JP7097014B2 - Algae and their use - Google Patents

Description

IPOD FERM P-22337

The present invention relates to algae and their use. Specifically, the present invention relates to algae, nutritional supplements, nutritional component supplementation compositions, and methods for producing nutritional components.

From the viewpoint of depletion of petroleum resources and reduction of greenhouse gases, there is a great deal of interest in the development of biomass fuels, and research is currently being conducted to produce fuel oils from microalgae (see, for example, Patent Document 1).

On the other hand, many microalgae produce functional substances such as linolenic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and other functional substances such as ω3 or ω6 unsaturated fatty acids, and these are various. Has excellent functionality. Therefore, attention is being paid to the use of microalgae in health and functional foods and pharmaceuticals.
In addition, microalgae species that produce biofuel oils or functional substances have been reported so far (see, for example, Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 9-009953

Traller JC, et al., “Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype.”, Biotechnol Biofuels, Vol. 9, No. 258, 2016.

Currently, the microalgae that are industrially put into practical use are limited to several kinds of algae, and there is a demand for new microalgae species that produce hydrocarbons, functional oils, or functional substances.

The present invention has been made in view of the above circumstances, and is a novel microalgae that can be industrially used, a novel nutritional supplement using the algae, a nutritional component supplementing composition containing the nutritional supplement, and the microalgae. Provided is a method for producing a nutritional component using.

As a result of diligent research to achieve the above object, the present inventors have found that Cyclotella sp. 3FG strain (FERM P-22337) is a hydrocarbon, α-linolenic acid, DHA, EPA, etc. They have found that they produce various useful substances such as functional oils of the above, and have completed the present invention.

That is, the present invention includes the following aspects.
The alga according to the first aspect of the present invention is Cyclotella sp. 3FG strain (FERM P-22337).

The nutritional supplement according to the second aspect of the present invention includes the cyclotera SP 3FG strain according to the first aspect and an extract thereof.

The nutritional component supplementing composition according to the third aspect of the present invention contains the nutritional supplement according to the second aspect.
In the nutritional component supplement composition according to the third aspect, the nutritional component may be an ω3 unsaturated fatty acid.
The ω3 unsaturated fatty acid may be one or more selected from the group consisting of α-linolenic acid, docosahexaenoic acid and eicosapentaenoic acid.
The composition for supplementing nutritional components according to the third aspect may be a food.
The nutritional component supplement composition according to the third aspect may be a functional food or a dietary supplement.
The nutritional component supplementing composition according to the third aspect may be feed or pet food.

The method for producing a nutritional component according to the fourth aspect of the present invention is a method comprising a culture step for culturing the cyclotera sp. 3FG strain according to the first aspect.
The method for producing a nutritional component according to the fourth aspect further comprises, after the culture step, a crushing step of crushing the Cyclotera SP 3FG strain to obtain a cell crushed product, and separating the nutritional component from the cell crushed product. The separation step may be provided in this order.
The nutritional component may be ω3 unsaturated fatty acid.
The ω3 unsaturated fatty acid may be one or more selected from the group consisting of α-linolenic acid, docosahexaenoic acid and eicosapentaenoic acid.

According to the above aspect, a novel microalgae that can be industrially used, a novel nutritional supplement using the algae, a nutritional component supplementing composition containing the nutritional supplement, and a method for producing a nutritional component using the microalgae are provided. can do.

It is a micrograph of the cyclotera SP 3FG strain. The image above is a photomicrograph in the bright field. The image below is a photomicrograph in the dark field. The molecular phylogenetic tree of the genus Cyclotera based on the base sequence of 18S ribosome is shown. The known cyclotera meneginiana G188D (AY496207) strain, cyclotera cryptoca (KY364697) strain and cyclotera cryptoca CCAP1070 / 2 (FR856514) strain were surrounded by a dotted line, and the 3FG strain was surrounded by a solid line. 3 is a graph showing the growth rate of the 3FG strain at a light intensity of 65 μmol / m ² / s or 150 μmol / m ² / s in Examples. It is a graph which shows the growth rate of the 3FG strain in fossil seawater or artificial seawater to which F medium or IMK medium was added in an Example. It is a graph which shows the growth rate of the Tahitian strain in the artificial seawater or fossil seawater to which F medium or IMK medium was added in an Example.

≪Algae≫
The algae according to one embodiment of the present invention is Cyclotella sp. 3FG strain (FERM P-22337) (hereinafter, may be referred to as "3FG").

The genus Cyclotera is taxonomically classified into the phylum Yellow, Bacillariophyceae, Centrales, Coscinodiscineae, and Thalassiosiraceae. The genus Cyclotera is also called the genus Diatom. As the algae belonging to the family Thalassiosila, in addition to the genus Cyclotera, the genus Stephanodiscus and the genus Thalassiosila are known. Algae belonging to the family Tarashioshira are widely inhabited in freshwater, ocean, etc. in nature.

The 3FG strain belongs to the genus Cyclotera and produces various useful substances such as hydrocarbons and functional oils such as α-linolenic acid, DHA and EPA. Conventionally, it has been known that known algae belonging to the genus Cyclotera produce useful substances as described above, but those producing high concentrations of DHA and EPA have not been known. Among the genus Cyclotera, those known to produce functional oils such as α-linolenic acid, DHA, and EPA together with hydrocarbons are Cyclotella cryptica and Cyclotera meneginiana. (Cyclotella meneghiniana) only. Therefore, the 3FG strain is distinguished from other algae belonging to the genus Cyclotera in that it produces high concentrations of DHA and EPA together with hydrocarbons.

The 3FG strain is a diatom isolated from the surface water at the mouth of the Jinzu River in Toyama Bay, Toyama Prefecture, Japan. The 3FG strain was deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-22337 on June 27, 2017. Has been done.

The 3FG strain has a light brown color at the initial stage of culture, but the color is almost indistinguishable unless colonies are mixed. On the other hand, as the culture time increases, colonies are formed and mixed, resulting in a dark brown color.
The shape and size of the cells are rectangular parallelepiped, columnar or spherical, and cells of different sizes are mixed. The difference in shape is presumed to be due to the cell cycle, and the spherical one is considered to be the mother cell before division. Further, as the culture time increases, cells escape from the cell shell and the number of spherical cells increases, and further, a plurality of cells are fused to form spherical cells larger than a single cell.
Specifically, in the case of a rectangular parallelepiped or columnar cell, the average major axis is about 10 to 15 μm, and the average minor axis is about 3 to 5 μm. The spherical shape has an average diameter of 8 to 11 μm, and the fused spherical shape has an average diameter of about 50 μm.

The 3FG strain grows in two divisions. The 3FG strain is suitably grown by using paleo-seawater.
The term "old seawater" as used herein means that old seawater is trapped and stored in a gap in the stratum. Paleo seawater is also called fossil seawater. In this stored seawater, it is considered that the components contained in the seawater cause a chemical reaction due to the stored stratum and the pressure and temperature due to its depth. Therefore, the components contained in the current seawater and the components contained in the old seawater are different.

The optimum temperature for culturing the 3FG strain is about 25 ° C., and the optimum pH is around 8.4.

As a result of molecular phylogenetic analysis based on the base sequence of 18S ribosomal DNA, the 3FG strain was classified into the genus Cyclotera. The nucleotide sequence of 18S ribosomal DNA of the 3FG strain is shown in SEQ ID NO: 1.

Examples of the algae that are closely related to the 3FG strain include algae having a base sequence of 18S ribosomal DNA having 90% or more identity with SEQ ID NO: 1. The identity between the base sequence of the 18S ribosomal DNA possessed by the algae and the base sequence set forth in SEQ ID NO: 1 is preferably 95% or more, more preferably 97% or more, and more preferably 98% or more. It is more preferable, and 99% or more is particularly preferable.

In addition, the identity (homology) between the base sequences is obtained by juxtaposing the two base sequences with a gap in the portion corresponding to the insertion and deletion so that the corresponding bases match most. It is calculated as the ratio of matching bases to the entire base sequence excluding the gap of. The identity between the base sequences can be determined by using various homology search software known in the art. For example, the identity value of the base sequence can be obtained by calculation based on the alignment obtained by the known homology search software BLASTn.

In addition, the base sequence of 18S ribosomal DNA possessed by algae can be obtained by a known method. For example, DNA is extracted from target algae cells by a known method, and a DNA fragment of 18S ribosomal DNA is amplified by a PCR method or the like. Next, by analyzing the base sequence of the amplified DNA fragment with a DNA sequencer, the base sequence of 18S ribosomal DNA of the target algae can be obtained. Examples of the primer for amplifying 18S ribosomal DNA include the primers used in the examples of the present specification.

Further, as the algae of the present embodiment, a mutant strain of the 3FG strain is also mentioned as a suitable example.
As used herein, the term "variant strain" means that the genome of the original algae strain (including nuclear genome, chloroplast genome, mitochondrial genome, hereinafter the same) is spontaneously or artificially mutated. Means an algae strain. The artificial method for causing a mutation in the genome is not particularly limited, and examples thereof include chemical treatment with ultraviolet irradiation, irradiation, nitrite and the like; genetic engineering methods such as gene transfer and genome editing.
In the present specification, the "variant strain of 3FG strain" is an alga strain in which the genome of the 3FG strain is mutated and has a nutritional component composition similar to that of the 3FG strain. Point to. For example, the content of EPA with respect to the total amount of oil components produced by the 3FG strain may be 10% by mass or more and 50% by mass or less, 20% by mass or more and 40% by mass or less, and 25% by mass or more and 35% by mass or less. It may be less than or equal to%. Further, for example, the content of DHA with respect to the total amount of oil components produced by the 3FG strain may be 0.5% by mass or more and 10% by mass or less, or 1% by mass or more and 7% by mass or less. It may be 5% by mass or more and 5% by mass or less.
As the mutant strain of the 3FG strain, the ratio of the mutation of the 3FG strain to the entire genome is preferably 10% or less, more preferably 5% or less, and further preferably 3% or less. It is preferably 2% or less, or particularly preferably 1% or less.

The algae of this embodiment can grow under medium temperature environment (15 to 35 ° C.) and weakly basic (pH 7.8 to 8.8) conditions. Therefore, it is possible to change the culture conditions according to the region and season, and it is suitable for outdoor mass culture.
Furthermore, it has high salt tolerance and can grow even under high salt conditions such as seawater (500 mM NaCl, etc.). Further, the light intensity can be grown in the range of 1500 to 5000 lux, and can be grown even under strong light.

The algae of the present embodiment contain hydrocarbons that can be fuel oils, and nutritional components such as amino acids, vitamins, proteins, lipids (particularly, functional oils such as α-linolenic acid, DHA, and EPA), and dietary fiber. Abundantly contained.
In particular, as shown in Examples described later, EPA is compared with the conventional EPA and DHA-producing algae known as Isochrysis sp. Tahiti strain "Zoyoshoku Research Institute Preservation Strain". It has been confirmed that it is contained in a high concentration.

In the present specification, the "functional oil" means an edible oil having functions such as a neutral fat lowering effect, a cognitive function improving effect, and a blood cholesterol lowering effect. Specific examples of the functional oil include ω3 fatty acids such as α-linolenic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), and ω6 fatty acids such as linoleic acid and arachidonic acid. Can be mentioned.
The algae of this embodiment contain DHA, EPA and the like as functional oils, as shown in Examples described later.

The algae of this embodiment contain a hydrocarbon that can be a fuel oil. Therefore, the biomass fuel can be produced by using the algae of the present embodiment.
Furthermore, the algae of the present embodiment contain various nutritional components such as functional oils such as DHA and EPA. Therefore, when the algae of the present embodiment are ingested by humans or non-human animals as a nutritional supplement described later, the above-mentioned nutritional components can be absorbed into the body. In addition, an extract containing a nutritional component can be appropriately prepared from the algae of the present embodiment.

The algae of the present embodiment can be cultivated using a medium for culturing microalgae. The medium is not particularly limited, and examples thereof include an inorganic salt medium containing a nitrogen source, a phosphorus source, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, etc.) and the like. For example, examples of the nitrogen source include ammonium salts, nitrates, nitrites, ureas, amines and the like. Further, examples of the phosphorus source include phosphates and the like. The medium may be a liquid medium or an agar medium.
Specific examples of the medium include the KWSW medium shown in Examples described later, Marine broth 2216 and Marine Agar 2216 manufactured by DIFCO.

As described above, the algae of the present embodiment can be grown under a relatively wide range of culture conditions. As the pH condition, pH 7.8 to 8.8 can be exemplified, and pH 8.0 to 8.6 is preferable.
As the temperature condition, 15 to 35 ° C. can be exemplified, and 20 to 30 ° C. is preferable.
As the light intensity, 1500 to 5000 lux can be exemplified, and 2500 to 3500 lux is preferable. When culturing outdoors, it may be cultivated in sunlight. When culturing indoors, culturing may be performed with continuous light, or a light-dark cycle (12L: 12D, etc.) may be provided.

As described above, the algae of the present embodiment contain abundant nutritional components and are suitable for outdoor mass culture, and therefore can be industrially used as a nutritional supplement described later. In addition, the nutritional component can be extracted from the algae of the present embodiment, and the nutritional component can be absorbed into the body even when ingested as it is by a human or a non-human animal.

≪Nutrition ≫
The nutritional supplement according to one embodiment of the present invention includes the Cyclotera SP 3FG strain of the above-mentioned embodiment or an extract thereof.

The algae contained in the nutritional supplement of the present embodiment are the algae of the above-mentioned embodiment. That is, a 3FG strain and a mutant strain thereof (hereinafter referred to as "this alga"). Preferred examples of the algae contained in the nutritional supplement of the present embodiment are the same as those exemplified above.
The algae can be used as the nutritional supplement of the present embodiment by culturing and growing them in an appropriate medium, recovering them by a known method such as centrifugation or filtration, and appropriately washing and drying them. ..

In addition, the nutritional supplement of the present embodiment may contain an extract of the algae in place of the algae or together with the algae.
As used herein, the term "extract of the algae" refers to cells of the algae that have been physically or chemically treated to extract intracellular components. For example, the extract of the algae may be a cell crushed product obtained by disrupting the cells of the algae by physical treatment or chemical treatment. Further, the extract of the algae may be one in which the cell crushed product is concentrated, or the product in which the solid content is removed from the cell crushed product, and some components are added from the cell crushed product. It may be separated.

The method of physical treatment or chemical treatment of cells is not particularly limited, and methods generally used for cell disruption can be used. Examples of the physical treatment include cell crushing with glass beads, a mortar, ultrasonic treatment, a French press, a homogenizer, and the like. Examples of the chemical treatment include a neutralization treatment, a hypotonic treatment, a freeze-thaw treatment, and the like.
When concentrating the cell disrupted product, the concentration method is not particularly limited, and a generally used concentration method may be used. Examples of the method for concentrating the cell crushed product include drying, freeze-drying, vacuum-drying and the like.
Further, when the solid content is removed from the cell crushed product, the method for removing the solid content is not particularly limited, and a method generally used for removing the solid content or the like can be used. Examples of the method for removing the solid content include filtration, centrifugation and the like.
When separating a part of the components from the cell disrupted product, the separation method is not particularly limited, and a method generally used for separation and purification of biochemical substances can be used. Examples of the separation method include salting out, dialysis, solvent extraction, adsorption, various types of chromatography (gas chromatography, thin layer chromatography, ion exchange chromatography, high performance liquid chromatography, adsorption chromatography, etc.) and the like. These methods may be used alone or in combination of two or more treatments. However, those purified into a single component are excluded from the "extract of this algae". The extract of this alga preferably contains 2 or more types of cellular components of this alga, more preferably 4 or more types, and further preferably 6 or more types.

As described above, since the algae contain a large amount of nutritional components such as functional oil, the extract of the algae also contains a large amount of nutritional components such as functional oil. Therefore, it can be used as a nutritional supplement of the present embodiment.

The nutritional supplement of the present embodiment may contain other components as appropriate in addition to the algae or an extract thereof. Examples of other components include, but are not limited to, pharmaceutically acceptable carriers and the like.
The "pharmaceutically acceptable carrier" means a carrier that does not inhibit the function of the nutritional component contained in the algae and does not show substantial toxicity to the administration target thereof.
Further, "does not show substantial toxicity" means that the component does not show toxicity to the administration subject at the dose normally used.
The pharmaceutically acceptable carrier is not particularly limited, and is, for example, an excipient, a binder, a disintegrant, a lubricant, an emulsifier, a stabilizer, a diluent, an oily base, a thickener, and an antioxidant. , Reducing agents, oxidizing agents, chelating agents, solvents and the like. As the pharmaceutically acceptable carrier, one type may be used alone, or two or more types may be used in combination.

The content of the algae or its extract in the nutritional supplement of the present embodiment is not particularly limited, and can be appropriately selected in the range of, for example, 1 to 100% by mass. The content of the algae or its extract in the nutritional supplement of the present embodiment is preferably 50 to 100% by mass, more preferably 60 to 99% by mass, still more preferably 70 to 99% by mass.
The algae or its extract can be appropriately mixed with other components to form dry powder, granules, tablets, jellies, liquids, capsules and the like according to a conventional method.

Since the nutritional supplement of the present embodiment contains a large amount of nutritional components such as functional oil, it can be used as a nutritional supplement for humans and animals other than humans. Examples of the nutritional component supplied by the nutritional supplement of the present embodiment include amino acids, vitamins, proteins, lipids (particularly, functional oils), dietary fiber and the like. Above all, the nutritional supplement of the present embodiment can be suitably used for supplementing functional oil (DHA, EPA, etc.).
The nutritional supplement of the present embodiment can be suitably used, in particular, for supplementing DHA and EPA.

Moreover, the nutritional supplement of this embodiment may be blended and used in the composition for supplementing nutritional components described later. By blending the nutritional supplement of the present embodiment, a composition rich in nutritional components such as functional oil can be prepared.

≪Nutrition ingredient supplement composition≫
The nutritional component supplementing composition according to one embodiment of the present invention contains the nutritional supplement of the above embodiment.

As used herein, the term "nutrition component supplementing composition" refers to a composition used by a human or a non-human animal to take in a nutritional component into the body. The uptake of nutritional components into the body may be oral or parenteral.

Examples of the nutritional component contained in the nutritional component supplement composition of the present embodiment include amino acids, vitamins, proteins, lipids (particularly, ω3 unsaturated fatty acids such as DHA and EPA), dietary fiber and the like. These components are nutritional components contained in a large amount of the above-mentioned nutritional supplements. Among these, the composition for supplementing nutritional components of the present embodiment is characterized in that it contains a large amount of ω3 unsaturated fatty acids.
In particular, among the ω3 unsaturated fatty acids, it is characterized in that it contains a large amount of DHA and EPA.
For example, DHA is known to have a cognitive function improving effect. In addition, EPA is known to have a triglyceride lowering effect.
Therefore, by ingesting the nutritional component supplementing composition of the present embodiment, the physical condition improving effect and the like possessed by the nutritional component as described above can be obtained.

The nutritional component supplementing composition of the present embodiment is not particularly limited as long as it is used by a human or a non-human animal to take in the nutritional component into the body. Examples of the nutritional component supplementing composition of the present embodiment include foods, feeds, pet foods, cosmetics and the like.

<Food>
The nutritional component supplement composition of the present embodiment may be a food.
When the nutritional component supplementing composition of the present embodiment is a food product, the above-mentioned nutritional supplement may be added to the food product as a food additive. By adding the above-mentioned nutritional supplement to a food product, it is possible to prepare a food product in which the nutritional component contained in the above-mentioned nutritional supplement is fortified. Therefore, the food additive according to the embodiment of the present invention includes the algae or an extract thereof.
The food of the present embodiment can be produced according to a known method according to the type of food by adding the above-mentioned nutritional supplement to the food raw material and adding other food additives as appropriate.

In the food of the present embodiment, the type of food is not particularly limited. Foods include, for example, various noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles, cup noodles; carbohydrates such as bread, wheat flour, rice flour, hot cakes, mashed potatoes; green juice, soft drinks, carbonated drinks, etc. Beverages such as nutritional drinks, fruit drinks, vegetable drinks, lactic acid drinks, milk drinks, sports drinks, tea and coffee; bean products such as tofu, okara and natto; various soups such as curry roux, stew roux and instant soup; ice cream Cold confectionery such as cream, ice sherbet, shaved ice; confectionery such as candy, cookies, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and other baked confectionery; kamaboko, hampen, ham, sausage Fisheries or processed livestock foods such as; processed milk, fermented milk, butter, cheese, yogurt and other dairy products; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils and fat processed foods; sauces, dressings , Miso, soy sauce, sauce and other seasonings; various retort foods, sprinkles, other processed foods such as pickles, etc. can be mentioned, but are not limited thereto.

In the above-mentioned foods, the content of the above-mentioned nutritional supplement is not particularly limited, and the content may be appropriately set according to the type of food. For example, in consideration of the flavor of the food, the content of the above-mentioned nutritional supplement in the food may be 0.01 to 30% by mass as the content of the algae or its extract. From the viewpoint of food flavor and the like, 0.05 to 20% by mass is preferable, 0.1 to 15% by mass is more preferable, 0.1 to 10% by mass is further preferable, and 0.1 to 5% by mass is particularly preferable. preferable.

Alternatively, the nutritional supplement composition of the present embodiment may be a functional food or a dietary supplement. The functional food or dietary supplement may be in the form of general foods as described above, or may be in the form of dry powder, granules, tablets, jellies, drinks and the like. In this case, the above-mentioned nutritional supplement and other components may be mixed as appropriate to form a dry powder, granules, tablets, jelly preparations, drink preparations and the like according to a conventional method.
The other components are not particularly limited, and examples thereof include pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carrier include the same carriers as those mentioned in the above-mentioned "<< nutritional supplement >>". Further, in order to improve the flavor and the like, sweeteners, flavoring agents, various seasonings, flavorings, oils and fats, other food additives and the like may be used as other ingredients. As for the other components, one kind may be used alone, or two or more kinds may be used in combination.

In the above-mentioned functional foods or dietary supplements, the content of the above-mentioned nutritional supplements is not particularly limited, and the content may be appropriately set according to the type of the functional food or the dietary supplement. For example, when the functional food or dietary supplement is in the form of dry powder, granules, tablets, etc., the content of the above-mentioned nutritional supplement in the functional food or dietary supplement is the content of the algae or its extract. The amount is exemplified by 0.1 to 99% by mass. From the viewpoint of efficient supplementation of flavor and nutritional components, 1 to 90% by mass is preferable, 10 to 85% by mass is more preferable, 20 to 85% by mass is further preferable, and 25 to 85% by mass is particularly preferable. When the functional food or dietary supplement is in the form of a jelly or drink, the content of the above-mentioned nutritional supplement in the functional food or dietary supplement is the content of the algae or its extract. , 0.05-80% by mass is exemplified. From the viewpoint of efficient supplementation of flavor and nutritional components, 1 to 75% by mass is preferable, 10 to 70% by mass is more preferable, 15 to 70% by mass is further preferable, and 20 to 70% by mass is particularly preferable.

The food of the present embodiment can be ingested in order to efficiently replenish the above-mentioned nutritional components contained in the algae.

<Feed, pet food>
The nutritional component supplementing composition of the present embodiment may be feed or pet food.
When the composition for supplementing nutritional components of the present embodiment is feed or pet food, the above-mentioned nutritional supplement may be added to feed or pet food as a feed additive or pet food additive. By adding the above-mentioned nutritional supplement to the feed or pet food, a feed or pet food enriched with the nutritional components contained in the above-mentioned nutritional supplement can be prepared. Therefore, the feed additive or pet food additive according to the embodiment of the present invention includes the algae or an extract thereof.

In the feed or pet food of the present embodiment, the above-mentioned nutritional supplement is added to the feed raw material or pet food raw material, and other feed additives or pet food additives are appropriately added, depending on the type of feed raw material or pet food. It can be manufactured according to a known method.

The type of animal to which the feed or pet food of this embodiment is given is not particularly limited. Examples thereof include livestock (cattle, pigs, chickens, horses, sheep, goats, etc.), fish, shellfish, pet animals (dogs, cats, hamsters, rabbits, inco, tropical fish, reptiles, amphibians, insects, etc.). , Not limited to these.

In the feed or pet food of the present embodiment, the content of the above-mentioned nutritional supplement is not particularly limited, and the content may be appropriately set according to the type of feed or pet food. For example, the content of the above-mentioned nutritional supplement in feed or pet food is preferably 0.01 to 90% by mass, preferably 0.1 to 80% by mass, as the content of the algae or its extract. 70% by mass is more preferable, and 1 to 60% by mass is particularly preferable.

The feed or pet food of the present embodiment can be used to efficiently supply the animal with the above-mentioned nutritional components contained in the algae.

≪Manufacturing method of nutritional components≫
The method for producing a nutritional component according to an embodiment of the present invention is a method comprising a culture step for culturing the cyclotera sp. 3FG strain of the above embodiment.
Hereinafter, the process of the manufacturing method of the present embodiment will be described.

<Culture process>
The culturing step is a step of culturing the algae.

Preferable examples of the algae used in this step include the same as those described in << Algae >> above.
In addition, the culture step can be performed by the method described in the above-mentioned << algae >>.

The production method of the present embodiment further comprises, after the culture step, a crushing step of crushing the Cyclotera SP 3FG strain to obtain a cell crushed product, and a separation step of separating a nutritional component from the cell crushed product. You may prepare in this order.

<Crushing process>
The crushing step is a step of crushing the cells of this algae to obtain a cell crushed product.

The method for disrupting the cells of this alga is not particularly limited, and a known method can be used. Examples of the cell crushing method include physical treatments such as glass beads, mortars, ultrasonic treatments, French presses, homogenizers; and chemical treatments such as neutralization treatments, hypotonic treatments, and freeze-thaw treatments. These treatments may be performed alone or in combination of two or more kinds of treatments.

Examples of the neutralization treatment method include a method of immersing the cells of this algae in a neutralizing solution having a pH of about 7 to 10. Since the algae are adapted to the pH in the acidic range, the cells are destroyed by immersing them in a neutral to basic neutralizing solution. The composition of the neutralizing solution is not particularly limited, but for example, a buffer solution such as a phosphate buffer solution or a Tris buffer solution can be used. The time for immersing the cells in the neutralizing solution may be such that the cells are destroyed, and for example, about 10 to 30 minutes is exemplified.

Examples of the hypotonic treatment method include a method of immersing the cells of this alga in a hypotonic solution such as water. Although this alga has a putamen, it has many holes and is considered to be brittle, and cells burst when immersed in a hypotonic solution. The composition of the hypotonic liquid is not particularly limited, and may be any hypotonic liquid to the extent that the cells of this alga burst. Examples of the hypotonic solution include water, a low-concentration buffer solution, and the like. The time for immersing the cells in the hypotonic solution may be such that the cells rupture, and for example, about 1 to 30 minutes is exemplified.

Examples of the freeze-thaw treatment method include a method in which the cells of this alga are subjected to a freeze-thaw cycle at least once. The number of freeze-thaw cycles is not particularly limited, and may be any number as long as the cells of this alga are crushed. The number of freezing and thawing cycles is exemplified by, for example, about 1 to 5 times. Each time of freezing and thawing is not particularly limited, and for example, about 10 to 30 minutes each is exemplified.

By crushing the cells of this alga by the method as described above, a cell crushed product of this alga can be obtained.

<Separation process>
The separation step is a step of separating nutritional components from the cell disrupted product of this algae.

In this step, the nutritional component to be separated is not particularly limited as long as it is the nutritional component of the algae. As described in the above-mentioned << nutritional supplement >>, this alga contains a large amount of nutritional components such as amino acids, vitamins, proteins, lipids (particularly, ω3 unsaturated fatty acids), and dietary fiber. Therefore, examples of the nutritional components to be separated in this step include ω3 unsaturated fatty acids and the like.

Among the nutritional components, examples of the ω3 unsaturated fatty acid include at least one selected from the group consisting of α-linolenic acid, DHA and EPA. Preferably, ω3 unsaturated fatty acids include DHA and EPA.

The nutritional components separated in this step may be one kind or two or more kinds. Further, it may be separated according to the type of ω3 unsaturated fatty acid (α-linolenic acid, DHA, EPA, etc.).

The method for separating the nutritional component from the cell disruption is not particularly limited, and an appropriate method may be selected according to the type of the nutritional component. As a method for separating nutritional components, a method generally used for separation / purification of biochemical substances can be appropriately combined and used. Separation methods include, for example, centrifugation, washing, salting, dialysis, recrystallization, reprecipitation, solvent extraction, adsorption, concentration, filtration, gel filtration, ultrafiltration, and various types of chromatography (gas chromatography, thin layer chromatography). (Graphic, ion exchange chromatography, high-speed liquid chromatography, adsorption chromatography, affinity chromatography, etc.) and the like), but are not limited thereto.

<Arbitrary process>
The production method of the present embodiment may include other steps in addition to the culture step, the crushing step and the separation step. Examples of other steps include a step of recovering the algae from the culture solution (recovery step), a step of washing the cells of the algae (washing step), and the like. These steps can be performed after the culture step described above and before the crushing step.

The recovery step can be performed by a known method such as filtration or centrifugation. The washing step can be performed by suspending the cells in a washing solution (buffer solution or the like) having a pH of 0.1 to 5.0, and then recovering the cells from the washing solution by a method such as filtration or centrifugation.

The nutritional components produced by the production method of the present embodiment can be used for various uses such as nutritional supplements, foods, feeds, pet foods, pharmaceuticals, and reagents.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

1. 1. Isolation of new microalgae The estuary surface water was sampled at the Jinzu River in Toyama Bay, Toyama Prefecture, Japan. Next, the estuary surface water was filtered under reduced pressure with a 0.45 μm polycarbonate membrane filter, and microorganisms containing microalgae were captured on the filter. Next, the whole filter was placed in a culture medium containing KWSW medium, SGI medium (Sager & Granic Medium I), IMK medium and Dunaliella medium, and cultured under a white fluorescent lamp while aerating. The composition of the KWSW medium is shown in Table 1 below, the composition of the SGI medium is shown in Table 2 below, the composition of the IMK medium is shown in Tables 3 and 4 below, and the composition of the medium for Dunaliella is shown in Tables 5 and 2 below. Shown in 6.

As a result, microalgae proliferated, and a part of the culture solution that became turbid in green or brown was applied on the 1.5% (w / v) agar medium prepared in each of the above media and cultured under a white fluorescent lamp. did. The microalgaes applied on the agar medium proliferated, and the microalgaes were selected and isolated (single species) by isolating the individual colonies formed. As a result, the 3FG strain was isolated.

Next, the observation of the 3FG strain under a microscope (manufactured by Olympus Corporation, BX51N-33-PH) was performed (magnification: 40 times). A micrograph of the 3FG strain is shown in FIG. The image above is a photomicrograph in the bright field. The image below is a photomicrograph in the dark field.
From FIG. 1, it was confirmed that the morphology of the cells of the 3FG strain was rectangular parallelepiped, columnar or spherical, and that cells of different sizes were mixed.

2. Phylogenetic analysis based on 18S ribozomal DNA sequence Using DNA extracted from cells of 3FG strain as a template, the overall length of 18S ribozomal DNA is amplified by PCR using the primers of SR-1 and SR-12 shown in Table 7 below. did. The nucleotide sequence of 18S ribozomal DNA of the 3FG strain is shown in SEQ ID NO: 1.
Furthermore, using the obtained PCR product as a template and using the primers shown in Table 7 below in pairs (SR-1, SR-5, SR-4 and SR-9, and SR-6 and SR-12). , Nested PCR was performed.

Based on the base sequence of 18S ribozomal DNA of 3FG strain, molecular phylogenetic analysis by maximum likelihood method was performed. Specifically, the sequences of closely related species obtained from the database were aligned using MEGA5.05 software (http://www.megasoftware.net/), and a molecular phylogenetic tree was prepared by the neighborhood joining method. The molecular phylogenetic tree based on the base sequence of 18S ribozomal DNA is shown in FIG.
From FIG. 2, it was found that the 18S ribozomal DNA of the 3FG strain was closely related to the known algae species shown in Table 8 below.

From the results of the above molecular phylogenetic analysis, it was determined that the 3FG strain belongs to the genus Cyclotera.

3.3 Component analysis of 3FG strain (1) Culturing and temperature examination of 3FG strain The 3FG strain was aerated and cultured using KWSW medium. The culture temperature was 25 ° C. or 30 ° C., the light intensity was 60 μmol / m ² / s, and the cells were aerated and cultured for 9 days while aerating.

As a result, there was no significant difference in the growth rate between 25 ° C and 30 ° C. However, since the period in which the concentration of the 3FG strain is high continues for a long time at 25 ° C., it was decided to carry out the culture temperature at 25 ° C. in the subsequent tests.

-Examination of medium The 3FG strain was cultured using an F medium or IMK medium prepared using artificial seawater or fossil seawater using a 1 L glass bottle. The culture temperature was 25 ° C. and the light intensity was 90 μmol / m ² / s. The irradiation cycle was repeated with the condition of continuously irradiating light for 18 hours and leaving it in a dark place for 6 hours as one cycle. In addition, the cells were cultured for 9 days while being aerated for 24 hours. The composition of the F medium is shown in Table 9 below. The F medium is a medium (modified medium of Guillard F) prepared with reference to Guillard F medium (Guillard and Ryther, 1962). As the artificial seawater, 33 g of Instant Ocean (product name) manufactured by Napco Limited was diluted with 1 L of water. The composition of fossil seawater is shown in Table 10. The fossil seawater was passed through a 0.45 μm filter and simply sterilized. As a control, Isocrisis SP (Tahitian strain) "Zoyoshoku Research Institute Preservation Strain" (hereinafter referred to as "Tahitian strain"), which is an alga that produces DHA and is known to have a relatively high growth rate. In some cases), the cells were cultured under the same conditions.

Then, the absorbance (OD680) at 680 nm was measured over time, and the growth rate was observed. FIG. 4A is a graph showing the growth rate of 3FG. Further, FIG. 4B is a graph showing the growth rate of the Tahitian strain. After the culture was completed, the algae were freeze-dried and the dry weight was measured. As a result of culturing, it was clarified that the 3FG strain could not be cultivated in artificial seawater and could be cultivated well in fossil seawater. On the other hand, it was clarified that the Tahitian strain could not be cultivated in fossil seawater and could be cultivated well in artificial seawater.

From FIGS. 4A and 4B, there was no significant difference between the 3FG strain and the Tahitian strain in terms of the culture rate up to the 9th day of culture, and they were equivalent. The dry weight of 3FG cultured in F medium prepared using fossil seawater was 0.21 g / L, and the dry weight of 3FG cultured in IMK medium prepared using fossil seawater was 0.26 g / L. there were. On the other hand, the dry weight of the Tahitian strain cultured in the F medium prepared using artificial seawater is 0.26 g / L, and the dry weight of the Tahitian strain cultured in the IMK medium prepared using artificial seawater is 0.28 g / L. It was L. The dry weight of the Tahitian strain was slightly heavier than that of the 3FG strain, but it was almost the same.

Further, although not shown, when the 3FG strain and the Tahitian strain were similarly cultured using the F medium prepared using deep seawater, the 3FG strain could not be cultured and the Tahitian strain could be cultivated well. .. Furthermore, when the Tahitian strain was cultured using an F medium (without a trace metal solution) prepared using deep seawater, it could not be cultured.

From the above, it was clarified that the 3FG strain can be cultivated well by using fossil seawater. It was also confirmed that a trace amount of metal was required for culturing the Tahitian strain.

-Examination of light intensity (photon bundle density) The 3FG strain was cultured in a 1 L volume glass bottle using F medium under two types of light conditions (65 μmol / m ² / s or 150 μmol / m ² / s). The irradiation cycle was repeated with the condition of continuously irradiating light for 18 hours and leaving it in a dark place for 6 hours as one cycle. The cells were cultured at a culture temperature of 25 ° C. for 24 hours while being aerated for 9 days. The absorbance (OD680) at 680 nm was measured over time to estimate the growth rate. The results are shown in FIG. In addition, the culture was terminated when OD680 reached a plateau. After the culture was completed, the algae were freeze-dried and the dry weight was measured.

From FIG. 3, no significant difference was observed in the growth rate at 65 μmol / m ² / s and 150 μmol / m ² / s, and the growth was good in both cases. The dry weight of the algae cultured at 65 μmol / m ² / s was 0.41 g / L, and the dry weight of the algae cultured at 150 μmol / m ² / s was 0.42 g / L. There was no big difference in.

In addition, since the turbidity has subsided 6 days after the start of culture, assuming that the above dry weight algae have grown so far, the growth rate of 3FG is about 70 mg / L / day (≈0.4 g). / L / 6day). When the fossil seawater was changed to artificial seawater using the Tahitian strain "Zoyoshoku Research Institute Preservation Strain" and almost the same test was performed, it was 75 mg / L / day. Therefore, it was confirmed that the 3FG strain had a growth rate almost the same as that of the Tahitian strain.

Separation and analysis of fatty acids Next, 1 to 10 mg of each freeze-dried algae was weighed and placed in a pressure-resistant test tube with a cap. 0.5 mL of a 2% methanol sulfate solution was added thereto, and the mixture was reacted at 80 ° C. for 3 hours and then cooled. Then, 0.3 mL of saturated sodium carbonate 1/4 solution and 0.3 mL of hexane were added, and after vortexing, centrifugation was performed. Then, the hexane layer of the supernatant and the fatty acid methyl ester contained therein were recovered and measured by gas chromatography.

Next, the content of fatty acid in the sample was measured by capillary gas chromatography (GC-2010, manufactured by Shimadzu Corporation). The analysis conditions for capillary gas chromatography are shown below.

(Analysis conditions for capillary gas chromatography)
Column: FAMEWAX (manufactured by GL Science, 30 m, inner diameter 0.25 μm)
Gas: Helium gas (He, 75.8 kPa)
Detector: FID

The measurement results of the obtained algae fatty acids are shown in Table 11 below.

From Table 11, the DHA content was 1.1% per algae, the EPA content was 5.2 to 5.6%, and the total oil and fat content was about 22 to 24%. When the fossil seawater was changed to artificial seawater using the Tahitian strain and almost the same test was performed, the DHA content was 2 to 2.5%, the EPA was less than 0.1%, and the total fat and oil content was 10 to 13. %, Less than 3FG strains. Therefore, in terms of the combined content of EPA and DHA, the 3FG strain was found to be superior to the Tahitian strain.

From the above, it was clarified that the 3FG strain can be cultivated well by using fossil seawater. It was also confirmed that a trace amount of metal was required for culturing the Tahitian strain.

From the above, it was confirmed that a sufficient amount of EPA and DHA can be obtained from the 3FG strain.

According to the present embodiment, a novel microalgae that can be industrially used, a new nutritional supplement using the algae, a nutritional component supplementing composition containing the nutritional supplement, and a method for producing a nutritional component using the microalgae are provided. Can be provided.

Claims (12)

Cyclotella sp. 3FG strain (FERM P-22337). A nutritional supplement containing the cyclotera sp. 3FG strain according to claim 1 and an extract thereof. A nutritional component supplementing composition comprising the nutritional supplement according to claim 2. The composition for supplementing nutritional components according to claim 3, wherein the nutritional component is an ω3 unsaturated fatty acid. The nutritional component supplement composition according to claim 4, wherein the ω3 unsaturated fatty acid is at least one selected from the group consisting of α-linolenic acid, docosahexaenoic acid and eicosapentaenoic acid. The composition for supplementing nutritional components according to any one of claims 3 to 5, which is a food product. The nutritional component supplement composition according to claim 6, which is a functional food or a dietary supplement. The nutritional component supplement composition according to any one of claims 3 to 5, which is a feed or pet food. A method for producing a nutritional component, comprising a culturing step of culturing the cyclotera sp. 3FG strain according to claim 1. Further, after the culture step,
A crushing step of crushing the Cyclotera SP 3FG strain to obtain a cell crushed product, and
The separation step of separating the nutrient component from the cell crushed product, and
9. The method for producing a nutritional component according to claim 9. The method for producing a nutritional component according to claim 9 or 10, wherein the nutritional component is an ω3 unsaturated fatty acid. The method for producing a nutritional component according to claim 11, wherein the ω3 unsaturated fatty acid is at least one selected from the group consisting of α-linolenic acid, docosahexaenoic acid and eicosapentaenoic acid.

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