JP7576815B2 - Food and drink composition for green juice - Google Patents

Description

The present invention relates to an edible composition for green juice that uses barley stems and/or leaves.

Barley is said to be native to Central Asia and is an annual or biennial herb belonging to the family Poaceae. Barley stems and/or leaves (hereinafter referred to as "stems and leaves") are known as ingredients of health foods rich in vitamins, minerals, dietary fiber, amino acids, chlorophyll, SOD enzymes, etc., and are used in edible and drinkable compositions such as green juice, jelly, cookies, ginger drinks, yogurt, and supplements. Green juice is a product that contains green leaves of plants and is processed into various products such as dried powder and juice, and is used as a health food that allows vegetable ingredients to be easily ingested.
It is known that barley stems and leaves have a lactic acid bacteria proliferation effect (Patent Document 1). This lactic acid bacteria proliferation effect is thought to lead to an intestinal regulation effect when barley stems and leaves are consumed.

JP 2013-031426 A

There is an increasing demand for health foods to have greater functionality, and barley stems and leaves and edible compositions for green juice that use them are no exception. However, in the past, edible compositions for green juice that use barley stems and leaves have been made multifunctional by adding other ingredients besides the barley stems and leaves, and the composition of barley required to obtain other functions while maintaining its intestinal regulating effect has not been fully examined.

The inventors therefore investigated various methods for obtaining barley for use in edible compositions for green juice that provides new effects in addition to its intestinal regulating effect. In particular, they focused on the relationship between the determination using genetic markers, a simple method for identifying barley, and the properties of barley as an edible composition for green juice, and carried out research. As a result, they discovered that barley that showed specific results in determination using specific genetic markers surprisingly provided excellent intestinal regulating effects as well as high cosmetic effects when its stems and leaves were used, leading to the completion of the present invention.

The present invention is based on the above findings, and provides a composition for consumption or drinking for green juice using barley stems and/or leaves, comprising:
The present invention provides an edible composition for green juice, wherein the barley satisfies two or three of the following conditions (A) to (D):
(A) The strain has a low expression of the gene for the serine protease Cxp1 as determined by the genetic marker Te945.
(B) The strain contains the yellow mosaic disease resistance gene rym5 as determined by the genetic marker E31/M41.
(C) The strain contains the powdery mildew resistance gene ml-o as determined by the genetic marker om2.
(D) The strain possesses the yellow mosaic disease resistance gene rym3 as determined by the genetic marker k09554-AvaI.

The present invention provides a composition that has cosmetic effects in addition to intestinal regulation.

FIG. 1 shows the nucleotide sequence of the low-expression Cxp1 gene in barley. FIG. 2 shows the base sequences of primers for determining the type (low expression type or high expression type) of the Cxp1 gene. FIG. 3 shows the nucleotide sequence of the eIF4E gene at the rym5 locus in barley. FIG. 4 shows the base sequence of a primer for determining the rym5 gene. Figure 5 shows the base sequence of the region including the om2 marker in the upstream region of the ml-o gene in barley. FIG. 6 shows the base sequences of primers for determining the ml-o gene. FIG. 7 shows the nucleotide sequence of a region containing k09554-AvaI, a CAPS marker for determining the rym3 gene in barley. FIG. 8 shows the base sequence of a primer for determining the rym3 gene. FIG. 9 shows the evaluation results regarding the proliferation and survival of bifidobacteria for the Examples and Comparative Examples. FIG. 10 shows the evaluation results of collagenase inhibition rates for the Examples and Comparative Examples.

The following describes the composition for consumption of green juice of the present invention based on a preferred embodiment.

One of the characteristics of the edible composition for green juice of this embodiment is the genetic makeup of the barley used.

Specifically, the barley used in this embodiment is characterized in that it meets two or three of the following conditions (A) to (D). Hereinafter, such barley is also referred to simply as "specific barley."
(A) The strain has a low expression of the gene for the serine protease Cxp1 as determined by the genetic marker Te945.
(B) The strain contains the yellow mosaic disease resistance gene rym5 as determined by the genetic marker E31/M41.
(C) The strain contains the powdery mildew resistance gene ml-o as determined by the genetic marker om2.
(D) The strain contains the yellow mosaic disease resistance gene rym3 as determined by the genetic marker k09554-AvaI.

Serine protease Cxp1 is said to be involved in the breakdown of malt proteins. Serine protease Cxp1 is also called serine carboxypeptidase Cxp1. In the present invention, it is simply called Cxp1. It is known that high expression of Cxp1 leads to excessive breakdown of malt proteins, which has a negative effect on growth.

rym5 and rym3 are said to be involved in barley yellow mosaic disease, which causes yellow-green, elongated, smudge-like spots and brown necrotic spots. rym5 is said to have resistance to barley yellow mosaic virus types I, II, IV, and V, which cause barley yellow mosaic disease, while rym3 is said to have resistance to barley yellow mosaic virus types I to III. Barley yellow mosaic disease is known to cause serious damage to barley quality and yield, such as reduced tillers, shorter plant height, and death in plants with severe symptoms.

ml-o is a powdery mildew resistance gene at the Mlo locus. Powdery mildew is caused by a type of mold called powdery mildew fungus. When infected, white powdery spots appear, and as the symptoms progress, they spread over the entire leaf, making the plant worthless as an ingredient for green juice. There are several known powdery mildew resistance loci, including the Mla locus, Mlo locus, MJg locus, and MJk locus.

As described above, the barley genes described in conditions (A) to (D) are not known to have any relationship to the intestinal regulating and beauty effects of the edible composition for green juice.

In this embodiment, the case where the number of applicable conditions among the following conditions (A) to (D) is two includes the following cases.
(i) The above conditions (A) and (B) are satisfied, but conditions (C) and (D) are not satisfied.
(ii) The above conditions (A) and (C) are satisfied, but conditions (B) and (D) are not satisfied.
(iii) The above conditions (A) and (D) are satisfied, but conditions (B) and (C) are not satisfied.
(iv) The above conditions (B) and (C) are satisfied, but conditions (A) and (D) are not satisfied.
(v) The above conditions (B) and (D) are satisfied, but conditions (A) and (C) are not satisfied.
(vi) The above conditions (C) and (D) are satisfied, but conditions (A) and (B) are not satisfied.

In this embodiment, the case where the number of applicable conditions among the following conditions (A) to (D) is three includes the following cases.
(vii) The above conditions (A), (B), and (C) are satisfied, but condition (D) is not satisfied.
(viii) The above conditions (B), (C), and (D) are satisfied, but condition (A) is not satisfied.
(ix) The above conditions (A), (C), and (D) are met, but condition (B) is not met.
(x) The above conditions (A), (B), and (D) are met, but condition (C) is not met.

The specific barley used in this embodiment may be any of the above (i) to (x), or may be a combination of barley that satisfies multiple different conditions (i) to (x).

Among the above, it is preferable that the specific barley used in this embodiment satisfies condition (A) in order to more effectively achieve both the intestinal regulation effect and the beauty effect.

The method for confirming the presence or absence of each gene will be described in detail below.
<Low expression gene of serine protease Cxp1>
In this embodiment, whether or not barley satisfies condition (A) is determined based on a CAPS (Cleaved Amplified Polymorphic Sequences) marker called Te945.
Te945 is a genetic marker that utilizes a single nucleotide polymorphism (SNP) in the serine protease Cxp1 gene, and has been reported as an eQTL marker related to the expression level of serine protease Cxp1 mRNA (Funct. Integr. Genomics, (2006) 6: pp. 25-35). The position of this single nucleotide polymorphism (SNP) is shown by double underline in the serine protease Cxp1 gene sequence shown in FIG. 1. The sequence in FIG. 1 is a part of the sequence disclosed in the DNA DataBank of Japan (DDBJ) under Accession No. CAJV010037927. The above-mentioned document (Funct. Integr. Genomics, (2006) 6: p25-35) shows that the barley gene is divided into type II, in which the double underlined portion and its surrounding sequence in FIG. 1 are "atgacat t cttg" as shown in FIG. 1, and type I, in which the sequence is "atgacat c cttg", and that the expression level of CxpI is type I>type II. In this embodiment, the primers shown in FIG. 2 (F primer: SEQ ID NO: 2, R primer: SEQ ID NO: 3) are used as primers that sandwich this SNP position, and the region sandwiched by these primers is subjected to polymerase chain reaction.
The DNA fragments are amplified by PCR and treated with BseGI, a restriction enzyme that specifically cleaves the "catcc" sequence, and the DNA fragments after the enzyme treatment are subjected to electrophoresis to confirm the base length of the DNA fragments to confirm whether or not they have a serine protease Cxp1 low expression type gene (type II). In the sequence of FIG. 1, the positions corresponding to the primers of FIG. 2 are single underlined. In the sequence shown in FIG. 1, the fragment amplified by PCR using the primers of FIG. 2 is 382 bp, and in the case of a low expression type, a fragment of this base length is observed even after the above restriction enzyme treatment. On the other hand, in the case of a high expression type (catcc) sequence, a band with a smaller base length (178 bp) is detected by cleavage with the above restriction enzyme. For a more clear determination, when the above-mentioned band with a smaller base length is detected, it is preferable that another DNA fragment (band) is observed at the position of 202 bp.

As described above, in this embodiment, in order to determine whether or not the condition (A) of having a low-expression Cxp1 gene is met, it is not necessary to analyze the entire base sequence of the Cxp1 gene by DNA sequence analysis. However, when such an analysis is performed, it is preferable that the barley has, as the base sequence of the Cxp1 gene, the sequence from base 2176 to base 2187 in the gene sequence disclosed in DDBJ under Accession No. CAJV010037927.

<Rym5 gene, a resistance gene to yellow mosaic disease>
In this embodiment, whether or not barley satisfies condition (B) is determined based on E31/M41, an STS marker derived from an AFLP polymorphism that has been reported to be linked to rym5 at 0.034 cM.
A part of the base sequence of the eIF4E gene at the rym5 locus is shown in FIG. 3. The sequence shown in FIG. 3 is described in DDBJ under Accession No. AY661558. In this embodiment, PCR is performed using the primers shown in FIG. 4 (reported in Theor. Appl. Genet. (2005) 110: p283-293) derived from E31/M41, and the PCR product is subjected to electrophoresis to confirm the presence or absence of a band that is a DNA fragment and the base length of the band, thereby confirming whether or not the gene rym5 is present. In FIG. 3, the part corresponding to the primers shown in FIG. 4 is underlined. In the sequence shown in FIG. 3, the fragment amplified by PCR using the primers in FIG. 4 is 436 bp. On the other hand, barley that does not have the gene rym5 does not usually produce an amplified product by PCR. When the amplified product obtained by PCR amplification using the primers shown in FIG. 4 using barley DNA as a template is subjected to electrophoresis, if a fragment of a length corresponding to 436 bp is detected, it is considered to have the yellow mosaic disease resistance gene rym5.

As described above, in this embodiment, the determination of the gene rym5 does not require the analysis of the entire base sequence of the gene rym5 by DNA sequence analysis. However, when DNA sequence analysis is performed on the barley used in this embodiment, it is preferable that the base sequence of the eIF4E gene at the rym5 locus is the sequence from base 44169 to base 44604 in the gene sequence disclosed in DDBJ under Accession No. AY661558.

<Powdery mildew resistance gene ml-o>
In this embodiment, whether or not the condition (C) is satisfied is determined using om2, which has been reported as a genetic marker for detecting an insertion/deletion mutation (In/del mutation) in the 5' upstream region (-2.3 kb) of the ml-o gene. In this embodiment, having the ml-o gene means having an insertion mutation detected by om2. An example of a barley gene sequence in which this insertion mutation has been made is shown in FIG. 5. The gene sequence shown in FIG. 5 is a part of the gene with Accession No. Y14573.1 in DDBJ. In FIG. 5, the double underlined part is the inserted sequence. Furthermore, a primer proposed in Genome.Vol.49,2006,p864-872 as a primer used to determine an insertion/deletion mutation by the genetic marker om2 is shown in FIG. 6, and further, the sequence corresponding to this primer is shown in FIG. 5 with a single underline. In the example shown in FIG. 5, the base length of the DNA fragment when a gene having an insertion mutation is amplified with the primer in FIG. 6 is about 1000 bp, and when no insertion mutation is present, it is about 750 bp. The presence or absence of ml-o can be determined based on the difference in base length of this DNA fragment. Specifically, the base length of the amplified product can be confirmed by subjecting the amplified product to electrophoresis, which is amplified by PCR using barley DNA as a template and the primers shown in Figure 6. If a fragment of about 1000 bp is detected when the amplified product obtained by PCR amplification using the primers is subjected to electrophoresis, it is deemed to have the ml-o gene.

As described above, in this embodiment, it is not necessary to analyze the entire base sequence of gene ml-o by DNA sequence analysis to determine whether gene ml-o is present. However, when such an analysis is performed, it is preferable that barley has the base sequence of gene ml-o from base 8106 to base 9198 in the gene sequence disclosed in DDBJ under Accession No. Y14573.1.

<rym3 gene for resistance to yellow mosaic disease>
In this embodiment, whether barley satisfies the condition (D) is determined based on k09554-AvaI, which is a CAPS marker linked to rym3. Specifically, when the amplification product amplified by the primers shown in FIG. 8 is treated with the restriction enzyme AvaI, the DNA is not cut by this enzyme treatment, and it is determined that the barley has the yellow mosaic disease resistance gene rym3. FIG. 7 shows the gene sequence amplified by this primer. In the sequence of FIG. 7, the band length amplified using the primers of FIG. 8 is 365 bp. In FIG. 7, the part corresponding to the primers shown in FIG. 8 is underlined. More specifically, when PCR is performed using the primers shown in FIG. 8 with barley DNA as a template, the obtained amplification product is treated with the restriction enzyme AvaI, and the treated product is subjected to electrophoresis, if a fragment corresponding to a length of 205 bp and a fragment corresponding to a length of 156 bp are confirmed, it is determined that the barley does not have the gene rym3.

As described above, in this embodiment, the presence of the rym3 gene does not require the entire rym3 gene base sequence to be analyzed by DNA sequence analysis. However, when such an analysis is performed, it is preferable that the barley has the rym3 gene base sequence from base 148 to base 512 in the gene sequence disclosed in DDBJ under Accession No. AV929051.1.

When determining the presence or absence of conditions (A) to (D) using the above genetic markers, the parts of barley from which the barley DNA to be determined is extracted may include flowers, seeds, fruits, leaves, stems, and roots, and it is preferable to use barley seeds.

Specific test methods include, for example, the following methods.
<Gene presence/absence determination test>

(1) Extract DNA from barley. This extraction can be carried out using a commercially available kit, etc. The DNA solution obtained is preferably adjusted to a DNA concentration of 1 ng/μL to 20 ng/μL before being subjected to PCR.
(2) PCR is carried out using the DNA solution obtained in (1) as a template. PCR is preferably carried out under the conditions shown in Table 1 below. Primers that can be used are those shown in Figures 2, 4, 6 and 8 above.

(3) When the PCR product obtained in (2) is to be examined for the presence or absence of the low-expression gene of Cxp1 and rym3, the PCR product is subjected to enzyme treatment with the restriction enzymes listed in Table 1. The restriction enzyme treatment is preferably performed at 30° C. to 40° C. for 30 minutes to 24 hours. As the restriction enzymes, FastDigest BseGI (Thermo Fisher) was used for BseGI, and AvaI 2,000 units (New England Biolabs) was used for AvaI.
The resulting treated product is used as a sample for electrophoresis. Electrophoresis can be carried out by any known method.

The particular barley may be botanically classified as six-row barley, two-row barley, or naked barley, and may be a wild species, a hybrid, or the like.

In addition, in this embodiment, whether or not conditions (A) to (D) are satisfied may deviate from the actual traits of barley. For example, barley that satisfies condition (A) may not necessarily have the trait of low expression of serine protease Cxp1, barley that satisfies condition (B) or (D) may not necessarily have the trait of yellow mosaic disease resistance, and barley that satisfies condition (C) may not necessarily have the trait of powdery mildew resistance.

The stalks and leaves of the specific barley mentioned above are preferably harvested before maturity, that is, from the start of tillering to the start of heading. Specifically, although this differs depending on the variety, it is generally preferable to harvest the stalks and leaves from barley that is 10 cm or more tall, particularly about 10 to 90 cm, and especially about 30 to 60 cm tall, but this is not limited thereto. It is preferable to process the barley stalks and leaves immediately after harvesting. If time is required before processing, they are stored by a storage method commonly used by those skilled in the art, such as low-temperature storage, to prevent deterioration of the barley stalks and leaves.

In the edible composition for green juice of this embodiment, various processed products obtained from the specific barley leaves can be used as the barley leaves. Examples of such processed products include crushed barley leaves and dried powders thereof (crushed powder), shredded barley leaves and dried powders thereof (shredded powder), squeezed juice of barley leaves and dried powders thereof (squeezed juice powder), and extract of barley leaves and dried powders thereof (extract powder).

For example, a conventionally known method can be used to pulverize barley stalks and leaves into powder. As such a method, a method in which a drying process and a pulverization process are combined for barley stalks and leaves can be used. Either the drying process or the pulverization process may be performed first, but it is preferable to perform the drying process first. For pulverization into powder, this method may be further combined with one or more processes selected from a blanching process, a sterilization process, etc., as necessary. In addition, the number of times the pulverization process is performed may be one or a combination of two or more processes, but it is preferable to combine a coarse pulverization process with a fine pulverization process for more fine pulverization.

The blanching treatment is a treatment for keeping the green color of the stems and leaves vivid, and examples of the blanching treatment include hot water treatment and steam treatment. The blanching treatment is preferably performed by treating the stems and leaves in hot water or steam at 80 to 100°C, preferably 90 to 100°C, for 60 to 180 seconds, preferably 90 to 120 seconds. In addition, when performing hot water treatment as the blanching treatment, it is preferable to dissolve a carbonate such as magnesium carbonate or a bicarbonate such as sodium bicarbonate in the hot water, since this can make the green color of the stems and leaves more vivid. In addition, as the steaming treatment, an intermittent steaming treatment in which the process of steaming the stems and leaves with steam and the process of cooling are repeated under normal pressure or pressure is preferable. In the intermittent steaming treatment, the steaming treatment with steam is preferably performed for 20 to 40 seconds, more preferably for 30 seconds. Cooling treatment after the steaming treatment is preferably carried out immediately, and the method is not particularly limited, but includes immersion in cold water, refrigeration, cooling with cold air, evaporative cooling with hot air, and evaporative cooling combining hot air and cold air. Of these, evaporative cooling combining hot air and cold air is preferred. Such cooling treatment is carried out so that the product temperature of the stems and leaves is preferably 60° C. or less, more preferably 50° C. or less, and most preferably 40° C. or less. In order to produce a powder of stems and leaves rich in nutritional components such as vitamins, minerals, and chlorophyll, it is preferable to repeat the intermittent steaming treatment 2 to 5 times.

Sterilization is a process that usually uses temperature, pressure, electromagnetic waves, chemicals, etc. to physically and chemically kill microbial cells. When blanching is performed in addition to drying and crushing, it is preferable that the blanching is performed before the drying. When sterilization is performed in addition to drying and crushing, it is preferable that the sterilization is performed after the drying, or before or after the crushing.

The drying process is preferably a process in which the stems and leaves are dried so that their moisture content is 10% by mass or less, and particularly 5% by mass or less. This drying process can be carried out by any method known to those skilled in the art, such as hot air drying, high-pressure steam drying, electromagnetic wave drying, or freeze drying. Drying by heating can be carried out at a temperature and for a time that does not cause the stems and leaves to discolor due to heating, preferably at 40°C to 140°C, more preferably 80°C to 130°C.

The crushing process may be performed by any method commonly used by those skilled in the art, using a crusher, mill, blender, stone mill, etc. The crushed stems and leaves are sieved as necessary, and it is preferable to use those that pass through a 30 to 250 mesh sieve as the stem and leaf powder. By using particles with a particle size of 250 mesh or less, the stem and leaf powder is easy to handle during further processing, and by using particles with a particle size of 30 mesh or more, the stem and leaf powder can be easily mixed uniformly with other materials.

Specific examples of methods for pulverization include cutting barley stalks and leaves, blanching them, drying them so that the moisture content is 10% by mass or less, preferably 5% by mass or less, and then pulverizing them (see JP 2004-000210 A). Other examples include cutting barley stalks and leaves, blanching them, rolling them, drying them, and pulverizing them (see JP 2002-065204 A and JP 3428956 A). Other examples include drying barley stalks and leaves, coarsely pulverizing them, heating them at 110°C or higher, and then finely pulverizing them (see JP 2003-033151 A and JP 3277181 A).

As a method for shredding barley stalks and leaves, methods that are commonly used by those skilled in the art when shredding plants, such as slicing, crushing, and chopping, can be used. As an example of shredding, it may be slurried. Slurrying is performed by putting barley stalks and leaves into a mixer, juicer, blender, mass colloider, etc., and turning the barley stalks and leaves into a mushy gruel-like substance (a suspension of liquid and solids). By slurried in this way, the stalks and leaves are shredded so that 80% or more by mass of the shredded pieces preferably have an average diameter of 1 mm or less, more preferably 0.5 mm or less, even more preferably 0.1 mm or less, and most preferably 0.05 mm, and the shredded pieces become fluid. The shredded material obtained can be freeze-dried, hot air dried, or other processing to produce a dry powder (shredded powder).

Examples of the method for squeezing barley stems and leaves include squeezing barley stems and leaves or shredded barley stems and leaves, or centrifuging or filtering shredded barley stems and leaves. A representative example is a method in which the juice is squeezed by mechanical crushing means such as a mixer or juicer, and if necessary, the crude solid content is removed by means of sieving, filtration, or the like to obtain a squeezed juice. Specifically, the methods described in JP-A-08-245408, JP-A-09-047252, JP-A-5-7471, JP-A-4-341153, and the like can be mentioned, and those skilled in the art can appropriately select and carry out these known methods. The obtained squeezed juice may be concentrated as necessary, or may be subjected to a process such as freeze-drying, hot air drying, or spray drying to obtain a dried powder (squeezed juice powder). When obtaining a squeezed juice powder, the squeezed juice alone may be dried, or it may be dried together with an excipient as necessary.

One method for obtaining an extract of barley stems and leaves is to add an extraction solvent normally used by those skilled in the art, such as ethanol, water, or aqueous ethanol, to barley stems and leaves or shredded barley stems and leaves, and then extract by stirring or heating as necessary. The extract obtained may be concentrated as necessary, or may be freeze-dried, hot air dried, spray-dried, or otherwise processed to produce a dried powder (extract powder). When producing an extract powder, the extract alone may be dried, or it may be dried together with an excipient as necessary.

In the present invention, it is preferable to use dried powder (pulverized powder, shredded powder, squeezed powder, extract powder) from the processed barley stems and leaves, and crushed powder or squeezed powder is more preferable. In particular, it is preferable to use crushed powder obtained by drying and crushing the stems and leaves, because it can make the edible composition for green juice have a brighter color and a better flavor, can make it rich in dietary fiber, and has a high bifidobacteria proliferation effect and cosmetic effect. Crushed powder is produced without going through a juice squeezing process.

The edible composition for green juice of the present invention may contain only barley stems and leaves, or may contain other ingredients as described below. The content of barley stems and leaves in the solid content of the edible composition for green juice of the present invention is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, even more preferably 1.0% by mass or more, particularly preferably 5.0% by mass or more, and most preferably 10.0% by mass or more, by dry mass. The upper limit is preferably 99.9% by mass or less, more preferably 90.0% by mass or less, and particularly preferably 80.0% by mass or less.

Furthermore, the proportion of the specific barley stems and leaves among the barley stems and leaves contained in the edible composition for green juice is preferably 10.0% by mass or more, more preferably 30.0% by mass or more, particularly preferably 50.0% by mass or more, and most preferably 70.0% by mass or more.

There is no particular limit to the amount of intake of the dietary composition of the present invention, but from the viewpoint of more significantly exerting the effects of the present invention, it is preferable for an adult to ingest 10 mg/day or more of the stems and/or leaves of the barley of the present invention, more preferably 50 mg/day or more, and even more preferably 100 mg/day or more.

The composition for consumption for green juice may contain other ingredients in addition to the barley stems and leaves. Examples of the other ingredients include vitamins, proteins, oligosaccharides, minerals, polysaccharides, dairy products, processed plant products, microorganisms such as lactic acid bacteria, sugars, sweeteners, citric acid, acidulants, colorants, thickeners, glossing agents, as well as manufacturing agents such as talc, silicon dioxide, cellulose, and calcium stearate. Other ingredients include various excipients, binders, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, colorants, flavorings, food additives, and seasonings. The content of the other ingredients can be appropriately selected depending on the form of the food or beverage. In this embodiment, it is particularly preferable that the composition for consumption for green juice contains oligosaccharides, water-soluble dietary fiber, and lactic acid bacteria as ingredients other than the processed barley stems and leaves.

The composition for eating and drinking for green juice of this embodiment can be in any form. Examples of the form of the composition for eating and drinking for green juice include forms suitable for oral intake such as eating and drinking, specifically, powder, granules, tablets, rods, plates, blocks, solids, rounds, liquids, candies, pastes, creams, capsules such as hard capsules and soft capsules, caplets, tablets, gels, jellies, gummies, wafers, biscuits, cookies, cakes, chewables, syrups, sticks, and the like.

The composition for consumption or drinking for green juice of this embodiment can include green juice, juices, shakes, smoothies made by adding fruit juice, vegetables, dairy products, etc. to green juice, or soft drinks, carbonated drinks, or the bases for these drinks.

The edible composition for green juice is preferably in a powder form (powder, granules, etc.) and in a form in which the mixture mixed with water is taken orally, since this prevents spoilage and is suitable for long-term storage, and the color of this edible composition is bright when mixed with water. If the edible composition for green juice is in a solid form, as described above, it can be mixed with water to make a liquid and taken orally by drinking the liquid, but it may also be taken orally in solid form depending on the preferences of the person taking it. It may also be added to not only water, but also milk, soy milk, fruit juice drinks, whey drinks, soft drinks, yogurt, pancake mix, etc. for use. It goes without saying that it may also be used as a food with nutritional function claims, a food for specified health uses, or a food with functional claims.

As described above, the composition for eating and drinking for green juice of this embodiment uses barley leaves that satisfy the above conditions in the judgment by the above gene markers, and therefore has improved intestinal regulating effects such as the proliferation of bifidobacteria, and also has improved cosmetic effects such as collagenase inhibition. Collagenase is an enzyme that breaks down collagen. Collagen, like hyaluronic acid and elastin, is contained in the connective tissue of mammals and is a component that contributes to the water retention, lubrication, and flexibility of the skin. Collagen is enhanced in collagen due to inflammation and aging, and the amount of collagen decomposed increases. As a result, the balance between collagen synthesis and decomposition is lost, and problems such as enhanced inflammatory reactions, skin wrinkles due to aging, and joint pain occur. Therefore, it is expected that a component that inhibits the activity of the collagen enzyme can prevent and prevent inflammation, wrinkles, joint pain, etc. associated with a decrease in collagen. In addition, the intestinal regulating effect in the present invention includes the effect of improving bowel movements.

Therefore, the composition for consumption or drinking of green juice of this embodiment can be used for one or more applications selected from the following: proliferation of bifidobacteria, improvement of intestinal flora (intestinal environment), intestinal regulation, inhibition of collagen decomposition enzymes, suppression of collagen reduction, collagen retention, reduction of joint pain, inflammation, etc., prevention of skin wrinkles, improvement of lubrication, flexibility or water retention of skin, and cosmetic applications.

Specifically, the use of the composition for eating or drinking of the present invention is as medicines (including quasi-drugs) and so-called health foods. Examples of health foods that promote intestinal regulation include those labeled with claims such as "For those concerned about stomach health," "For those concerned about bowel movements," "Regulates stomach condition," "Protects stomach health," "Improves intestinal environment," "Maintains good bowel movements," "Improves bowel movements," and "Rebalances intestinal bacteria." Examples of health foods that promote beauty include those labeled with claims such as "Maintains skin moisture," "Protects skin," "Protects skin," "For those concerned about blemishes," "For those concerned about wrinkles," "For skin that can withstand stress," "Good for beauty," and "Good for skin."

When the composition for eating and drinking for green juice is used for the purpose of growing bifidobacteria, it may be used for either the growth of bifidobacteria in the living body (e.g., in the intestine) or the growth of bifidobacteria outside the living body. The bifidobacteria are not particularly limited as long as they are bacteria that produce a large amount of lactic acid from sugars, and can be appropriately selected according to the purpose, and examples thereof include Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium adolesentis, and Bifidobacterium mongoliense. These may be used alone or in combination of two or more.

The present invention will be explained in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

<Examples 1 to 4, Comparative Examples 1 and 2>
The above-mentioned "Gene presence/absence determination test" was carried out on the seeds of multiple barley varieties. That is, DNA extraction, PCR, restriction enzyme treatment as necessary, and electrophoresis were carried out by the above-mentioned method using the gene markers shown in Table 1. The specific conditions and procedures were as follows.
DNA extraction: Several seeds were collected from several barley plants of different varieties. DNA was extracted from some of the seeds derived from one plant. The Dneasy Plant Mini Kit (QIAGEN) was used for DNA extraction. The extraction method was according to the protocol attached to the kit.
PCR: The DNA obtained above was diluted with nuclease-free water to a DNA concentration of 7.5 ng/μL and used in PCR. PCR was performed using the DNA solution after the above concentration adjustment as a template and the following reagents in the amounts shown in Table 2 below, and the resulting solution was subjected to PCR under the conditions shown in Table 1 above. In Table 2, Ex Taq and its buffer are products of Takara Bio Inc.

- Restriction enzyme treatment: 20 μl of Nuclease Free Water was added to the obtained PCR product and diluted 2-fold. When determining the presence or absence of (A) the low expression type gene of Cxp1 and (D) the yellow mosaic disease resistance gene rym3, the restriction enzymes in Table 1 above were used, and each solution in Table 3 was mixed in a tube in the ratio shown in Table 3 below, and the sealed tube was submerged in a 37°C water bath and incubated for 1 hour, and then cooled on ice. In Table 3 below, the buffer attached to the enzyme was used as the restriction enzyme buffer. The obtained treated product was used as the sample for electrophoresis. When determining other genes, the PCR product was diluted 2-fold with Nuclease Free Water as above without restriction enzyme treatment and was used as the sample for electrophoresis as it was.

Electrophoresis: 0.5xTBE (Tris Borate EDTA) Buffer and agarose were added to the flask so that the agarose concentration was 1% or 2% by mass, and dispersed. The flask was autoclaved to dissolve the agarose, and then gently stirred. After the mixture had cooled, a small amount of GelGreen (BIOTIUM) was added and stirred again. Agarose gel (hereinafter referred to as AGE) was formed using a dedicated mold. 0.5xTBE Buffer and AGE were placed in an electrophoresis tank (Mupidα mini gel electrophoresis tank; Advance). 0.5xTBE Buffer was added until the gel was immersed. 1μL of 6x Loading Buffer (Takara Bio) and 5μL of sample were mixed, and then applied to each well of the AGE. Electrophoresis was performed at 100V at appropriate times.

Seeds obtained from the same plant as the seeds that gave the following results using the above method were used as barley for the examples and comparative examples according to the correspondence in Table 4 below. In Table 4 below, ○ indicates that the result satisfied the conditions, and × indicates that the result did not satisfy the conditions.

The seeds of the barley from each of the examples and comparative examples for which the above judgments were obtained were sown and cultivated by known methods, and the stems and leaves were harvested before maturity, i.e., from the time when tillers start to the time before ear emergence starts, and a ground powder was obtained by the method described below, which was used as a powdered composition for consumption and drinking for green juice.

<Method of producing dry powder>
The harvested barley stalks and leaves were washed with water to remove adhering mud, and then pre-treated by cutting them into pieces of about 5 cm in size. The pre-treated stalks and leaves were blanched once with hot water at 90-95°C for about 100 seconds, and then cooled with cold water. The obtained stalks and leaves were then dried in a dryer with hot air at 70-80°C until the moisture content was 5% by mass or less. The dried stalks and leaves were coarsely crushed to a size of about 1 mm using a mixer, and then finely crushed using a jet mill grinder to obtain crushed powder of barley stalks and leaves.

The obtained edible compositions for green juice of Examples 1 to 4 and Comparative Example 1 were subjected to the following Evaluation 1, and the edible compositions for green juice of Examples 1 to 4 and Comparative Examples 1 and 2 were subjected to the following Evaluation 2.
<Evaluation 1: Intestinal regulation effect>
The following test was carried out as a model experiment for the proliferation of bifidobacteria in the body.
Bifidobacterium (scientific name: Bifidobacterium bifidum) was inoculated into a medium (385 medium) with catalog number 385 provided by the National Institute of Technology and Evaluation in a 96-well plate, and cultured at 37° C. in an anaerobic atmosphere for 24 hours.
The resulting culture solution was diluted with 385 medium and the turbidity was adjusted to 0.5 McFarland.
The powdered composition for consumption was autoclaved, and then adjusted to 10 mg/mL with phosphate buffered saline (PBS) to prepare a sample solution with a concentration 10 times the final concentration.
20 μL of sample solution was added to a 96-well plate, and 180 μL of bacterial solution with turbidity adjusted to 0.5 McFarland was added to each well. For background correction, 180 μL of medium without bacteria was added to the wells containing the sample solution (control). After that, the wells were cultured in an anaerobic atmosphere at 37°C for 24 hours. 7 μL of stock solution of Cell counting kit-8 (Dojindo Chemical Industries) was added to the cultured solution.
After adding 1 L/well and allowing to stand until color developed, the absorbance at 450 nm, which is the absorption maximum of reduced WST-8, was measured. The ratio (%) of absorbance to the control was calculated from the measured values and the results are shown in Figure 9. Note that a higher absorbance indicates a higher number of bifidobacteria in the liquid after culture.

<Rating 2: Beauty effect>
(1) After autoclaving the powdered composition for consumption, it was adjusted to 4 mg/mL with phosphate buffered saline (PBS) to prepare a sample solution with a concentration four times the final concentration. This sample solution was added to a 96-well black plate at 50 μL/well. However, 50 μL of PBS was added to some wells as a control instead of the sample solution.
(2) 100 μL/well of enzyme solution (Test) or 0.1% by mass BSA-containing D-PBS(-) (Blank) was added to the 96-well black plate and incubated at 37°C for 10 minutes. D-PBS(-) is a solution prepared by dissolving one tablet of tablet-type PBS (model number T900) manufactured by Wako Pure Chemical Industries, Ltd. in 100 mL of pure water, to which albumin (bovine serum-derived corn fraction V, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved to a concentration of 0.1% by mass to produce 0.1% by mass BSA-containing D-PBS(-). The enzyme solution was prepared by diluting a D-PBS(-) solution containing 10 mg/mL of Collagenase B (Roche) with 0.1% by mass BSA-containing D-PBS(-) to a concentration of 10 μg/mL.
(3) After incubation, 50 μL/well of fluorescent substrate solution (1 mM DMSO solution of MOCAc-Pro-Leu-Gly-Leu- _A2pr (DNP)-Ala- _NH2 (Peptide Institute) adjusted to 5 μM in D-PBS(-) containing 0.1% by mass BSA) was added to the 96-well black plate and incubated at 37°C for 60 minutes in the dark.
(4) Using a microplate reader, the fluorescence intensity was measured at 405 nm with excitation at 320 nm.
(5) Using the measured fluorescence intensity, the collagenase inhibition rate was calculated according to the following formula. The results are shown in FIG.
Collagenase inhibition rate (%) = [1 - (Sample _test - Sample _blank )/(Control _test - Control _blank )] x 100
In the above formula, sample _test is the fluorescence intensity of a well to which sample solution and enzyme solution were added, sample _blank is the fluorescence intensity of a well to which sample solution was added but no enzyme solution was added (0.1 mass% BSA-containing D-PBS(-) was added), control _test is the fluorescence intensity of a well to which no sample solution was added (PBS was added) but enzyme solution was added, and control _blank is the fluorescence intensity of a well to which no sample solution or enzyme solution was added.

From Figures 9 and 10, it can be seen that the composition for consumption of green juice of the present invention, which has a specific genotype determined by a specific gene marker, has excellent bifidobacteria proliferation activity and collagenase inhibition activity, and is effective for intestinal regulation and beauty.

[Production of the composition of the present invention]
[Production Example 1] (Production of granules for instant beverage)
The following ingredients were mixed to produce 3 g of granules for instant drinks. The resulting granules for instant drinks can be dissolved in 100 mL of water and taken to provide excellent beauty and intestinal regulation effects.

Ground barley stalk and leaves that meets the conditions (A) and (B) but does not meet the conditions (C) and (D)
40% by mass
Resistant dextrin 5% by mass
Erythritol 5% by mass
Fragrance 2% by weight
Dextrin Remainder

[Production Example 2] (Production of granules for instant beverage)
The following ingredients were mixed to produce 4 g of granules for instant drinks. The resulting granules for instant drinks can be dissolved in 100 mL of water and taken to provide excellent beauty and intestinal regulation effects.

Barley stover juice powder that satisfies the conditions (C) and (D) but does not satisfy the conditions (A) and (B)
60% by mass
Kale powder 10% by weight
Matcha 3% by weight
Trehalose 20% by mass
Vitamin B 0.2% by mass
Vitamin C 0.2% by mass
Dextrin Remainder

Claims (1)

A composition for consumption or drinking for green juice using crushed powder of barley stems and/or leaves,
The barley is the following (i) and is used as a collagenase inhibitor in an edible composition for green juice (excluding those in which the barley is Ichibanboshi, Mannenboshi or Nishinohoshi):
(i) The following conditions (A), (C), and (D) are satisfied, but condition (B) is not satisfied.
(A) Possesses a low expression gene for the serine protease Cxp1 as determined by the genetic marker Te945.
(B) Contains the yellow mosaic disease resistance gene rym5 as determined by the genetic markers E31/M41.
(C) Carries the powdery mildew resistance gene ml-o as determined by the gene marker om2.
(D) Carries the yellow mosaic disease resistance gene rym3 as determined by the gene marker k09554-AvaI.

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