JP6998157B2 - How to make fortified foods, Euglena-containing food compositions and fortified foods - Google Patents

Description

The present invention relates to a method for producing a fortified food, a euglena-containing food composition, and a method for fortifying food.

Euglena (genus name: Euglena, Japanese name: Euglena) is attracting attention as a biological resource that is expected to be used as food, feed, fuel and the like.
Euglena is equipped with 59 kinds of nutrients such as vitamins, minerals, amino acids, unsaturated fatty acids, etc., which correspond to most of the nutrients necessary for human life, and supplements and foods for balanced intake of various kinds of nutrients. It has been proposed that it can be used as a food source in poor areas where necessary nutrients cannot be obtained.

Further, an attempt has been made to use food as a culture solution when culturing Euglena. For example, Patent Document 1 describes a technique for culturing Euglena in a liquid containing brewed liquor such as beer and sake. ing. Conventional techniques utilize foods as a nutrient source for culturing Euglena and have no intention of producing foods containing live Euglena.

Japanese Unexamined Patent Publication No. 2014-054240

Conventionally, most euglena-containing foods containing supplements are processed and cooked euglena dry powder. In addition, mass production of general microalgae requires large-scale equipment such as a large pool, and costs are required for the process of separating, recovering, and drying microalgae cells after culturing.

Therefore, if the technology to use Euglena as food without separating, recovering, and drying can be realized, the variation of Euglena-containing food can be increased, the production cost can be reduced, and the bioavailability (bioavailability) is high. It becomes possible to provide food.

The present invention has been made in view of the above problems, and an object of the present invention is to cultivate Euglena in a food product so that Euglena can be used as a food containing Euglena without being separated, recovered or dried. It is an object of the present invention to provide a possible method for producing a fortified food, a euglena-containing food composition, and a method for fortifying a food.

As a result of diligent research to solve the above problems, the present inventors have found that when Euglena cells are cultured using a specific food or food material, Euglena cells can grow well and enhance the nutrition of the food, and the nutrition-enriched food. It was found that the present invention can be manufactured, and the present invention has been completed.

Therefore, the subject is the step of preparing at least one kind of food selected from the group including gel-like foods, vegetable drinks, fruit drinks, and soybean foods according to the method for producing a fortified food of the present invention. It is solved by performing a cell addition step of adding euglena cells to a food and a culturing step of culturing the euglena cells together with the food.
At this time, the gelled food may be selected from tofu and egg yolk pudding.
At this time, the vegetable beverage may be tomato juice.
At this time, the fruit beverage may contain grape juice or prune juice.
At this time, the soybean food may be selected from the group containing soymilk drink, natto, and tofu.
At this time, the food may contain natto and at least one selected from seaweed or dried sardines .

According to the method for producing a nutritionally fortified food of the present invention, the nutritional components contained in the food can be fortified by culturing Euglena cells in the food. Further, it is possible to provide a euglena-containing food composition enriched with nutritional components without separating, recovering, and drying euglena.

Further, the above-mentioned problems include a step of preparing at least one kind of food selected from the group including gel-like foods, vegetable drinks, fruit drinks, and soybean foods according to the method for fortifying foods of the present invention, and the above-mentioned foods. It is solved by performing a cell addition step of adding euglena cells to the food and a culturing step of culturing the euglena cells together with the food.
According to the method for fortifying foods of the present invention, the nutritional components contained in foods can be fortified by culturing Euglena cells in the foods.

Further, according to the method for culturing Euglena of the present invention, the subject is a culture medium preparation step for preparing a culture medium containing at least one food product selected from the group containing gelled foods, vegetable drinks, fruit drinks, and soybean foods. The problem is solved by performing a cell addition step of adding Euglena cells to the medium and a culture step of culturing the Euglena cells in the medium.
According to the method for culturing Euglena of the present invention, Euglena cells can be cultured using the nutritional components contained in food.

Further, according to the Euglena dyeing method of the present invention, the above-mentioned problems are a step of preparing at least one kind of food selected from the group including vegetable drinks and fruit drinks, and cell addition to add Euglena cells to the foods. It is solved by carrying out a step and a culturing step of culturing the Euglena cells together with the food .
According to the method for staining Euglena of the present invention, it is possible to provide Euglena containing a pigment derived from vegetables or fruits, specifically, a polyphenol such as anthocyanin or a carotenoid in the cell. Euglena cells can be stained with a desired color by selecting a vegetable or fruit containing a pigment corresponding to the desired color.

Further, the above-mentioned problem is solved by containing natto and at least one selected from seaweed or boiled seaweed and used for culturing Euglena according to the medium composition for Euglena of the present invention.
The medium composition for Euglena of the present invention can be used as a medium suitable for the growth of Euglena, and in particular, Euglena grown with a medium composition of a combination of natto and seaweed is composed only of non-animal foods. It can be a source of vitamin B ₁₂ (V. B ₁₂ ), which tends to be deficient in strict vegetarian (vegan) diets.

Further, according to the method for producing a fermented vegetable food of the present invention, the subject is a step of mixing soymilk, plant-derived lactic acid bacteria, and Euglena cells to prepare a raw material mixture, and a step of fermenting the raw material mixture. , Is solved .

According to the present invention, a method for producing a fortified food, a food composition containing euglena, and a food can be used as a food without separating, recovering, and drying euglena by culturing euglena in the food. A method of fortification can be provided.
Therefore, according to the present invention, it is possible to provide foods and dietary supplements that reduce the costs required for large-scale culture equipment, recovery, drying, and processing processes, and that utilize the nutrients of Euglena.

Further, according to the present invention, by selecting an appropriate food and culturing Euglena, it is possible to supply nutrients necessary for the growth of Euglena to effectively grow Euglena and to enhance the nutrition of food. , It becomes possible to produce fortified foods.

It is a flow chart which shows the manufacturing method of the fortified food which concerns on one Embodiment of this invention, the fortification method of a food, and the dyeing method of Euglena. It is a flow chart which shows the culture method of Euglena which concerns on one Embodiment of this invention. After re-adding the amounts of V.B ₁ and V.B ₁₂ initially contained in the CM medium to the CM medium in which the Euglena cells used in the test grew poorly (left), only V.B ₁₂ was added. It is a photograph after re-addition (middle) and after re-addition of only V.B ₁ (right). It is a photograph showing the growth state of Euglena cells using the culture solution containing natto examined in Test 1. Natto only (left), natto and dried sardines (middle), natto and seaweed (right). It is a graph which shows the Euglena cell density of each sample in the stationary phase using the culture solution containing natto examined in Test 1. It is a graph which shows the growth curve of each sample in the case of using the culture solution containing natto examined in Test 1. It is a photograph showing the growth state of Euglena cells after culturing for 9 days using the milk and yogurt (Caspian sea yogurt containing Cremoris bacterium) examined in Test 2. The numbers in the figure indicate the water: milk ratio (volume ratio). It is a photograph showing the growth state of Euglena cells after culturing for 24 days using soymilk and soymilk yogurt (containing plant-derived lactic acid bacteria) examined in Test 2. The numbers in the figure indicate the soymilk: water ratio (volume ratio). It is a photograph showing the growth state of Euglena cells after culturing for 8 days using the tomato juice examined in Test 3. It is a photograph showing the change in hue when euglena cells were cultured by adding boiled dried tomato juice examined in Test 3 (each photograph was taken every other day). It is a micrograph showing the staining of Euglena cells after culturing using the grape juice or prune juice examined in Test 3 (left: grape juice, right: prune juice). It is a graph which shows the Euglena cell density of each sample after culturing for 9 days using the culture solution containing tomato juice examined in Test 3. It is a graph which shows the growth curve when the culture solution containing tomato juice examined in Test 3 was used. Photograph showing the growth state of Euglena cells after culturing using the egg yolk pudding examined in Test 4 (right), 0.2% agar in the solution of "soy milk: water = 1: 9", and 0. It is a photograph showing the growth state of Euglena cells after culturing using a gel medium containing 1% gelatin (left) or a gel medium containing only 0.2% agar in the same solution (middle). It is a photograph (left) showing the growth state of Euglena cells after culturing using the tofu examined in Test 4, and a photograph (right) showing the state of breaking the tofu. It is a graph which shows the ratio (%) of each amino acid to the total amount of amino acids in each food in Euglena, tomato, natto, and yogurt. It is a graph which shows the density of Euglena cells after culturing using various beverages which examined in test 5. It is a graph showing the density of Euglena cells after culturing under dark culture conditions using various beverages using various beverages, which was examined in Test 5, and after culturing after switching to light culture irradiation conditions. .. It is a graph showing the density of Euglena cells after culturing under light irradiation culture conditions using various beverages using various beverages, which was examined in Test 5, and after culturing after switching to dark culture conditions. .. 6 is a graph showing the density of Euglena cells after culturing using various beverages under predetermined conditions, which was examined in Test 6. It is a graph which shows the growth curve when the culture solution containing wine or grape juice examined in Test 7 was used. It is a graph which shows the Euglena cell density of each sample after culturing for 9 days using the culture medium containing the wine examined in Test 7. 6 is a photomicrograph showing the staining of Euglena cells after culturing with the grape juice examined in Test 8. It is a photograph showing the growth state of Euglena cells after culturing for 6 days using the red paprika juice squeezed as examined in Test 9. It is a photograph showing the change in hue when euglena cells were cultured using the red paprika juice squeezed examined in Test 9 (each photograph was taken every day from the 3rd day). It is a photograph showing the growth state of Euglena cells after culturing for 12 days using the red paprika juice squeezed as examined in Test 9. It is a graph showing the Euglena cell density in a steady state after culturing for 12 days using the red paprika juice squeezed as examined in Test 9.

Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 27.
This embodiment includes a method for producing a fortified food, a food composition containing euglena, a method for fortifying food, a method for culturing euglena, a method for dyeing euglena fermented vegetable food, a medium composition for euglena, and producing fermented vegetable food. It concerns methods, fermented vegetable foods.

<Euglena>
In embodiments, "Euglena" includes microorganisms taxonomically classified in the genus Euglena (Euglena), varieties thereof, variants thereof and closely related species of the family Euglena (Euglenaceae).
Here, the genus Euglena (Euglena) is a group of eukaryotes belonging to Excavata, Euglenozoa, Euglenidae, Euglena, and Euglena.

Specific examples of the species included in the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis and the like.
As Euglena, Euglena gracilis (E. gracilis), in particular, Euglena gracilis (E. gracilis) Z strain can be used, but in addition, a mutant strain SM-ZK strain of Euglena gracilis (E. gracilis) Z strain can be used. (Chloroplast-deficient strains), variants E. gracilis var. Bacillaris, gene mutants such as chloroplast variants of these species, and other Euglena species such as Astaia longa.

The genus Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any genus Euglena that has already been isolated may be used.
The genus Euglena includes all its variants. In addition, among these mutant strains, those obtained by genetic methods such as recombination, transduction, transformation and the like are also included.

In culturing Euglena cells, the culture medium is, for example, a culture medium to which nutrient salts such as a nitrogen source, a phosphorus source, and minerals are added, for example, a modified Kramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L. , KH ₂ PO ₄ 1.0 g / L, י4.7H ₂ O 0.2 g / l, CaCl 2.2H ₂ O 0.02 g / l, Fe _{2 (SO 2) 3.7H 2 O 3 mg /} l, MnCl 2.4H ₂ O 1.8 mg / l, CoSO _{4.7H 2} O 1.5 mg / l, ZnSO _{4.7H 2} O _0.4 mg / l, Na ₂ MoO _4.2 H ₂ O 0.2 mg / l, CuSO _{4.5H 2 O} 0.02 g / l, thiamine hydrochloride (vitamin B ₁ ) 0.1 mg / l, cyanocobalamine (vitamin B ₁₂ ), (pH 3.5)) can be used. It should be noted that (NH ₄ ) ₂ HPO ₄ can also be converted into (NH ₄ ) ₂ SO ₄ or NH ₃ aq. In addition, known Hutener medium and Korean-Hutner medium prepared based on the description of Euglena physiology and biochemistry (edited by Shozaburo Kitaoka, Publishing Center of the Society) may be used.

The pH of the culture broth is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, photosynthetic microorganisms can grow predominantly over other microorganisms, so that contamination can be suppressed.
Euglena cells can be cultured by a method using a culture device that directly uses sunlight, a method that sends sunlight focused by a light condensing device with an optical fiber, etc., and irradiates it in a culture tank to use it for photosynthesis. good.
In addition, the euglena cells can be cultured using, for example, a feed batch method, but a flask culture, a culture using a fermenter, a batch culture method, a semi-batch culture method (feeding culture method), or a continuous culture method (perfusion). Any liquid culture method such as (culture method) may be used.
Separation of Euglena cells can be performed, for example, by centrifugation, filtration or simple sedimentation of the culture medium, but in this embodiment, the cells do not have to be separated.

<Foods subject to fortification or foods used in the culture medium>
In the present embodiment, the food to be fortified for nutrition, the food used as the medium for culturing Euglena, and the food added to the medium for culturing Euglena are not particularly limited as long as the food is capable of proliferating Euglena cells. Examples thereof include liquid foods, gel foods, sol foods and solid foods, and liquid foods such as vegetable drinks or fruit drinks, gel foods and soybean foods are preferable.

(Liquid food)
Liquid foods refer to, for example, vegetable beverages, fruit beverages, milk, soy milk, rice milk, coffee, tea beverages and other beverages, various soups such as corn soup, liquid cold confectionery such as juice powder, and other liquid foods. , Refers to foods that are liquid when eaten.
Examples of vegetable beverages include beverages containing green-yellow vegetables such as tomatoes, carrots, spinach, and kale.
Examples of fruit beverages include beverages containing fruit juices such as grape juice, prune juice, pine juice, orange juice, apple juice, almond milk, coconut milk and acerola.

The vegetable beverage and fruit beverage are not limited to commercially available beverages, and fruit juice obtained by squeezing, grated, or crushing vegetables and fruits can also be used. It is also possible to use a mixed solution in which vegetable and fruit juices are mixed.

(Gel-like food)
The gel-like food refers to a food that is hardened into a gel by the gelling ability of a thickener and a gelling agent, and is also referred to as a jelly-like food or a semi-solid food. Examples include tofu, gel pudding, jelly, yokan, boiled sardines, agar and gelatin coagulated with other foods.

(Sol-like food)
The sol-like food refers to a food that does not have shape retention and has fluid physical characteristics at room temperature (25 ° C.), and is a viscous liquid or a viscous liquid food, for example, yogurt. Can be mentioned.

(Solid food)
The solid food refers to a solid food such as natto, dried sardines, and seaweed, and refers to a food having a solid shape at the time of eating.

(Soy food)
The soybean food refers to a food produced from soybean as a raw material, and examples thereof include soymilk drinks, natto, tofu, miso, and soy sauce.

<Manufacturing method of fortified foods>
The method for producing a fortified food according to the present embodiment includes a step of preparing at least one kind of food selected from the group including gel-like food, vegetable drink, fruit drink, and soybean food, and addition of Euglena cells to the food. It is characterized by performing a cell addition step and a culturing step of culturing the Euglena cells together with the food.
Hereinafter, each step will be described in detail with reference to FIG.

In the step of preparing foods, at least one kind of food selected from the group including gelled foods, vegetable drinks, fruit drinks, and soybean foods is prepared (step S1). The food prepared at this time is not limited to the finished food as a product, and may be a food in the process of being manufactured. In addition, media usually used for culturing Euglena, such as Kramer-Myers medium (CM medium) and Korean-Hutner medium (KH medium), may be added to foods, and the components contained in these media may be appropriately selected. It can also be added to foods.

In the cell addition step, Euglena cells are added to the food prepared in the step of preparing the food (step S2). At this time, the amount of Euglena cells added to the food may be appropriately selected according to the type of the food and its properties (solid or liquid, viscosity, pH, composition, contained components, etc.). ..

In the culturing step, the Euglena cells are cultured together with the food (step S3). The culture conditions such as temperature, period, light irradiation, and presence / absence of shaking may be appropriately selected according to the type and properties of the food and the final state of the desired food.

According to the method for producing a nutritionally fortified food according to the present embodiment, it is possible to provide a euglena-containing food composition enriched with nutritional components. Further, it is possible to provide a euglena-containing food composition enriched with nutritional components without separating, recovering, and drying euglena.

<Food fortification method>
The method for fortifying foods according to the present embodiment includes a step of preparing at least one kind of food selected from the group including gel-like foods, vegetable drinks, fruit drinks, and soybean foods, and adding Euglena cells to the foods. It is characterized by performing a cell addition step and a culturing step of culturing the Euglena cells together with the food.
Hereinafter, each step will be described in detail with reference to FIG.

In the step of preparing foods, at least one kind of food selected from the group including gelled foods, vegetable drinks, fruit drinks, and soybean foods is prepared (step S1). The food prepared at this time is not limited to the finished food as a product, and may be a food in the process of being manufactured. Further, it is also possible to add a medium usually used for culturing Euglena, such as a CM medium or a KH medium, to the food, or to appropriately select and add the components contained in these media to the food.

In the cell addition step, Euglena cells are added to the food prepared in the step of preparing the food (step S2). At this time, the amount of Euglena cells added to the food may be appropriately selected according to the type of the food and its properties (solid or liquid, viscosity, pH, composition, contained components, etc.). ..

In the culturing step, the Euglena cells are cultured together with the food (step S3). The culture conditions such as temperature, period, light irradiation, and presence / absence of shaking may be appropriately selected according to the type and properties of the food and the final state of the desired food.

According to the method for fortifying foods according to the present embodiment, the nutritional components contained in the foods can be fortified.

<Euglena dyeing method>
The method for staining Euglena according to the present embodiment includes a step of preparing at least one kind of food selected from the group including vegetable drinks and fruit drinks, a cell addition step of adding Euglena cells to the food, and the Euglena cells. It is characterized by carrying out a culturing step of culturing with the food.
Hereinafter, each step will be described in detail with reference to FIG.

In the step of preparing the food, at least one kind of food selected from the group including the vegetable drink and the fruit drink is prepared (step S1). The food (vegetable beverage or fruit beverage) prepared at this time is not limited to the finished food as a product, and may be a food in the process of production (vegetable or fruit juice). It is also possible to add a medium usually used for culturing Euglena, such as a CM medium or a KH medium, to the food, or to appropriately select and add the components contained in these media to the food.

In the cell addition step, Euglena cells are added to the food prepared in the step of preparing the food (step S2). At this time, the amount of Euglena cells added to the food may be appropriately selected according to the type of the food and its properties (viscosity, pH, composition, contained components, etc.).

In the culturing step, the Euglena cells are cultured together with the food (step S3). The culture conditions such as temperature, period, light irradiation, and presence / absence of shaking may be appropriately selected according to the type and properties of the food and the desired degree of Euglena staining.

According to the method for staining Euglena according to the present embodiment, it is possible to provide Euglena containing a pigment derived from vegetables or fruits, specifically, a polyphenol such as anthocyanin or a carotenoid in the cell. Euglena cells can be stained with a desired color by selecting a vegetable or fruit containing a pigment corresponding to the desired color.

<Euglena culture method>
The method for culturing Euglena according to the present embodiment includes a medium preparation step of preparing a medium containing at least one food selected from the group including gel-like food, vegetable beverage, fruit beverage, and soybean food, and Euglena in the medium. It is characterized by performing a cell addition step of adding cells and a culturing step of culturing the Euglena cells in the medium.
Hereinafter, each step will be described in detail with reference to FIG.

In the culture medium preparation step, a culture medium containing at least one food product selected from the group containing gelled foods, vegetable drinks, fruit drinks, and soybean foods is prepared (step S11). The food prepared at this time is not limited to the finished food as a product, and may be a food in the process of being manufactured. In addition, the prepared medium is prepared using general medium components other than food.

In the cell addition step, Euglena cells are added to the medium prepared in the medium preparation step (step S12). At this time, the amount of Euglena cells added to the medium may be appropriately selected according to the properties of the medium (solid or liquid, viscosity, pH, composition, contained components, etc.).

In the culturing step, the Euglena cells are cultured in the medium (step S13). The culture conditions such as temperature, period, light irradiation, and presence / absence of shaking may be appropriately selected according to the properties of the medium and the like.

According to the method for culturing Euglena according to the present embodiment, Euglena cells can be cultured using the nutritional components contained in food.

<Medium composition for Euglena (medium composition containing solid food)>
The medium composition for Euglena (solid food-containing medium composition) according to the present embodiment contains natto and at least one selected from seaweed or dried sardines, and is characterized by being used for culturing Euglena. ..
The euglena medium composition (solid food-containing medium composition) according to the present embodiment can be used as a medium suitable for the growth of euglena, and in particular, euglena grown with a medium composition of a combination of natto and seaweed is not. Since it is composed only of animal foods, it can be a source of _V.B12 , which tends to be deficient in strict vegetarian (vegan) diets.

<Manufacturing method of fermented vegetable food>
The method for producing a fermented vegetable food according to the present embodiment is characterized by performing a step of mixing soymilk, a vegetable lactic acid bacterium, and Euglena cells to prepare a raw material mixture, and a step of fermenting the raw material mixture. And.
According to the method for producing a fermented vegetable food according to the present embodiment, since the vegetable lactic acid bacterium contained in soymilk yogurt is used as the vegetable lactic acid bacterium, it is completely non-animal for vegans (strict vegetarians). It is possible to provide a vegetable fermented food that can be used as a raw food containing V.B ₁₂ .

Hereinafter, the present invention will be specifically described based on specific examples, but the present invention is not limited thereto.
In the following examples, Euglena was cultured using various foods, and the growth state of Euglena cells was examined.

<Euglena used in the test>
The Euglena gracilis used in the test used cells maintained in KH medium (Koren-Hutner medium) having the composition shown in Table 1 for a period of 1 month or longer, and the CM medium (CM medium) shown in Table 2 ( Precultured in an independent nutritional state using (Cramer-Myers medium) was used (white fluorescent lamp constant irradiation 40-60 μmol / m ² · sec, temperature 24-27 ° C (generally 26 ° C), static culture). .. It is known that the CM medium is essential for vitamin B ₁ (V.B ₁ ) and vitamin B (V.B ₁₂ ), which are the components thereof. For example, by repeating the autoclave treatment of the culture solution, the culture solution in which vitamin B ₁ (V.B ₁ ) and vitamin B (V.B ₁₂ ) are destroyed in the culture solution is used for about one month. When Euglena is cultured repeatedly by subculture, the cell density in the stationary phase is low, the color becomes yellowish green, and almost all cells are precipitated.
When inoculating Euglena cells prepared as described above into food, adjust the volume ratio of Euglena cells to 1% v / v or less in the volume ratio of the food to be inoculated, or inoculate with sterile water. By cleaning the nutrients in the original suspension, we adopted growing conditions that would be burdensome for Euglena.

When the amounts of _V.B1 and _V.B12 originally contained in the CM medium are added again to the CM medium in which Euglena cells have grown poorly due to vitamin deficiency, many Euglena cells float on the next day. It was seen, and after 3 days, it had a deep green color that could be visually confirmed (Fig. 3, left). However, when only one of V.B ₁ and V.B ₁₂ was added, remarkable cell motility recovery and proliferation were not observed (Fig. 3, center, right). When only _V.B12 was added, an increase in floating cells was confirmed, but the color remained pale yellowish green (center of FIG. 3). No cell recovery was observed with the addition of _V.B1 alone (Fig. 3, right).

From the above, it was confirmed that vitamin B (V.B ₁ , V.B ₁₂ ) was easily damaged by heat treatment, and it was suggested that raw food without heat during cooking is the most bioavailable method. .. In general, V.B ₁ is weak against heat and V.B ₁₂ is relatively strong, which seems to contradict the above results, but this is because V.B ₁₂ is acidic (pH 3. 5) And it is considered that it is caused by decomposition in the presence of light.

<Test 1 Culture using natto (1)>
(Test method of test 1)
In Test 1, Euglena cells were cultured using a culture medium containing natto.
Niboshi or seaweed as a source of V.B ₁₂ was added to the natto aqueous solution so that V.B ₁₂ was equivalent to the CM medium. In addition, the amount of natto added so that the total value of V.B ₁₂ in the food (dried sardines or seaweed) added as the source of V.B ₁₂ and V.B ₁₂ in natto is equal to that of the CM medium. Adjusted (Table 3). A 100 mL closed container was used as a culture container, and the volume of the medium was adjusted to 100 mL with purified water. For each food ingredient value, the Standard Tables of Food Composition in Japan 2015 (7th revision) was used.

For natto, groat natto (manufactured by Mizkan Co., Ltd., product name: Kinnotsubu Domestic Hikiwari 3P) is used, the temperature is 27.5 ° C, a white fluorescent lamp is used as a light source, and the light intensity is 40 to 50 μmol / m ² seconds. The euglena was cultivated by constantly irradiating with.

(Results and discussion of Test 1)
Four days after the transplantation of Euglena cells, cell proliferation could be visually confirmed on the surface of the natto settled at the bottom, and the steady state was reached 7 days after the transplantation. FIG. 4 shows a photograph showing the growth state after 7 days of culturing. Since _V.B12 is not contained in natto, which is a plant-based food, it was a factor that limited the growth of the culture solution containing only natto (Fig. 4, left). Euglena cells showed good growth by adding an appropriate amount of dried sardines and seaweed, which are high-content foods of _V.B12 (Fig. 4, center, right). Due to the bacteriostatic action of Bacillus natto (dipicolinic acid, etc.), even if Euglena cells were transplanted outside the clean bench, other microorganisms did not grow to the extent that they could be visually confirmed.

Bacillus natto converts various nutrients such as proteins in soybeans into a form that is easy for Euglena to use by using small molecules such as amino acids. On the other hand, Euglena grown in an inorganic medium at the planting source supplies oxygen to the aerobic bacterium Bacillus natto by photosynthesis. Since such a symbiotic system is realized, it is considered that good Euglena growth was realized only by sealing the culture vessel and allowing it to stand still.

Conventionally, euglena culture could not be realized without supplying and stirring CO ₂ to the medium with a pump or using an organic medium (KH medium) having a complicated composition. In addition, since Chlamydomonas, which is a member of the same unicellular microalgae as Euglena, hardly grows in a medium using Bacillus natto, it was found that the symbiotic partner of Bacillus natto is highly selective.

In addition, even if the content of natto in the medium was as small as 0.2% by mass / mL (50 mL of water for 0.1 g of natto), good growth was observed, although not as much as in the example shown in FIG. rice field. When the natto content was 20% w / v or more, the growth was suppressed. Therefore, it was found that the concentration of natto added to the medium is preferably 0.2% by mass / mL or more and 20% by mass / mL or less.

Euglena, grown in a combination of natto and seaweed as a nutrient source in the medium, is a source of V.B ₁₂ that tends to be deficient in strict vegetarian (vegan) diets because it consists only of non-animal foods. can do. In particular, methylcobalamin, which is a type of _V.B12 , has high activity in the body and has high added value, so it may be contained more than other _V.B12 -containing foods such as chlorella and seaweed. .B ₁₂ Can be used as a supplement for supplementation. Euglena grown from a combination of natto and seaweed can be used as plant-based whole food and raw food, which are becoming mainstream in cutting-edge nutrition in Europe and the United States.

<Test 1-2 Culture using natto (2)>
In Test 1-2, when euglena cells were cultured using a culture medium containing natto, light irradiation conditions, V.I. With or without _B12 source addition, and V. The difference in the growth of Euglena cells depending _on the type of B12 source was investigated.

○ Experiment 1
Euglena cells were grown in CM medium (initial pH 3.5) to a steady phase, and the Euglena cell suspension was washed twice with sterile water (6000 rpm, 5 minutes, 4 ° C). To a test tube (straight mouth: φ15 × 105 mm, model number: A-Wassermann), add 10 mL of sterile water, and as shown in Table 4, V. _B12 source was added. Only "natto (CM medium)" was added with CM medium (pH 3.5) instead of water.
The CM medium used was once sterilized using an autoclave, and V.I. A non-sterile _B12 source was used.

As for natto, "Niwanatto (manufactured by Mizkan)" was used, and 0.44 g of each was added.
Cyanocobalamin (Wako Pure Chemical Industries, Ltd.) added 0.55 μL of a 0.1 g / L solution.
As the seaweed, "Kizami Nori (Omoriya)" was used, and a piece was added with a width of 2 mm and a length of 10 mm or less.
For the "natto (without beans)" sample, sterile water was added and the mixture was stirred well, and then the beans were collected.
Only seaweed was added to "Nori only", and natto was not added.

1 mL of the washed Euglena cell suspension was added to the test tubes of each sample, and the initial cell density was 2.5 × 10 ⁴ cell / mL.
The test tube was sealed with a silicone rubber stopper (No. 1).
The series of work was performed in a non-sterile environment outside the clean bench.

Each sample was statically cultured for 9 days under light irradiation culture conditions or dark culture conditions.
Under the light irradiation culture conditions, the test tube was set horizontally on a metal plate at constant irradiation with a white fluorescent lamp, a light amount of 60 μmol / m ² · sec, and a temperature of 24 ° C., and light was irradiated from above.
Under dark culture conditions, the test tubes were placed horizontally in a metal box, covered and allowed to stand in the vicinity of the sample under light irradiation conditions.

○ Result 1
FIG. 5 shows the results of measuring the cell density of each sample 9 days after culturing (stationary phase) using a plankton counter (MPC-200, Matsunami Glass Ind.). Each sample was diluted with a solution of benzalkonium chloride solution (Taiyo Seiyaku) diluted to 2% with water to fix the motility of Euglena cells for counting.

○ Consideration 1
When culturing using natto, it was found that light irradiation is necessary for the growth of Euglena cells.
Nori is V. As a _B12 source, it showed the same effect as cyanocobalamin. Considering the amount of seaweed added (corresponding to 1/10 of KH medium-containing V.B ₁₂ (= CM medium equal amount)), the limiting factor for growth in normal CM medium is V. It was suggested that it is not only _B12 but also other organic nutrients.
V. Even in the "natto only" culture medium containing no _B12 , good growth was shown in this test. This depends on the type of natto, V. It is shown that it can contribute to Euglena growth to some extent without the addition of _B12 .

○ Experiment 2
Euglena cells were grown in CM medium (initial pH 3.5) to a steady phase, and the Euglena cell suspension was washed 3 times with sterile water (6000 rpm, 5 minutes, 4 ° C.). Test tube (straight mouth:
Add 29 mL of sterile water to φ18 × 180 mm, model number: A.18), and as shown in Table 5, V.I. _B12 source was added. V. A non-sterile _B12 source was used.

As for natto, "Niwanatto (manufactured by Mizkan)" was used, and 1.2 g of each was added.
Cyanocobalamin (Wako Pure Chemical Industries, Ltd.) added 15 μL of a 0.001 g / L concentration solution.
As the seaweed, "Kizami Nori (Omoriya)" was used, and a piece was added with a width of 2 mm and a length of 10 mm or less.

1 mL of the washed Euglena cell suspension was added to the test tubes of each sample, and the initial cell density was 2.6 × 10 ⁴ cell / mL.
The test tube was sealed with a silicone rubber stopper (No. 3).
The series of work was performed in a non-sterile environment outside the clean bench.

Each sample is constantly irradiated with a white fluorescent lamp, the amount of light is 60 μmol / m ² · sec, the test tube is set horizontally on a metal plate at a temperature of 24 ° C., and the test tube is statically cultured for 11 days under the light irradiation culture condition of irradiating light from above. did.

A syringe was inserted into a silicon rubber stopper to extract several tens to 200 μL of the sample, and the cell density was calculated using a cell counter without making the inside of the test tube aerobic. When extracting the sample, it was simply disinfected with 70% isopropyl alcohol and performed outside the clean bench.

○ Results and discussion 2
The growth curve of each sample is shown in FIG. From the 6th day to the 7th day of the culture, the culture was carried out under dark culture conditions for about 9 hours.

The sample supplemented with seaweed showed a growth curve almost equal to that of the medium supplemented with cyanocobalamin. Nori is described in the food composition table as V.I. Although it is supposed to contain B ₁₂ , it is actually a pseudo-V. This is the result of the detection of _B12 . Pseudo V. _B12 is actually V. in the human body. Since it cannot take _on the function of B12 (methylmalonic acid metabolism), Nori seaweed is V. B ₁₂ Not a source. On the other hand, V. It is known that B ₁₂ includes a form that can be used by humans. For growth V. Euglena, which requires _B12 as an essential vitamin, is used in the seaweed-added medium. Since the growth was almost the same as that of the _B12 -added medium, Euglena was a pseudo-V. V. of the form in which B ₁₂ can be used. It is suggested that it is converted to B ₁₂ . In this case, by using Euglena, V.I. B ₁₂ It is possible to add value as a source. Since Euglena grows densely on Nori seaweed, it is possible to produce Euglena seaweed by growing Euglena on the surface of the seaweed and drying it.

At a high cell density of about ¹⁰⁶ orders, blocking light causes great stress on the cells. It was also found that the cells continued to decrease for about 28 hours even when the light irradiation was resumed. The recovery of cell density was better in "natto + seaweed" than in "natto + V.B ₁₂ ". This suggests that photosynthesis must be maintained in a high cell density environment. It is considered that the good recovery was shown in the sample to which seaweed was added because it contained a large amount of vitamins and minerals necessary for photosynthesis and growth.
^On the other hand, it was found that the growth was not inhibited by light blocking at a cell density as low as about 105 orders.

In Experiment 2, V.I., which is an essential vitamin, is also used. Good growth was obtained in the "natto only" culture medium containing no _B12 source (although the growth rate was slower than that with the _V.B12 source). It is possible that the cause of this is the use of "gravy" for culturing Bacillus natto.
V. of Itohiki Natto (ordinary grain natto) in the Food Composition Table (Standard Tables of Food Composition in Japan 2015 (7th edition)). The B ₁₂ content is "Tr" and the groat natto is "0". Since Tr (trace) is contained but does not reach the minimum stated amount, and 0 indicates that it is less than 1/10 of the minimum stated amount in the food composition table or was not detected, V. It is considered that Euglena cells were able to grow due to the fact that _B12 was contained in a very small amount and was supplemented by other organic nutrients. Therefore, it is preferable to use threaded natto rather than groat natto as natto.

V. in Euglena. The main reason why B ₁₂ is required is that it is essential for methionine synthesis, and by adding a small amount of methionine to the medium, V. _B12 deficiency can be alleviated. On the other hand, it is also known that the presence of a large amount of methionine in the medium inhibits the growth. Therefore, by including natto in the culture medium, an appropriate amount of methionine or a methionine-containing peptide is eluted in the culture medium, whereby V.I. It is also possible that growth promotion under _B12 deficiency was achieved.

In addition, E. When glacilis is grown on Cbl (cobalamin) -restricted medium (0.05 μg Cbl / mL medium (Koren-Hunter medium)), it easily becomes Cbl-restricted cells ( ¹⁰⁶ cell / mL) and grows in proportion to the Cbl concentration in the medium. Is known to indicate (Shozaburo Kitaoka, "Eugrena", Society Publishing Center, 1989).

<Test 2 Culture using yogurt>
(Test method of test 2)
In Test 2, Euglena cells were cultured using yogurt.
In this test, Euglena cells grown in KH medium were used as a sample. Unadjusted milk or unadjusted soymilk was diluted and dispensed in 10 vol% increments using purified water so that the total volume was 10 mL in a test tube. 100 μL of culture medium containing Euglena cells, 12 μL of _V.B , and 0.5 μL of V.B ₁₂ were added to each test tube. As a starter, drop about 0.2 g of Caspian Sea yogurt (delicious Caspian Sea: Glico) or soymilk yogurt (manufactured by Marusanai Co., Ltd., product name: soymilk glut), seal with a silicon rubber stopper, and mix well. Using a fluorescent lamp as a light source at 27 ° C., continuous irradiation was performed at a light intensity of 35 to 50 μmol / m ² seconds, and the growth of Euglena cells was observed.

(Results and discussion of Test 2)
Euglena is acid resistant and can coexist with lactic acid bacteria. Lactic acid produced by lactic acid bacteria lowers the pH of the surrounding environment (pH 4.1 to 4.3), so that storage stability is improved and coagulation occurs due to protein denaturation. FIG. 7 is a photograph showing the state after 9 days have passed since Euglena was transplanted to a medium using milk as a substrate and Caspian Sea yogurt (Cremoris bacterium) as a starter. The numbers in FIG. 7 indicate the water: milk ratio (volume ratio), and the substrate (milk) concentration increases from left to right. There was a correlation between growth and milk concentration, and the balance between the growth rate and the amount of Euglena cells was good in the milk concentration range of 30 to 50 vol% (water: milk 3: 7 to 5: 5). In addition, within the range of the milk concentration, the yogurt drink is finished to the same hardness as the yogurt to be drunk, so that it can be used as a yogurt drink (at a higher substrate concentration, the hardness is the same as that of normal yogurt). .. Euglena showed the same cell morphology (rod-like) and motility as when grown in a normal liquid medium even in yogurt.

Also, when soymilk was used as the substrate and the starter was soymilk yogurt (vegetable lactic acid bacterium), good growth of Euglena cells was observed (Fig. 8 shows the state 24 days after transplantation, and the numbers in the figure are soymilk. : Indicates the ratio of water (volume ratio)). However, 8-10% of the soybean solid component of soymilk was suitable, and 14% was too hard, and Euglena cells remained on the bottom surface of the test tube. Fermented vegetable foods obtained using euglena, soymilk and soymilk yogurt can be used as a completely non-animal V.B ₁₂ -containing raw food for vegans. In addition, when soymilk was used as a substrate, the color of Euglena cells in the stationary phase was brighter and deeper green than when milk was used as a substrate, and the state was maintained for a long time.

Since the optimum temperature for growth of Cremoris bacterium contained in Caspian Sea yogurt is about 30 ° C. (close to the growth temperature of Euglena), it is preferable to use Cremoris bacterium as a starter. When Cremoris bacterium was used, the substrate coagulated in less than one day, whereas in other high-temperature lactic acid bacteria (optimum temperature 40 to 45 ° C), it took three days or more for the substrate to coagulate (substrate). The higher the concentration, the faster the coagulation rate). When yogurt was fermented in a temperature range of 40 to 45 ° C., it coagulated in one day, but Euglena did not grow.

When growing in a medium using yogurt, an unpleasant odor was exhibited when the cell concentration became excessively high or when the cells were left in a normal temperature environment for a long time. The development of unpleasant odors was prevented by stopping the growth of Euglena cells at appropriate stages (stored at low temperatures) or by using appropriate fragrances. Specifically, the odor was suppressed by growing using banana or coffee-flavored soymilk. Further, when the yogurt contained in the medium deteriorated, the color changed from white to ocher (FIG. 8), but the deterioration of the medium could be prevented for a long period of time by placing it in a low temperature environment such as a refrigerator. When culturing by irradiating light in a refrigerator (10 to 15 ° C) (light intensity 1.5 to 35 μmol / m ² seconds), the growth rate of Euglena cells is extremely slow and the deterioration of the medium is also slow, so quality control (Growth management) was possible. The growth of Euglena cells was also confirmed in a test in which a suspension of Euglena cells grown in CM medium instead of KH medium was used as a sample and cultured.

<Test 3 Culture using vegetable drinks or fruit drinks>
○ Experiment 1
In Experiment 1, Euglena cells were cultured using a vegetable drink or a fruit drink.
Table 6 shows the composition and culture conditions of the culture solution using tomato juice (manufactured by ITO EN Co., Ltd., product name: ideal tomato), which is a vegetable beverage. In addition, V. _Niboshi or seaweed was added to tomato juice as a source of B12.

In addition, grape juice, prune juice, pineapple juice, orange juice, and apple juice were used as acidic fruit drinks, and V.B ₁ and V.B ₁₂ were added.

○ Results and discussion 1
When tomato juice was used, Euglena cells showed particularly good growth even without the addition of V.B ₁ and V.B ₁₂ (Fig. 9). Since tomato has a wide range of applications as a food material, it is a highly versatile culture method. In addition, when tomato juice was used, a dramatic change in hue from red to green was observed during the logarithmic growth phase of Euglena cells (8 days after planting). The change in hue was more pronounced in the shake-cultured sample than in the static-cultured sample.

When a medium obtained by adding dried tomato juice to tomato juice was used, a change in hue from red to yellow was observed, and within 24 hours thereafter, a deep green color was exhibited (FIG. 10).

Euglena also showed good growth in pine juice, orange juice, and apple juice, which are acidic fruit drinks, when _V.B1 and _V.B12 were added.

When euglena was cultivated using grape juice or prune juice, it was observed that the cells were stained reddish purple, indicating that euglena was well stained with anthocyanin, which is a kind of polyphenol (Fig. 11). Cremoris, a lactic acid bacterium contained in Caspian sea yogurt, also stains well with anthocyanins, but does not stain lycopene in tomatoes or carotene in carrots visually.

Euglena provides euglena with high nutritional value because it can actively accumulate antioxidants (pigments) such as polyphenols in its cells by culturing it in vegetable drinks and fruit drinks. be able to. In addition, Euglena cells can be stained with a desired color by selecting a vegetable or fruit containing a pigment corresponding to the desired color.

In addition, Euglena cells showed good growth in various diluted solutions of almond milk, coconut milk, acerola juice, prune juice, sweet sake, corn soup, rice milk, green juice, milk, soy milk, and melted pudding (V). .B ₁ , V.B ₁₂ added).
On the other hand, no remarkable proliferation of Euglena cells was observed in maple water (maple sap) and coconut water.

○ Experiment 2
Euglena cells were grown to a steady phase using CM medium (initial pH 3.5), and the Euglena cell suspension was washed 3 times with sterile water (6000 rpm, 5 minutes, 4 ° C.).
In a vial (FS media vial (φ55 × 85), model number: T-150), a mixture of tomato juice and water (sterile water) at the volume ratio (vol%) shown in Table 7 below is used as the growth medium. did. The volume of water also includes the washed cell suspension. The total volume was 100 mL.
For tomato juice, "Kagome tomato juice salt-free" was used, and the tomato juice was opened in a clean bench and used as it was without autoclaving.
The initial cell density was 2.9 × 104 ⁽ cell / mL).

Each sample was statically cultured for 9 days under constant irradiation with a white fluorescent lamp, a light intensity of 40 μmol / m ² · sec, a temperature of 25 ° C., lightly loosening the lid of the vial, and aerobic conditions.

○ Results and discussion 2
The logarithmic growth phase cell density on the 9th day of culture is shown in FIG.
V. in all samples. It was found that the cell density was on the order of ¹⁰⁶ even though B ₁₂ was not added, and good growth was obtained. As a characteristic of the growth of Euglena cells when statically cultured, a tomato juice precipitate layer (lower layer) and a water tank (upper layer) were formed in the bottle, and the cells proliferated around the boundary.

○ Experiment 3
In a vial (500 mL), 100 mL of purified water, V.I. B ₁ , V. B ₁₂ and a stirrer (φ8 × 30 mm, model: C8 × 30) were added and sterilized by autoclave.
V.B ₁ : 0.5 g / L x 100 μL = 50 μg (equivalent amount of CM medium)
V.B ₁₂ : 0.001 g / L × 250 μL = 0.25 μg (equivalent amount of CM medium)

Tomato juice (manufactured by ITO EN Co., Ltd., product name: ideal tomato) was opened in a clean bench, and 100 ml of tomato juice was injected into a vial after sterilization.
Euglena cells grown to a steady state in CM medium (initial pH 3.5) were precipitated using a centrifuge (6000 rpm, 5 minutes, 4 ° C). After discarding the supernatant, Euglena cells were suspended in sterile water and injected into a vial, and the volumetric flask was increased to 500 mL with sterile water (corresponding to a volume ratio of substrate food: water = 2: 8). The initial cell density was 3.4 × 104 ⁽ cell / mL). The series of work was performed in a sterile environment in a clean bench.

Each sample was constantly irradiated with a white fluorescent lamp, the amount of light was 10 to 15 μmol / m ² · sec, the temperature was 26 ° C., the lid of the vial was lightly loosened, and the cells were cultured under aerobic conditions for 15 days while stirring with a stirrer.
Samples for cell counting were taken in a clean bench, and counting was performed using a bacterial calculator (Sunlead glass, model A-161) until the 10th day of culture, and a plankton calculator after the 11th day.

○ Results and discussion 3
FIG. 13 shows a growth curve. It showed an increase until the 7th day of culturing, but after that, it was not stable due to fluctuations in cell density, but good growth was observed with the lapse of culturing days.

<Test 4 Culture using gel-like food>
(Test method of test 4)
In Test 4, Euglena cells were cultured using a gelled food.
Specifically, soymilk diluted solution coagulated with agar or gelatin, egg yolk pudding coagulated by heating egg yolk, and tofu were used as a medium for culturing Euglena cells.

A sample (Fig. 14, left) to which 0.2 wt% of agar powder and 0.1 w% of gelatin powder were added and 0.2 wt of agar powder were added using a soymilk diluted solution of soymilk: water = 1: 9 (volume ratio). A sample (center of FIG. 14) was prepared, sterilized by autoclave, and then 50 μL of a constant phase CM medium suspension of Euglena cells was added and cultured for 8 days.
In addition, the egg yolk solution was diluted with a mixture of milk: water = 7: 3 (volume ratio) and heated to adjust the egg yolk pudding (Fig. 14, right). 50 μL of the solution was added and the cells were cultured for 5 days.

(Results and discussion of Test 4)
In agar and gelatin (diluted soymilk), the growth rate of Euglena cells was slower and the cell density in the stationary phase was lower than in Tests 1 to 3. As a result of microscopic observation, it was found that Euglena cells became spherical and did not show motility. Agar and gelatin have a wide range of applications as foods, so their utility value is considered to be high. Moreover, considering the application to food, it may not be preferable that Euglena cells grow too much. In this test, essential vitamins were not added to agar or gelatin, but the addition of essential vitamins promotes the growth of Euglena cells.

The right side of FIG. 14 shows a pipette piercing splicing of Euglena culture solution into the yolk pudding, and good growth was observed in the pipette trajectory and the liquid part on the upper surface, but it penetrated into the yolk pudding (inside the gel). It diffused and did not grow. In addition, good growth was not observed even in the mayonnaise medium containing high α-linolenic acid obtained by emulsifying flaxseed oil using egg yolk as an emulsifier (Fig. 14, left). In addition, the growth in the medium obtained by gelling the diluted egg white solution by heating was also not good (center of FIG. 14).

In the medium using tofu, the growth of Euglena cells was observed in the surrounding aqueous solution and the surface of the tofu (Fig. 15, left), but when the tofu was broken, uniform growth of Euglena cells was also observed inside the tofu (Fig. 15, left). FIG. 15 right). As with natto (test 1), soymilk yogurt (test 2), and soymilk (test 3), euglena cells grew well in tofu and showed a bright green color, so the compatibility between euglena and soybean foods was good. all right.

<Consideration of tests 1 to 4>
Among the foods handled in each test, tomato juice (unsalted), natto (hikiwari natto), and yogurt, which showed particularly good growth, were compared with the amino acid composition of Euglena as shown in FIG. ..
The numbers on the horizontal axis of FIG. 16 indicate 18 kinds of amino acids, and the table below is the corresponding table. The vertical axis of the figure shows the percentage of each of the 18 amino acids in the food to the total weight of the 18 amino acids contained in each food. (Example: Although the content of tomato in 15 glutamic acid is large, it is inferior to natto in terms of absolute amount because it is relative to the total amino acid content of tomato). For the amino acid composition of Euglena, the analysis results of the Japan Food Research Laboratories were used (http://www.eu-glena.net/eiyou/). For other foods, refer to the Amino Acid Composition Table of the Standard Tables of Food Composition in Japan 2015 (7th edition), Chapter 2, Table 1, Amino Acid Composition Table per 100g of edible portion. Since "natto, tofu, soymilk" or "yogurt, ordinary milk" showed almost the same composition ratio, "natto" and "yogurt" were used as representatives. In Tests 1 to 4, each food product was diluted to an appropriate concentration before use.

Natto and yogurt showed an amino acid composition almost equal to that of Euglena. It can be said that natto and yogurt, which are foods having an amino acid composition close to that of Euglena itself, are substrates with a good amino acid balance in the growth of Euglena.
On the other hand, tomatoes with the best growth of Euglena cells have a significantly higher glutamic acid (No. 15) content (twice as much as other foods), but do not contain much other amino acids. In the growth of Euglena cells, glutamic acid can be used as both a carbon source and a nitrogen source. Therefore, the abundance of glutamic acid indicates that it is advantageous for the growth of Euglena cells. Furthermore, the low content of isoleucine, leucine, phenylalanine, and tyrosine (Nos. 1, 2, 6, and 7), which have an inhibitory effect on the growth of Euglena cells, also shows good growth in a medium using tomato juice. It is considered that the cause showed. In addition, lycopene contained in tomato was rapidly decomposed during the logarithmic growth phase, and the color of the culture medium was maintained well for a long period of time thereafter. Therefore, it is considered that lycopene has a great influence on the growth of Euglena cells. It was also suggested that some substance that compensates for the V.B ₁₂ deficiency is present in the tomato components.

As described above, in Tests 1 to 4, the course of Euglena cells grown in a CM medium containing a small amount of nutrients was observed in the sample in which the Euglena cells were transplanted so as to have a ratio of 1% or less of the food as the medium. This was due to the intention of making the difference in the growth pattern of Euglena cells remarkable in each food by adopting the condition that puts a heavy load on the growth of Euglena cells. Therefore, by appropriately increasing the amount of Euglena cells to be transplanted to the food as a medium, it is possible to grow Euglena cells to a stationary phase at a growth rate higher than that obtained in Tests 1 to 4.

<Test 5 Culture using beverages>
In Test 5, Euglena cells were cultured using vegetable drinks, fruit drinks and other drinks.

(Test method of test 5)
(1) V.B ₁ (thiamine hydrochloride) and V.B ₁₂ (cyanocobalamin) were added to an autoclave-sterilized laboratory screw tube bottle (No. 5, 20 mL).
V.B ₁ : 0.5 g / L x 3 μL = 1.5 μg (equivalent amount of CM medium)
V.B ₁₂ : 0.1 g / L × 0.75 μL = 0.075 μg (equal amount of KH medium)
Each reagent was dissolved in purified water and was not autoclaved to prevent heat damage.

(2) Various beverages (unheated) as substrates, sterilized water in which purified water was autoclaved, and cell suspension were put into a screw tube bottle.
The volume ratio of various beverages used as substrates and sterilized water was as follows.
・ Tomato juice: Sterilized water = 4: 6
・ Coconut water: Sterilized water = 14: 1
・ Maple water: Sterilized water = 14: 1
・ Grape juice (concentrated): Sterilized water = 7: 3
・ Carrot juice (concentrated): Sterilized water = 7: 3
・ Other beverages: Sterilized water = 3: 7
The sterile water also contained a suspension of Euglena cells, and the Euglena cells were washed twice with sterile water (centrifugation conditions: 6000 rpm, 5 minutes, 4 ° C.).

Beverage as a substrate was poured into a screw tube bottle, sterile water was added to the total amount of about 8 minutes, and the mixture was lightly stirred. The washed Euglena cell suspension was injected and the volume was increased to 15 mL with sterile water. Diluted beverages were used because high concentrations of substrate beverages can damage Euglena cells. After measuring with sterile water, the lid of the screw tube bottle was closed and the mixture was lightly stirred.
The initial cell density of each sample was 1.6 × 10 ⁴ cell / mL.

(3) The lid of the screw tube bottle of each prepared sample was loosened, and the sample was statically cultured under aerobic conditions.
Light irradiation Culture conditions: White fluorescent lamp constant irradiation, light intensity 100 μmol / m ² · sec, temperature 26 ° C
Culture conditions in the dark: In the dark, the temperature is 23 ° C.

The above operations (1) and (2) were performed in a clean bench.
Euglena cells at the source of transplantation were cultured using CM medium (initial pH 3.5). The initial pH of the CM medium-only sample used as a inverse proportion was also set to 3.5.

(Results and discussion of Test 5)
FIG. 17 shows the results of culturing each sample under light irradiation culture conditions or dark culture conditions and measuring the cell density in the stationary phase (when 11 days have passed since the start of culturing).

In almost all samples, the density of Euglena cells tended to be higher in the light irradiation culture condition than in the dark culture condition. In particular, good growth was confirmed in tomatoes, oranges, pineapples, grapes, carrots, grapefruits, and prunes.
In addition, when culturing in a dark place, Euglena cells do not show green but show white, so there is an advantage that the nutrition of food can be strengthened without significantly changing the hue of the substrate food. all right.

Next, the light and dark conditions of each sample were changed, the culture was continued, and the cell density was measured 15 days after the light and dark conditions were changed. The results are shown in FIGS. 18 and 19.
FIG. 18 shows the cell densities in various samples when culturing under dark culture conditions until the stationary phase and then culturing under light irradiation culture conditions.
The color of each sample changed from white to green in about 1.5 days after switching to the light irradiation culture conditions. In particular, citrus pineapples, oranges, grapefruits, and tomatoes changed to green quickly, but apples showed almost no green color. Euglena cells showed good growth in the sample using tomato, which is considered to be due to the abundance of glutamic acid and lycopene in tomato. In addition, amazake is V. B ₁ , V. B ₂ , glucose is abundant.

FIG. 19 shows the cell densities in various samples when culturing under light irradiation culture conditions until the stationary phase and then culturing under dark culture conditions.
By switching from the light irradiation culture condition to the dark place culture condition, the color of each sample changed from bright green to dull green, but it did not turn white.

<Test 6 Culture in sterile beverage medium>
In Test 6, Euglena cells were grown to a steady phase under the growth conditions of "light or dark" and "open or sealed" using an autoclave-sterilized beverage-containing medium, and the Euglena cell density was measured and sensory test (tasting). ), And the effect of light and airtightness of the container on the growth of Euglena was examined.

(Test method of test 6)
(1) The following amounts of V.B ₁ (thiamine hydrochloride) and V.B ₁₂ (cyanocobalamin) were added to an autoclave-sterilized screw tube bottle (No. 5, φ27 × 55 mm) (the total amount of sample solution was 15 mL). did).
V.B ₁ : 0.5 g / L x 3 μL = 1.5 μg (equivalent amount of CM medium)
V.B ₁₂ : 0.1 g / L × 0.75 μL = 0.075 μg (equal amount of KH medium)
Each reagent was dissolved in purified water and was not autoclaved to prevent heat damage.

(2) As the beverage, grape juice, pine juice, apple juice, sweet sake, carrot juice, tomato juice, orange juice and prune juice were used.
For tomato juice, two types of volume ratio with sterilized water were prepared: tomato juice: sterilized water = 3: 7 and tomato juice: sterilized water = 4: 6.
Add the beverage as a substrate, add purified water to about half (10 mL) of the screw tube bottle, close the lid of the screw tube bottle, and use a high-pressure steam sterilizer (manufactured by Alp Co., Ltd., model: KT-2322). , 121 ° C., 20 minutes, autoclaved. Here, the lid of the screw tube bottle was closed to prevent the aroma component from being dissipated and mixed during the autoclave. In other tests as well, the autoclave sterilization treatment was performed under the relevant conditions.

(3) Euglena cells were grown in CM medium (initial pH 3.5) until a steady phase. The Euglena cell suspension was washed twice with sterile water (6000 rpm, 5 minutes, 4 ° C.). Washed cells were added to each sterilized medium, and the volume was increased to 15 mL with sterile water.
The initial cell density was 1.1 × 10 ⁴ cell / mL.
The series of work was performed in a sterile environment in a clean bench.

(4) Each sample was statically cultured for 9 days under four types of growth conditions: "light irradiation / opening", "light irradiation / sealing", "dark place / opening", and "dark place / sealing".
-Light irradiation conditions: White fluorescent lamp constant irradiation, light intensity 100 μmol / m ² seconds, temperature 26 ° C
・ Culture conditions in the dark: In the dark, the temperature is 23 ° C.
・ Opening condition: Lightly loosen the lid of the screw tube bottle ・ Sealing condition: Tighten the screw tube bottle lid tightly

(Results and discussion of Test 6)
FIG. 20 shows the results of culturing each sample under predetermined conditions and measuring the cell density in the stationary phase (when 11 days have passed since the start of culturing).
Comparing the graph of FIG. 20 with the graph of FIG. 17, it was found that the Euglena cell density in the stationary phase did not change significantly depending on the presence or absence of the autoclave sterilization treatment. Therefore, it can be said that it is beneficial to autoclave sterilize the food-containing medium in consideration of hygiene.

For samples other than tomatoes, good growth was obtained even under the "light irradiation / sealing" condition, which was almost the same as the "light irradiation / opening" condition. It can be said that good growth was obtained because the cell density of "light irradiation / sealing" of tomato is larger than that of other samples.
Since Euglena cells showed motility and were confirmed to be alive in all samples, it is possible to provide a nutritionally enriched food composition in which Euglena cells are alive.

The color of the cells in the appearance of the culture solution by visual inspection was green under light irradiation conditions and white under dark conditions regardless of whether the cells were closed or open. As a result of the sensory test of each sample after culturing, both the taste and aroma were not unpleasant and were suitable for eating.

A common tendency for all samples was that as Euglena cells proliferated, they became less sour and had a milder, rounded sweetness compared to the original substrate beverage. This tendency was remarkable under the condition of "light irradiation" in which the Euglena cell density increased. The odor of Euglena algae was masked by the scent of the substrate beverage, but the odor mask was remarkable under "dark / closed" conditions where the Euglena cell density was low.

As a sample that exhibited a scent different from that of the substrate beverage after culturing, the tomatoes under the "light irradiation / sealing" condition exhibited the scent of canned oranges. Amazake had a lily of the valley flower scent, and orange had a kumquat scent. Grape and pineapple had almost the same aroma as the substrate drink.

From the above, if it is desired to retain the characteristics (color, taste, aroma) of the substrate beverage (substrate food), it is advisable to culture under "dark place / closed" conditions. In addition, when it is desired to increase the characteristics of Euglena and the cell density of Euglena, it is advisable to culture under "light irradiation, hermetically sealed" conditions.

Even if the medium containing food is autoclaved, Euglena can be properly grown and the growth of other microorganisms can be prevented.
In addition, even in a closed container used in general beverage products, since Euglena has grown appropriately, it is possible to provide a fortified beverage product containing live Euglena cells in the form of a closed container. ..

<Test 7 Culture using wine or grape juice>
In Test 7, Euglena cells were cultured using wine containing grape juice or a medium containing grape juice.

(Test method of test 7)
As the wine, "delicious antioxidant-free wine (puffy and thick), made by KIRIN" was used, a predetermined amount of wine was injected into a culture vessel, and alcohol was removed by boiling water.
"Red grape (manufactured by Nagoya Dairy Co., Ltd.)" was used as the grape juice.
A 30 mL sample container (screw tube No. 6, φ30 × 65) or a 50 mL sample container (FS media vial, φ40 × 75, model: T-50” was used as the culture container.
In each culture vessel, V. B ₁ (equivalent amount of CM medium), V.I. B ₁₂ (equal amount of KH medium) was added, and water was added until about 5 minutes and autoclaved.
The formulation of each sample is shown in Table 8 (water in the table also includes cell suspensions).

Euglena cells grown to a steady state using CM medium (initial pH 3.5) were precipitated using a centrifuge (6000 rpm, 5 minutes, 4 ° C.). After discarding the supernatant, Euglena cells were suspended in sterile water and injected into a culture vessel. The volume was increased to a predetermined total amount with sterile water. The initial cell density was 1.5 × 10 ⁴ cell / mL.

Each sample was constantly irradiated with a white fluorescent lamp, the amount of light was 40 μmol / m ² · sec, the temperature was 26 ° C., the lid of the vial was lightly loosened, and the cells were statically cultured under aerobic conditions.
The cell density measurement in the wine medium was performed on the 9th day of culture using a plankton calculation plate.

(Results and discussion of Test 7)
The growth curve of each sample is shown in FIG. 21, and the cell density on the 9th day of culture in the wine medium is shown in FIG. 22.
It was found that when alcohol was removed, good growth similar to that of grape juice was obtained even in a medium containing wine. On the other hand, when cultured in grape juice, it exhibited a wine-like aroma and masked the algae odor.
Regarding the concentration of wine, the best growth was obtained when the volume ratio of wine: water was 3: 7, and the tendency was similar to that of other fruit juice beverages.

<Test 8 Staining of Euglena cells with fruit drink>
In Test 8, Euglena cells were cultured using a culture medium using grape juice as a fruit drink, and the hue of Euglena cells was observed.

(Test method of test 8)
Euglena cells were cultured for 1 day using a CM medium, a medium having a volume ratio of grape juice: water = 3: 7, and a medium having a volume ratio of grape juice: water = 7: 3.
Euglena cells cultured with grape juice were washed twice with sterile water under centrifugation conditions of 8000 rpm, 5 minutes and 5 ° C.

Euglena cells after washing were observed by using a bacterial calculator (sample thickness 20 μm) to shorten the optical path length so that the purple background color was not emphasized.
The results are shown in FIG.

In Euglena cells after culturing with a culture solution of grape juice: water = 3: 7, the purple-stained part was uniformly dispersed in the cells before washing, but after washing (centrifugation). In the cells, the stained part was localized, so it was found that the specific gravity was different between the stained part and the unstained part. All cells were spherical or ellipsoidal in shape and were slowly expanding and contracting.

In Euglena cells after culturing in a culture solution of grape juice: water = 7: 3, the whole cells were stained, and the cells were rod-shaped and had reduced motility.
In addition, Euglena cells after culturing in CM medium were green.

From the above results, it was found that when Euglena cells are cultured using a fruit drink, it is possible to stain the cells with anthocyanins contained in grape juice.

<Test 9 Culture using vegetable juice>
In Test 9, Euglena cells were cultured using a culture medium containing vegetable juice.
(Test method of test 9)
(1) The red paprika seeds and seeds were removed, and the juice was squeezed using a whole slow juicer (manufactured by Kuvings, model: JSG-621). A tea strainer was used to remove the solid components of the red paprika juice.
The following amounts of V.B ₁ (thiamine hydrochloride) and V.B ₁₂ (cyanocobalamin) were added to an autoclave-sterilized screw tube bottle (No. 5, φ27 × 55 mm) (the total amount of the sample solution was 15 mL).
V.B ₁ : 0.5 g / L x 2 μL = 1.0 μg (equivalent amount of CM medium)
V.B ₁₂ : 0.001 g / L × 50 μL = 0.05 μg (equal amount of KH medium)

Red paprika juice was poured into each screw tube bottle. From 10% to 100% of the total volume, the juice concentration was adjusted to be different every 10%.
Inject purified water to about half of the screw tube bottle (a little less than 10 mL), close the lid, and use a high-pressure steam sterilizer (manufactured by Alp Co., Ltd., model: KT-2322) to sterilize by autoclave at 121 ° C for 20 minutes. did.

(2) Euglena cell suspension grown to a steady stage in CM medium was added to the sterilized culture medium, and the cells were squeezed up to 10 mL with sterilized water.
The initial cell density was 6.3 × 10 ³ cell / mL.

The cells were statically cultured for 12 days at a temperature of 26 ° C. under constant irradiation with a white fluorescent lamp and light irradiation conditions of 45 μmol / m ² seconds.

(Results and discussion of Test 9)
FIG. 24 shows the growth state of Euglena cells on the 6th day of culture (the numbers in the figure represent the volume ratio of juice: water). In addition, FIG. 25 shows the color change of the culture solution every other day from the third day (left) of the culture (sample is juice solution: water = 1: 9, 2: 8, 3: 7). Furthermore, FIG. 26 shows the Euglena growth state after 12 days of culturing. FIG. 27 shows the cell density of Euglena in the culture medium 12 days after culturing.

Euglena cell proliferation was observed in all samples, but good initial growth was obtained when the ratio of "substrate food: water" was "3: 7" or "4: 6" as in the case of commercially available beverages. Was done. On the other hand, the higher concentration of the juice had a higher final cell concentration. Therefore, it is particularly preferable that the ratio of substrate food (squeezed liquid): water is in the range of 3: 7 to 4: 6 when it is provided as a food immediately, and 4 when it takes a longer time to provide it. It is preferable to prepare the juice at a concentration in the range of 6: 6 to 10: 0. In addition, dramatic color changes in the culture medium were observed at all juice juice concentrations, similar to tomatoes. The red pigment contained in red paprika is mainly derived from the xanthophylls capsanthin and has high antioxidant power similar to that of lycopene contained in tomatoes. It can be provided.

Claims (11)

The process of preparing at least one food product selected from the group including gelled foods, vegetable beverages, fruit beverages, soybean foods, and
The cell addition step of adding Euglena cells to the food, and
A culture step of culturing the Euglena cells together with the food,
A method for producing a fortified food, which is characterized by performing. The method for producing a fortified food according to claim 1, wherein the gel-like food is selected from tofu and egg yolk pudding. The method for producing a fortified food according to claim 1, wherein the vegetable beverage is tomato juice. The method for producing a fortified food according to claim 1, wherein the fruit beverage contains grape juice or prune juice. The method for producing a fortified food according to claim 1, wherein the soybean food is selected from the group containing a soymilk drink, natto, and tofu. The foods are natto and
The method for producing a fortified food product according to claim 1, further comprising at least one selected from seaweed or dried sardines. The process of preparing at least one food product selected from the group including gelled foods, vegetable beverages, fruit beverages, soybean foods, and
The cell addition step of adding Euglena cells to the food, and
A culture step of culturing the Euglena cells together with the food,
A method of fortifying foods, characterized by doing so. A medium preparation step for preparing a medium containing at least one food product selected from the group containing gelled foods, vegetable beverages, fruit beverages, and soybean foods.
A cell addition step of adding Euglena cells to the medium, and
A culture step of culturing the Euglena cells in the medium, and
A method for culturing Euglena, which comprises performing. The process of preparing at least one food product selected from the group including vegetable and fruit beverages, and
The cell addition step of adding Euglena cells to the food, and
A culture step of culturing the Euglena cells together with the food,
Euglena dyeing method characterized by performing. Natto and
Including at least one selected from seaweed or dried sardines,
A medium composition for Euglena, which is used for culturing Euglena. The process of mixing soymilk, plant-derived lactic acid bacteria, and Euglena cells to prepare a raw material mixture,
A method for producing a fermented vegetable food, which comprises a step of fermenting the raw material mixture and a step of fermenting the raw material mixture.

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