JP6681065B2 - Food, heat treatment method of food, phycocyanin-containing material manufacturing method, and food manufacturing method - Google Patents

Description

  The present invention relates to a food, a heat treatment method for food, a method for producing phycocyanin, a method for producing an organic acid, and a method for producing hydrogen, which are related to phycocyanins such as red algae sizon.

  Recently, cyanidioschyzon merolae (Cyanidioschyzon merolae) isolated from an acidic hot spring has attracted attention as a unicellular red alga. Due to its small genome size and easiness of culturing, sizon is mainly interested in its use as a research object, and its industrial use is scarcely examined.

  Phycocyanin, which is a pigment protein, is a photosynthetic pigment produced by algae such as cyanobacteria. Phycocyanin has a beautiful blue color and is highly safe when taken orally. Due to these excellent properties, it is often used as a food coloring agent and has a very high market value. As a method for producing phycocyanin, a production method using cyanobacteria is known (Patent Document 1), and among others, a production method using Spirulina, which is a kind of cyanobacteria, has been put to practical use.

  When recovering phycocyanin from Spirulina, spirulina is recovered from the culture pond, spirulina is separated and concentrated, and phycocyanin is extracted from the dried and powdered product.

On the other hand, in recent years, due to concerns about global warming and resource depletion, expectations for biomass, which is a renewable resource, have increased. Above all, the development of alternative oil resources is an important issue.
Organic acids are highly useful compounds. For example, polylactic acid, which is an excellent biodegradable plastic, can be produced from lactic acid, which is an organic acid. Among them, succinic acid is a general-purpose chemical and industrial raw material, and is widely used as a raw material for polybutylene succinate (PBS), a chemical intermediate, a solvent, and a plasticizer. For example, polybutylene succinate produced using succinic acid as a raw material is used for agricultural mulch films, packaging materials, farm / civil engineering materials, etc., and the shipment amount in 2011 is about 3100 tons.
Currently, succinic acid is mainly produced from petroleum as a raw material, but there is a trend to use biomass instead of petroleum. For example, Non-Patent Document 1 discloses a technique for producing succinic acid using a heterotrophic microorganism. Some companies have begun producing succinic acid by fermentation of bacteria or yeast.

  On the other hand, expectations for the use of hydrogen as a new energy source to replace petroleum are increasing. Unlike petroleum, hydrogen does not generate carbon dioxide when burned, so it is also an excellent measure against global warming.

JP 62-6691A JP-A-11-299450 JP, 2005-295829, A

Sanchez AM, Bennett GN, San KY (2005) Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity.Metab Eng 7: 229-239.

The above phycocyanin derived from cyanobacteria has a problem of poor heat resistance. Therefore, the main target for blending the cyanobacteria phycocyanin is limited to frozen desserts and frozen desserts. On the other hand, conventionally, there has been known an attempt to improve the heat resistance of phycocyanin by combining phycocyanin of cyanobacteria and a saccharide (Patent Documents 2 and 3). However, very high concentrations of sugar have to be used, and the situation in which it can be used is limited.
Further, in the conventional method for producing phycocyanin using cyanobacteria, in order to recover phycocyanin, cyanobacteria must be dried and powdered, and equipment and cost therefor are required.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a food containing a phycocyanin-containing material having excellent heat resistance, and a heat treatment method for the food.
Moreover, this invention makes it a subject to provide the manufacturing method of phycocyanin which can manufacture phycocyanin efficiently.
Another object of the present invention is to provide a method for producing an organic acid and a method for producing hydrogen by a red alga belonging to the genus Cyanidiophyceae.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that an acidic phycocyanin-containing material containing phycocyanin, which is a red alga belonging to the genus Cyanidiophyceae, has excellent heat resistance. Further, the present inventors have found that phycocyanin can be produced very easily by using a red alga belonging to the genus Ideyugogome. Further, the present inventors have found that the phycocyanin of the red alga belonging to the genus Hydrangea genus obtained by the above method has excellent heat resistance, even compared to the phycocyanin derived from cyanobacteria that has been conventionally used.

  Further, the present inventors have found that it is possible to produce an organic acid using a red alga belonging to the genus Lepidoptera, and it is possible to produce hydrogen using a red alga belonging to the genus Lepidoptera.

  That is, the present invention provides a food, a heat treatment method for a food, a method for producing an organic acid, and a method for producing hydrogen having the following characteristics.

(1) A food containing a phycocyanin-containing substance having a pH of 0 or more and 6 or less , which contains phycocyanin of a red alga belonging to the genus Cyanidiophyceae (Cyanidioschyzon) .
(2) The food according to (1), which is a beverage.
(3) A heat treatment method for food, comprising heat-treating the food according to (1) or (2) above.
(4) The heat treatment method for food according to (3), wherein the temperature of the heat treatment is 60 ° C. or higher and 85 ° C. or lower.
( 5 ) A method for producing the phycocyanin-containing product contained in the food according to (1) or (2) above,
Red algae belonging to the genus Cyanidiophyceae (Cyanidioschyzon) are cultivated in a culture to produce phycocyanin, and a recovery medium having a salt concentration of the culture or less is contacted with the red algae. A method for producing a phycocyanin- containing product , comprising: obtaining a pigment recovery product in which phycocyanin produced by red alga is extracted in the recovery medium, and collecting phycocyanin from the red alga.
(6) The method for producing a food according to claim 1 or 2, which comprises using the phycocyanin-containing material obtained by the method for producing a phycocyanin-containing material according to (5).

According to the present invention, it is possible to provide a food containing a phycocyanin-containing material having excellent heat resistance.
Further, according to the present invention, it is possible to provide a method for producing phycocyanin capable of producing phycocyanin excellent in heat resistance with high efficiency.
Further, according to the present invention, it is possible to provide a method for producing an organic acid and a method for producing hydrogen, which are produced by a red alga belonging to the genus Lepidoptera.

It is a schematic diagram explaining the manufacturing method of the phycocyanin which concerns on one Embodiment of this invention. It is a schematic diagram which shows the outline of a structure of the phycocyanin manufacturing apparatus which concerns on one Embodiment of this invention. 3 is a graph showing the amount of phycocyanin contained in the supernatant after culturing red algae in Examples. 3 is a photograph showing phycocyanin contained in the supernatant after culturing red algae in Examples. It is a graph of the result of having compared the heat resistance with the schizon phycocyanin and spirulina phycocyanin in an Example. It is a graph of the result of having compared the heat resistance with the schizon phycocyanin and spirulina phycocyanin in an Example. It is the graph which showed the organic acid amount of the extracellular (in culture) of red alga in an Example.

<< Food >>
The food of the present invention contains a phycocyanin-containing material having a pH of less than 7 and containing phycocyanin of red alga belonging to the class Cyanidiophyceae.
The phycocyanin according to one aspect of the present invention is a phycocyanin of the red alga belonging to the genus Cyanidiophyceae. Examples of the phycocyanin include those described in the method for producing phycocyanin described below, and examples include those obtained by the method for producing phycocyanin of the present invention described below.
Hereinafter, the red algae belonging to the class Cyanidiophyceae may be simply referred to as "red algae".

As shown in Examples described later, the present inventors have found that the red alga phycocyanin belonging to the genus Lepidoptera has superior heat resistance as compared to the conventionally used cyanobacteria-derived phycocyanin.
Phycocyanin according to one aspect of the present invention, relative to the phycocyanin-containing material containing phycocyanin according to one aspect of the present invention, the dye residual rate after heat treatment may be 50% or more compared to before heat treatment, It may be 70% or more, 80% or more, or 90% or more. The conditions for the heat treatment include the conditions exemplified in the << heat treatment method for foods >> described below. As shown in the examples of the present specification, the dye residual rate can be determined by the rate of decrease in the absorbance with the absorbance at 620 nm for the phycocyanin-containing substance as an index. The absorbance of the base at which the residual rate is 0% may be obtained, for example, from the absorbance at 620 nm for the composition of the phycocyanin-containing material excluding phycocyanin, and the absorbance at 620 nm for the phycocyanin-modified phycocyanin-containing material. You may ask from

The phycocyanin-containing product according to one aspect of the present invention contains phycocyanin of the red alga belonging to the genus Lepidoptera, and has a pH of less than 7.
The inventors have found that the phycocyanin of the red alga belonging to the genus Lepidoptera has superior heat resistance under acidic conditions. From such a viewpoint, the pH of the phycocyanin-containing product may be pH 0 or higher and lower than pH 7, pH 1 or higher and pH 6 or lower, pH 2 or higher and pH 5 or lower, or pH 3 or higher and pH 4.5 or lower. The pH may be 3 or more and less than pH 4. Among them, the phycocyanin-containing material having a pH of less than 4 is preferable because it is less likely to rot and the heat treatment condition for storage is relaxed.

  The phycocyanin-containing material refers to a phycocyanin solution in which phycocyanin is dissolved in a solvent, and examples of the phycocyanin-containing material include an aqueous phycocyanin solution using water as a solvent. The phycocyanin-containing material is not limited to a liquid state, and may be, for example, a gelled state, a frozen state, or a state in which the carrier is impregnated. The phycocyanin-containing material may contain any substance other than phycocyanin.

The amount of phycocyanin in the phycocyanin-containing material may be, for example, 50 mg / L or more, 50 mg / L to 10000 mg / L, 80 mg / L to 1000 mg / L, or 100 mg / L. It may be up to 500 mg / L.
The amount of phycocyanin in the phycocyanin-containing material can be determined by a method described in Examples described later using a spectrophotometer or the like. Also, phycocyanin can be separated by SDS-PAGE to estimate the approximate amount of phycocyanin.

  The phycocyanin-containing product according to one aspect of the present invention is characterized in that it can exhibit excellent heat resistance without using sugar or sugar alcohol. From such a viewpoint, the phycocyanin-containing material may have a sugar or sugar alcohol content of less than 70 (w / v)%, less than 50 (w / v)%, or 30 (w / v). )% Or less than 10 (w / v)%.

The phycocyanin-containing product according to one aspect of the present invention may be contained in reagents, foods, or pharmaceuticals.
As one embodiment of the present invention, there is provided a food containing a phycocyanin-containing substance having a pH of less than 7 and containing phycocyanin of the red alga belonging to the class Cyanidiophyceae.
As one embodiment of the present invention, there are provided reagents including a phycocyanin-containing substance having a pH of less than 7 and containing phycocyanin of the red alga belonging to the class Cyanidiophyceae.
As one embodiment of the present invention, there is provided a pharmaceutical product containing a phycocyanin-containing substance having a pH of less than 7 and containing phycocyanin of the red alga belonging to the class Cyanidiophyceae.
These may be foods, reagents, or medicines containing a phycocyanin-containing substance having a pH of less than 7 and containing phycocyanin of the red alga belonging to the class Cyanidiophyceae.

  The phycocyanin-containing substance contained in the reagents, foods, or pharmaceuticals may be liquid, gelled, frozen, or impregnated with a carrier. The form of the phycocyanin-containing material in the reagents, food, or medicine is not particularly limited, and for example, the phycocyanin-containing material may be in a state of being encapsulated in a capsule, or an emulsion containing droplets of the phycocyanin-containing material. It may be in a state.

The food of the present invention contains a phycocyanin-containing material having a pH of less than 7 and containing phycocyanin of red alga belonging to the class Cyanidiophyceae. The phycocyanin according to one embodiment of the present invention is excellent in the dye residual rate after heat treatment. Therefore, it can be suitably used for foods and food raw materials that include heat treatment in the manufacturing process. For example, as shown in Examples below, the phycocyanin-containing product according to one embodiment of the present invention can withstand heat treatment at 60 ° C. for 1 hour, and thus can be provided after heat sterilization.
The food of the present invention is particularly suitable as a drink among foods. Here, the pH of the phycocyanin-containing material is preferably less than 4. For example, the manufacturing standard for soft drinks having a pH of less than 4 may require heat sterilization treatment at 65 ° C. for 10 minutes or at a temperature equal to or higher than that. The phycocyanin-containing material having a pH of less than 4 can achieve sufficient sterilization in terms of food hygiene while improving the coloring of the pigment by heat treatment at 65 ° C. for 10 minutes. It can be served as a beverage as it is.

For example, when the phycocyanin-containing substance is an aqueous phycocyanin solution, examples of foods include beverages, soft drinks, jams, shaved ice, frozen desserts such as ice, and confectioneries such as jelly. Examples of the drink include carbonated drinks such as cider and carbonated water; alcoholic drinks such as beer, liqueur and rum, and sports drinks, mineral water, vegetable drinks, fruit drinks and the like. Food ingredients also include food ingredients such as colorants.
Examples of the reagents include cell labeling reagents and the like. Reagent raw materials such as colorants are also included in the reagents.
The medicine may be either an internal preparation or an external preparation, and examples thereof include oral preparations, injection preparations, drink preparations, suppositories, eye drops and jelly preparations. Phycocyanin contained in a drug may be an active ingredient or an additive other than the active ingredient.

The phycocyanin-containing product according to one aspect of the present invention can be produced by the phycocyanin production method of the present invention described below. That is, the phycocyanin-containing material, phycocyanin is produced by culturing red algae belonging to the class Ideyugogome (Cyanidiophyceae) in a culture, and the salt medium is brought into contact with the red algae and a recovery medium having the culture or less, The recovery medium may contain a phycocyanin collected from the red alga by obtaining a pigment recovery product in which the phycocyanin produced by the red alga is extracted.
The phycocyanin-containing material may or may not contain red algae. Examples of the phycocyanin-containing material containing red algae include dye recovery products containing phycocyanin extracted from red algae in the phycocyanin production method of the present invention described below. Examples of the phycocyanin-containing substance containing no red alga include a recovery medium containing phycocyanin extracted from red alga in the phycocyanin production method of the present invention described below. The phycocyanin-containing product in the present specification is a phycocyanin solution in which phycocyanin is dissolved in a solvent, and a liquid containing only red alga in which phycocyanin has been accumulated in the body is not treated as a phycocyanin-containing product.

Further, as one embodiment of the present invention, there is provided phycocyanin obtained by the method for producing phycocyanin of the present invention.
Moreover, as one embodiment of the present invention, there is provided a food containing phycocyanin obtained by the method for producing phycocyanin of the present invention.
As one embodiment of the present invention, there is provided a method for producing a food, which comprises using the phycocyanin obtained by the method for producing phycocyanin of the present invention as a raw material.
As one embodiment of the present invention, a beverage containing phycocyanin obtained by the method for producing phycocyanin of the present invention is provided.
As one embodiment of the present invention, there is provided a method for producing a beverage, which comprises using the phycocyanin obtained by the method for producing phycocyanin of the present invention as a raw material.
As one embodiment of the present invention, a reagent containing phycocyanin obtained by the method for producing phycocyanin of the present invention is provided.
As one embodiment of the present invention, there is provided a method for producing a reagent, which comprises using the phycocyanin obtained by the method for producing phycocyanin of the present invention as a raw material.
As one embodiment of the present invention, there is provided a medicine containing phycocyanin obtained by the method for producing phycocyanin of the present invention.
As one embodiment of the present invention, there is provided a method for producing a drug, which comprises using the phycocyanin obtained by the method for producing phycocyanin of the present invention as a raw material.
Examples of the food, beverage reagent, and drug include those described above.

The red alga according to the present invention is a red alga that belongs to the genus Lepidoptera and is capable of producing phycocyanin. As the name implies, red algae are typically a group of red-colored algae. Among them, red algae that belong to the class Ideyugogome are also called hot spring algae, and are algae that can grow even in high-temperature acidic environments such as hot springs.
Examples of red algae belonging to the genus Lepidoptera include Cyanidioschyzon merolae, Cyanidioschyzon red algae, Cyanidium caldarium, and other red algae, and Gardieria sulfuraria. ) And other red algae of the genus Gardieria. In the present specification, the red alga belonging to the genus Lepidoptera may be a mutant or recombinant thereof other than those listed above. Red algae belonging to the newly-identified Lepidoptera genus or closely related taxa that can grow in a high temperature acidic environment shall be treated as red algae belonging to the genus Lepidoptera.
The high-temperature acidic environment refers to an environment equivalent to the high-temperature acidic environment in which the red alga belonging to the above-mentioned Leptococcidae can grow, and is, for example, an environment of pH 1 to 6 and preferably pH 1 to 3 at a temperature of 40 ° C or higher. As an example, the optimal growth conditions for cyanidiocizone melorae are a temperature of 42 ° C. and a pH of 2.5.

Phycocyanin is a kind of photosynthetic pigment possessed by algae such as cyanobacteria and red algae, and is a complex of pigment compound and protein. It is known that there are two types of subunits, α and β, in the protein that constitutes phycocyanin. In vivo, the dimer further associates with each other to form a disk of 3 or hexamer. There is.
The phycocyanin according to the present invention is produced from the red alga belonging to the above-mentioned Lepidoptera family, that is, the phycocyanin of red alga belonging to the Lepidoptera family. Among them, the phycocyanin of red algae belonging to the genus Cyanidiocison is preferable, and the phycocyanin of cyanidiocizone melorae is more preferable.
Cyanidiosison Melorae has completed the decoding of genomic information. Based on that information, the α subunit of phycocyanin of cyanidiocison melorae is a protein having the amino acid sequence represented by SEQ ID NO: 1, and the β subunit has the amino acid sequence represented by SEQ ID NO: 2. It is called protein.

In general, a protein having an amino acid sequence similar to the original amino acid sequence has properties equivalent to those of the protein having the original amino acid sequence.
As an example, the phycocyanin protein produced in the phycocyanin production method of the present embodiment may be a partially modified version of the phycocyanin protein of cyanidiocison melorae, for example, the following (a) or It may be the protein of (b).
(A) A protein having an amino acid sequence in which one to several amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and capable of constituting a phycocyanin dye (b) A protein having an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 or 2 and capable of constituting a phycocyanin dye

In the amino acid sequence of (a), “1 to several” bases may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 bases. Good.
In the polypeptide of (a), "a nucleotide sequence in which a base has been deleted, substituted, inserted or added" means that the amino acid sequence shown in SEQ ID NO: 1 or 2 has an amino acid deleted, substituted, inserted or It may be an amino acid sequence added / or, by at least one modification or mutation selected from the group consisting of deletion, substitution, insertion and addition, to the amino acid sequence shown in SEQ ID NO: 1 or 2 before modification Thus, a difference of 1 to several amino acids may occur.

In the polypeptide of (b), the sequence identity with the amino acid sequence shown by SEQ ID NO: 1 or 2 is 80% or more and less than 100%, for example, 85% or more, 90% or more, 95% or more, or 98 % Or more.
The sequence identity between amino acid sequences can be calculated using a known sequence alignment algorithm, BLAST (Basic Local Alignment Search Tool) or blastp.

≪Food heat treatment method≫
The heat treatment method for food according to the present invention includes heat treatment for the food according to the present invention.
Examples of the food of the present invention include those exemplified in the above << Food >>.
The heat treatment method of the phycocyanin-containing material according to one aspect of the present invention includes heat-treating the phycocyanin-containing material of one aspect of the present invention.

  The temperature of the heat treatment is preferably 60 ° C or higher and 85 ° C or lower, more preferably 60 ° C or higher and 70 ° C or lower, and further preferably 60 ° C or higher and 65 ° C or lower.

  The time of the heat treatment may be appropriately set, but may be 5 minutes or more and 90 minutes or less, 10 minutes or more and 60 minutes or less, or 20 minutes or more and 45 minutes or less.

  The pH of the phycocyanin-containing material may be pH 0 or higher and lower than pH 7, pH 1 or higher and pH 6 or lower, pH 2 or higher and pH 5 or lower, pH 3 or higher and pH 4.5 or lower, pH 3 It may be above pH 4 and below.

  The heat treatment method for a phycocyanin-containing material according to one aspect of the present invention is suitable as a heat treatment method for a phycocyanin-containing material in food.

The heat treatment method for food according to the present invention includes heat treatment for the food according to the present invention. That is, the method for heat-treating food according to the present invention includes heat-treating a food containing phycocyanin-containing material having a pH of less than 7 and containing phycocyanin of red alga belonging to the class Cyanidiophyceae. Among them, it is preferable to heat-treat a food containing a phycocyanin-containing substance having a pH of less than 4 and containing phycocyanin of red alga belonging to the class Cyanidiophyceae.
The heat treatment method for a phycocyanin-containing material according to one embodiment of the present invention can be used as a heat sterilization method for a phycocyanin-containing material. Among them, it is suitable as a heat sterilization method for food-containing phycocyanin-containing materials, and is suitable as a heat sterilization method for food-containing phycocyanin-containing materials having a pH of less than 4.

  As one embodiment of the present invention, there is provided a heat treatment method for a phycocyanin-containing material, which comprises heat treatment of the phycocyanin obtained by the method for producing phycocyanin of the present invention. Examples of the temperature and time of the heat treatment include those mentioned in the above heat treatment method.

  According to the method for heat-treating a phycocyanin-containing material according to one aspect of the present invention, it is possible to heat-treat a phycocyanin-containing material while improving the color development of the phycocyanin.

  According to the method for heat treatment of food of the present invention, heat treatment of food can be performed while improving the color development of phycocyanin.

≪Method for producing phycocyanin≫
The method for producing phycocyanin of the present invention is to produce phycocyanin by culturing red algae belonging to the genus Lepidoptera (Cyanidiophyceae) in a culture, and bringing the recovery medium having a salt concentration not higher than that of the culture into contact with the red algae. And obtaining a pigment recovery product in which phycocyanin produced by the red alga is extracted in the recovery medium, and collecting phycocyanin from the red alga.
Hereinafter, a method for producing phycocyanin according to an embodiment of the present invention will be described with reference to the drawings. Descriptions of parts common to the phycocyanin, the phycocyanin-containing material, and the food are omitted.

  FIG. 1 is a schematic diagram illustrating the method for producing phycocyanin according to this embodiment. First, the red alga 30 belonging to the genus Lepidoptera is cultured in a culture medium 40 (culture). By culturing the red alga 30 in the culture solution 40, the red alga 30 can be proliferated, and since phycocyanin is biosynthesized by the red alga 30, phycocyanin can be produced. Next, the red alga 30 cultivated in the culture solution 40 is introduced into the recovery solution 50 (recovery medium), the red alga 30 and the recovery solution 50 are brought into contact with each other, and the dye recovery including the red alga 30 and the recovery solution 50 is performed. Liquid 60 (a dye recovery product) is obtained. In the dye recovery liquid 60, when the red alga 30 and the recovery liquid 50 come into contact with each other, phycocyanin is extracted from the red alga 30 into the recovery liquid 50, and the recovery liquid 50 becomes the recovery liquid 51 containing phycocyanin. The dye recovery liquid 61 (color recovery product) includes a red alga 31 from which phycocyanin is extracted, and a recovery liquid 51 in which the phycocyanin extracted from the red alga 31 is dissolved in the recovery liquid 50. Then, the phycocyanin is recovered from the red alga 30 by collecting the recovery liquid 51 or the color recovery liquid 61 from the color recovery tank 20.

Thus, to obtain a pigment recovery product containing a red alga belonging to the class Ideyugogome and a recovery medium, and by collecting the phycocyanin extracted in the recovery medium from the red alga, phycocyanin having excellent heat resistance can be efficiently used. Can be obtained. This is because phycocyanin is easily extracted into the recovery medium when a red alga belonging to the class Ideyugogome is immersed in the recovery medium.
When phycocyanin is extracted from red algae, the cells of red algae may or may not be destroyed.
As the red alga belonging to the genus Hydrangeae, which is dipped in the recovery medium, red alga belonging to the genus Cyanidioschyzon is preferable, and cyanidiozone melorae is more preferable. Phycocyanin can be obtained more easily by using Cyanidioschyzon merolae. This is considered to be because cyanidiocisone melorae does not have a strong cell wall, so that phycocyanin can be recovered from the body of algae into the recovery medium very easily by immersing it in the recovery medium.

There is no limitation on the method for extracting phycocyanin from the red alga into the recovery medium. If phycocyanin is extracted from the red alga in the recovery medium by only immersing the red alga in the recovery medium, it is sufficient to immerse the red alga in the recovery medium.
Further, in order to enhance the recovery efficiency of phycocyanin, in addition to immersing the red alga in the recovery medium, another treatment may be performed. For example, a dye recovery product including red algae and a recovery medium may be kept under anaerobic conditions.
In the specification, holding under anaerobic conditions means, for example, bringing the inside of a container holding the dye recovery product into an anaerobic state. The anaerobic state means that the oxygen concentration in the gas phase in the culture system may be 0 to 1% by volume, 0 to 0.5% by volume, or 0 to 0.2% by volume. Or 0% by volume.
An example of a method for making the container holding the dye recovery product in an anaerobic state is, for example, by culturing red algae in a culture container that is sealed and shielded from light irradiation, and oxygen in the culture container by breathing red algae. Can be consumed. Therefore, in the phycocyanin production method of the present embodiment, the pigment recovery product containing red algae and the recovery medium may be kept under dark conditions, and the pigment recovery product containing red algae and the recovery medium may be retained under dark anaerobic conditions. You may keep it.
As another example, for example, there is a method of flowing nitrogen gas into the culture container and replacing oxygen in the culture container with nitrogen.
By holding the dye recovery product under anaerobic conditions, the recovery efficiency of phycocyanin can be increased.

Any recovery medium may be used as long as it can recover phycocyanin from red algae.
Examples of cultures for culturing red algae include those in which red algae can grow, and examples include media such as M-Allen medium and MA-2 medium.

  The culture and the recovery medium may be liquid or solid, but when producing phycocyanin, it is preferable to use liquid because red algal or phycocyanin can be easily recovered.

  The method of transferring red algae from the culture to the recovery medium is not particularly limited. For example, a method of culturing the red alga in the culture and then mixing the red alga and the culture with a recovery medium can be mentioned. Alternatively, after culturing the red alga in a culture, the liquid content and the red alga are separated from the cultured culture by centrifugation or the like, and the separated red alga and the recovery medium are mixed. Alternatively, after culturing the red alga in the culture, a method of mixing the red alga and the culture with a liquid having a composition different from that of the culture, and using the mixed liquid as a dye recovery product, and the like can be mentioned. . Further, it is also possible to cultivate the red alga in a culture and then separate the liquid component and the red alga from the cultured culture by centrifugation or the like. The separated red algae may or may not undergo cell processing treatment such as dry powderization or crushing treatment other than contact with a recovery medium. From the advantage that phycocyanin having excellent heat resistance can be produced with high efficiency, it is preferable that the separated red algae is not subjected to cell processing treatment before being brought into contact with the recovery medium.

Here, the salt concentration of the recovery medium is lower than the salt concentration of the culture.
The medium for culturing red alga usually contains a salt, and the salt concentration of the culture is preferably 10 to 80 mM, more preferably 20 to 40 mM, further preferably 20 to 30 mM. preferable. For example, the salt concentration of the above M-Allen medium is 25 mM.
The salt concentration of the recovery medium is preferably 0 to 20 mM, more preferably 0 to 10 mM, and further preferably 5 mM or less. Examples of the recovery medium having a salt concentration lower than that of the culture include a buffer, and a buffer having a salt concentration of 5 mM or less. For example, the salt concentration of Hepes buffer is 1 mM or less. The pH of the buffer is not particularly limited, but an example is a pH of about 6-8.
Here, the salt concentration does not refer to the salt concentration of only sodium chloride, but refers to the salt concentration of all salts.

  By making the salt concentration of the recovery medium lower than the salt concentration of the culture, the recovery efficiency of phycocyanin can be enhanced. It is considered that this is because by contacting the red alga with a liquid having a low salt concentration, phycocyanin is easily extracted from the cells of the red alga under the conditions of osmotic pressure and the like.

  Water is mentioned as a recovery medium having a salt concentration lower than that of the culture. The water may be substantially salt-free water. By using water as the recovery medium, the efficiency of recovery of phycocyanin can be improved, and the purity of phycocyanin in the recovery medium can be increased.

  The dye recovery product containing the red algae and the recovery medium may be held in a stationary state, or the dye recovery product may be subjected to treatments such as shaking and stirring. In addition, in order to enhance the recovery efficiency of phycocyanin, an alga cell disruption method applicable in the art can be appropriately used.

  The temperature of the pigment recovery product containing the red alga and the recovery medium may be a temperature at which phycocyanin can be recovered, and may be -80 to 75 ° C, preferably 0 to 65 ° C, and more preferably Can be 15 to 40 ° C., and more preferably 20 to 25 ° C.

  Regarding recovery of phycocyanin, referring to FIG. 1, for example, when recovering phycocyanin from the dye recovery liquid 61, separation treatment such as centrifugation or filtration is performed to separate the red alga 30 and the recovery liquid 51. May be collected, or the pigment recovery liquid 61 from which the red alga 30 has not been separated may be collected. The red alga 30 may not be completely removed in the separated recovery liquid 51.

The phycocyanin may be collected and manufactured at the same time or separately.
The method for producing phycocyanin of the present embodiment may include purifying the collected phycocyanin. Purification may be performed at the same time as the collection or after the collection. Examples of the purification method include crystallization purification, chromatography by HPLC, and column purification of an ion exchange resin and the like.

  Examples of the red alga used in the method for producing phycocyanin of the present invention and the phycocyanin produced by the red alga include those exemplified above in the section of << Foods >>.

The phycocyanin obtained by the method for producing phycocyanin of the present embodiment has excellent heat resistance.
This is thought to be because, first of all, the red alga phycocyanin itself, which belongs to the genus Ideyukogome, has excellent heat resistance. It is considered that the protein derived from the red alga belonging to the genus Lepidoptera is adapted to the high temperature acidic environment which is the growth environment of the red alga.
Secondly, a method of immersing the red alga belonging to the class Ideyugogome in a recovery medium and collecting the phycocyanin extracted from the red alga in the recovery medium may enhance the heat resistance of the obtained phycocyanin. Conceivable. In this method, it is possible to recover phycocyanin without going through the step of denaturing the protein, and it is possible to collect phycocyanin without inhibiting the binding state and function of molecular chaperones that contribute to thermal stability. Conceivable.

  According to the method for producing phycocyanin of the present embodiment, it is possible to highly efficiently produce phycocyanin having excellent heat resistance.

<< Phycocyanin production equipment >>
The phycocyanin production apparatus according to one aspect of the present invention is a culturing tank for cultivating the red alga belonging to the genus Ideyugogome (Cyanidiophyceae), and the phycocyanin is extracted from the red alga in the recovery medium containing the red alga and the recovery medium And a dye recovery tank for holding the dye recovery product.

According to the manufacturing apparatus of this embodiment, an example of the method for manufacturing phycocyanin of the above-described embodiments can be carried out. For example, the phycocyanin production apparatus of one embodiment is a red alga belonging to the genus Cyanidiophyceae (Cyanidiophyceae) is cultured in a culture to produce phycocyanin, and the recovery medium having a salt concentration of the culture or less is contacted with the red alga. Then, to obtain a pigment recovery product obtained by extracting phycocyanin produced by the red alga in the recovery medium, is used in the method for producing phycocyanin to collect phycocyanin from the red alga, and to culture the red alga A culture tank and a dye recovery tank that contains the red alga and a recovery medium and holds a dye recovery product obtained by extracting phycocyanin from the red alga into the recovery medium. Note that the method for producing phycocyanin of the present invention is not limited to the method using the apparatus for producing phycocyanin according to one aspect of the present invention.
Hereinafter, the phycocyanin production apparatus of the embodiment will be described with reference to the drawings. Descriptions of parts common to the method for producing phycocyanin are omitted.

FIG. 2 is a schematic diagram showing an outline of the configuration of the phycocyanin production apparatus 1 according to this embodiment. As shown in FIG. 2, the phycocyanin production apparatus 1 includes a culture tank 10, a dye recovery tank 20, and a pipe 11 (transfer means) for sending red algae 30 from the culture tank 10 to the dye recovery tank 20.
In the culture tank 10, the red alga 30 is cultivated in the culture solution 40. In the culture tank 10, red alga 30 grows. The cultured red alga 30 and the culture solution 40 are sent to the dye recovery tank 20 through the pipe 11. A liquid having a composition different from that of the culture liquid 40 is introduced into the dye recovery tank 20, and the liquid and the culture liquid 40 are mixed to form a recovery liquid 50 (recovery medium). In this way, the dye recovery tank 20 holds the dye recovery liquid 60 (color recovery product) containing the red alga 30 and the recovery liquid 50.
In the dye recovery liquid 60, when the red alga 30 and the recovery liquid 50 come into contact with each other, phycocyanin is extracted from the red alga 30 into the recovery liquid 50. The dye recovery liquid 61 (color recovery product) includes the red alga 31 in which phycocyanin is extracted, and the recovery liquid 50 includes a recovery liquid 51 in which the phycocyanin extracted from the red alga 31 is dissolved. Then, the phycocyanin is recovered from the red alga 30 by collecting the recovery liquid 51 or the color recovery liquid 61 from the color recovery tank 20.

Examples of the red alga 30, the culture solution 40, and the recovery solution 50 include those described in the method for producing phycocyanin.
The dye recovery tank 20 may be capable of holding the dye recovery liquids 60 and 61 under anaerobic conditions, and may be configured, for example, so that air communication with the outside of the tank can be blocked. Further, the dye recovery tank 20 may be capable of holding the dye recovery liquids 60 and 61 under dark conditions, and may be configured to be capable of blocking the light irradiation to the dye recovery liquid 60, for example. .

  In the above description, the case where the red alga 30 is introduced into the pigment recovery tank 20 together with the culture liquid 40 and the recovery liquid 50 includes the culture liquid 40 has been described, but the culture liquid 40 and the red alga 30 are separated in advance. Alternatively, the separated red alga 30 may be sent to the pigment recovery tank 20. The phycocyanin production apparatus 1 may further include a separating means as a suitable device for carrying out the phycocyanin production method. The separation means separates the red alga 30 cultivated in the culture tank 10 and the culture solution 40. Examples of the separating means include a centrifugal separator and the like.

  The phycocyanin production apparatus of the present embodiment can produce phycocyanin with high efficiency. Moreover, according to the phycocyanin production apparatus of the present embodiment, the above-described phycocyanin production method can be carried out, and phycocyanin excellent in heat resistance can be easily obtained.

<< Method for producing organic acid >>
The method for producing an organic acid according to the present invention comprises culturing a red alga belonging to the genus Hydrangea (Cyanidiophyceae) in a culture solution to produce an organic acid, and collecting the organic acid from the red alga and / or the culture. Including doing.

  In the present specification, the “organic acid” refers to an acid of an organic compound biosynthesized by red algae according to the embodiment. The organic acid may be an organic acid biosynthesized by the red alga according to the embodiment by anaerobic respiration, or an organic acid biosynthesized by the red alga according to the embodiment by a biochemical reaction of a glycolytic system and / or a TCA cycle. May be Examples of the organic acid include lactic acid, pyruvic acid, citric acid, α-ketoglutaric acid (2-oxoglutaric acid), succinic acid, fumaric acid, malic acid and the like. The organic acid is preferably succinic acid and / or lactic acid.

<Culture>
In one embodiment of the present invention, the method for producing an organic acid comprises culturing the red alga in a culture to produce an organic acid, and collecting the organic acid from within the red alga and / or the culture after the culture. Including that.

  In the present specification, the culture is used for culturing red algae, and is a substance in which red algae can grow. The culture is, for example, a medium, a buffer, water, an aqueous solution, or the like. The culture may be a liquid or a solid, but when producing an organic acid, a liquid is preferable because red algal or organic acid can be easily recovered.

From the viewpoint of efficient organic acid production, the culture for causing red algae to produce an organic acid is preferably an anaerobic culture. This is because when red algae is exposed to anaerobic conditions, the glycolytic reaction preferentially proceeds in the red algae, which is considered to enable the red algae to efficiently produce organic acids. . In the present specification, anaerobic culture refers to culture in a culture system capable of anaerobic fermentation in red algae. The inside of the culture system refers to, for example, the inside of a container for culturing red algae. Conditions under which anaerobic fermentation of red algae is possible are known, and for example, the oxygen concentration in the gas phase in the culture system may be 0 to 1% by volume, or 0 to 0.5% by volume. , 0 to 0.2% by volume, or 0% by volume.
Examples of the method for preparing the culture system for cultivating red algae under conditions in which red algae can be anaerobically fermented include, for example, culturing red algae in a culture container that is sealed and shielded from light irradiation. A method of consuming oxygen in the culture container by respiration of. Therefore, in the method for producing an organic acid according to this embodiment, it is preferable that the anaerobic culture is performed under dark conditions.
As another example, for example, there is a method of flowing nitrogen gas into the culture container and replacing oxygen in the culture container with nitrogen.

Culturing the red alga in a culture preferably includes aerobically culturing the red alga followed by anaerobic culturing. First, aerobically culturing red algae has the advantages of promoting the growth and proliferation of red algae and accumulating organic acid raw materials in red algae. Aerobic culture is preferably performed under bright conditions, and carbon dioxide is preferably present in the culture system. By culturing red algae in a culture container irradiated with light, the carbon source that is a raw material of the organic acid is synthesized by the photosynthesis of red algae. The conditions under which red algae can undergo photosynthesis are known, and examples thereof include conditions under which red algae are irradiated with light of about 20 to 200 μmol photons m ⁻² s ⁻¹ .

  In the anaerobic culture, it is preferable to carry out the anaerobic culture under a nitrogen-deficient condition. In anaerobic culture, by culturing red algae in a nitrogen-deficient state, the ability of red algae to produce organic acids can be improved. This mechanism is not clear, but it is considered that when nitrogen in the culture is reduced, accumulation of carbon sources such as glycogen and starch proceeds in the red alga.

The culture for culturing red algae may be any one that can cultivate red algae, and a medium that can be used as a medium for red algae may be used. Examples of the medium include M-Allen medium and MA-2 medium. These media are rich in nutrient sources available to red algae.
The medium as exemplified above can be used for both aerobic culture and anaerobic culture, but in the anaerobic culture, the production of organic acid is mainly carried out using the carbon source accumulated in the red algae. Therefore, it is not necessary to use a medium containing a nutrient source. In anaerobic fermentation, for example, a buffer capable of allowing red algae to survive can be used as a culture instead of the medium.

The temperature for culturing the red alga may be appropriately determined in consideration of the species of the red alga to be used and other conditions, but may be about 15 to 50 ° C, for example, 25 ° C to 50 ° C. Of course, it is preferable to set the temperature to about 30 to 45 ° C.
The period for culturing the red alga may be appropriately determined according to the concentration of the red alga in the culture and the amount of organic acid produced. The aerobic culture period may be about 1 hour to 20 days, 1 day to 10 days, or 1 to 3 days. Aerobic culture may be performed by shaking culture or aeration culture. The period of the anaerobic culture may be about 1 hour to 20 days, about 1 day to 10 days, or about 1 to 3 days.

When the red alga accumulates an organic acid in the red alga body, the organic acid may be collected from the red alga. For example, the red alga is recovered from the culture after the culturing and the red alga cells are crushed. Then, the organic acid may be collected from the crushed material.
When the red alga releases the organic acid to the outside of the red alga, the organic acid may be collected from the culture after the culture. For example, the red alga of the above embodiment may be cultured in a culture to produce an organic acid, and succinic acid and / or lactic acid may be collected from the culture after the culture. The culture after the culture may be one in which red algae has been separated by a separation treatment such as centrifugation or filtration, or may be a state in which red algae has not been separated. The red algae may not be completely removed in the separated culture.

  Collection and production of the organic acid may be performed simultaneously or separately. Examples of the case where the organic acid is collected and produced at the same time include a case where the supernatant of the culture solution of the red alga producing the organic acid is collected and the organic acid is collected.

  The method for producing an organic acid according to this embodiment may include purifying the collected organic acid. Purification may be performed at the same time as the collection or after the collection. Examples of the purification method include crystallization purification, chromatography by HPLC, and column purification of an ion exchange resin and the like.

  The red alga according to the present embodiment can directly convert carbon dioxide and light energy into resources by photosynthesis and is excellent in the ability to produce organic acids. According to the method for producing an organic acid of the present embodiment, it is possible to produce an organic acid with high efficiency using red algae. Therefore, it is a method that is economically and environmentally superior to the conventional method.

≪Hydrogen production method≫
The method for producing hydrogen according to the present invention includes culturing red alga belonging to the genus Cyanidiophyceae in a culture to produce hydrogen, and collecting the hydrogen.

<Culture>
In the present specification, the culture is used for culturing red algae, and is a substance in which red algae can grow. The culture is, for example, a medium, a buffer, water, an aqueous solution, or the like. The culture may be liquid or solid, but when hydrogen is produced, liquid is preferable because red algal or hydrogen can be easily recovered.

From the viewpoint of efficient hydrogen acid production, the culture for causing red algae to produce an organic acid is preferably an anaerobic culture. In the present specification, anaerobic culture refers to culture in a culture system capable of anaerobic fermentation in red algae. The inside of the culture system refers to, for example, the inside of a container for culturing red algae. Conditions under which anaerobic fermentation of red algae is possible are known, and for example, the oxygen concentration in the gas phase in the culture system may be 0 to 1% by volume, or 0 to 0.5% by volume. , 0 to 0.2% by volume, or 0% by volume.
Examples of the method for preparing the culture system for cultivating red algae under conditions in which red algae can be anaerobically fermented include, for example, culturing red algae in a culture container that is sealed and shielded from light irradiation. A method of consuming oxygen in the culture container by respiration of. Therefore, in the method for producing an organic acid according to this embodiment, it is preferable that the anaerobic culture is performed under dark conditions.
As another example, for example, there is a method of flowing nitrogen gas into the culture container and replacing oxygen in the culture container with nitrogen.

Culturing the red alga in a culture preferably includes aerobically culturing the red alga followed by anaerobic culturing. First, aerobically culturing red algae has the advantages of promoting the growth and proliferation of red algae and accumulating hydrogen raw materials in the red algae. Aerobic culture is preferably performed under bright conditions, and carbon dioxide is preferably present in the culture system. The conditions under which red algae can undergo photosynthesis are known, and examples thereof include conditions under which red algae are irradiated with light of about 20 to 200 μmol photons m ⁻² s ⁻¹ .

The culture for culturing red algae may be any one that can cultivate red algae, and a medium that can be used as a medium for red algae may be used. Examples of the medium include M-Allen medium and MA-2 medium. These media are rich in nutrient sources available to red algae.
The medium as exemplified above can be used for both aerobic culture and anaerobic culture, but in the anaerobic culture, the production of organic acid is mainly carried out using the carbon source accumulated in the red algae. Therefore, it is not necessary to use a medium containing a nutrient source. In anaerobic fermentation, for example, a buffer capable of allowing red algae to survive can be used as a culture instead of the medium.

The temperature for culturing the red alga may be appropriately determined in consideration of the species of the red alga to be used and other conditions, but may be about 15 to 50 ° C, for example, 25 ° C to 50 ° C. Of course, it is preferable to set the temperature to about 30 to 45 ° C.
The period for culturing the red alga may be appropriately determined according to the concentration of the red alga in the culture and the amount of organic acid produced. The aerobic culture period may be about 1 hour to 20 days, 1 day to 10 days, or 1 to 3 days. Aerobic culture may be performed by shaking culture or aeration culture. The period of the anaerobic culture may be about 1 hour to 20 days, about 1 day to 10 days, or about 1 to 3 days.

Since red algae release hydrogen to the outside of red algae, hydrogen may be collected from the gas phase in the culture vessel after culturing.
Hydrogen collection and production may be performed simultaneously or separately. An example of the case where hydrogen is collected and produced simultaneously is, for example, the case where hydrogen is collected by recovering the gas phase in the culture vessel of the red alga producing hydrogen.

  The red alga according to this embodiment can directly convert carbon dioxide and light energy into resources by photosynthesis. According to the method for producing hydrogen of the present embodiment, hydrogen can be produced with high efficiency by red algae. Therefore, it is a method that is economically and environmentally superior to the conventional method.

  Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Further, in the following Examples and Comparative Examples, phycocyanin obtained from sizon is called sizon phycocyanin, and phycocyanin obtained from spirulina is called spirulina phycocyanin.

1. Production of schizon phycocyanin (1)
<Example 1>
Sizon (NIES collection strain number: NIES-3377) was suspended in 10 mL M-Allen medium (pH 2.5) so that OD ₇₃₀ = 20, 40 ° C., air (1% CO ₂ ), white light 40 to 40. Aerobic culture was carried out for 4 to 10 days under a bright condition of 60 μmol photons m ⁻² s ⁻¹ .
After that, the medium after the aerobic culture was centrifuged to collect schizon, and the cells were resuspended in 10 mL of pure water (collection medium) so that OD ₇₃₀ = 20. This resuspended product (recovered dye) was placed in a gas chromatography vial, and sealed with butyl rubber, and two syringes were connected to the stopper. N ₂ gas was blown into the gas chloride vial for 10 minutes from one syringe to replace the air in the bottle with N ₂ gas. The N ₂ gas was stopped, the syringe was removed from the stopper, the bottle was covered with aluminum foil, and shake culture was carried out at 40 ° C. in the dark for 3 days in a sealed state. The supernatant (collection medium) after shaking culture was blue, and it could be confirmed that schizon phycocyanin was extracted in the supernatant.
The absorption spectrum of the supernatant after shaking culture was measured, and the amount of schizon phycocyanin (mg / L) in the supernatant was calculated from the absorbance at a wavelength of 620 nm. Cultivation was performed in duplicate, and these were designated as Example 1-1 and Example 1-2. The results are shown in Table 2 and FIG. The composition of M-Allen medium is shown in Table 1 below.

<Example 2>
Culturing was performed in the same manner as in Example 1 except that the air in the bottle was not replaced with N ₂ gas in Example 1 above, and the amount of sizon phycocyanin (mg / L) in the supernatant was calculated. Cultivation was performed in duplicate, and these were designated as Example 2-1 and Example 2-2. The results are shown in Table 2 and FIG.

<Example 3>
Culturing was carried out in the same manner as in Example 1 except that shaking culture was carried out under light conditions instead of shaking culture under dark conditions in Example 1 above, and the amount of sizon phycocyanin in the supernatant (mg / L ) Was calculated. Cultivation was performed in duplicate, and these were designated as Example 3-1 and Example 3-2. The results are shown in Table 2 and FIG.

<Example 4>
In the above-mentioned Example 1, the culture was performed in the same manner as in Example 1 except that the air in the bottle was not replaced with N ₂ gas, and the shaking culture was performed under light conditions instead of the shaking culture under dark conditions. Then, the amount of schizon phycocyanin in the supernatant (mg / L) was calculated. Cultivation was performed in duplicate, and these were designated as Example 4-1 and Example 4-2. The results are shown in Table 2 and FIG.

  The method for calculating the amount of phycocyanin was calibrated by measuring the absorbance at 620 nm using a multipurpose spectrophotometer (Shimadzu Corporation) using phycocyanin (Funakoshi Co., Ltd.) extracted from commercially available cyanobacteria Spirulina as a standard substance. Calculated by the line.

The results are shown in Table 2 and FIG. The supernatant after shaking culture of Examples 1 to 4 contained a large amount of schizon phycocyanin, and it was shown that sizon phycocyanin can be obtained from schizon by a simple operation of culturing the sison with shaking in pure water. Was done.
Comparing Examples 1 to 2 and Examples 3 to 4, in Examples 1 to 2 in which sizon was cultured under shaking under dark anaerobic conditions, Examples 3 to 3 in which sizon was cultured under shaking under clear anaerobic conditions were used. Compared with 4, more schizon phycocyanin was contained in the supernatant after shaking culture.

2. Manufacture of schizon phycocyanin (2)
<Example 5>
In Example 1 above, the cells were resuspended in pure water (recovery medium), the resuspension (recovered dye) was transferred to a 1.5 mL tube, and left standing at room temperature for 3 days in a sealed state.
The supernatant (recovery medium) after standing was blue (Table 3), and it was confirmed that schizon phycocyanin was extracted in the supernatant.
After that, the mixture was allowed to stand still at room temperature for 1 month under the same conditions in a sealed state. FIG. 4 (right) shows a sample one month after resuspension in pure water. The supernatant remained a deep blue color even after 1 month. This indicates that sizon phycocyanin has excellent storage stability.
The supernatant of Example 5 after standing contains a large amount of schizon phycocyanin, and schizon phycocyanin can be obtained from schizon by a very simple operation of introducing schizon into pure water and allowing it to stand. Was shown.

<Comparative Example 1>
In Example 1 above, the cells were resuspended in M-Allen medium instead of pure water (collection medium), then the resuspension was transferred to a 1.5 mL tube, and allowed to stand at room temperature for 3 days in a sealed state. . The supernatant after standing was not blue (Table 3). After that, the mixture was allowed to stand still at room temperature for 1 month under the same conditions in a sealed state.
FIG. 4 (left) shows a sample one month after resuspension in M-Allen medium. Even after 1 month, the supernatant did not show blue color.

3. Evaluation of heat resistance of schizon phycocyanin (1)
<Example 6>
The recovery medium containing schizon phycocyanin obtained in Example 1 was heat-treated at 30 ° C. for 1 hour.

<Example 7>
The heat treatment was performed in the same manner as in Example 6 except that the heat treatment condition in Example 6 was changed from 30 ° C. for 1 hour to 60 ° C. for 1 hour.

<Example 8>
In Example 5, a recovery medium containing schizon phycocyanin was obtained in the same manner as in Example 1 except that pure water was replaced with 20 mM Hepes-KOH (pH 7.8), and the mixture was heated at 30 ° C for 1 hour. Processed.

<Example 9>
The heat treatment was performed in the same manner as in Example 8 except that the heat treatment conditions in Example 8 were changed from 30 ° C. for 1 hour to 60 ° C. for 1 hour.

<Comparative example 2>
Spirulina phycocyanin (product: C-Phycocyanin, manufactured by Japan Aldi Co., Ltd.) final concentration of 0.3 mg / mL was suspended in 20 mM Hepes-KOH (pH 7.8), and heat-treated at 30 ° C. for 1 hour.

<Comparative example 3>
The heat treatment was performed in the same manner as in Comparative Example 2 except that the heat treatment condition in Comparative Example 2 was changed from 30 ° C. for 1 hour to 60 ° C. for 1 hour.

  The absorption spectra of the liquids after the heat treatments of Examples 6 to 9 and Comparative Examples 2 to 3 were measured.

  Results are shown in FIG. The absorption maximum of phycocyanin is around 620 nm. Comparing Examples 6 to 9 with Comparative Examples 2 to 3, in Spirulina phycocyanin of Comparative Examples 2 to 3 (spirulina PC), the absorbance is reduced by half by the heat treatment at 60 ° C as compared with the heat treatment at 30 ° C. On the other hand, with the schizon phycocyanins of Examples 6 to 9 (schizon PC), the decrease in absorbance was slight even after the heat treatment at 60 ° C. From these results, it became clear that the heat resistance of schizon phycocyanin is superior to that of spirulina phycocyanin.

  In addition, in Examples 6 to 7 in which pure water was used as the recovery medium, absorption maximums other than around 620 nm were observed as compared with Examples 8 to 9 in which Hepes-KOH (pH 7.8) was used as the recovery medium. There wasn't. For example, in Examples 8 to 9, the maximum was observed near 400 nm and around 680 nm. From these results, it became clear that the purity of the recovered schizon phycocyanin is increased by using pure water as the recovery medium.

4. Evaluation of heat resistance of schizon phycocyanin (2)
<Example 10>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM Hepes-KOH (pH 7.8) and heat-treated at 30 ° C. for 1 hour.
<Example 11>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM acetate buffer (pH 4) and heat-treated at 30 ° C. for 1 hour.
<Example 12>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM acetate buffer (pH 3.5) and heat-treated at 30 ° C. for 1 hour.
<Example 13>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM Hepes-KOH (pH 7.8) and heat-treated at 65 ° C. for 1 hour.
<Example 14>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM acetate buffer (pH 4) and heat-treated at 65 ° C. for 1 hour.
<Example 15>
0.1 mL of the recovery medium containing schizon phycocyanin obtained in Example 1 was suspended in 20 mM acetate buffer (pH 3.5) and heat-treated at 65 ° C. for 1 hour.

<Comparative example 4>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Algae) was suspended in 20 mM Hepes-KOH (pH 7.8) and heat-treated at 30 ° C for 1 hour.
<Comparative Example 5>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Aldi Co., Ltd.) was suspended in 20 mM acetate buffer (pH 4) and heat-treated at 30 ° C. for 1 hour.
<Comparative example 6>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Aldi Co., Ltd.) was suspended in 20 mM acetate buffer (pH 3.5) and heat-treated at 30 ° C. for 1 hour.
<Comparative Example 7>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Aldi Co., Ltd.) was suspended in 20 mM Hepes-KOH (pH 7.8) and heat-treated at 65 ° C for 1 hour.
<Comparative Example 8>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Aldi) was suspended in 20 mM acetate buffer (pH 4) and heat-treated at 65 ° C for 1 hour.
<Comparative Example 9>
A final concentration of 0.1 mg / mL of spirulina phycocyanin (product C-Phycocyanin, manufactured by Japan Aldi Co., Ltd.) was suspended in 20 mM acetate buffer (pH 3.5) and heat-treated at 65 ° C for 1 hour.

The absorption spectra of the liquids after the heat treatments of Examples 10 to 15 and Comparative Examples 4 to 9 were measured.
FIG. 6 shows the results. In FIG. 6, the absorption maximum of phycocyanin is around 620 nm.

  Comparing Examples 10 to 15 with Comparative Examples 4 to 9, in the Spirulina phycocyanin of Comparative Examples 4 to 9 (spirulina PC), while the absorbance was significantly decreased by the heat treatment at 65 ° C., With 10 to 15 schizon phycocyanin (schizon PC), the decrease in absorbance was slight even after the heat treatment at 65 ° C. From these results, it became clear that the schizon phycocyanin has a higher heat resistance than the spirulina phycocyanin.

  In addition, comparing Examples 10 and 13 with Examples 11-12 and 14-15, it was found that pH 3 was higher than that of Examples 10 and 13 in which heat-treatment of schizon phycocyanin was performed in Hepes-KOH buffer having pH 7.8. In Examples 11 to 12 and 14 to 15 in which schizon phycocyanin was heat-treated in an acetate buffer of 0.5 or pH 4, the decrease in absorbance was slight even after the heat treatment at 65 ° C. From these results, it became clear that the shizon phycocyanin exhibits more excellent heat resistance under acidic conditions.

5. Production of Organic Acid Using Sison <Example 16>
(Aerobic culture)
Sizon (NIES collection strain number: NIES-3377) was suspended in 10 mL M-Allen medium (pH 2.5) so that OD ₇₃₀ = 20, 40 ° C., air (1% CO ₂ ), white light 40 to 40. Aerobic culture was carried out for 4 to 10 days under a bright condition of 60 μmol photons m ⁻² s ⁻¹ .
(Anaerobic culture)
After that, the medium after the aerobic culture was centrifuged to collect schizon, and the cells were resuspended in 10 mL of 20 mM Hepes-KOH (pH 7.8) so that OD ₇₃₀ = 20. The resuspended material was placed in a gas bottle and sealed with butyl rubber, and two syringes were connected to the stopper. N ₂ gas was blown into the gas chloride vial from one syringe for 10 minutes to replace the air in the bottle with N ₂ gas. The N ₂ gas was stopped, the syringe was removed from the stopper, the bottle was covered with aluminum foil, and shake culture was carried out at 40 ° C. in the dark for 3 days in a sealed state.

<Example 17>
In Example 16, except that the aerobic culture was performed using a medium in which nitrogen was removed from the M-Allen medium (pH 2.5) instead of the M-Allen medium (pH 2.5) during the aerobic culture. The culture was performed in the same manner as in Example 16. Table 4 below shows the medium obtained by removing nitrogen from the M-Allen medium.

Each of the culture solutions after the anaerobic culture obtained in Examples 16 to 17 above was centrifuged to collect 1 ml of the supernatant. The supernatant was freeze-dried, the obtained solid component was dissolved in 100 μl of a mobile phase of 3 mM HClO ₄ solution, and these were subjected to high performance liquid chromatography (HPLC) LC-2000 Plus (JASCO Corporation) according to a standard method. The components were analyzed.

The HPLC measurement conditions are as follows.
Mobile phase: 3 mM aqueous solution of perchloric acid reaction: 0.2 mM bromothymol blue (BTB), 15 mM sodium hydrogen phosphate _{(Na 2 HPO 4 · 12H 2} O)
Column: Shodex RSpak KC-811 x2

The results are shown in FIG. 7 and Table 5. The vertical axis of the graph shown in FIG. 7 is a value obtained by converting the measured value of each organic acid obtained by HPLC into the amount (mg / L) of the organic acid per 1 L of the culture solution.
From the results shown in FIG. 7, it was shown that extracellular production of organic acids (succinic acid, lactic acid, acetic acid) by schizon is possible. It was possible to increase the amount of succinic acid in the culture medium by culturing under a nitrogen-deficient condition during aerobic culture. Further, it was shown that culturing under a nitrogen-deficient condition during aerobic culturing is effective for obtaining lactic acid in the culture solution. In order to obtain acetic acid in the culture medium, it was shown that it is effective to culture under a nitrogen-sufficient condition during aerobic culture.
In particular, lactic acid could be produced at a high concentration exceeding 1 g / L.

6. Production of hydrogen using sizon <Example 18>
(Aerobic culture)
Sizon (NIES collection strain number: NIES-3377) was suspended in 10 mL M-Allen medium (pH 2.5) so that OD ₇₃₀ = 20, 40 ° C., air (1% CO ₂ ), white light 40 to 40. Aerobic culture was performed for 4 to 10 days under a bright condition of 70 μmol photons m ⁻² s ⁻¹ .
(Anaerobic culture)
After that, the medium after the aerobic culture was centrifuged to collect schizon, and the cells were resuspended in 10 mL of 20 mM Hepes-KOH (pH 7.8) so that OD ₇₃₀ = 20. The resuspended material was placed in a gas bottle and sealed with butyl rubber, and two syringes were connected to the stopper. N ₂ gas was blown into the gas chloride vial from one syringe for 10 minutes to replace the air in the bottle with N ₂ gas. The N ₂ gas was stopped, the syringe was removed from the stopper, the bottle was covered with aluminum foil, and shake culture was carried out at 40 ° C. in the dark for 3 days in a sealed state.

<Example 19>
In Example 18 described above, except that the aerobic culture was performed using a medium in which nitrogen was removed from the M-Allen medium (pH 2.5) instead of the M-Allen medium (pH 2.5) during the aerobic culture. The culture was performed in the same manner as in Example 16.

<Example 20>
In Example 18, except that Hepes-KOH (pH 7.8) was used instead of M-Allen medium (pH 2.5) at the time of aerobic culture, the same as Example 16 except that the aerobic culture was performed. Were cultured.

<Example 21>
In Example 18, except that the aerobic culture was performed using Hepes-KOH (pH 7.8) with nitrogen removed, instead of the M-Allen medium (pH 2.5) during aerobic culture. Culture was performed in the same manner as in Example 16.

  The amount of hydrogen was measured using GC-TCD (Gas Chromatograph-Thermal Conductivity Detector) for each gas phase in the hermetically sealed container after the anaerobic culture obtained in Examples 18 to 21 described above.

  Table 6 shows the results. From the results shown in Table 6, it was shown that hydrogen production by sizon is possible.

  The configurations and combinations thereof in each of the embodiments described above are merely examples, and additions, omissions, substitutions, and other changes can be made to the configurations without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, but is limited only by the scope of claims (claims).

DESCRIPTION OF SYMBOLS 1 ... Phycocyanin production apparatus, 10 ... Culture tank, 11 ... Piping, 20 ... Pigment collection tank, 30, 31 ... Red algae, 40 ... Culture solution, 50, 51 ... Collection solution, 60, 61 ... Pigment collection solution

Claims (6)

A food containing a phycocyanin-containing substance, which contains phycocyanin of the red alga belonging to the genus Cyanidiophyceae (Cyanidioschyzon) and has a pH of 0 or more and 6 or less .   The food according to claim 1, which is a beverage.   A heat treatment method for food, comprising heat-treating the food according to claim 1 or 2.   The heat treatment method for food according to claim 3, wherein the temperature of the heat treatment is 60 ° C or higher and 85 ° C or lower. A method for producing the phycocyanin-containing product contained in the food according to claim 1 or 2,
Red algae belonging to the genus Cyanidiophyceae (Cyanidioschyzon) are cultivated in a culture to produce phycocyanin, and a recovery medium having a salt concentration of the culture or less is contacted with the red algae. A method for producing a phycocyanin- containing product , comprising: obtaining a pigment recovery product in which phycocyanin produced by red alga is extracted in the recovery medium, and collecting phycocyanin from the red alga.   The manufacturing method of the foodstuff of Claim 1 or 2 including using the said phycocyanin containing material obtained by the manufacturing method of the phycocyanin containing material of Claim 5.