WO2021015087A1

WO2021015087A1 - Method for producing rubisco

Info

Publication number: WO2021015087A1
Application number: PCT/JP2020/027659
Authority: WO
Inventors: 育夫遠山; ジャンピエールベリエ; 加藤　智子; 明穗横田
Original assignee: 国立大学法人滋賀医科大学; 株式会社ＭｅＤｒｅａｍ
Priority date: 2019-07-19
Filing date: 2020-07-16
Publication date: 2021-01-28
Also published as: JP2022130752A; TW202116351A

Abstract

Disclosed is a method for producing rubisco which is derived from plant leaves, the method comprising: (1) a step for heat-treating plant leaves at 50-58°C for 3 minutes to 1 hour; (2) a step for crushing the plant leaves obtained in step (1); (3) a step for removing an insoluble substance from the crushed liquid obtained in step (2); and (4) a step for heat-treating the crushed liquid obtained in step (3) for 5 minutes to 1 hour at 60-100°C, and obtaining agglomerates. Also disclosed are: a method for producing a food or pharmaceutical composition containing rubisco, the method being characterized by using rubisco obtained by said production method; and a food or a pharmaceutical composition which is for promoting serotonin synthesis in the brain and includes the rubisco as an active ingredient.

Description

Rubisco manufacturing method

The present invention relates to a method for producing rubisco derived from plant leaves and a method for producing a food or pharmaceutical composition using rubisco obtained by the production method. Furthermore, the present invention relates to foods or pharmaceutical compositions for promoting serotonin synthesis in the brain.

Photosynthesis of plants including crops begins with the synthesis of chemical energy using solar energy by taking in carbon dioxide in the atmosphere. Chlorophyll protein complex is present in the membrane system of chloroplasts that perform photosynthesis, and the enzyme that organicizes carbon dioxide in the solution phase (Rubilos 1,5-bisphosphate carboxylase / oxygenase (commonly known as RuBisCO)) Exists. Considering plants, there are 100 billion to 1 trillion tons of rubisco on the earth, and it can be said that the protein called rubisco is the most abundant protein on the earth. For example, spinach leaves contain 6 g of rubisco per gram of chlorophyll (Yokota, A and Canvin, D. T. (1985) Plant Physiol. 77, 735-739.). Spinach leaves contain 138 mg to 225 mg of chlorophyll per 100 g fresh weight (Toledo, M.E.A. et al. (2003) Sci. Rep. Grad. Sch. Agric. Biol. Sci., Osaka Prefecture University 55, 1-6; Downton, W. J. S. (1985) Plant Physiol. 78, 85-88.), 0.83 to 1.35 g is included when converted to rubisco amount. It will be.

It has become clear that this rubisco protein contains a large amount of essential amino acids, and it is expected to be used as a highly nutritious food (Non-Patent Documents 1 and 2). Furthermore, it has been clarified that among these ideal essential amino acids, tryptophan, which is a raw material for serotonin in the brain, is abundantly contained.

Serotonin, a neurotransmitter in the brain, is synthesized from tryptophan, which is one of the essential amino acids, and is said to have a physiological function such as sleep and thermoregulation, as well as a function to stabilize the mind. On the other hand, it is known that a decrease in serotonin in the brain causes depressive symptoms and insomnia. Therefore, foods that promote the synthesis of serotonin in the brain are considered to have high added value as functional foods.

Expecting such high nutritional value of rubisco protein, a method for easily purifying rubisco protein from crops has been devised (for example, Patent Documents 1-3). However, these are purification methods limited to specific plants, safety for use as food materials, and the possibility that rubisco protein is partially decomposed during the manufacturing process to cause quality deterioration. There are problems such as.

On the other hand, Patent Document 4 reports a method for preparing a rubisco protein. Specifically, a step of heat-treating a cabbage, a step of crushing the heat-treated cabbage with a buffer added, and crushing. A method for preparing rubisco, which comprises a step of removing an insoluble substance from the liquid and a step of purifying rubisco from a crushed liquid from which the insoluble substance has been removed. In the method reported in Patent Document 4, it is described that the rubisco protein could be recovered from the outer leaves of cabbage without being cleaved at all.

U.S. Pat. No. 4,268,632 U.S. Pat. No. 4,347,324 U.S. Pat. No. 4400471 Japanese Patent Application Laid-Open No. 2018-131414

However, the product purified by the method reported in Patent Document 4 was contaminated with polysaccharides and had a foul odor due to the contaminated vitamin U-related compound dimethyl sulfide of cabbage leaves.

The present invention is a method for producing rubisco derived from plant leaves, which can obtain rubisco with high purity without being hydrolyzed from plant leaves, and production of a food or pharmaceutical composition using rubisco obtained by the production method. The purpose is to provide a method. Furthermore, an object of the present invention is to provide a food or pharmaceutical composition having an action of promoting the synthesis of serotonin in the brain.

As a result of intensive research to achieve the above object, the present inventors heat-treated the cabbage outer leaf at 55 ° C. for 5 minutes, immediately ice-cooled, added a buffer solution, and crushed and squeezed the cabbage outer leaf. After removing insoluble substances such as cell walls with a centrifuge, heat treatment was performed at 68 ° C for 10 minutes, and it was found that rubiscoproteins aggregated almost completely and were not hydrolyzed. It was also found that by washing the rubisco protein aggregate with water, the purity of the rubisco protein can be increased and dimethyl sulfide, which causes an offensive odor, can be removed.

The present invention has been completed by further studies based on these findings, and is a method for producing rubisco derived from the leaves of the following plants, a method for producing a food or pharmaceutical composition containing rubisco, and promotion of serotonin synthesis in the brain. It provides food products or pharmaceutical compositions.

Item 1. Method for producing rubisco derived from plant leaves, which comprises the following steps (1) to (4):
(1) A process of heat-treating plant leaves at 50 to 58 ° C. for 3 minutes to 1 hour.
(2) A step of crushing the leaves of the plant obtained in the step (1),
(3) A step of removing insoluble substances from the crushed solution obtained in step (2), and (4) The crushed solution obtained in step (3) is heat-treated at 60 to 100 ° C. for 5 minutes to 1 hour to aggregate. The process of getting things.
Item 2. Item 2. The production method according to Item 1, further comprising the following step (5):
(5) A step of washing the agglomerates obtained in the step (4).
Item 3. Item 2. The production method according to

Item

1 or 2, wherein the SPAD value of the plant is 55 or more.
Item 4. The plants are cabbage, broccoli, pumpkin, watermelon, yugao, turnip, cauliflower, daikon, Chinese cabbage, carrot, gobo, sweet potato, taro, spinach, lettuce, bok choy, shungiku, cerule, pepper, taro, banana, moroheiya, cassaba, The production method according to any one of Items 1 to 3, which is at least one selected from the group consisting of zucchini, bitter gourd, and coffee tree.
Item 5. A method for producing a food or pharmaceutical composition containing Rubisco, which comprises using Rubisco obtained by the production method according to any one of Items 1 to 4.
Item 6. A food or pharmaceutical composition for promoting serotonin synthesis in the brain containing rubisco as an active ingredient.
Item 7. Item 6. The composition according to Item 6, which is for treating, improving or preventing mood disorders, insomnia, autism or attention deficit hyperactivity disorder.
Item 8. A method of promoting serotonin synthesis in the brain, comprising the step of administering Rubisco to a mammal in need thereof.
Item 9. Item 8. The method according to Item 8, which is a method for treating, improving or preventing mood disorders, insomnia, autism or attention deficit hyperactivity disorder.
Item 10. Use of Rubisco in the manufacture of foods or pharmaceutical compositions for promoting serotonin synthesis in the brain.
Item 11. Item 10. The use according to Item 10, wherein the composition is for treating, ameliorating or preventing mood disorders, insomnia, autism or attention deficit hyperactivity disorder.

According to the method for producing rubisco derived from plant leaves of the present invention, rubisco can be obtained with high purity without being hydrolyzed from plant leaves.

In addition, since Rubisco has a remarkably excellent effect of promoting serotonin synthesis in the brain, it is used for promoting serotonin synthesis in the brain (particularly, mood disorders such as depression, insomnia, autism or attention deficit). It is useful as an active ingredient in food or pharmaceutical compositions (for the treatment, amelioration or prevention of dyskinesia).

It is a photograph which shows the result of SDS-PAGE of an Example. 1: Cabbage outer leaf crushed solution (supernatant) 2: Aggregates obtained by heat-treating the outer leaf crushed solution at 68 ° C for 10 minutes 3: Cabbage outer leaf crushed solution (insoluble substance) It is a graph which shows the result of the test example. A: 5-HT (5-hydroxytryptamine, also known as serotonin) amount (pg / μl homogenate) in the brain stem, B: 5-HIAA (5-hydroxyindoleacetic acid) amount (pg / μl homogenate) value in the brain stem are average ± Standard error ^** p <0.01, ^*** p <0.001 vs. Free intake group

Hereinafter, embodiments of the present invention will be described in detail.

In addition, in this specification, "comprise" also includes the meaning of "essentially consist of" and the meaning of "consist of".

In the present specification, "Rubisco" shall mean protein unless otherwise specified.

The method for producing rubisco derived from plant leaves of the present invention is characterized by including the following steps (1) to (4) or steps (1) to (5).
(1) A process of heat-treating plant leaves at 50 to 58 ° C. for 3 minutes to 1 hour.
(2) A step of crushing the leaves of the plant obtained in the step (1),
(3) Step of removing insoluble substances from the crushed solution obtained in step (2),
(4) The step of heat-treating the crushed solution obtained in step (3) at 60 to 100 ° C. for 5 minutes to 1 hour to obtain agglomerates, and (5) washing the agglomerates obtained in step (4). Process.

RuBisCO (EC 4.1.1.39) is a common name for ribulose 1,5-bisphosphate carboxylase / oxygenase (also called ribulose-bisphosphate carboxylexase) and is responsible for the carboxylation of the reducing pentose phosphate pathway in photosynthesis. , Ribulose 1,5-bisphosphate is added with CO ₂ to catalyze the reaction to produce two molecules of 3-phosphoglycerate. In higher plants, it has a molecular weight of about 500,000 and is composed of a 16-mer consisting of 8 L (large) subunits and 8 S (small) subunits.

The plant used as a raw material in the present invention is not particularly limited as long as it is a plant containing Rubisco, and examples thereof include the following. Leafy vegetables (eg Chinese cabbage, spinach, turnip, lettuce, komatsuna, bok choy, shungiku, cerule, onion, onion, broccoli, cabbage, asparagus, moroheiya), root vegetables (eg carrots, radish, gobo, sweet potato, sweet potato, potato) , Taloymo, Renkon, Turnip, Cassaba), Fruit vegetables (eg, tomatoes, cherry tomatoes, eggplants, peppers, yugao, cucumbers, zucchini, bitter gourd, watermelons, strawberries, pumpkins, melons, corn, sweet corn), etc. Fruit trees such as pears, grapes, turnips, bananas, coffee trees; beans such as soybeans, edible beans, small beans, Chinese cabbage, peanuts, green beans; tea; tobacco; cotton; wheat such as barley, wheat, swallow, rye; rice; pansy, marie Gold, salvia, petunia, nichinichisou, kiku, carnation, rose, lindow, root vegetable, gerbera, statice, turkish sardine, alstroemeria, lily, tulip, cyclamen, vegetableus, freesia, primula, dendrobium, begonia, symbidium , Daisy, Scovaria, Calibrachoa and other flowers; Cactus and other succulent plants; Shrubs, conifers, broad-leaved trees, deciduous trees and other trees; grasses, etc.

As the plant used as a raw material in the present invention, a plant having a high Rubisco content is particularly desirable. Generally, a plant with a dark green color is considered to have a high Rubisco content. Therefore, plants with a SPAD value of 55 or more are preferable (the SPAD value can be measured with a chlorophyll meter such as Konica Minolta). In addition, a plant with a large yield of leaves per area is also preferable because more rubisco can be obtained.

Preferred plants include cabbage, pumpkin, watermelon, yugao, turnip, cauliflower, radish, Chinese cabbage, broccoli, taro, banana, cassaba, zucchini, bitter melon, coffee tree, carrot, gobo, sweet potato, taro, spinach, lettuce, bok choy, shungiku. , Cellully, peppers, moroheiya, etc. Of these, cabbage and broccoli are particularly preferable because they yield a large amount of leaves per area.

The leaves of the plant used as a raw material include only the leaves of the plant, or in addition to the leaves of the plant, other parts of the plant (for example, stems, bark, fruits, trunks, flowers, roots, seeds, etc.). You can also use what you have.

In step (1), the leaves of the plant are heat-treated. The temperature in this heat treatment is usually 50 to 58 ° C., preferably 53 to 56 ° C., and the heat treatment time is usually 3 minutes to 1 hour, preferably 5 to 10 minutes. The heat treatment can be performed, for example, by immersing in hot water. By heat-treating in this way, it is possible to prevent the decomposition of Rubisco by various proteases contained in plant cells. In addition, the heat treatment softens the leaves so that they can be easily crushed. After the heat treatment, it is desirable to cool with ice.

In step (2), the leaves of the plant obtained in step (1) are crushed. Crushing of plant leaves can be carried out using equipment used in the food sector. Examples of such a device include various choppers such as a meat chopper, various cutters such as a silent cutter, various mills such as a cutter mill and a feather mill, and devices such as a micromeister, a grinder, and a comitroll. When performing the crushing treatment, it is desirable to add an additive having a buffering action that can be used in foods in order to prevent acidification of the extract. After crushing the leaves of the plant, the juice may be further squeezed with a juice squeezing machine or the like. As the juice squeezing machine, a screw press, a filter press, a decanter, or the like that is usually used for juice squeezing in the food and drink field can be used.

In step (3), insoluble substances such as cell walls are removed from the crushed solution obtained in step (2). The removal of such an insoluble substance is not particularly limited, and can be performed by, for example, centrifugation, filtration, or the like.

In the step (4), the crushed liquid obtained in the step (3) is heat-treated to recover the agglomerates. The temperature in this heat treatment is usually 60 to 100 ° C., preferably 68 to 75 ° C., and the heat treatment time is usually 5 minutes to 1 hour, preferably 5 to 20 minutes. By performing such a heat treatment, Rubisco is almost completely aggregated. For the crushed liquid heat-treated in this way, the agglomerates are held on top, but the liquid can be recovered by pouring the liquid onto a filter paper, cloth, sieve, etc., centrifuging, or tilting. it can.

In step (5), the agglomerates obtained in step (4) are washed. Since the heat-treated Rubisco is insoluble in water, it can be washed with a liquid. The liquid used for cleaning is not particularly limited, and examples thereof include water, lower alcohol (for example, ethanol), a mixed solution thereof (for example, hydrous ethanol), and a buffer solution (particularly, a buffer solution used at the time of crushing). .. When hydrous alcohol (hydrous ethanol) is used as the solvent for washing, the alcohol (ethanol) concentration includes, for example, 20 to 90% by volume and 40 to 80% by volume. Examples of the water used for cleaning include tap water, groundwater, purified water and the like. Examples of the cleaning method include a method of collecting Rubisco aggregates on filter paper, cloth, a sieve, or the like, and then pouring a liquid such as water from above to perform cleaning. By performing such cleaning, impurities such as dimethyl sulfide and polysaccharides that cause offensive odors can be removed, so that the purity (content) of Rubisco can be increased and odorless can be achieved. .. The agglomerates after washing may be further dried by natural drying such as ventilation drying and sun drying, forced drying by heating with electricity and drying, freeze drying and the like.

According to the method for producing rubisco derived from plant leaves of the present invention, rubisco can be obtained with high purity without being hydrolyzed from plant leaves. Furthermore, when the leaves of the plant contain dimethyl sulfide or the like that causes an offensive odor, it can be made odorless. Further, since the present invention is a simple method that does not use an expensive reagent and does not perform a complicated treatment, it is also advantageous in terms of cost. As described above, Rubisco obtained by the production method of the present invention has high purity and no odor, so that it can be mixed with various foods. In addition, cabbage wastes up to 1 million tons of outer leaves annually, so if this discarded tissue can be used, it will lead to effective use of food resources and reduction of waste.

In addition, since Rubisco has a remarkably excellent effect of promoting the synthesis of serotonin in the brain, it is used for promoting the synthesis of serotonin in the brain (particularly, mood disorders such as depression, insomnia, autism, and attention deficit). It is useful as an active ingredient of foods or pharmaceutical compositions (for treatment, improvement or prevention of dyskinesia, etc.). The ratio (100 × W / LNAA) of tryptophan contained in rubisco protein to a large molecular neutral amino acid (LNAA) is extremely high as a food protein.

The method for producing a food or pharmaceutical composition containing rubisco of the present invention is characterized by using rubisco obtained by the above method for producing rubisco derived from plant leaves. In addition, the food or pharmaceutical composition for promoting serotonin synthesis in the brain of the present invention is characterized by containing rubisco as an active ingredient.

Food compositions include any food or drink that can be consumed by mammals (including humans), such as dairy products; fermented foods (yogurt, cheese, etc.); beverages (coffee, juice, cocoa, tea beverages, sports drinks). , Soft drinks such as nutritional drinks, dairy drinks, lactic acid bacteria drinks, lactic acid bacteria-containing drinks, yogurt drinks, carbonated drinks, sake, western liquor, liquor such as fruit liquor, etc. Paste etc.); Western confectionery (chocolate, donut, pie, cream puff, gum, gummy, jelly, candy, cookie, cake, pudding, biscuits, etc.); Japanese confectionery (Daifuku, rice cake, bun, castella, anmitsu, sheep syrup, etc.); Iced drinks (ice cream, ice drinks, sherbets, etc.); Foods (curry, beef bowl, miscellaneous dishes, miso juice, soup, meat sauce, pasta, pickles, jam, ham, sausage, bacon, etc.); Seasonings (dressing, sprinkle, etc.) , Taste seasoning, soup base, miso, soy sauce, sauce, ketchup, oyster sauce, etc.).

The method for producing the food composition is not particularly limited, and a known method can be appropriately followed.

Examples of food compositions include health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, specified health foods, nutritionally functional foods, and foods with functional claims. The form of the administration unit when used as a supplement is not particularly limited and may be appropriately selected. Examples thereof include tablets, capsules, granules, liquids and powders.

In addition, the food composition also includes the meaning of an additive that imparts an action of promoting serotonin synthesis in the brain.

Gluten, the main protein of wheat, has a lysine content, which is one of the essential amino acids, of only about 40% of the FAO recommended level. This becomes a limiting amino acid, which makes the protein nutritional value in wheat flour extremely low. Therefore, rubisco can also be added to food substrates such as flour and corn to improve nutritional value.

Food compositions include excipients, vitamins, minerals, flavonoids, polyphenols, amino acids, nucleic acids, fatty acids, refreshing agents, binders, sweeteners, acidulants, disintegrants, glides, as needed. Agents, colorants, fragrances, pH adjusters, stabilizers, preservatives, sustained release adjusters, surfactants, brighteners, solubilizers, wetting agents and the like can be blended.

Examples of the proportion of rubisco contained in the food composition include 1 to 99% by mass, 1 to 80% by mass, and 1 to 70% by mass.

The food composition preferably contains sugar (particularly glucose) in order to promote the transport of rubisco into the brain. For example, rubisco is added to a glucose solution of 10 to 30% (w / v). Examples include those that have been dissolved.

The intake amount of the food composition can be appropriately set according to various conditions such as the weight, age, sex, and symptoms of the ingestor.

Rubisco alone can be used in the pharmaceutical composition, or it can be mixed with other pharmaceutical ingredients such as vitamins and crude drugs described in the Japanese Pharmacopoeia.

When prepared as a pharmaceutical composition, Rubisco is mixed with non-toxic carriers, diluents or excipients allowed in pharmaceuticals, tablets (bare tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.). (Including), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, etc. (for example, for oral use) It can be a pharmaceutical formulation.

Examples of the ratio of Rubisco contained in the pharmaceutical composition include 1 to 99% by mass, 1 to 80% by mass, and 1 to 70% by mass.

The dose of the pharmaceutical composition can be appropriately determined according to various conditions such as the patient's weight, age, sex, and symptoms. In addition, the pharmaceutical composition of the present invention is administered to mammals including humans.

The pharmaceutical composition of the present invention also includes quasi-drugs.

As shown in Examples described later, the present inventors have found that ingestion of rubisco isolated and purified from plant leaves increases serotonin in the brain, and thus the food product obtained by the above production method or The pharmaceutical composition can be expected to have an action of promoting the synthesis of serotonin in the brain. Since rubisco is contained in cells guarded by a tough cell wall, it cannot be sufficiently digested and absorbed by humans simply by eating the leaves of plants containing rubisco as it is, and the action of promoting the synthesis of serotonin in the brain cannot be expected.

The use for promoting serotonin synthesis in the brain is not particularly limited, and is preferably for the treatment, amelioration or prevention of diseases caused by a decrease in serotonin in the brain, and more preferably for mood disorders such as depression, insomnia, etc. It is for treatment, improvement or prevention of autism, attention deficit hyperactivity disorder, etc.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

Example The outer leaves of cabbage were heat-treated at 55 ° C. for 5 minutes and then immediately ice-cooled. The outer leaves of cabbage were crushed by adding a 10 mM phosphate buffer solution commonly used in food production. Next, the outer leaf crushed solution was squeezed with a juicer using a triple cotton cloth as a strainer and a centrifuge to remove insoluble substances such as cell walls (8,200 x g, 15 minutes). Then, the outer leaf crushed solution from which the insoluble substance was removed was heat-treated at 68 ° C. for 10 minutes to aggregate Rubisco.

The crushed liquid after the heat treatment was allowed to stand at 5 to 10 ° C and waited for the aggregated Rubisco to sufficiently precipitate. The brown liquor of the supernatant was discarded by the tilting method, and the aggregated Rubisco was collected on cooking paper. When the supernatant was sufficiently removed, the leaves were washed several times with tap water or the buffer used for crushing the leaves, and the washing was repeated until the color of the filtrate completely disappeared. After the washing liquid was no longer discharged, the collected Rubisco was cryopreserved together with cooking paper at -20 ° C or lower, and then freeze-dried to obtain the desired Rubisco standard.

Figure 1 shows the results of SDS-PAGE. 1 in FIG. 1 is a cabbage outer leaf crushed solution (supernatant), 2 is an aggregate obtained by heat-treating the outer leaf crushed solution at 68 ° C. for 10 minutes, and 3 is a cabbage outer leaf crushed solution (insoluble substance). 2 and 3 were dissolved in 8M urea having the same volume as the mother liquor to perform SDS-PAGAE.

The dark band in the upper part of Fig. 1 is the L subunit of Rubisco, and the dark band in the lower part is the S subunit. Also, the thin band in the middle is the chlorophyll-binding protein. From the results shown in FIG. 1, it can be seen that the chlorophyll-binding protein and the like can be removed and purified by heat-treating the outer leaf crushed solution from which the insoluble substance has been removed at 68 ° C. for 10 minutes.

The cabbage rubisco obtained in this example was used in the following rat brain serotonin synthesis function evaluation test example.

Test Example 16 male Wistar rats aged 6-7 weeks were used. Five animals were freely fed and watered to form a free feeding group. The remaining 4 rats were fasted for 24 hours and then administered Rubisco dissolved in 20% glucose solution to 250 mg / kg (2.5 ml / kg volume). As a control experiment, the same amount of bovine serum albumin was dissolved in 20% glucose solution and administered. Specifically, the following measures were taken.

1) Free feeding group (5 animals): Freely fed food and water for 24 hours.
2) Fasting group (3 animals): After fasting for 24 hours, an oral tube was placed in the stomach and physiological saline was administered to a liquid volume of 2.5 ml / kg. A sample was taken 1 hour after administration.
3) Rubisco administration group dissolved in 20% glucose solution (4 animals): After fasting for 24 hours, an oral tube was placed in the stomach and 20% glucose solution (1 g Rubisco) mixed with 10% cabbage rubisco was added. Was dissolved in 10 ml of 20% glucose solution) to a rubisco amount of 250 mg / kg (liquid volume of 2.5 ml / kg). A sample was taken 1 hour after administration.
4) Bovine serum albumin-administered group dissolved in 20% glucose solution (4 animals): After fasting for 24 hours, an oral tube was placed in the stomach and 20% glucose mixed with 10% bovine serum albumin (BSA). The solution (1 g of BSA dissolved in 10 ml of 20% glucose solution) was administered so that the amount of BSA was 250 mg / kg (2.5 ml / kg of solution). A sample was taken 1 hour after administration.

Rats were euthanized under isoflurane anesthesia 1 hour after administration, and blood was collected from the heart. Then, about 300 ml of 10 mM PBS (pH = 7.4) was perfused systemically from the heart to remove blood, and then the brain was removed. The brain was cut in half on the spot, and the right brain was divided into the cerebral cortex (frontal lobe), cerebellum, brain stem, and others, placed in a cryotube, rapidly frozen in liquid nitrogen, and then frozen and stored at -80 ° C until measurement. Frozen brains were homogenized with perchloric acid and then centrifuged. Supernatants were collected and the concentrations of serotonin (5HT) and 5HIAA in the supernatant were measured using high performance liquid chromatography (HPLC).

The results shown in FIG. 2 were obtained in the brain stem. Serotonin was significantly elevated only in the glucose-dissolved rubisco-treated group compared to the free-feeding control group ( ^** p <0.01), but no significant change was observed in the other groups ( ^** p <0.01). FIG. 2A). Serotonin in the glucose-dissolved Rubisco-administered group was also significantly increased as compared with the glucose-dissolved BSA-administered group (Fig. 2A).

5HIAA was significantly reduced by fasting (* ^*** p <0.001). In the glucose-dissolved Rubisco-administered group, 5HIAA was significantly increased as compared with the fasting group, and recovered to a level at which there was no significant difference from the free-feeding group (Fig. 2B). On the other hand, the glucose-dissolved BSA-administered group also had an increase of 5 HIAA compared to the fasting group, but the degree of increase was lower than that of the glucose-dissolved Rubisco-administered group, and it was still significant compared to the free-feeding group ( ^** It was a low value at p <0.01) (Fig. 2B).

Claims

Method for producing rubisco derived from plant leaves, which comprises the following steps (1) to (4):
(1) A process of heat-treating plant leaves at 50 to 58 ° C. for 3 minutes to 1 hour.
(2) A step of crushing the leaves of the plant obtained in the step (1),
(3) A step of removing insoluble substances from the crushed solution obtained in step (2), and (4) The crushed solution obtained in step (3) is heat-treated at 60 to 100 ° C. for 5 minutes to 1 hour to aggregate. The process of getting things.
The production method according to claim 1, further comprising the following step (5):
(5) A step of washing the agglomerates obtained in the step (4).
The production method according to claim 1 or 2, wherein the SPAD value of the plant is 55 or more.
The plants are cabbage, broccoli, pumpkin, watermelon, yugao, turnip, cauliflower, daikon, Chinese cabbage, carrot, gobo, sweet potato, taro, spinach, lettuce, bok choy, shungiku, cerule, pepper, taro, banana, moroheiya, cassaba, The production method according to any one of claims 1 to 3, which is at least one selected from the group consisting of zucchini, bitter gourd, and coffee tree.
A method for producing a food or pharmaceutical composition containing rubisco, which comprises using rubisco obtained by the production method according to any one of claims 1 to 4.
A food or pharmaceutical composition for promoting serotonin synthesis in the brain containing rubisco as an active ingredient.
The composition according to claim 6, which is for treating, improving or preventing mood disorders, insomnia, autism or attention deficit hyperactivity disorder.