JP7197813B2 - A method for increasing the amount of stilbenoids and/or TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof in plants, a method for producing a plant having an increased content of said compound, and an increased content of said compound plants and foods or beverages using said plants - Google Patents

Description

The present disclosure provides a method for increasing the amount of stilbenoids and/or TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof in plants, a method for producing plants having an increased content of said compounds, It relates to plants with increased compound content, foods and beverages with increased stilbenoid content.

Patent Document 1 describes a method for increasing the resveratrol content by storing at room temperature after irradiating grapes after harvesting with ultraviolet light (especially UV-C).

WO 02/085137

A method that can safely and/or efficiently increase the amount of stilbenoids such as resveratrol and/or other useful substances in plants is desired.

According to the present disclosure, a plant or a portion thereof (especially a grape skin or a grape fruit containing at least the skin), a crushed product thereof, or a plant cultured cell is irradiated with light in the wavelength range of 275 to 295 nm in an amount of 50,000 to 2,500,000 μmol / m ² and the irradiation amount of light in the wavelength range of 200 to 270 nm is less than 20% of the irradiation amount of light in the wavelength range of 275 to 295 nm, and the plant after the irradiation or stilbenoids in plants or parts thereof (particularly grape skins or grape fruits) or crushed products thereof, characterized in that the parts thereof (especially pericarp or fruit) or crushed products thereof are stored in the dark for at least one day is provided.
In addition, according to the present disclosure, the irradiation amount of light in the wavelength range of 275 to 295 nm is 50,000 to 2,500,000 μmol/m ² and the wavelength range of 200 to 270 nm is applied to the plant or its parts, crushed products thereof, or cultured plant cells. The amount of light irradiation is less than 20% of the amount of light in the wavelength range of 275 to 295 nm, and the plant or its parts or crushed material after the irradiation or after the irradiation and storing cultured plant cells in the dark for one day or longer. Provided is a method of extending one or more compounds selected from the group consisting of acids and salts thereof.
In addition, according to the present disclosure, the plant or its parts (particularly, the grape skin or the grape fruit containing at least the fruit skin), the crushed product thereof, or the plant cultured cells are irradiated with light in the wavelength range of 275 to 295 nm at a dose of 50,000 to 2,500,000. μmol/m ² and the irradiation amount of light in the wavelength range of 200 to 270 nm is less than 20% of the irradiation amount of light in the wavelength range of 275 to 295 nm, and after the irradiation A plant or part thereof with increased stilbenoids (particularly, (Grape pericarp or fruit) or a crushed product thereof or a method for producing cultured plant cells.

Further, according to the present disclosure, the plant or parts thereof (especially grape skins or grape fruits containing at least the skins) or crushed products thereof are irradiated with light in the wavelength range of 275 to 295 nm in an amount of 50,000 to 2,500,000 μmol/m ² and irradiating the plant or its part after the irradiation ( in particular, dark storage of the pericarp or fruit) or crushed material thereof for one day or more, and processing of the resulting plant or part thereof (especially grape skin or fruit) or crushed material thereof. A method for producing a food or beverage is provided.
Also according to the present disclosure, a mature plant or part thereof (especially a fruit comprising at least the pericarp of grapes), wherein resveratrol and phenylalanine in the plant or part thereof (especially the pericarp) by mass spectrometry and the signal intensity ratio (resveratrol signal intensity / phenylalanine signal intensity) is 0.5 or more, and the cells of the plant or its parts (especially pericarp) express enzymes involved in the resveratrol biosynthetic pathway Plants or parts thereof (especially grape berries or pericarp thereof) are provided that have not been genetically engineered to increase their abundance.
Furthermore, according to the present disclosure, the signal intensity ratio between resveratrol and phenylalanine by mass spectrometry (resveratrol signal intensity/phenylalanine signal intensity) is 0.5 or more, and genetically modified plants or parts thereof are used as raw materials. (especially grape)-free food or beverages (especially wine when the plant is grape) are provided.

According to the bulking method of the present disclosure, the content of stilbenoids such as resveratrol and/or other useful substances in plants can be safely and/or efficiently increased.

Figure 1 shows the irradiation dose (2,250, 22,500 or 225,000 μmol/m ² ) of LED light (peak wavelength: 290 nm) irradiated to the fruit (fruit) of harvested mature grapes (variety: Kyoho), and following light irradiation 1 is a graph showing the relationship between resveratrol content (μg/g dry weight) in pericarp after storage in the dark at 15° C. for one day. Fig. 2 shows the period of storage in the dark at 15°C after irradiating the harvested mature grapes (cultivar: Kyoho) with LED light (peak wavelength: 290 nm) (irradiation amount: 225,000 μmol/m ² ). It is a graph showing the relationship between (1 day or 2 days) and the resveratrol content (μg/g dry weight) in the pericarp. Figure 3 shows that the fruits (bundles) of harvested mature grapes (variety: Muscat Bailey A) were irradiated with LED light (peak wavelength: 290 nm) (irradiation amount: 225,000 μmol/m ² ), and the fruit was in a dark state. It is a graph showing the relationship between the storage period (1 day, 2 days or 5 days) and the resveratrol content (μg/g dry weight) in the pericarp. Figure 4 shows the resin in the pericarp of mature grapes (variety: Muscat Bailey A) stored in the dark for 2 days in the bunch after irradiation with LED light (peak wavelength: 290 nm; irradiation amount: 225,000 μmol/m ² ). FIG. 10 is a graph showing veratrol content (μg/g dry weight) compared to non-irradiated controls that were kept in dark post-harvest bunches for 2 days. Figure 5 shows the content in the pericarp of mature grapes (variety: Muscat Bailey A) stored in the dark for 2 days in the bunch after irradiation with LED light (peak wavelength: 290 nm; irradiation amount: 225,000 μmol/m ² ). Shows the ratio between the detected amount of some phenolic compounds and the detected amount of the said several phenolic compounds contained in the non-irradiated control pericarp that was stored in the dark for 2 days (= 48 hours) in the bunch after harvesting. graph. Figure 6 shows the irradiation dose (0, 2,250, 22,500 or 225,000 μmol/m ² ) of LED light (peak wavelength: 290 nm) irradiated to the fruit (fruit) of purchased mature grapes (cultivar: Shine Muscat) and the light 1 is a graph showing the relationship between resveratrol content (μg/g dry weight) in pericarp stored in the dark for 2 days at 15° C. without irradiation or following irradiation with light. Figure 7 shows the fruits (fruits) of purchased mature grapes (cultivar: Shine Muscat) stored in the dark at 15 ° C for 5 days after irradiation with LED light (peak wavelength: 290 nm; irradiation amount: 225,000 μmol / m ² ) ) is a graph showing the resveratrol content (μg/g dry weight) in the pericarp. Figure 8 shows the temperature of dark storage after irradiation of LED light (peak wavelength: 280 nm; irradiation amount: 216,000 μmol / m ² ) to the fruits (fruits) of purchased mature grapes (variety: Delaware) (5, 15, 25 or 40° C.) and period (1 day or 5 days) and the resveratrol content (μg/g dry weight) in the peel. Figure 9 shows several TCA cycle metabolites, spermidine and 4-aminobutyric acid contained in Arabidopsis thaliana stored in the dark for 2 days after irradiation with LED light (peak wavelength: 280 nm; irradiation dose: 6,750 μmol/m ² ). and the detected amount of the compound in the non-irradiated control stored in the dark for 2 days. 10 is a photograph showing white callus (left) and red callus (right) derived from the leaf tissue of "Bailey Alicante A" used in Example 12. FIG. Figure 11 shows the ^{thresveratrol} content (μg /g dry weight) and resveratrol content in non-irradiated callus stored in the dark for 2 days.

In this specification, the numerical range "a to b" (a and b are specific numerical values) means a range including the values "a" and "b" at both ends. In other words, "a to b" is synonymous with "a or more and b or less".

From a first aspect, the present disclosure provides a method for increasing the amount of stilbenoids in a plant or a part thereof (particularly, a grape skin or a grape fruit containing at least the skin), a crushed product thereof, or a plant cultured cell, Part (particularly, pericarp or fruit) or its crushed material or the plant cultured cells is irradiated with light in the wavelength range of 275 to 295 nm at a dose of 50,000 to 2,500,000 μmol/m ² and light in the wavelength range from 200 to 270 nm. Light irradiation so that the irradiation dose is less than 20% of the irradiation dose of light in the wavelength range of 275 to 295 nm, and the plant or its part (especially pericarp or fruit) after irradiation, or crushed material thereof and storing in the dark for one day or more (hereinafter also referred to as "first volume increasing method of the present disclosure").
From the second point of view, the present disclosure provides a plant or its parts, crushed products thereof, or cultured plant cells with an irradiation amount of light in the wavelength range of 275 to 295 nm of 50,000 to 2,500,000 μmol/m ² and The irradiation amount of light in the wavelength range of is less than 20% of the irradiation amount of light in the wavelength range of 275 to 295 nm, and the plant or its part after irradiation or its crushed material or Storing the irradiated plant cultured cells in the dark for at least one day, the plant or its parts, crushed products thereof, or TCA cycle metabolites, polyamine alkaloids, and 4-amino in plant cultured cells. A method for increasing the amount of one or more compounds selected from the group consisting of butyric acid, abscisic acid and salts thereof (hereinafter also referred to as "second increasing method of the present disclosure"). Hereinafter, the first weight increasing method of the present disclosure and the second weight increasing method of the present disclosure will be collectively referred to as the “weight increasing method of the present disclosure”.
From another aspect, the present disclosure provides a method for producing a stilbenoid-enriched plant or part thereof (especially grape skin or grape fruit containing at least the skin) or a crushed product thereof, comprising: (Particularly, pericarp or fruit) or its crushed material, the irradiation amount of light in the wavelength range of 275 to 295 nm is 50,000 to 2,500,000 μmol / m ² , and the irradiation amount of light in the wavelength range of 200 to 270 nm is the above 275 Irradiate light so that the irradiation amount of light in the wavelength range of ~ 295 nm is less than 20%, and the plant or its part (especially pericarp or fruit) after irradiation or its crushed material is kept in a dark place for 1 day or more. The method is characterized by storing.
From another aspect, the present disclosure applies the first volume-increasing method of the present disclosure to plants or parts thereof (particularly, grape skins or grape fruits containing at least the skins), crushed products thereof, or plant culture cells. A method for producing stilbenoid-enriched plants or parts thereof (especially grape skins or fruits), crushed products thereof, or cultured plant cells. Hereinafter, this production method and the production method described above are collectively referred to as the "production method of the present disclosure".

As shown in the examples below, the present disclosure irradiates a harvested plant or part thereof (particularly grape skin) or plant cultured cells with light in the wavelength range of about 275 to 295 nm, and then stores in a dark place for one day or more. It is based on the new knowledge that the stilbenoid content in the plant or its parts (particularly pericarp) or cultured plant cells can be remarkably increased by doing so. Therefore, according to the present disclosure, while reducing adverse effects such as DNA damage and production of allergens due to exposure to ultraviolet light (especially UV-C), plants or parts thereof (especially grape skins) or plant culture cells Since it is possible to irradiate light in a specific wavelength range effective for increasing the amount of stilbenoids in plants or their parts (especially grape skins) or stilbenoids in plant culture cells (especially resvera trolls) can be safely and/or effectively increased.

As used herein, the plant is not particularly limited, but is, for example, a plant capable of producing stilbenoids and/or polyamine alkanoids.
In certain embodiments, the plant is not particularly limited as long as it can naturally produce stilbenoids (especially resveratrol), and can be, for example, angiosperms, especially dicotyledons.
In another specific embodiment, the plant is not particularly limited as long as it can produce polyamine alkanoids, and can be, for example, an angiosperm, especially a dicotyledonous plant.
Dicotyledonous plants suitable for the method of the present invention include, for example, the family of Grapes (particularly Grapes), Ericaceae (particularly Vaccinium), Solanaceae (particularly Solanaceae), Currantaceae (particularly Currants), Theaceae (particularly Camellia), Malvaceae (especially cacao genus), Polygonaceae (especially buckwheat genus and freckle genus), Rutaceae (especially citrus, kumquat and trifoliate genus), amaryllidaceae (especially allium genus), Poaceae (especially Poaceae), Fabaceae (especially Soybean and Arachis), Zingiberaceae (especially Zingiber and Curcuma), Rubiaceae (especially Coffea), Brassicaceae (especially Brassicaceae and Radish), Berberaceae (especially Rhododendron) genus), Rosaceae (especially Orchid, Prunus and Apple), Asteraceae (especially Lactobacillus), Labiatae (especially Labiatae), Gnetaceae (especially Gnetum), Lythraceae (especially Pomegranate) Contains plants. It also includes medicinal herbs such as plantain, dokudami, and gardenia, and edible flowers such as marshmallow and edible chrysanthemum. Specific examples of plants that can be used in the present invention include grapes (which produce resveratrol and anthocyanins), mountain cranberries (which produce resveratrol), cowberries, lingonberries, santa berries, knotweed, melinjo, and peanuts. (peanuts), almonds, apples, pomegranates, strawberries, blueberries (producing anthocyanins), eggplant and cassis, tea trees (producing catechins), cacao (producing cocoa polyphenols), buckwheat (producing rutin), citrus fruits (plants belonging to the genus citrus, kumquat or trifoliate) and onions, rice (producing ferulic acid), legumes (producing isoflavones), turmeric (producing curcumin), (producing shogaol) Ginger, as well as coffee trees (which produce coffee polyphenols), and the like.
In the present disclosure, a plant may be a young plant, an adult plant, a plant in the vegetative growth stage, or a plant in the reproductive growth stage. In the present disclosure, the term "plantlet" refers to a plant from post-germination (more specifically, after rooting) to, for example, leaf age 10, more specifically leaf age 7, more specifically leaf age 5. More specifically, it refers to a plant with a leaf age of 3 or less. In the present disclosure, the term "adult plant" refers to a plant with a leaf age of 3 or later, more specifically a leaf age of 5 or later, more specifically a leaf age of 7 or later, more specifically a leaf age of 10 or later. say. In certain embodiments, the plant is a post-harvest plant.
In the present disclosure, cultured plant cells may be cultured cells of the plant. Plant cultured cells may be in the form of suspension cultures or callus cultures.
It is preferable that the plant or its part or its crushed product that can be used in the method of the present invention per se or its extract or processed product can be used for food, drink or medicine.

In a preferred embodiment, the plant is Vitis spp.
Advantageously, therefore, the present disclosure provides a method for increasing the amount of stilbenoids in grape skins or grape fruit comprising at least pericarp or crushed material thereof, producing grape skins with increased stilbenoid content or grape fruit comprising at least pericarp or crushed material thereof , grape skins with increased stilbenoid content or grape fruit containing said skins or crushed products thereof, and foods and beverages with increased stilbenoid content (eg fruit wines, especially wine).

As used herein, "plant" includes plant parts, unless the context clearly indicates that only the whole plant is meant. A "part" of a plant is one or more organs selected from root system, stem, leaf, flower and fruit, or one or more of the components of said organs (e.g. pericarp, which is a component of fruit), e.g. It may be a shoot system (stem and leaves and optionally flowers and/or fruit), stem, leaf, fruit or pericarp.
Preferably, the plant or part thereof has not been genetically engineered so that its cells increase the expression of enzymes involved in the biosynthetic pathway of the compound to be increased.
A “debris” of a plant or part thereof is the result obtained by applying physical means of disruption, such as cutting and/or compression, to a plant or part thereof, in which at least some of the plant cells contained are viable. , such as a piece of tissue or debris.

In the present specification, "grape" is not particularly limited as long as it is a plant of the genus Vitis (Vitis spp.), and may be a hybrid between species of the genus Vitis. The grapes belong, for example, to the species Vitis vinifera, Vitis labrusca, Vitis amurensis, Vitis mustangensis, Vitis riparia or Vitis rotundifolia, or hybrids derived from at least one of these (preferably between said species crossbreds). Preferably, the grape belongs to the species Vitis vinifera, or is a combination of Vitis vinifera and one or more species selected from Vitis labrusca, Vitis amurensis, Vitis mustangensis, Vitis riparia and Vitis rotundifolia. It is a hybrid between
Specific examples of varieties include: Alieniko/Aglianico, Cabernet Sauvignon, Cabernet Franc, Gamay, Carmenere, Garnacha/Grenache, Sangiovese. Sangiovese, Syrah, Zinfandel, Tempranillo, Nebbiolo, Barbera, Pinot Noir, Malbec, Merlot, Montepulciano Montepulciano, Albarino, Viognier, Gewurztraminer, Koshu, Chardonnay, Chenin blanc, Semillon, Sauvignon blanc ), Trebbiano, Torrontes, Pinot gris, Furmint, Pedro Ximenez, Muscat of Alexandria, Macabeo, Muscadet (Muscadet), Moscato, Riesling, Concord, Kyoho, Pione, Muscat Bailey A, Shine Muscat, Ruby Roman Roman), Delaware, etc., but not limited to these.

Preferably, the grapes used herein, at least in the pericarp cells, have not been genetically engineered to increase the expression of enzymes involved in the resveratrol biosynthetic pathway. Enzymes involved in the resveratrol biosynthetic pathway are typically phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) and stilbene synthase (STS). is.
As used herein, "genetic manipulation" generally refers to the use of recombinant DNA technology, particularly the use of recombinant DNA technology, genome editing technology or chemical mutagenesis. does not include the use of cross-breeding methods. Genetic manipulation includes, for example, introduction of exogenous genes, coding sequences and/or regulatory elements (e.g. promoters, enhancers, suppressors, silencers), modification of endogenous genes, coding sequences and/or regulatory elements and/or endogenous It may be a deletion of all or part of the gene, coding sequence and/or regulatory element.
More preferably, the grape is non-genetically engineered.

As used herein, "pericarp" more specifically refers to the exocarp. The pericarp may be separated from the pulp. Pericarp includes pericarp pieces (eg, splits). In certain embodiments, the pericarp is the pericarp of (the fruit of) the plant that has been harvested. In a more specific embodiment, the pericarp is that of harvested grape berries.
As used herein, "fruit" may or may not contain seeds as long as it contains at least pericarp. In the latter case, the fruit may be either a seedless fruit or a seed-free fruit. Fruit includes fruit pieces (eg, splits). In certain embodiments, the fruit is harvested fruit. In a more specific embodiment, the fruit is a harvested grape berry. In this case, the grapes may be undestemmed (bundle) or destemmed (fruit).
A "crushed" fruit may be the fruit after pressing or juicing, including at least the pericarp. The crushed product includes, for example, a mixture of peel, juice, pulp, and seed, such as "must", and a mixture of peel and seed, such as "pomace".

The fruit is preferably a mature fruit and the pericarp is preferably of a mature fruit. As used herein, "mature fruit" generally refers to fruit harvested for raw consumption or for processing or brewing.
In some embodiments, the mature grapes have a sugar content of 15oBrix or higher, more specifically 16oBrix or higher, more specifically 17oBrix or higher, more specifically 18oBrix or higher, more specifically 19oBrix or higher, More specifically, it can be 20oBrix or more. In this case, the upper limit of sugar content is not particularly defined, but may be, for example, 25oBrix.
In some other embodiments, the mature grape fruit has an acid content (titratable acid: converted to tartaric acid) of 0.9% (or g/100 mL) or less, more specifically 0.8% (or g/100 mL) or less, More specifically, it may be 0.7% (or g/100 mL) or less, more specifically 0.6% (or g/100 mL) or less, more specifically 0.5% (or g/100 mL) or less.
In some other embodiments, the mature grapes are 70 days or more after full bloom, more specifically 75 days or more, more specifically 80 days or more, more specifically 85 days or more, more specifically Typically, it may have been harvested after 90 days. In this case, the upper limit of the number of days after full bloom is not particularly defined, but may be, for example, 125 days, 120 days, 115 days or 110 days. As used herein, "full bloom" with respect to grapes refers to the time when 80% or more of all flower buds of the flower spike have opened, and "blooming" refers to the state in which the corolla has fallen off the flower buds.
Whether or not the pericarp is that of a mature grape fruit may be determined based on the pericarp color, for example, with reference to a color chart created exclusively for grapes of the cultivar.

In the present disclosure, light to irradiate plants or parts thereof (particularly, grape skins or fruits), crushed products thereof, or plant cultured cells includes light in the wavelength range of 275 to 295 nm, preferably light in the wavelength range of 285 to 295 nm. is. By irradiating with light in this wavelength range, while suppressing photodenaturation of DNA and/or production of allergens, stilbenoids and/or TCA cycle in plants or their parts (especially grape skins) or plant cultured cells The content of metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof can be increased safely and/or efficiently.
On the other hand, plants or their parts (especially grape skins) or plant cultured cells contain many other phenolic compounds, and these compounds absorb light in the wavelength range of 275 to 295 nm, so light in the wavelength range of 275 to 295 nm is , plants or parts thereof (especially grape skins) or plant cultured cells need to be irradiated with relatively high doses.

The lower limit of the irradiation amount of light in the wavelength range of 275 to 295 nm irradiated to plants or parts thereof (especially, grape skins or fruits), crushed products thereof, or plant culture cells is 50,000 μmol/m ² , for example, 60,000 μmol/ m ² , more specifically 70,000 μmol/m ² , more specifically 80,000 μmol/m ² , more specifically 90,000 μmol/m ² , more specifically 100,000 μmol/m ² , more specifically can be 150,000 μmol/m ² . Stilbenoids and/or metabolites of the TCA cycle via activation of the photoreceptor UVR8, polyamine alkaloids, 4-aminobutyric acid, abscisin when the irradiation dose of light in the wavelength region of 275-295 nm is less than 50,000 μmol/m ² . Metabolites of the stilbenoid and/or TCA cycle in plants or parts thereof (especially grape skins) or plant cultured cells, polyamine alkaloids, because they do not result in significant activation of the biosynthetic pathways of acids and/or their salts , 4-aminobutyric acid, abscisic acid and/or salts thereof cannot be achieved.

The upper limit of the irradiation amount of light in the wavelength range of 275 to 295 nm is not particularly limited, but is, for example, 2,500,000 μmol/m ² , more specifically 2,000,000 μmol/m ² , more specifically 1,500,000 μmol/m ² , more specifically 1,500,000 μmol/m 2 Specifically 1,000,000 μmol/m ² , more specifically 900,000 μmol/m ² , more specifically 800,000 μmol/m ² , more specifically 700,000 μmol/m ² , more specifically 600,000 μmol /m ² , more specifically 500,000 μmol/m ² . When the irradiation amount of light in the wavelength range of 275 to 295 nm exceeds 2,500,000 μmol/m ² , the possibility of severe damage to plants or their parts (especially grape skins or fruits) or plant cultured cells is increased, so stilbenoids and / or to obtain a plant or part thereof (especially grape skin or fruit) or crushed product thereof or cultured plant cells enriched with TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof There is a high possibility that it will not be possible, and even if it is obtained, it is often not suitable for food.

The range of irradiation amount of light in the wavelength region of 275 to 295 nm may be any combination of the above lower and upper limits. Specific examples of the irradiation dose range are 50,000 to 2,500,000 μmol/m ² , 50,000 to 2,000,000 μmol/m ² , 50,000 to 1,500,000 μmol/m ² , 50,000 to 1,000,000 μmol/m ² , 60,000 to 1,000,000 μmol/m ² , 70,000 μmol/m 2 . ~1,000,000 µmol/ ^m2 , 70,000-800,000 µmol/ ^m2 , 80,000-800,000 µmol/ ^m2 , 80,000-500,000 µmol/ ^m2 , 100,000-500,000 µmol/ ^m2 and 120,000-500,000 µmol/ ^m2 , but not limited to.

On the other hand, since the maximum absorption wavelength of DNA and RNA is around 260 nm, there is concern that light with a wavelength around 260 nm has a great adverse effect on plants. Therefore, the irradiation amount of light in the wavelength range of 200 to 270 nm is preferably less than 20%, more preferably less than 15%, and less than 10% of the irradiation amount of light in the wavelength range of 275 to 295 nm. more preferably less than 5%, most preferably less than 1%.

In addition, it is known that light in the wavelength range of 300 to 400 nm does not contribute to increasing the amount of phenolic compounds in plants, but rather acts on plant damage (International Publication No. 2018/199307). When the irradiation dose is 50% or more of the irradiation dose of light in the wavelength range of 275 to 295 nm, the amount of stilbenoids and/or TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof increases. It becomes impossible to efficiently obtain a plant or a part thereof (particularly, a grape skin or a fruit), a crushed product thereof, or a cultured plant cell. From the viewpoint of avoiding adverse effects on plants or their parts (especially grape skins or fruits), their crushed products, or plant culture cells, the irradiation amount of light in the wavelength range of 300 to 400 nm is the light in the wavelength range of 275 to 295 nm. is preferably less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20% More preferably less than 15%, more preferably less than 10%, more preferably less than 5%.

Light in the wavelength range of 275 to 295 nm is applied at a photon flux density of, for example, 0.1 to 1,000 μmol/m ² /s. stilbenoids and/or metabolites of the TCA cycle in plants (especially grape skins) when less than 0.1 μmol/m ² /s, while maintaining the freshness of the plant or parts thereof (especially grape skins or fruits); Increasing amounts of polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof may not be achieved efficiently. If it exceeds 1,000 μmol/m ² /s, it may induce damage to plants or parts thereof (particularly grape skins or fruits) or plant cultured cells. Light in the wavelength range of 275 to 295 nm is preferably 1 to 1,000 μmol/m ² /s, more preferably 1 to 800 μmol/m ² /s, more preferably 2 to 800 μmol/m ² /s, more preferably 2.5 μmol/m 2 /s. 600 μmol/m ² /s, more preferably 5 to 600 μmol/m ² /s, more preferably 10 to 600 μmol/m ² /s, more preferably 20 to 600 μmol/m ² /s. be.

The light source used in the present disclosure is not particularly limited as long as it can irradiate light in the wavelength range of 275 to 295 nm. For example, commonly used ultraviolet light sources such as UV lamps can be used. Examples of UV lamps include light-emitting diodes (LED), laser diodes (LD), xenon lamps, fluorescent lamps, incandescent lamps, metal halide lamps, and high-pressure mercury lamps. In addition, as the ultraviolet light, the light extracted from sunlight through an optical filter or the like may be used.
If the light source used emits light in the wavelength range of 275 to 295 nm and light in the wavelength range of 200 to 270 nm at 20% or more of the photon flux density of light in the wavelength range of 275 to 295 nm, 275 By using a filter with a higher transmittance for light in the wavelength range of 200 to 270 nm for light in the wavelength range of 295 nm, the grape skin or fruit or crushed material thereof or plant cultured cells are irradiated 200 ~ The photon flux density of light in the wavelength range of 270 nm may be less than 20% of the photon flux density of light in the wavelength range of 275 to 295 nm, more specifically less than 15%, less than 10%, less than 5%, or It may be less than 1%.
Additionally or alternatively, the light source used emits light in the wavelength range of 300-400 nm together with light in the wavelength range of 275-295 nm at a photon flux density of 50% or more of the photon flux density of light in the wavelength range of 275-295 nm. If it emits light, the plant or its part (especially the grape skin) is used in combination with a filter whose transmittance for light in the wavelength range of 275 to 295 nm is greater than that for light in the wavelength range of 300 to 400 nm. or fruit) or its crushed product or plant cultured cells, the photon flux density of light in the wavelength range of 300 to 400 nm may be less than 50% of the photon flux density of light in the wavelength range of 275 to 295 nm, and more Specifically, it may be less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.

From the viewpoint of energy efficiency, light in the wavelength range of 275 to 295 nm is irradiated as light having a main peak wavelength within, for example, 285±7 nm, more preferably within 285±5 nm. Preferably, the second peak does not exist, or if it does exist, its intensity is 1/10 or less of that of the main peak. As used herein, the term "main peak wavelength" refers to the peak wavelength at which the intensity is maximized. For light such as LED light whose spectrum exhibits a single peak, the “peak wavelength” is synonymous with the “main peak wavelength”.
The full width at half maximum of the main peak (within a peak wavelength of 275-295 nm) is preferably 5-15 nm. The full width at half maximum of the main peak is 15 nm or less, stilbenoids and/or TCA cycle metabolites, polyamine alkaloids, 4- Stilbenoids in plants or parts thereof (particularly grape skins or fruits) or crushed products thereof or cultured plant cells while avoiding irradiation with light in a wavelength range that does not contribute to increasing the amount of aminobutyric acid, abscisic acid and/or salts thereof and/or energy efficiency in addition to being able to selectively irradiate light in a wavelength range effective for increasing the amount of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof. will also improve. Although light with a main peak full width at half maximum of less than 5 nm can also be used in the method of increasing and production of the present disclosure, it is currently preferred to use light with a main peak with a full width at half maximum of 5 nm or more from a cost-effectiveness point of view. preferable. In some preferred specific embodiments, the light irradiated to plants or parts thereof (particularly grape skins or fruits), crushed products thereof, or plant cultured cells has a peak wavelength of 285 ± 5 nm and a full width at half maximum of 5 to 15 nm. It is light with a spectrum of wavelengths.

In the present disclosure, light sources for irradiating plants or parts thereof (particularly, grape skins or fruits), crushed products thereof, or plant cultured cells include light emitting diodes (LEDs) or laser diodes (LDs) having a single peak in the emission spectrum. is particularly preferred. When LEDs or LDs are used as light sources, plants or parts thereof (especially grape skins or fruits) or crushed products thereof or stilbenoids in plant cultured cells and/or metabolites of the TCA cycle, polyamine alkaloids, 4-aminobutyric acid, abscisin Stilbenoids and/or TCA in plants or parts thereof (particularly grape skins or fruits) or crushed products thereof or plant cultured cells while avoiding irradiation with light in wavelength ranges that do not contribute to increasing the amount of acids and/or their salts. Selective irradiation of light in a wavelength range effective for increasing the amount of cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and/or salts thereof can be easily realized. Also, the use of LEDs or LDs is preferable from the viewpoint of energy efficiency and economy due to their low heat generation, low power consumption, and long life. In addition, it facilitates control/manage of irradiation dose and/or photon flux density.
LEDs capable of emitting light in the wavelength range of 275 to 295 nm may use, for example, AlGaN-based materials or InAlGaN-based materials.

The irradiation amount of light in the wavelength range of 275 to 295 nm to plants or parts thereof (especially grape skins or fruits), crushed products thereof, or plant cultured cells is controlled, for example, by turning on and off the light source (e.g., plants or Parts thereof (especially grape skins or fruits) or crushed products thereof or plant cultured cells are allowed to stand), or plants or parts thereof (especially grape skins or fruits) or crushed products thereof pass through the irradiation area. It can be set between 50,000 and 2,500,000 μmol/m ² by controlling the time required to do so (for example, when the plant or its parts (especially grape skins or fruits) or crushed products thereof are conveyed in a conveying device).

Light in the wavelength range of 275 to 295 nm may be applied to plants or parts thereof (especially grape skins or fruits), crushed products thereof, or plant cultured cells as continuous light, intermittent light, or a combination thereof. good. Light in the wavelength range of 275 to 295 nm is preferably applied as intermittent light. Intermittent light makes it possible to avoid or reduce the temperature rise of plants or parts thereof (especially grape skins or fruits) or crushed products thereof or plant cultured cells and/or light sources. A specific example of the intermittent light has a pulse width of 100 ms or less, more specifically 50 ms or less, more specifically 20 ms or less, more specifically 10 ms or less, more specifically 5 ms or less, and a duty ratio of 50 ms. %, more specifically 40% or less, more specifically 30% or less, more specifically 20% or less, more specifically 10% or less, more specifically 5% or less.

During light irradiation, the plant or parts thereof (especially grape skins or fruits) or crushed material thereof or plant culture cells may be stationary or in motion, for example rotating, vibrating or floating. may be mixed or stirred. In addition, plants or parts thereof (especially grape skins or fruits) or crushed products thereof or plant cultured cells may be irradiated with light during transportation. In a specific embodiment, the plant or part thereof (especially grape skin or fruit) or crushed product thereof is subjected to quality inspection such as appearance (e.g. size and/or presence of blemish or rot), color and/or sugar content. You may light-irradiate in between.
During light irradiation, the plant or parts thereof (especially grape skins or fruits) or crushed material thereof or plant cultured cells may be cooled at all times, periodically or as required. Cooling can be done, for example, by water jetting or immersion in water.
Irradiation may be from one direction or from two or more directions. When irradiation is performed from two directions, the two directions are preferably two opposite directions such as the vertical direction, the horizontal direction, or the front-rear direction, but are not limited thereto.
Light in the wavelength range of 275 to 295 nm does not need to irradiate the entire plant or its parts (particularly, grape skins or fruits (including peel pieces or fruit pieces)) to be irradiated, and it is irradiated with the necessary irradiation dose. Limited areas of plants or parts thereof (particularly grape skins or fruits) may be irradiated as long as possible. For example, the plant or part thereof to be irradiated (especially grape skin or fruit) may be covered with a mesh, striped mesh, or the like during irradiation.
When irradiating cultured plant cells, it is preferable to irradiate so that light in the wavelength region of 275 to 295 nm does not penetrate the medium in order to avoid absorption by the medium. More specifically, for plant cells being cultured in a solid medium, it is preferable to irradiate light with a wavelength range of 275 to 295 nm from above the medium, and for plant cells being cultured in a liquid medium, It is preferable to irradiate light with a wavelength range of 275 to 295 nm while the cells are spread on the surface of the liquid.
The plant treatment apparatus disclosed in International Publication No. WO2021/006342 can be used to irradiate the plant or its parts with light in the wavelength range of 275-295 nm.

After irradiation with light in the wavelength region of 275-295 nm, the plant or its parts (particularly grape skins or fruits), crushed products thereof, or plant cultured cells are stored in a dark place for 24 hours or more. As used herein, the term “dark place” refers to a level where the photosynthetic photon flux density does not cause photosynthesis in the cells in the pericarp, more specifically, a place where the photosynthetic photon flux density is ≦10 μmol/m ² /s. say. Dark place storage time is more specifically 36 hours or more, more specifically 48 hours or more, more specifically 50 hours or more, more specifically 60 hours or more, more specifically 72 hours or more. , more specifically 84 hours or more, more specifically 96 hours or more, more specifically 100 hours or more.

The upper limit of the dark storage time is the plant or its parts (particularly, grape skin or fruit) or its crushed product stored in the dark after light irradiation, or stilbenoids and/or TCA cycle metabolites, polyamines in plant cultured cells. The content of alkaloids, 4-aminobutyric acid, abscisic acid and/or their salts is stored in the dark under the same conditions without irradiating light in the wavelength range of 275 to 295 nm or parts thereof (especially grape skin or There is no particular limitation as long as it increases relative to the non-irradiated control, which is fruit) or its crushed product or plant cultured cells. For example, when fermenting a plant or its parts (especially grape skins or fruits) after light irradiation, or its crushed product as a raw material for fermented food (including beverages), storage in the dark is more specifically until the end of the fermentation. For example, up to 3 weeks, 2 weeks, 1 week or 5 days until pressing or juicing. Also, for example, when irradiated plants or parts thereof (especially grape skins or fruits) or crushed products thereof are used as raw materials for non-fermented foods (including beverages), storage in the dark is Pericarp or fruit) or its crushed material, more specifically, depending on the temperature, for example, 10 days (240 hours), 9 days (216 hours), 8 days (192 hours), 7 days (168 hours), 6 days (144 hours) or up to 5 days (120 hours).

The temperature during dark storage is not particularly limited, but may be, for example, 8 to 38°C, more specifically 10 to 35°C, more specifically 10 to 30°C. For example, when the plant or its parts (especially, grape skin or fruit) after light irradiation, or its crushed product is subjected to fermentation as a raw material for fermented food or beverage, dark storage should be at 20 to 35 ° C, more specifically It may be carried out at 21-33°C, more specifically 22-30°C, more specifically 23-28°C. Also, for example, when using irradiated plants or parts thereof (especially grape skins or fruits) or crushed products thereof as raw materials for non-fermented foods or beverages, store in the dark at 10 to 25 ° C, more specifically It may be carried out at 11-23°C, more specifically 12-20°C, more specifically 13-18°C.
A dark place is, for example, a storage tank containing barrels, a storage or storeroom containing refrigerators (chambers), the inside of a shipping container or freight car, an aircraft, a ship or a truck, or a light-tight packaging material such as cardboard. Or it can be inside the packaging.

As used herein, "stilbenoid" is not particularly limited as long as it can be naturally synthesized in plants or parts thereof (e.g., grapes, especially grape skins). veratrol, piceido (also called piceed), ε-viniferin, δ-viniferin, piceatannol, astringin and the like. A preferred stilbenoid herein is resveratrol, especially trans-resveratrol. In the present specification, when simply referred to as "resveratrol", both trans- and cis-forms are included.

As used herein, "increased amount of stilbenoids" or "increased stilbenoid content" refers to non-genetically modified plants or parts thereof (especially grape skins or fruits) cultivated under natural light, or crushed products thereof. , the amount of stilbenoids is increased, for example, 100% or more, more preferably 200% or more, more preferably 300% or more, more preferably 400% or more, more preferably 700% or more, more preferably 900% or more , more preferably 1400% or more, more preferably 1900% or more, more preferably 2400% or more.
Similarly, as used herein, "increased amount of resveratrol" or "increased resveratrol content" refers to non-genetically modified plants or parts thereof (especially, grape skins or fruits) grown under natural light, or Compared to the crushed product, the amount of resveratrol is increased, for example, 100% or more, more preferably 200% or more, more preferably 300% or more, more preferably 400% or more, more preferably 700 % or more, more preferably 900% or more, more preferably 1400% or more, more preferably 1900% or more, more preferably 2400% or more.

Stilbenoids (particularly resveratrol) can be measured, for example, by chromatography. Chromatography includes, but is not limited to, liquid chromatography (eg, HPLC).
Liquid chromatography for stilbenoid separation can be any liquid chromatography known in the art as long as it is a suitable method for separating stilbenoids, and the separation mode is not limited. Examples of separation modes can include reverse phase chromatography, normal phase chromatography, hydrophilic interaction chromatography (HILIC), ion exchange chromatography, size exclusion chromatography. Preferably, the liquid chromatography separation mode is reversed-phase chromatography.

Specific conditions of liquid chromatography for stilbenoid separation can be appropriately determined as appropriate for the separation of stilbenoids in the separation mode used.
Liquid chromatography for stilbenoid separation may use a gradient elution method.
When the liquid chromatography is based on the gradient elution method, water optionally containing 0.01 to 10% acid is used as eluent A, and a polar solvent optionally containing 0.01 to 10% acid is used as eluent B. can be used with a gradient between A:B = 100:0 to 0:100, for example, in 30-60 minutes. Although the flow rate is not particularly limited, it can be, for example, 0.1-3 ml/min.
Stilbenoids can be detected, for example, by measuring absorbance at 190-800 nm.

By applying the first bulking method of the present disclosure, stilbenoids (especially , resveratrol) is 3 times or more, for example 4 times or more, more preferably 5 times or more, more preferably 8 times or more, more preferably 10 times or more, more preferably 15 times or more, more preferably 20 times It is possible to obtain plants that are more than 25 times higher, and more preferably more than 25 times higher. Therefore, according to the volume-increasing method of the present disclosure, harvested plants or parts thereof (especially grape skins or fruits) can be inexpensively provided as high-value-added foods or raw materials for high-value-added foods or beverages.

In some embodiments of the first bulking method of the present disclosure, in plants or parts thereof (especially grape skins or fruits) to which the first bulking method of the present disclosure is applied, or crushed products thereof, additionally , metabolites of the TCA cycle, polyamine alkaloids, 4-aminobutyric acid and/or abscisic acid may be increased.
In addition, by applying the second method of increasing the amount of the present disclosure, compared to plants (especially non-genetically modified plants) or parts thereof cultivated under natural light, metabolites of the TCA cycle, polyamine alkaloids, 4-amino Plants enriched in butyric acid and/or abscisic acid can be obtained.
As used herein, "TCA cycle metabolites" include products and intermediates in the TCA cycle (or citric acid cycle or Krebs cycle). Metabolites of the TCA cycle are not particularly limited as long as they can be naturally produced in plants (e.g., grapes, especially their pericarp), and examples include pyruvate, acetyl-CoA, citric acid, cis-aconitic acid, D-isocitric acid, α-ketoglutaric acid (2-oxoglutaric acid), succinyl CoA, succinic acid, fumaric acid, L-malic acid, oxaloacetic acid and the like. If the TCA cycle metabolite is an acid, it may be in the free acid form or in the salt form. Typically, the TCA cycle metabolites are pyruvate, acetyl-CoA, citrate, cis-aconitate, D-isocitrate, α-ketoglutarate (2-oxoglutarate), succinyl-CoA, succinate, fumarate. , L-malic acid and oxaloacetic acid and salts thereof.

As used herein, a "polyamine alkaloid" is an organic basic compound (preferably a linear aliphatic hydrocarbon) having two or more amino groups, which is naturally occurring in plants (e.g., grapes, especially the pericarp). Examples include agmatine, putrescine, cadaverine, spermidine, N'-acetylspermidine, carboxyspermidine, norspermidine, homospermidine, aminopropyl homospermidine, spermine, N'-acetyl spermine, norspermine, thermospermine, homospermine, canavarmine, 1,3-diaminopropane and the like. Typically, polyamine alkaloids are one or more compounds selected from the group consisting of putrescine, spermidine and spermine. Polyamine alkaloids may be in the form of free acids or salts.
4-Aminobutyric acid and abscisic acid may be in the form of free acids or salts.

In the present specification, "increasing the amount of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid and/or abscisic acid" or "increasing the content of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid and/or abscisic acid" "Increase" means metabolites of the TCA cycle, polyamine alkaloids, 4-aminobutyric acid and/or abscisin compared to non-transgenic plants (particularly grape skins or fruits) or crushed products grown under natural light. The amount of acid is increased, for example 20% or more, preferably 50% or more, more preferably 100% or more, more preferably 200% or more, more preferably 300% or more, more preferably 400% or more, more Preferably, it means an increase of 500% or more.

Determination of compounds of interest such as TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid and/or abscisic acid can be performed, for example, by chromatography and/or mass spectrometry. Chromatography includes, but is not limited to, liquid chromatography (eg, HPLC) or gas chromatography.
Liquid chromatography (LC) is as described above for stilbenoid separation.
Gas chromatography (GC) can be any method known in the art suitable for separating compounds of interest. Specific conditions for gas chromatography can be appropriately determined so as to be suitable for separation of the target compound.
Mass spectrometry (MS) can use any mass spectrometry method known in the art, as long as it is an appropriate method for separating compounds of interest, and is not limited to ionization mode and mass separation mode. Examples of ionization modes may include electron ionization, electrospray ionization, chemical ionization, and the like. Examples of mass separation modes include magnetic field type, quadrupole type, and ion trap type.
Mass spectrometry may be combined with liquid chromatography or gas chromatography.

Plants or parts thereof (especially grape skins or fruits) produced by applying the first method for increasing the volume of the present disclosure or the production method of the present disclosure, or crushed products thereof, have a high content of stilbenoids (especially resveratrol) Suitable as a raw material for producing grape-derived foods or beverages containing in.
Therefore, from another aspect, the present disclosure also
Plants or parts thereof (especially grape skins or grape fruits containing at least the skins) or crushed products thereof are irradiated with light in the wavelength range of 275 to 295 nm in an amount of 50,000 to 2,500,000 μmol/m ² and with a wavelength of 200 to 270 nm. irradiating light so that the irradiation amount of light in the wavelength range is less than 20% of the irradiation amount of light in the wavelength range of 275 to 295 nm;
Storing the irradiated plants or parts thereof (especially grape skins or fruits) or crushed products thereof in the dark for one day or longer, and storing the resulting plants or parts thereof (especially grape skins or fruits) or crushed products thereof A method for producing a food or beverage (hereinafter also referred to as “method for producing a food or beverage according to the present disclosure”) is also provided, comprising processing.
The food or beverage manufacturing method of the present disclosure includes a step of applying the first bulking method of the present disclosure to a plant or a portion thereof (especially a grape skin or a grape fruit containing at least the skin) or a crushed product thereof, and It may be a method for producing a food or beverage, comprising a step of processing a plant or its parts (particularly, grape skins or fruits) or crushed products thereof.

The food or beverage may be a non-fermented food or beverage or a fermented food or beverage. The food or drink can be, for example, dried fruits (eg raisins if the plant is grapes), jams, juices, soft drinks, fruit vinegars or wines. When the plant is grapes, specific examples of fruit wines include, but are not limited to, wine and pomace brandy (eg, grappa).
Processing to produce dried fruits includes, for example, drying stilbenoid-enriched plants or parts thereof (especially grape berries) by artificial means such as sun drying or hot air.
Processing to produce jams includes, for example, boiling stilbenoid-enriched plants or parts thereof (especially grape berries) or crushed products thereof.
Processing to produce juices and soft drinks includes, for example, squeezing from stilbenoid-enriched plants or parts thereof (particularly grape berries) or crushed products thereof (preferably seed-free). , enzymatic treatment (pectinization), filtration and tartar removal.
Processing to produce fruit wines or fruit vinegars includes, for example, fermentation of stilbenoid-enriched plants or parts thereof (especially grape skins or fruits) or crushed products thereof, including pressing, aging, racking, It may further include one or more of filtering and distillation.

In some embodiments, the food or beverage manufacturing method of the present disclosure does not include the step of adding stilbenoids other than those derived from the plant or parts thereof (especially grape skins or fruits) used as raw materials.

Plants or parts thereof (especially grape skins or fruits) produced by the production method of the present disclosure contain a high content of stilbenoids (especially resveratrol). Additionally or alternatively, plants or parts thereof (particularly grape skins or fruits) produced by the production methods of the present disclosure may contain metabolites of the TCA cycle, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and their It contains a high content of one or more compounds selected from the group consisting of salts.
Therefore, from another aspect, the present disclosure provides a mature plant or part thereof (particularly, a grape skin or a grape fruit containing at least the skin) by applying the first bulking method of the present disclosure to produce Also provided are plants or parts thereof (especially grape-ripening pericarp or fruits) that are capable of

From another aspect, the present disclosure also provides a mature plant or a part thereof (particularly, a fruit containing at least a grape skin), wherein the signal intensity of resveratrol and phenylalanine in the skin by mass spectrometry The ratio (resveratrol signal intensity/phenylalanine signal intensity) is 0.5 or more, and the cells of the plant or its parts (particularly pericarp) increase the expression level of enzymes involved in the resveratrol biosynthetic pathway. Also provided are plants or parts thereof (especially grape berries or pericarp thereof) that have not been genetically engineered for the purpose (hereinafter also referred to as "plants or parts thereof of the present disclosure").
In certain embodiments, the mature plant or part thereof is a post-harvest plant or part thereof.

The plant or part thereof (particularly, grape skin or fruit) of the present disclosure has a signal intensity ratio of resveratrol and phenylalanine in the skin by mass spectrometry of 0.50 or more, more preferably 0.51 or more, and more preferably is 0.52 or more, more preferably 0.53 or more, and more preferably 0.54 or more.

Mass spectrometry (MS) can be any mass spectrometry method known in the art suitable for the analysis of resveratrol and phenylalanine, and is not limited in ionization mode and mass separation mode. Examples of ionization modes may include electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization. Examples of mass separation modes include electric field focusing, quadrupole, time-of-flight, ion trap, orbitrap, ion cyclotron resonance, and tandem and hybrid modes thereof. In some embodiments, mass spectrometry is by MS/MS where the ionization mode is electrospray ionization and the mass separation mode is a hybrid quadrupole-orbitrap.
The analysis conditions for mass spectrometry can be appropriately determined so as to be suitable for the analysis of resveratrol and phenylalanine in the ionization mode and mass separation mode used.

Preferably, prior to mass spectrometry, a fraction containing resveratrol and/or phenylalanine is separated from a test sample, such as a pericarp extract, by a known separation method (e.g., liquid chromatography (LC)). To separate.
The liquid chromatography can be any liquid chromatography known in the art that is suitable for separating fractions containing resveratrol and/or phenylalanine, and is not limited in terms of separation mode. Examples of separation modes can include reverse phase chromatography, normal phase chromatography, hydrophilic interaction chromatography (HILIC), ion exchange chromatography, size exclusion chromatography. In some embodiments, liquid chromatography is reversed-phase chromatography.

Liquid chromatography conditions can be determined as appropriate to separate fractions containing resveratrol and/or phenylalanine in the separation mode used.
The column used for liquid chromatography can be appropriately selected from known columns according to the separation mode. For reversed-phase chromatography, octadecylated silica gel (ODS) columns (also called C18 columns) can generally be used, but are not limited thereto.
As a mobile phase (eluent), water, a polar organic solvent, and/or a mixed solvent thereof (a mixed solvent of water and one or more polar organic solvents, a mixed solvent of two or more polar organic solvents) can be used. . Polar organic solvents include, for example, methanol, ethanol, n- or iso-propanol, acetonitrile, acetone, hexanedioxane, ethyl acetate, dimethylsulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran. Acids such as formic acid, acetic acid, trifluoroacetic acid, phosphoric acid and trichloroacetic acid or bases such as ammonia, ammonium formate, ammonium acetate and ammonium bicarbonate may be added to the eluent, for example at 0.01 to 10M.
Although the flow rate of the mobile phase is not particularly limited, it can be, for example, 0.1 to 2 ml/min.

A pericarp extract can be subjected to liquid chromatography. Extraction can be performed by any known method, for example by solvent extraction.
The solvent used for solvent extraction can be appropriately selected from known solvents. Solvents include, for example, water (normal temperature to boiling water), water-miscible organic solvents, or mixed solvents thereof (mixed solvents of water and one or more water-miscible organic solvents, two or more water It is possible to use a mixed solvent of an organic solvent miscible with Water may be used as hot or boiling water. The water-miscible organic solvent can be a polar organic solvent such as methanol, ethanol, n- or iso-propanol, acetonitrile, acetone, dioxane, ethyl acetate, dimethylsulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran. be done. An acid may be added to the polar organic solvent. Acids can be, for example, hydrochloric acid, sulfuric acid, formic acid, acetic acid, phosphoric acid, trichloroacetic acid, trifluoroacetic acid or perchloric acid. The acid may be included in the organic solvent in a weight ratio of eg 0.05-5%, preferably 0.1-3%.

Extraction may be performed at elevated temperature (eg, 80-90° C.) and/or under pressure. The extraction may be reflux extraction. The extraction time is not particularly limited and can be determined appropriately from the viewpoint of extraction efficiency.
The pericarp may be used for extraction as it is, or may be used after pulverization. The plant may be dried and/or frozen prior to extraction or grinding. Drying may be performed by any method, such as hot air drying, normal temperature drying, reduced pressure drying, or freeze drying.
The extract may be filtered through an appropriate filter or subjected to centrifugation, for example, to remove contaminants.

In some embodiments, the abundance ratio of resveratrol and phenylalanine in plants or parts thereof (especially grape skins) or plant cultured cells is determined by an LC-MS/MS method. In a specific embodiment, LC is reverse phase chromatography and MS/MS is a quadrupole-orbitrap hybrid. In a more specific embodiment, LC is reverse phase chromatography using a C18 column and MS/MS is quadrupole-orbitrap MS/MS where the ionization mode is electrospray ionization.

The plant or part thereof (particularly, the grape skin or fruit) of the present disclosure is such that the cells of the plant or part thereof (when the plant part is the grape skin or fruit, at least the grape skin cells) are in the resveratrol biosynthetic pathway. It has not been genetically engineered to increase expression of the enzymes involved. Enzymes involved in the resveratrol biosynthetic pathway are typically phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) and stilbene synthase (STS). is.
Plants or parts thereof (particularly grape skins or fruits) of the present disclosure are preferably non-genetically engineered.

Grape skins or fruits of the present disclosure, for example, wherein the skins contain resveratrol at least 1500 μg/g dry weight, more specifically at least 1550 μg/g dry weight, more specifically at least 1600 μg/g dry weight, more specifically at least 1600 μg/g dry weight, more specifically Typically included in an amount of at least 1650 μg/g dry weight.
The grape skins or fruits of the present disclosure may be the skins or fruits of grapes belonging to any species (eg, the above species) or hybrids (eg, hybrids between the above species). The grape skins or fruits of the present disclosure can also be the skins or fruits of any grape variety, more specifically the grape skins or fruits of the above varieties. The grape skin or fruit of the present disclosure preferably belongs to Vitis vinifera species, or is selected from Vitis vinifera species and Vitis labrusca species, Vitis amurensis species, Vitis mustangensis species, Vitis riparia species and Vitis rotundifolia species. A hybrid between two or more species. In some embodiments, the grape skin or fruit of the present disclosure is Muscat Bailey A fruit or skin.

Plants or parts thereof (especially grape skins or fruits) of the present disclosure can be produced, for example, in mature plants or parts thereof (particularly grape skins or grape fruits comprising at least the skins) by the production method of the present disclosure. It is possible, but not limited to those produced by the method.

Since the plant or part thereof (especially grape skin or fruit) of the present disclosure contains a high content of stilbenoids (especially resveratrol), it can be used as a raw material for foods or beverages (e.g., fruit wine) containing a high content of stilbenoids. preferred.
Therefore, from another point of view, the present disclosure also provides that the signal intensity ratio between resveratrol and phenylalanine by mass spectrometry (resveratrol signal intensity/phenylalanine signal intensity) is 0.5 or more, and genes as raw materials A fruit wine (hereinafter also referred to as “fruit wine of the present disclosure”) that does not use recombinant plants or parts thereof (especially grapes) is also provided.

When the plant is grape, the fruit wine (ie, grape wine) of the present disclosure can be, but is not limited to, wine (preferably red wine) or pomace brandy (eg, grappa).
The fruit wine (especially, grape wine) of the present disclosure has a signal intensity ratio of resveratrol and phenylalanine by mass spectrometry of 0.50 or more, more preferably 0.51 or more, more preferably 0.52 or more, It is more preferably 0.53 or more, more preferably 0.54 or more.
The mass spectrometric method for measuring the signal intensity ratio between resveratrol and phenylalanine in the fruit wines of the present disclosure is as described above for the plants or parts thereof of the present disclosure.

The fruit wines (especially grape wines) of the present disclosure contain, for example, resveratrol of at least 15 μg/mL, more specifically at least 15.5 μg/mL, more specifically at least 16 μg/mL, more specifically in an amount of at least 16.5 μg/mL.
The grape wine of the present disclosure may be brewed using grapes belonging to any species (e.g., the above species) or hybrids (e.g., hybrids between the above species), but is preferably belongs to the species Vitis vinifera or is a hybrid between Vitis vinifera species and one or more species selected from Vitis labrusca species, Vitis amurensis species, Vitis mustangensis species, Vitis riparia species and Vitis rotundifolia species It is brewed using grapes from In some embodiments, the grape wine of the present disclosure is a wine brewed using Muscat Bailey A.

Plants or parts thereof (especially grape skins or fruits) produced by the production methods of the present disclosure may additionally or alternatively maintain freshness and/or maintain anti-mold properties for long periods of time after harvest. can be enhanced.
Therefore, from another aspect, the present disclosure provides a method for maintaining freshness and/or maintaining or enhancing antifungal properties of a post-harvest plant, comprising: Or, the crushed material has an irradiation amount of light in the wavelength range of 275 to 295 nm of 50,000 to 2,500,000 μmol/m ² and an irradiation amount of light in the wavelength range of 200 to 270 nm is equal to the light in the wavelength range of 275 to 295 nm. (hereinafter also referred to as the "post-harvest plant treatment method of the present disclosure").
In some embodiments, the method of treating a post-harvest plant of the present disclosure further comprises storing the irradiated plant or part thereof (especially pericarp or fruit) or crushed material thereof in the dark for one day or more. .
The plant treatment apparatus disclosed in International Publication No. WO2021/006342 can be used in the post-harvest plant treatment method of the present disclosure.

"plant", "part of plant", "debris", "light in the wavelength range of 275-295 nm" (including its light source and irradiation mode), "light in the wavelength range of 200-270 nm", "irradiance" and "dark storage" are as described above for the method of increasing the volume of the present invention and/or the production method of the present invention.
Evaluation of freshness can be performed by any method known in the art, for example, based on one or more of visual observation, component measurement and physical measurement. Appearance observations can be made, for example, on items such as weight loss rate, shrinkage, spoilage, color (including color of fruit and/or pericarp). Component measurements may include, for example, measurements of sugar content, acidity, vitamin C concentration and/or respiration rate. Physical measurements may include, for example, hardness, color and/or texture measurements.
Antifungal evaluation can be performed by any method known in the art. For example, antifungal properties can be evaluated by adhering fungi (for example, spores) to the epidermis of a target plant and then determining whether or not the fungi grow.

In the examples below,
Wavelength measurement was performed by measuring the emission spectrum using a multichannel detector (model: PMA-11 C7473; Hamamatsu Photonics).
The illuminance [W/m ² ] was measured using a photodiode sensor (model: PD300-UV; Ophir) whose sensitivity was calibrated at the peak wavelength of the LED used.
The photon flux density was obtained from the measured illuminance by the following conversion formula.
Photon flux density [mol/m ² /s] = Illuminance [W/m ² ] / (Avogadro's number [mol-1] x Planck's constant [J s] x speed of light [m/s] ÷ wavelength [m])
The irradiation dose [mol/m ² ] was calculated as the product of photon flux density [mol/m ² /s] and irradiation time [sec].

(Example 1)
^{2.5, 25 or 250 μmol/m 2 /} s illuminance (photon flux density ) was irradiated with LED light (peak wavelength: 290 nm), and then the fruit was stored in a dark place at 15° C. for 1 day (24 hours). The process from light irradiation to moving to a dark place was carried out on the day of harvest.
After storage for 1 day, only the pericarp was freeze-dried, crushed, and subjected to solvent extraction using 80% methanol. The resulting extract was subjected to high performance liquid chromatography (HPLC: Prominence, Shimadzu Corporation) under the following analysis conditions to quantify resveratrol. As a control, the same analysis was performed on fruits (fruits) stored for one day in a dark place at 15°C without irradiation with light. Analysis was performed on 3 samples, with 3 berries as 1 sample, for each experimental group.

<HPLC conditions>
・Column: ODS column (Triart C18 (150×4.6mm, S-5μm), YMC)
・Column temperature: 40℃
・Flow rate: 1 ml/min ・Injection volume: 10 μl
・Mobile phase: Eluent A 0.1% formic acid aqueous solution
Eluent B 0.1% formic acid acetonitrile solution
Linear gradient: B: 1% to 100% over 30 minutes
・Detection: 190 to 800 nm

result:
FIG. 1 shows the amount of resveratrol (μg) contained in 1 g (dry weight) of the peel obtained by HPLC analysis. In FIG. 1, the vertical axis indicates the resveratrol content in the pericarp, the upper portion of the horizontal axis indicates the illuminance (photon flux density), and the lower portion indicates the irradiation dose.
The amount of resveratrol in the grape skin increased depending on the amount of irradiation of light with a wavelength peak around 290 nm to the grape fruit from which the skin was derived.
The amount of resveratrol in grapes irradiated with light having a wavelength of about 290 nm at an irradiation dose of 225,000 μmol / m ² is about 4.5 compared to the fruit not irradiated with the light ("Cont" in the figure). While it increased to 10-fold, it hardly increased in the grape berries irradiated with the light at the irradiation dose of 2,250 μmol/m ² .

These results indicated that light with a peak wavelength of around 290 nm increased the amount of resveratrol in the grape skin when the irradiation dose was 22,500 μmol/m ² or higher.
Without being bound by theory, considering that light around 280 nm can promote the shikimate pathway via activation (monomerization) of the photoreceptor UVR8 (WO2018/199307), It is presumed that the activation of the photoreceptor UVR8 is involved in the increase in the amount of stilbenoids in grape skins by irradiation with light in the wavelength region of 275-295 nm.
When the irradiation dose of light in the wavelength range of 275 to 295 nm is too low, the activation of the photoreceptor UVR8 is not sufficiently induced due to the shading effect of phenolic compounds, which are plant pigments, and the biosynthesis of stilbenoids in the peel is activated. (See the data for the dose of 2,250 μmol/m ² in Figure 1). On the other hand, if the illuminance is too high, the synthesized phenolic compounds (including stilbenoids) are thought to be decomposed (the irradiation dose at the illuminance of 250 μmol/m ² /s in Fig. 1 is 225,000 μmol/m ² ). ” data).
Therefore, it is considered that the amount of stilbenoids in the irradiated grape skin can be increased efficiently by irradiating light with a wavelength range of 275 to 295 nm for a long period of time at an illuminance at which the amount of stilbenoid photolysis does not exceed the amount of biosynthesis.

(Example 2)
Harvested mature grapes (cultivar: Kyoho) fruits (berries) were irradiated with LED light (peak wavelength: 290 nm) with an illuminance of 250 μmol/m ² /s so that the irradiation amount was 225,000 μmol/m ² . The fruit was then stored in the dark at 15°C for 1 day (24 hours) or 2 days (48 hours). The process from light irradiation to moving to a dark place was carried out on the day of harvest.
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1. Analysis was performed on 3 samples, with 3 berries as 1 sample, for each experimental group.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 2, the vertical axis indicates the resveratrol content in the pericarp, the upper portion of the horizontal axis indicates the number of days stored in a dark place, and the lower portion indicates the irradiation dose.
In the pericarp of grapes stored in the dark for 48 hours after light irradiation, the amount of resveratrol increased about 31-fold compared to the fruit not irradiated with light (“Cont” in the figure).
From this result, it can be understood that the amount of resveratrol in the grape skin is further increased by storing in a dark place for a longer period of time after irradiation with light in the wavelength range of 275 to 295 nm.
Without being bound by theory, long-term storage in the dark induced upregulation of genes of enzymes involved in the biosynthetic pathway of stilbenoids (particularly resveratrol) induced by irradiation with light in the wavelength range of 275-295 nm. was continued for a long period of time, and as a result, it is presumed that the synthesis amount of stilbenoids (especially resveratrol) increased.

(Example 3)
The fruits (bundles) of mature grapes (cultivar: Muscat Bailey ^A ) harvested for winemaking ^were irradiated with LED light (peak wavelength: 290 nm) in one direction for 7.5 minutes and in the opposite direction for an additional 7.5 minutes, then the fruit is placed in a dark place at 15 ° C for 1 day (24 hours), 2 days (48 hours) or Stored for 5 days (120 hours). The process from light irradiation to moving to a dark place was carried out on the day of harvest.
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1. Analysis was performed on 3 samples, with 3 berries as 1 sample, for each experimental group.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 3, the vertical axis indicates the resveratrol content in the pericarp, the upper horizontal axis indicates the number of days stored in a dark place, and the lower horizontal axis indicates the irradiation dose.
In Muscat Bailey A, similarly to Kyoho, the amount of resveratrol in the pericarp increased by irradiation with light in the wavelength range of 275 to 295 nm. The amount of resveratrol increased to about 2.7 times that of the non-irradiated control after 1 day, about 12.57 times after 2 days, and 29.1 times after 5 days after light irradiation. In other words, with light irradiation, the amount of resveratrol in grape skins continued to increase during at least 5 days of dark storage.
From these results, irradiation with light in the wavelength range of 275 to 295 nm can increase the amount of stilbenoids (especially resveratrol) in the grape skin, regardless of the variety, and that the stilbenoid biosynthetic pathway by light irradiation can be increased. It can be seen that activation continues for at least 5 days with dark storage.

(Example 4)
LED light (peak wavelength: 290 nm) at an illuminance of 250 μmol/m ² /s to the fruits (bundles) of harvested mature grapes (variety: Muscat Bailey A) so that the irradiation amount is 225,000 μmol/m ² was irradiated for 7.5 minutes in one direction and for another 7.5 minutes in the opposite direction, and then the fruits were stored in bunches in the dark at 15°C for 2 days (48 hours). The process from light irradiation to moving to a dark place was carried out on the day of harvest.
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1. As a control, the fruit stored in the dark for 2 days without exposure to light was also analyzed in the same manner. Analysis was performed on one sample (3 berries) for each experimental group.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 4, the vertical axis indicates the resveratrol content in the pericarp, and the horizontal axis indicates the irradiation dose.
The amount of resveratrol in the pericarp after being exposed to light in the bunch and stored in the dark for 2 days was about 6.5 times that of the pericarp after being stored in the dark for 2 days without being exposed to light. Increased.
In addition, the amount of resveratrol in the pericarp after dark storage in the bunch was equivalent to the amount in the pericarp after dark storage in the fruit.

(Example 5)
An LED light with an illuminance of ^{250 μmol/m 2 /} s (peak Wavelength: 290 nm) in one direction for 7.5 minutes and in the opposite direction for another 7.5 minutes, then the fruits were stored in the dark at 15° C. for 2 days (48 hours). The process from light irradiation to moving to a dark place was carried out on the day of harvest.
After storage for 2 days, only the pericarp was freeze-dried, crushed, and subjected to solvent extraction using 80% methanol. The resulting extract was subjected to LC-MS (LC: Ultimate™ 3000, Thermo Fisher Scientific; MS: Q Exactive™, Thermo Fisher Scientific) under the following analysis conditions to measure the amount of phenolic compounds including stilbenoids. As a control, the same analysis was performed on the fruits that had been stored in the dark for 2 days after harvesting without being exposed to light. Analysis was performed on one sample (3 berries) for each experimental group.

<LC conditions>
・ Column: ODS column (InertSustain (registered trademark) AQ-C18 (2.1 × 150 mm, 3 μm-particle), GL Sciences)
・Column temperature: 40℃
・Flow rate: 0.2ml/min ・Injection volume: 2μl
・Mobile phase: Mobile phase A 0.1% formic acid aqueous solution
Mobile phase B Acetonitrile solution, LC gradient program:
Time (minutes) 0.0 3.0 30.0 35.0 35.1 40.0
Mobile phase A (%) 98 98 2 2 98 98
Mobile phase B (%) 2 2 98 98 2 2

<MS conditions>
・Measurement time: 3 to 30 minutes ・Ionization method: Electrospray ionization (ESI)
・Measurement mass range: m/z: 80 to 1,200
・Full scan resolution: 70,000
・MS/MS scan resolution: 17,500
・MS/MS precursor selection method: Data Dependent Scan (Top 10)
・Dynamic Exclusion: 20 seconds

result:
The analysis results are shown in Table 1 below and FIG. In FIG. 5, the vertical axis indicates the detection amount ratio of each phenolic compound, and the horizontal axis indicates the phenolic compound.

The abundance ratio of resveratrol and phenylalanine in grape skins irradiated with light having a peak wavelength of around 290 nm (ratio of intensity of resveratrol/intensity of phenylalanine) was 0.54, compared with the abundance ratio of 0.04 in the non-irradiated control. increased significantly.
In addition to resveratrol, cis-piceid and ε-viniferin (both types of stilbenoids), cyanidic acid glucoside (a type of anthocyanin) and epicatechin gallate (pro A kind of anthocyanidin) was confirmed to increase.

From these results, irradiation with light with a peak wavelength of around 290 nm not only activates the biosynthetic pathway of phenolic compounds overall, but particularly significantly activates the biosynthetic pathway of stilbenoids (especially resveratrol) in grape skins. It is understandable to be activated.

(Example 6)
LED light with an illuminance of ^{250 μmol/m 2 /} s (peak wavelength: 290 nm), the fruit was stored in the dark at 15° C. for 2 days (48 hours).
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1. As a control, the fruit stored in the dark for 2 days without exposure to light was also analyzed in the same manner. Analysis was performed on 3 samples, with 3 berries as 1 sample, for each experimental group.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 6, the vertical axis indicates the resveratrol content in the pericarp, and the horizontal axis indicates the irradiation dose.
In the case of white grapes, as in the case of red grapes, the amount of resveratrol in the pericarp increased by about 3 times compared to the control by irradiation with light with a wavelength range of 275 to 295 nm (irradiation amount: 225,000 μmol/m ² ). .
From this result, it can be understood that the irradiation of light with a wavelength range of 275 to 295 nm according to the present disclosure can increase the amount of stilbenoids (especially resveratrol) in the grape skin regardless of the color of the skin.

(Example 7)
The fruits (fruits) of purchased mature grapes (cultivar: Shine Muscat) were irradiated with LED light (peak wavelength: 290 nm) with an illuminance of 250 μmol/m ² /s so that the irradiation amount was 225,000 μmol/m ² . After that, the fruit was stored in a dark place at 15°C for 5 days (120 hours).
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 7, the vertical axis indicates the resveratrol content in the pericarp, the upper portion of the horizontal axis indicates the number of days stored in a dark place, and the lower portion indicates the irradiation dose. In the figure, the data for the control (Cont) and the storage period of 2 days (48 hours) after light irradiation are the data of Example 6.
In white grapes, as with red grapes, the amount of resveratrol after 5 days of dark storage after irradiation with light in the wavelength range of 275 to 295 nm increased more than 2 days (compared to the control). approximately 12.9 times).
From this result, it can be understood that the amount of stilbenoids (especially resveratrol) in the grape skin increases as the dark storage period after irradiation with light in the wavelength range of 275 to 295 nm increases, regardless of the color of the skin.

(Example 8)
The fruits (berries) of purchased mature grapes (variety: Delaware) were irradiated with LED light (peak wavelength: 280 nm) with an illuminance of 600 μmol/m ² /s so that the irradiation amount was 216,000 μmol/m ² . The fruits were then stored in the dark at 5, 15, 25 or 40°C for 1 day (24 hours) or 5 days (120 hours).
After dark storage, HPLC analysis of resveratrol content in pericarp was performed as in Example 1. As a control, fruits that were not exposed to light and stored in the dark were analyzed in the same manner. Analysis was performed on one sample (3 berries) for each experimental group.

result:
The HPLC analysis results obtained are shown in FIG. In FIG. 8, the vertical axis indicates the resveratrol content in the pericarp, the upper horizontal axis indicates the temperature of dark storage, the middle indicates the irradiation dose, and the lower indicates the storage days.
Dark storage at 15 and 25°C for 5 days after light irradiation increased the amount of resveratrol in the pericarp more than 60-fold.
On the other hand, storage in the dark at 5°C for 1 day did not increase the amount of resveratrol in the pericarp, and storage in the dark for 5 days increased it to approximately double. Storage in the dark at 40°C for 1 day and 5 days increased the amount of resveratrol in the pericarp more than 2-fold and about 3-fold or more, respectively.
From this result, even if the temperature during storage in the dark is too low or too high, the rate of increase in the amount of resveratrol can be suppressed, and it is preferable to store in the dark at a temperature of 15 ° C. to 25 ° C. is understood.

(Example 9)
Gene expression analysis in grapes Harvested mature grapes ⁽ cultivar: Muscat Bailey ^A ) were irradiated with LED light ( peak wavelength: 290 nm) was irradiated from one direction for 7.5 minutes and from the opposite direction for another 7.5 minutes.
Immediately after the second irradiation, only the pericarp was frozen in liquid nitrogen. Total RNA from pericarp was then prepared using the Maxwell® simply RNA plant kit (Promega) according to the manufacturer's instructions.
RNA-seq analysis was performed on the prepared total RNA (Macrogen Japan Co., Ltd.). The NovaSeq6000 system (Illumina) was used for sequence analysis. Analysis was performed on one sample (3 berries) for each experimental group.

Table 2 shows genes whose expression levels were 1.2 times or more.

By irradiating a plant with light in the wavelength region of 275 to 295 nm at a predetermined dose, genes of enzymes involved in the TCA cycle, polyamine alkaloid synthesis and metabolic pathways, and abscisic acid synthesis pathway are upregulated in the plant. It was confirmed that
From these results, it is speculated that the TCA cycle, the polyamine alkaloid synthesis and metabolic pathways, and the abscisic acid synthesis pathway are activated in the plant, thus increasing the amount of products in these pathways.

In addition to the genes listed in Table 2, genes associated with hypersensitivity reactions (RIN4, RPM1, RPS2, EDS1, PAR1, HSP90) and transcription factors associated with the induction of defense-related genes (WRKY22, PIT6) It was upregulated more than 1.2 times.
From this result, in plants irradiated with light in the wavelength range of 275 to 295 nm at a predetermined irradiation amount, phytoalexin and infection-specific protein increased, and therefore the plant was a pathogen (or pathogen) (for example, filamentous It is presumed that the resistance to fungi, bacteria and viruses is increasing.

(Example 10)
Gene expression analysis in Arabidopsis thaliana Arabidopsis thaliana was irradiated with LED light (peak wavelength: 280 nm; full width at half maximum: 10 nm; Deep UV-LED manufactured by Nichia Corporation) at an illuminance of 2.5 μmol/m ² /s for 45 minutes (irradiance : 6,750 μmol/m ² ).
Shoots of some Arabidopsis thaliana were frozen in liquid nitrogen immediately after irradiation, and shoots of some other Arabidopsis thaliana were stored in the dark for 2 days (48 hours) after irradiation. After that, using NucleoSpin (registered trademark) RNA plant (Takara Bio Inc.) according to the instruction manual, total RNA was prepared.
RNA-seq analysis was performed on the prepared total RNA (Takara Bio Inc.). The NovaSeq6000 system (Illumina) was used for sequence analysis. Analysis was performed on 3 samples for each experimental group.

Table 3 shows genes whose expression levels were 1.2 times or more.

Even if the species is different, the genes of the enzyme group involved in the TCA cycle, polyamine alkaloid synthesis and metabolic pathways, and abscisic acid synthesis pathway are upregulated in plants irradiated with light in the wavelength range of 275 to 295 nm at a predetermined dose. It was confirmed that
In addition, this upregulation was observed immediately after irradiation (day 0 of storage) and after storage in the dark for at least two days.
The results show that the light irradiation disclosed herein activates the TCA cycle, the polyamine alkaloid synthesis and metabolism pathways, and the abscisic acid synthesis pathway in plants, regardless of species, and thus also increases the amount of products in these pathways. I can understand that.

(Example 11)
Arabidopsis thaliana was irradiated with LED light (peak wavelength: 280 nm; full width at half maximum: 10 nm; Deep UV-LED manufactured by Nichia Corporation) with an illuminance of 2.5 μmol/m ² /s for 45 minutes (irradiance: 6,750 μmol/m ² ).
After 2 days of dark storage, the shoots were frozen in liquid nitrogen and ground, followed by solvent extraction.
The resulting extract is subjected to LC-MS analysis (LC: UltimateTM3000 RSLC, Thermo Fisher Scientific; MS: Q ExactiveTM, Thermo Fisher Scientific) under condition 1 or 2 below, or GC-MS under condition 3 below It was subjected to analysis (QP2010 Ultra and AOC-5000 plus, Shimadzu). Alternatively, to the obtained extract, methoxyamine/pyridine is added (methoximation), followed by the addition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (trimethylsilylation), and the resulting trimethylsilylated form was subjected to GC-MS analysis (QP2010 Ultra and AOC-5000 plus, Shimadzu) under condition 4 below. Analysis was performed on 3 samples per experimental group.

Solvent extraction was performed as follows. For LC-MS analysis under analytical condition 1, 75% methanol was used. 10 mixed solvent was added in an amount of 6.5 times and homogenized, followed by adding ultrapure water in an amount of 1.25 times the amount of the sample and collecting the methyl tert-butyl ether fraction. For GC-LC analysis under analytical condition 3, add 13 volumes of a 3:10 mixed solvent of methanol:methyl tert-butyl ether to the sample, homogenize, and then add 2.5 volumes of ultrapure water to the sample. was added to collect the methyl tert-butyl ether fraction, then 10% boron trifluoride methanol was added (methyl esterification), and then ultrapure water and hexane were added to collect the hexane fraction. rice field. Furthermore, 75-80% methanol was used for GC-LC analysis under analysis condition 4.
As a control, Arabidopsis thaliana stored in the dark for 2 days without light irradiation was also analyzed in the same manner.

<LC-MS analysis conditions 1>
<LC conditions>
・ Column: ODS column (InertSustain (registered trademark) AQ-C18 (2.1 × 150 mm, 3 μm-particle), GL Sciences)
・Column temperature: 40℃
・Flow rate: 0.2ml/min ・Injection volume: 2μl
・Mobile phase: Mobile phase A 0.1% formic acid aqueous solution
Mobile phase B Acetonitrile solution, LC gradient program:
Time (minutes) 0.0 3.0 30.0 35.0 35.1 40.0
Mobile phase A (%) 98 98 2 2 98 98
Mobile phase B (%) 2 2 98 98 2 2

<MS conditions>
・Measurement time: 3 to 30 minutes ・Ionization method: Electrospray ionization (ESI)
・Measurement mass range: m/z: 80 to 1,200
・Full scan resolution: 70,000
・MS/MS scan resolution: 17,500
・MS/MS precursor selection method: Data Dependent Scan (Top 10)
・Dynamic Exclusion: 20 seconds

<LC-MS analysis conditions 2>
<LC conditions>
・Column: ODS column (SunShell C18 (2.1×150mm, 2.6μm-particle), ChromNik Technologies)
・Column temperature: 40℃
・Flow rate: 0.2ml/min ・Injection volume: 2μl
・Mobile phase: Mobile phase A with 0.1% formic acid and 10 mM ammonium formate, acetonitrile/water (60:40)
Mobile phase B 2-propanol/acetonitrile (90:10) with 0.1% formic acid and 10 mM ammonium formate
・LC gradient program:
Time (min) 0.0 10.0 20.0 35.0 45.0 55.0 55.1 60.0
Mobile phase A (%) 70 65 45 35 0 0 70 70
Mobile phase B (%) 30 35 55 65 100 100 30 30

<MS conditions>
・Measurement time: 3 to 30 minutes ・Ionization method: Electrospray ionization (ESI)
・Measurement mass range: m/z: 80 to 1,200
・Full scan resolution: 70,000
・MS/MS scan resolution: 17,500
・MS/MS precursor selection method: Data Dependent Scan (Top 10)
・Dynamic Exclusion: 20 seconds

<GC-MS Analysis Condition 3>
・Column: DB-5ms (Agilent Technologies)
・Vaporization chamber temperature: 280℃
・Oven temperature: 40°C (2 minutes) to 320°C (5 minutes), heating rate 6°C/minute ・Connection part temperature: 280°C
・Ion source temperature: 280℃
・Ionization method: electron ionization (EI)
・Flow rate: 39cm/min (1.1mL/min)
・Scanning speed: 2,500u/sec ・Measuring mass range: m/z: 45 to 500
・Injection volume: 0.5 μl

<GC-MS Analysis Condition 4>
・Column: DB-5 (30m×0.250mm×1.00μm; Agilent Technologies)
・Vaporization chamber temperature: 280℃
・Oven temperature: 100°C (4 minutes) to 320°C (8 minutes), heating rate 4°C/minute ・Connection temperature: 280°C
・Ion source temperature: 280℃
・Ionization method: electron ionization (EI)
・Flow rate: 39cm/min (1.1mL/min)
・Scanning speed: 2,000u/sec ・Measured mass range: m/z: 45 to 600
・Injection volume: 0.5 μl

The analysis results are shown in FIG. 9 and Table 4.
Table 4 shows compounds with a detection amount ratio (irradiated/unirradiated) of 1.2 or higher.

In Arabidopsis thaliana irradiated with light in the wavelength region of 275 to 295 nm at a predetermined dose, an increase in the amount of products of the TCA cycle, polyamine alkaloid synthesis and metabolic pathways, and abscisic acid synthesis pathway was confirmed, corresponding to the upregulation of genes. rice field.
Considering this result and the result of Example 9 together, even in grapes (for example, pericarp) irradiated with a predetermined irradiation amount of light in the wavelength range of 275 to 295 nm, the TCA cycle, corresponding to the upregulation of genes, It can be inferred that the products of the polyamine alkaloid synthesis and metabolism pathway and the abscisic acid synthesis pathway are increased.

To summarize the above results, by irradiating light in the wavelength range of 275 to 295 nm with a predetermined irradiation amount and then storing it in a dark place for at least one day, regardless of the species, in the plant or its parts or crushed material stilbenoids (especially resveratrol), additionally or alternatively selected from the group consisting of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and salts thereof can be increased in amount of one or more compounds.

(Example 12)
Induction of Callus Leaves of 'Bailey Alicante A' were immersed in 70% ethanol and then immersed in a sterile aqueous solution (0.5% sodium hypochlorite, 0.01% Tween 20) for 20 minutes. Thereafter, the leaves were washed three times with sterilized water, cut into 1 cm squares, and cultured in the following medium at 24°C in the dark to induce callus. While continuing the culture at 24° C. in the dark, the cells were subcultured to a fresh medium every 14 days, and white callus and red callus were separated at that time.
Medium composition 1 bag of mixed salt for Murashige and Skoog medium (4.6g)/L
2% sucrose
0.01% myo-inositol 1×10 ^-4 % thiamine-HCl,
6 × 10 ^-5 % kinetin 5 × 10 ^-6 % 2,4-dichlorophenoxyacetic acid
1.0% agar, pH 5.8

UV irradiation and dark storage The plant cultured cells ⁽ callus) obtained as described above ^were irradiated with LED light ( Peak wavelength: 280 nm; LED: NCSU334B, manufactured by Nichia Corporation), and immediately stored in a dark place at 23°C for 2 days. As a control, non-irradiated plant cultures were stored in the dark at 23°C for 2 days.

Analysis of resveratrol content For the analysis, 1 white callus and 1 red callus of about 1 cm (length) x 1 cm (width) x 0.5 cm (height) (volume: about 0.5 cm ³ ) were used (Fig. 10). .
After storage in the dark, it was lyophilized, pulverized and extracted with 80% methanol.
The obtained extract was subjected to HPLC analysis under the following conditions (Prominence, manufactured by Shimadzu Corporation).
<HPLC conditions>
・Column: ODS column (Triart C18 (150×4.6mm, S-5μm), YMC)
・Column temperature: 40℃
・Flow rate: 1 ml/min ・Injection volume: 10 μl
・Mobile phase: Eluent A 0.1% formic acid aqueous solution
Eluent B 0.1% formic acid acetonitrile solution
Linear gradient detection: 190 - 800 nm

<Results>
The results of resveratrol content analysis are shown in FIG.
Irradiation of 50,000 μmol/m ² light with a wavelength of 280 nm increased the resveratrol content of white callus by 3.8 times.
Similar irradiation increased resveratrol content in reddish callus more than 49.9-fold.
From this result, light irradiation and dark storage according to the present disclosure can increase the stilbenoid content in cultured plant cells as well as in plant bodies.

Examples of the use of the present disclosure include store lighting, showcase lighting, food storage lighting, and downlights. When used in a store, after the business is closed, the vegetables, fruits, fresh flowers, etc. displayed in the store and/or stored in the warehouse are irradiated with UV under the conditions of this disclosure, and after UV irradiation, stored in a dark place until the start of business. By doing so, it is possible to increase the amount of useful components in the plant during business hours. When used at home, by irradiating vegetables and fruits stored in a food storage cabinet, fresh flowers arranged in a vase, etc. with UV light under the conditions of the present disclosure at bedtime, useful in plants when waking up Ingredients can be increased.
A distribution container may be equipped with a device for UV irradiation under the conditions of the present disclosure. By irradiating the plants with UV light and storing them in a dark place during transportation, it is possible to increase the amount of useful components in the plants when they are transported.

Claims (14)

Plants or their parts, crushed products thereof, or cultured plant cells are irradiated with light in the wavelength range of 275 to 295 nm in an amount of 50,000 to 2,500,000 μmol/m ² and in a wavelength range of 200 to 270 nm. Irradiate with light so that the irradiation amount of light in the wavelength range of 275 to 295 nm is less than 20%, and store the plant or its parts, crushed material thereof, or cultured plant cells after irradiation in a dark place for 1 day or more. matter,
A method for increasing the amount of stilbenoids in a plant or its crushed product or plant cultured cells, comprising: Grape skins or grape fruits containing at least the skins or crushed products thereof, the irradiation amount of light in the wavelength range of 275 to 295 nm is 50,000 to 2,500,000 μmol / m ² , and the irradiation amount of light in the wavelength range of 200 to 270 nm is Irradiating with light so that the irradiation amount of the light in the wavelength range of 275 to 295 nm is less than 20%, and storing the pericarp or fruit after the irradiation or the crushed product thereof in a dark place for 1 day or more;
A method for increasing the amount of stilbenoids in grape skins or grape fruits or crushed products thereof, comprising: Increasing amounts of stilbenoids and one or more compounds selected from the group consisting of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and salts thereof in the grape skins or grape berries or crushed products thereof 3. The method of claim 2. 4. The light irradiation according to any one of claims 1 to 3, wherein the irradiation amount of light in the wavelength range of 300 to 400 nm is less than 50% of the irradiation amount of light in the wavelength range of 275 to 295 nm. the method of. A method according to any one of claims 2 to 4, wherein the pericarp is that of a mature grape fruit. The method according to any one of claims 1 to 5, wherein the light in the wavelength range of 275-295 nm is irradiated with a photon flux density of 0.1-1,000 µmol/m ² /s. The method according to any one of claims 1 to 6, wherein the light irradiation has a wavelength spectrum with a peak wavelength of 285 ± 5 nm and a full width at half maximum of 5 to 15 nm. 8. The method according to any one of claims 1 to 7, wherein the light irradiation is performed using an LED as a light source. The method of any one of claims 1-8, wherein the stilbenoid is resveratrol. A method according to any one of claims 1 to 9, wherein said dark storage is carried out at a temperature between 15°C and 25°C. A stilbenoid-enriched grape skin or fruit or crushed product thereof, characterized in that the method according to any one of claims 2 to 10 is applied to the grape skin or grape fruit containing at least the skin or crushed product thereof. method of producing things. A step of applying the method according to any one of claims 2 to 10 to grape skins or grape fruits containing at least the skins or crushed products thereof, and processing the obtained grape skins or fruits or crushed products thereof. A method for producing a food or beverage, comprising: 13. The method of claim 12, wherein said food or beverage is raisins, jams, juices, soft drinks, fruit vinegars or fruit wines. Plants or their parts, crushed products thereof, or cultured plant cells are irradiated with light in the wavelength range of 275 to 295 nm in an amount of 50,000 to 2,500,000 μmol/m ² and in a wavelength range of 200 to 270 nm. Irradiate with light so that the irradiation amount of light in the wavelength range of 275 to 295 nm is less than 20%, and store the plant or its parts, crushed material thereof, or cultured plant cells after irradiation in a dark place for 1 day or more. One or more selected from the group consisting of TCA cycle metabolites, polyamine alkaloids, 4-aminobutyric acid, abscisic acid and salts thereof in plants or their crushed products or plant cultured cells, characterized by comprising A method for increasing the amount of a compound.

Images