

In one embodiment, the density of microalgae during the treatment to inactivate chlorophyllase is about 0.01-100 g / L, eg, about 100 g / L or less, about 70 g / L or less, about 50 g by dry weight. / L or less, about 40 g / L or less, about 30 g / L or less, about 20 g / L or less, about 15 g / L or less, about 10 g / L or less, about 7 g / L or less, about 5 g / L or less, about 4 g / L Below, about 3 g / L or less, about 2 g / L or less, about 1 g / L or less, about 0.5 g / L or less, about 0.1 g / L or less, about 0.01 g / L or more, about 0.05 g / L Above, about 0.1 g / L or more, about 0.2 g / L or more, about 0.5 g / L or more, about 0.7 g / L or more, about 1 g / L or more, about 2 g / L or more, about 3 g / L As mentioned above, it may be about 4 g / L or more, about 5 g / L or more, about 7 g / L or more, or about 10 g / L or more. If the density of microalgae exceeds, for example, 10 g / L, the overall inactivation of chlorophyllase may be inadequate. In one embodiment, the microalgae are not concentrated to the above concentrations after culturing until the treatment to inactivate chlorophyllase. In one embodiment, the microalgae are not diluted after culturing until the treatment to inactivate chlorophyllase.

In one embodiment, the microalgae are not subjected to advanced centrifugation before and / or during the treatment to inactivate chlorophyllase, eg, 50 G or higher, 100 G or higher, 200 G or higher, 500 G or higher, 700 G. Exposed to gravitational acceleration of 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, or 10000G or more. For example, about 10 seconds or more, about 30 seconds or more, about 1 minute or more, about 2 minutes or more, about 5 minutes or more, about 7 minutes or more, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, about 25 minutes or more, about 30 minutes or more, about 40 minutes or more, about 50 minutes or more, about 1 hour or more, about 1.5 hours or more, about 2 hours or more, about 2.5 hours or more, about 3 hours or more, about 4 Do not centrifuge for more than an hour, about 5 hours or more, about 7 hours or more, about 10 hours or more, or about 20 hours or more.

In one embodiment, the method for producing a microalgae product of the present disclosure comprises the step of concentrating the microalgae. Any suitable means known in the art can be used to concentrate the microalgae, including, for example, centrifugation, filtering, media removal (evaporation, etc.), use of flocculants or precipitants, and the like. However, it is not limited to these. Concentration operations can increase the amount of stress in microalgae. In particular, Euglena and Pavlova, which do not have a cell wall, are relatively soft, so the concentration operation may increase pheophorbide production. For Euglena and Pavlova microalgae, which do not have a cell wall and whose pheophorbide production is increased by the concentration operation, the problem that pheophorbide is increased by the cell concentration treatment was found for the first time in the present disclosure. Is. For example, the amount of chlorophyll a + b of chlorella / chlamydomonas depends on the culture conditions and timing, but is often about 25 mg per 1 g of dried algae, but the pavlova used in the examples is 35.3 mg per 1 g of dried algae. Yes, it turned out to be unexpectedly large. Therefore, the present disclosure addresses issues not envisioned in conventional methods involving the enrichment of microalgae, and also provides solutions thereof.

In one embodiment, no step of concentrating microalgae is performed prior to the treatment to inactivate chlorophyllase. When a medium containing unconcentrated microalgae is subjected to a treatment for inactivating chlorophyllase, more reagents and energy may be required as compared with the case of concentrating, which may result in a higher environmental load. However, the inventor has found a culture method capable of growing microalgae (for example, haptophytes) at a high density of 2 g / L or more (for example, the culture apparatus of the present disclosure described in detail below). The method used), the environmental load could be suppressed to the minimum even when the microalgae were subjected to the treatment of inactivating chlorophyllase without concentrating them.

In one embodiment, the step of treating microalgae comprises killing microalgae and / or other microorganisms. When the microalgae product is provided as a food or food additive, the product may be easier to handle in the absence of living organisms. For example, such killing treatment includes, but is not limited to, heat treatment, irradiation, and the like.

In one embodiment, the method for producing a microalgae product of the present disclosure comprises a step of drying the microalgae. It can be dried to the water content of the microalgae products of the present disclosure described above.

In one embodiment, the method for producing a microalgae product of the present disclosure comprises the step of separating the microalgae components. For microalgae, the algae themselves can be useful, but certain components can also be useful. Therefore, the specific component contained in the microalgae may be separated from the other microalgae components to increase the concentration of the specific component. In another embodiment, a specific component (harmful component, etc.) may be separated and removed from the microalgae. For example, since the present inventors have found that pavlova, which is a haptophyte, contains a large amount of fucoxanthin, fucoxanthin may be separated and purified to obtain the microalgae product of the present disclosure.

In one embodiment, the present disclosure may provide a refined product of fucoxanthin. Fucoxanthin is not limited to these, but alcohols such as methyl acetate, ethyl acetate, acetone, chloroform, toluenemethanol, ethanol, propanol, isopropanol and n-butanol, ketones such as methyl ethyl ketone and acetone, methyl acetate and ethyl acetate. Etc., organic chlorine hydrocarbons such as chloroform, aliphatic hydrocarbons such as hexane, and organic solvents such as aromatic hydrocarbons such as benzene and toluene are used alone or in combination of two or more. It can be extracted from algae. In one embodiment, fucoxanthin can be purified from the resulting extract using any known purification means such as chromatography, resin adsorption, crystallization and the like.

(General technology)
The molecular biological, biochemical, and microbiological methods used herein can be well known and commonly used in the art.

(Note)
As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When "within a range" of "two values" is specified in the present specification, the range also includes the two values themselves.

References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference in their entirety to the same extent as each specifically described.

The present disclosure has been described above with reference to preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of claims.

Examples are described below. Specifically, the products described in the examples were used as the reagents, but equivalent products of other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R & D Systems, USCN Life Science INC, etc.) can be substituted.

(Example 1: Discovery of new microalgae)
The inventor prepared and tested various microalgae, found OPMS30543 strain (Pavlova granifera) of the Pavlova family collected in the sea of Okinawa as a microalga that produces high fatty acids, and deposited this strain (consignment number). NBRC 114066). Further investigation of the characteristics of this strain revealed that it produces high fucoxanthin, and it was predicted that it is a microalga with useful properties not found in the past.

For the OPMS30543 strain, two primers for 18S rDNA were designed, two primers for 28S rDNA were designed, and sequence analysis was performed. As a result, the following nucleotide sequences were identified. As a result, OPMS30543 strain was found in P.M. It was suggested that it was granifera.
・ OPMS30543, 18S rDNA
(SEQ ID NO: 1)

OPMS30543, 28S rDNA
(SEQ ID NO: 2)

Since the microalgae of the Pavlova family were predicted to have useful properties, Pavlova microalgae were classified as Pavlova microalgae. gyrans (OPMS30543X strain), P. et al. pinguis and P. Luther was obtained, bred and the properties of these species were also investigated. As a result, it was confirmed that all of these species produce fucoxanthin. Here, the OPMS30543X strain is a strain based on the NBRC102809 strain (Pavlova gyrans).

Breeding was carried out as follows. The basic culture conditions are shown in the table below.
* IMK medium (Daigo IMK medium, Nihon Pharmaceutical Co., Ltd., Tokyo) is dissolved in Milli-Q water (1 bag is dissolved in 500 mL water) so as to be a 200-fold concentrated solution as used, and after dissolution, filter filtration (0.22 μm) is performed. ) The one that was sterilized and stored in a refrigerator and shaded was used.

Subculture was performed after culturing for 10 to 12 days. Here, this period may be changed according to the proliferative property. At the time of subculture, about 5% to 20% of the culture immediately before the subculture operation was transferred to a new medium while appropriately changing depending on the growth condition. During the subculture operation, stirring such as aeration was stopped and the cells were allowed to stand for a while, and the part close to the supernatant of the culture (where there were many swimming cells) was selectively collected.

When the condition of the culture deteriorated extremely, the surviving cells were picked up by micromanipulation or the like depending on the situation, and the return culture was carried out in an environment where the culture scale was lowered to a minimum of 2 mL. When the growth of mixed bacteria became intense, ampicillin and hypochlorous acid were sometimes added in appropriate amounts.

(Example 2: Culture of new microalgae)
OPMS30543 strain and OPMS30543X strain are dissolved in an aqueous solution of artificial seawater base Marine Art SF-1 (Tomita Pharmaceutical Co., Ltd., Tokushima) in water so that the concentration is 50% in seawater, so that the concentration is twice the usage regulation. In the culture solution prepared by adding the components of IMK medium (Nihon Pharmaceutical Co., Ltd., Osaka), the cells were cultured while adjusting the pH to be maintained at about 8 by adding CO ₂ . As the culture tank, an acrylic bioreactor as shown in FIG. 3 was used, and the culture was carried out outdoors.
The OPMS30543 strain and the OPMS30543X strain had low adhesion between the wall surface of the culture tank or algae cells, and could be stably cultured.

(Example 3: Component analysis of novel microalgae)
The microalgae cultured in Example 2 were collected when the density exceeded 2 g / L, and these were heated, concentrated, and lyophilized to prepare samples of OPMS30543 strain and OPMS30543X strain. For these samples, we requested the Japan Food Research Laboratories (Tokyo) to analyze the components of OPMS30543 strain and OPMS30543X strain (presumed to be Pavlova gyrans). As a result, the following components were found as typical components.

(Example 4: Microalgae that highly produce fucoxanthin)
By outdoor culturing of the Pavlova strain by the method described in Example 2, a high fucoxanthin productivity of 23.22 mg / g per dry weight of microalgae was sometimes confirmed.

(Example 5: Treatment for microalgae products)
As described above, the OPMS30543 strain contains about 2250 mg / 100 g (dry weight) of chlorophyll, and the haptophyte Pavlova may contain more chlorophyll than ordinary microalgae such as chlorella. It is known that chlorophyll is converted to pheophorbide by being metabolized in microalgae, and it is known that pheohorbide causes photosensitivity and the like, and it is desirable that intake by animals is restricted. Pheophorbide can be examined as follows.

-Quantitative method of existing pheophorbide The amount of chlorophyll decomposition product transferred from the ether extraction solution of the dye to 17% hydrochloric acid is converted into pheoformide a (mg%).
Weigh 100 mg of dried microalgae into a mortar, add about 0.5 g of sea sand and 20 ml of 85% (V / V) acetone, grind quickly, and then transfer the supernatant to a centrifuge tube. Further, 10 ml of acetone is added to the residual test and the same operation is performed, the supernatant is transferred to a centrifuge tube, and this operation is repeated once again. Then, the mixture is centrifuged (3000 rpm, 5 minutes), and the supernatant is transferred to a separating funnel containing 30 ml of ethyl ether. Then, 50 ml of a 5% sodium sulfate solution is added to this ether-acetone mixture, and the mixture is gently shaken to discard the sodium sulfate layer. Further, after repeating this washing operation three times, anhydrous sodium sulfate is added for dehydration, the ether layer is taken out, and the total amount is adjusted to 50 ml with ethyl ether to prepare a dye stock solution. 20 ml of this dye stock solution is sequentially shaken and extracted with 20 ml of 17% hydrochloric acid and then 10 ml of the same hydrochloric acid, and then the hydrochloric acid layer is transferred to a separatory funnel containing 150 ml of saturated sodium sulfate solution and 20 ml of ethyl ether. This is extracted by shaking, the ether layer is separated, and ethyl ether is added thereto to make the total volume 20 ml, which is used as a decomposition product extract. The decomposition product extract is diluted with ethyl ether to exactly the required concentration and the absorbance at 667 nm is measured. The amount of chlorophyll decomposition product is calculated from the absorbance of the standard pheoformide a, and the amount is used as the existing amount of pheoformide (mg%). The absorbance of the standard pheoformide a was determined by S. R. A 667 nm specific extinction coefficient of Pheoformide a from Brown (J. Fish Res. Bd. Canada 25, 523-540.1968) was used with a specific extinction coefficient of 70.2 (0.1% solution, 1 cm absorbance).

-Quantitative method of chlorophyllase activity Incubate in hydrous acetone and convert the increased amount of chlorophyll decomposition product production into the amount of pheophorbide a (mg%).
100 mg of dried microalgae is precisely weighed, 10 ml of cold M / 15 phosphate buffer (pH 8.0) and a mixture of acetone (7: 3) are added thereto, and the mixture is incubated at 37 ° C. for 3 hours. After that, it is weakly acidic with 10% hydrochloric acid, the amount of pheophorbide is measured by the same method as the method for quantifying existing pheophorbide, the amount of existing pheophorbide is subtracted from the measured value to obtain the amount of increase, and the increased amount is defined as the chlorophyllase activity.

10 L (0.516 g / L) of the Pavlova strain culture was centrifuged and concentrated 100-fold. The concentrate was then autoclaved (100 ° C. for 1 minute). Then, the existing pheophorbide and chlorophyllase activity were measured, respectively. Total pheophorbide amount = existing pheophorbide amount + chlorophyllase activity.

In the unconcentrated culture medium, both the existing pheophorbide and the total pheophorbide were low, but when the centrifugation treatment was performed, the amount of the existing pheophorbide increased. Since the chlorophyllase activity was suppressed by heating, the total amount of pheophorbide was equivalent to the amount of existing pheophorbide.

In addition, an increase in the amount of existing pheophorbide was observed when the culture of the Pavlova strain was concentrated 100-fold with an MF membrane (about 12 hours or more) and when the culture of the Pavlova strain was passed through a cascade pump. It was.

On the other hand, chlorophyllase, which catalyzes the production of pheophorbide, was considered to be inactivated by heating, so it was tested whether heating before concentration suppresses pheoformide production.

The culture of the Pavlova strain (0.592 g / L) was sent to an oil heater (105 ° C.) at a constant rate (10, 20, 40 or 80 mL / min) through a tube to adjust the heating time. The heating liquid sent from the oil heater was collected in a bottle on ice. The heat treatment time under each condition was about 8 minutes, about 4 minutes, about 2 minutes and about 1 minute. Each collected sample was centrifuged and the existing pheophorbide, total pheoformide and chlorophyllase activity were measured. As a result, it was found that the total amount of pheophorbide did not increase even if the centrifugation treatment was performed after sufficient heating in advance.

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

(Example 7: Acquisition of further Pavlova shares)
A related strain or an inducible strain can be obtained as follows.
Obtain microalgae from brackish water deposits or seawater. The properties such as fatty acid productivity and fucoxanthin productivity of the acquired microalgae strains will be investigated. If necessary, the acquired microalgae strain is bred (for example, the breeding method shown in Example 1) to obtain a new strain.
The breeding method (screening method for new strains) is as follows.
The following conditions ・ Seawater concentration: 50% seawater ・ Medium concentration: IMK medium double concentration ・ Culture solution volume: 800 mL
-Light source: Side: Fluorescent lamp (100 to 150 μmol)
・ Light-dark cycle: 12 hours each for light and dark ・ pH: pH about 7.4 at the start of each culture
・ Aeration: Implementation ・ Mechanical stirring: None ・ Culture period: Conducted for a period of about 10 days ・ Culture temperature: 25 ° C to 28 ° C
Repeated culture passage of Pavlova microalgae cell lines in
During each subculture, cells floating from the supernatant portion of the culture are selectively used, thereby concentrating only cell lines that survive well under the culture conditions.
Then, the step of repeating the above culture passage is carried out under conditions that satisfy the implementation with a period of at least: -seawater concentration: 50% seawater-medium concentration: IMK medium double concentration-culture period: about 10 days.
Then, when the cell density exceeds 2 g / L, the cell line is collected and the candidate cell lines fucoxanthin, eicosapentaenoic acid, γ-aminobutyric acid, hydroxyproline, calcium, dietary fiber, chlorophyll and amino acid scores are obtained. Measure according to the above-mentioned embodiment.
The measured candidate cell line (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 γ-aminobutyric acid per 100 g of dried cells.
(Iv) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(V) Produces 500 mg or more of calcium per 100 g of dried cells.
(Vi) Produces 5 g or more of dietary fiber per 100 g of dried cells,
(Vii) produces about 2 g or more of chlorophyll per 100 g of dried cells, and (viii) has an amino acid score of 85 or more.
A cell line satisfying at least one of the above conditions is selected as a further novel inducer.
According to this method, several strains may be obtained by one breeding.

(Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of claims. The patents, patent applications and other documents cited herein should be incorporated herein by reference in their content as they are specifically described herein. Is understood.

The present disclosure provides novel microalgae and microalgae products using the same, and since the microalgae produce various useful ingredients and are edible, they can be usefully used in various products, especially foods. it can.

OPMS30543 strain (NBRC 114066)

Claims (26)

A microalga of the Pavlova family that produces 1 g or more of fucoxanthin per 100 g of dried cells. A microalga of the Pavlova family that produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells. A microalga of the Pavlova family that produces 50 mg or more of γ-aminobutyric acid per 100 g of dried cells. A microalga of the Pavlova family that produces 100 mg or more of hydroxyproline per 100 g of dried cells. A microalga of the Pavlova family that produces 1 g or more of fucoxanthin per 100 g of dried cells and 50 mg or more of γ-aminobutyric acid per 100 g of dried cells and / or 100 mg or more of hydroxyproline per 100 g of dried cells. Pavlova microalgae that produce 500 mg or more of calcium per 100 g of dried cells. Microalgae of the Pavlova family that produce 5 g or more of dietary fiber per 100 g of dried cells. Pavlova microalgae that produce about 2 g or more of chlorophyll per 100 g of dried cells. Microalgae of the Pavlova family with an amino acid score of 85 or higher. 1 g or more of fucoxanthin is produced per 100 g of dried cells, and the following characteristics (a) to (g):
(A) Produces 3 g or more of eicosapentaenoic acid per 100 g of dried cells.
(B) Produces 50 mg or more of γ-aminobutyric acid per 100 g of dried cells.
(C) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(D) Produces 500 mg or more of calcium per 100 g of dried cells.
(E) Produces 5 g or more of dietary fiber per 100 g of dried cells.
(F) Produces about 2 g or more of chlorophyll per 100 g of dried cells, and (g) has an amino acid score of 85 or more.
Pavlova microalgae having one or more of the properties of. The microalga according to any one of claims 1 to 10, which belongs to the genus Pavlova. P. granifera or P.I. The microalgae according to any one of claims 1 to 10, which are gyrans. Microalgae which is OPMS30543 strain (strain specified by accession number NBRC 114066). Any of claims 1 to 12, which is an inducible strain of OPMS30543 strain (strain specified by accession number NBRC 114066) and has the characteristics of the microalgae according to any one or more of claims 1 to 10. The microalgae described in item 1. A food containing the microalgae according to any one of claims 1 to 14. The food according to claim 15, wherein the amount of pheophorbite per 100 g of the microalgae is 100 mg or less. A microalgae product comprising the purified microalgae according to any one of claims 1 to 14. A microalgae product comprising the fucoxanthin-purified microalgae according to any one of claims 1 to 14. The microalgae product according to claim 18, wherein the fucoxanthin purified product has a fucoxanthin purity of 5 to 70%. The microalgae product according to claim 18, wherein the fucoxanthin purity of the purified fucoxanthin product is about 70% or more. The microalgae product according to any one of claims 17 to 20, wherein the microalgae or a purified product thereof is immersed in oil. The microalgae product according to any one of claims 17 to 21, wherein the microalgae or a purified product thereof is encapsulated. A method for breeding microalgae of the order Pavlova under the following conditions: ・ Seawater concentration: 50% seawater ・ Medium concentration: IMK medium double concentration ・ Culture solution volume: 800 mL
-Light source: Side: Fluorescent lamp (100 to 150 μmol)
・ Light-dark cycle: 12 hours each for light and dark ・ pH: pH about 7.4 at the start of each culture
・ Aeration: Implementation ・ Mechanical stirring: None ・ Culture period: Conducted for a period of about 10 days ・ Culture temperature: 25 ° C to 28 ° C
A step of repeating the culture passage of a Pavlova microalgae cell line under the condition of satisfying at least one of the above.
Here, for each subculture, cells floating from the supernatant portion of the culture are selectively used, thereby concentrating only cell lines that survive well under the culture conditions. how to. Repeat the steps of repeating the culture passage at least: -Seawater concentration: 50% seawater-Medium concentration: IMK medium double concentration-Culture period: Conditions that satisfy one or more of the implementations with a period of about 10 days. 23. The method according to claim 23. When the cell density exceeds 2 g / L, the cell line is recovered and fucoxanthin, eicosapentaenoic acid, and γ-aminobutyric acid of the cell line are added to the group consisting of hydroxyproline, calcium, dietary fiber, chlorophyll, and amino acid score. The step of measuring at least one selected
(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 γ-aminobutyric acid per 100 g of dried cells.
(Iv) Produces 100 mg or more of hydroxyproline per 100 g of dried cells.
(V) Produces 500 mg or more of calcium per 100 g of dried cells.
(Vi) Produces 5 g or more of dietary fiber per 100 g of dried cells,
(Vii) produces about 2 g or more of chlorophyll per 100 g of dried cells, and (viii) has an amino acid score of 85 or more.
The method according to claim 23 or 24, which comprises the step of selecting a cell line satisfying at least one of the above conditions as a breeding strain. Microalgae of the order Pavlova produced by the method according to any one of claims 23 to 25.