JP6491775B1 - A new arabinogalactan protein from grapes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To have an action to promote skin cell proliferation, production of hyaluronic acid and type I collagen, a moisturizing action, etc., and excellent in action to improve skin roughening, wrinkles, skin elasticity, etc., melanin production suppressing action, pigmentation improving action, etc. Provision of a convenient method for obtaining arabinogalactan protein (AGP).
SOLUTION: An arabinogalactan protein-containing aqueous solution which is a sap overflowing from a pruned branch of an open-field grown grape before the germination period, and a powder obtained by drying the aqueous solution. A sorting step of collecting the sap, a contacting step of contacting the grape sap and the urine sugar test paper, and a sorting step of judging whether the grape sap is collected or not based on the coloration of the urine sugar test paper And a recovery step of recovering the grape sap when it is determined in the step that the grape sap is recovered, a method of recovering the arabinogalactan protein-containing grape sap.
【Selection chart】 None

Description

  The present invention relates to an arabinogalactan protein-containing aqueous solution, an arabinogalactan protein-containing powder, a cosmetic composition, a functional food, a method for recovering arabinogalactan protein-containing grape sap, a method for producing an arabinogalactan protein-containing aqueous solution, and arabic The present invention relates to a method for producing nogalactan protein-containing powder.

  In spring, when the soil temperature rises in early spring, the roots actively absorb water, and the water and nutrients are revived freshly spreading to every corner of the branch. At this time, the sap flows out from the cut end of the pruned branch of the vines. This phenomenon lasts one week to three weeks, and the vines grow and sprout with moisture and nutrients in the sap.

  Grape sap spilled from the cut end of a pruned branch of a grapevine includes sugars such as glucose, sucrose and fructose, organic acids such as malic acid, tartaric acid and citric acid, amino acids such as threonine, aspartic acid and glutamic acid, It is known that minerals such as potassium, calcium and magnesium, and vitamins such as inositol (vitamin group B) and the like are contained (Non-patent Document 1).

  By the way, in the stratum corneum located at the top of the skin, there are hydrophilic hygroscopic components such as natural moisturizing factor (NMF) called various natural sugars, amino acids and minerals. ing. Grape sap has many components similar to NMF that support the moisturizing function of the skin, and since it is weakly acidic as the skin, it has excellent permeability to the skin.

  Therefore, by using grape sap as a lotion or as a material of other basic cosmetics, it is possible to reinforce the moisturizing function that the skin originally has and to obtain a cosmetic that gives a natural feeling of use with less burden on the skin. (Patent Document 1).

  Furthermore, grape sap has a fibroblast activating activity that is one of the main cells producing collagen (Patent Document 2), and by enhancing a ceramide synthesis enzyme most important for maintaining the barrier function of the skin. It is also known to enhance ceramide synthesis (U.S. Pat. No. 5,677,859).

  In addition, it is possible to efficiently extract grape sap from a vine in which the thickness of the branches and the position and shape of the cut where the sap extravasates differ from tree to tree, and which can suppress the quality deterioration of the collected grape sap. A sap collection device and method have been proposed (Patent Document 4).

  In addition, the grape sap contains many compounds other than the above components, and the problems of turbidity, precipitation and coloration that are considered to be caused by these have been problems, but these problems are caused by heat treatment or clarification treatment. It is known that it can be solved (Patent Document 5).

  On the other hand, in recent years, it promotes the proliferation of skin cells and the production of hyaluronic acid and type I collagen, and also has high water retention capacity itself, and also confirms rough skin, wrinkles, skin elasticity, melanin production suppressing action and pigmentation improving action Proteoglycans have been noted as having an effect to keep the skin youthful.

Proteoglycan is a type of glycoprotein in which a sugar chain called glycosaminoglycan such as chondroitin sulfate or dermatan sulfate is bound to a core protein.
Conventionally, proteoglycan has been obtained by extraction from salmon nasal cartilage (ice head) and has been used as a raw material for cosmetics and health food, but the extraction process is complicated and the amount obtained is limited, so PG Could not supply cheaply.

  Apart from animal-derived proteoglycans, plant-derived proteoglycans are also known, and they are called arabinogalactan proteins because they have a structure in which a core protein is linked to a sugar chain called arabinogalactan such as arabinose or galactose. There is. Arabinogalactan protein is said to have the same functionality as proteoglycan from salmon nasal cartilage (ice head). In addition, arabinogalactan protein is a subconcept of proteoglycan, and proteoglycan is a subconcept of glycoprotein.

  Arabinogalactan protein is universally present in tissues of higher plants regardless of monocotyledonous and dicotyledonous plants, and is known to be localized in cell membranes and cell walls (Non-patent Document 2). In addition, it is known that arabinogalactan protein differs in molecular weight, sugar combination and structure of sugar chain (arabinogalactan) depending on the type of plant (Non-patent Document 3).

Japanese Patent Application Laid-Open No. 10-045564 International Publication No. 2007/046353 International Publication No. 2008/053960 Japanese Patent Application Laid-Open No. 10-295207 Unexamined-Japanese-Patent No. 11-139950 gazette

Imamura, Kazuyuki, Secret of Grapevine Liquor, Modern Agriculture, March 1, 2004, Vol. 83, No. 3, pp. 58-61 Tsuburaya Yoichi, structure and related enzymes of plant arabinogalactan-protein, CACS Forum, Saitama University Research Organization Scientific Analysis Support Center, December 2014, No. 5, pp. 2-10 Kozaki Makoto, structure of arabinogalactan and its physiological function-Comparison of structure of functional arabinogalactan and agricultural origin of arabinogalactan-, Applied Carbohydrate Science, Applied Carbohydrate Science Association, July 20, 2011, Volume 1 , No. 3, pages 238-243.

  Although the presence of arabinogalactan protein in grape sap was expected, the content of arabinogalactan protein in plants is generally low (Tsuriya, 2014), so is the amount of AGP in grape sap as well. It was expected that it would be impossible to obtain grape sap containing high concentrations of AGP.

  An object of the present invention is to easily obtain an arabinogalactan protein-containing aqueous solution in view of the above circumstances.

  The present inventors diligently studied to solve the above-mentioned problems, and it was found that the sap (duct fluid) extravasated from the pruned branches of the grape grown in the open field before the germination period of early spring is a high concentration of arabinogalactan protein. In addition, the present inventors have completed the present invention by unexpectedly knowing that a grape sap containing a high concentration of arabinogalactan protein can be selectively obtained using urine sugar test paper. That is, the present invention is as follows.

[1] An arabinogalactan protein-containing aqueous solution which is a sap extravasated from a pruned branch of an open-field grown grape before the germination period.
[2] An arabinogalactan protein-containing powder obtained by drying the arabinogalactan protein-containing aqueous solution according to the above [1].
[3] A cosmetic composition comprising the arabinogalactan protein-containing aqueous solution according to the above [1] or the arabinogalactan protein-containing powder according to the above [2].
[4] A functional food comprising the arabinogalactan protein-containing aqueous solution according to the above [1] or the arabinogalactan protein-containing powder according to the above [2].
[5] a collecting step of collecting grape sap extravasated from a pruned branch of the open-field grown grape before the germination period;
Contacting the grape sap with urine sugar test paper;
A sorting step of determining whether or not grape sap is to be recovered based on the coloration of the urine sugar test paper;
A recovery method of arabinogalactan protein-containing grape sap, including a recovery step of recovering grape sap when it is determined to be recovered.
[6] Each step of the method for recovering arabinogalactan protein-containing grape sap according to the above [5],
A low molecular weight component removing step of removing at least a part of a component having a molecular weight lower than that of the arabinogalactan protein from the collected grape sap, and a method of producing an aqueous solution containing arabinogalactan protein.
[7] Each step of the method for producing an arabinogalactan protein-containing aqueous solution according to the above [6],
And a pulverizing step of pulverizing an arabinogalactan protein-containing aqueous solution obtained by removing low molecular weight components, and a method of producing an arabinogalactan protein-containing powder.

  According to the present invention, it is possible to easily obtain an arabinogalactan protein-containing aqueous solution.

  Grape sap spontaneously drains from the pruned branches of open-grown grapes before the early spring germination period, and without recovery and utilization, it only drains and dissipates without any benefit. The present invention has been found that arabinogalactan protein is contained at a high concentration in such grape sap, and has been found to find use in cosmetics or functional foods.

  The arabinogalactan protein-containing aqueous solution of the present invention is a sap (conduit fluid) extravasated from a pruned branch of an open-cultivated grape before the germination period of early spring. From the pruned branches of the grape grown in the open field before the germinating season of spring, since the grape sap (drainage fluid) spontaneously flows out, it can be easily obtained by recovering the drained grape sap. Therefore, it is possible to obtain an aqueous solution containing a high concentration of arabinogalactan protein derived from grape at a much simpler cost and lower cost than extracting from grape fruits and other plants. The grape sap-derived arabinogalactan protein contained in the aqueous solution is a novel arabinogalactan protein which has not been known as described in the examples of the present invention, and known AGPs derived from grape fruits. And at least the sugar composition of the sugar chains are different.

  The arabinogalactan protein-containing powder of the present invention is a powder obtained by drying the above-mentioned arabinogalactan protein-containing aqueous solution of the present invention. The arabinogalactan protein-containing powder of the present invention is effective for storage without addition of preservatives and is easy to handle.

  The method for recovering arabinogalactan protein-containing grape sap according to the present invention is economical and efficient because a grape sap containing high concentration of arabinogalactan protein can be obtained by a simple method.

  The present invention has at least one of these effects.

FIG. 1 is a diagram showing a sugar chain structural model (expected) of grape sap-derived arabinogalactan protein. FIG. 2 is a graph showing evaluation of moisture retention in Examples and Comparative Examples.

The present invention will be described in detail.
Hereinafter, arabinogalactan protein may be abbreviated as "AGP" and arabinogalactan may be abbreviated as "AG".
In addition, the range represented using "-" in this specification means the range which includes the both ends described before and after "-" in the range.

[Arabinogalactan protein-containing aqueous solution]
The arabinogalactan protein-containing grape sap according to the present invention is a sap (conduit fluid) overflowing from a pruned branch of an open-grown grape before the germination period.

  The method for obtaining the arabinogalactan protein-containing grape sap of the present invention is not particularly limited, but the recovery method of the arabinogalactan protein-containing grape sap described later is preferable.

  The arabinogalactan protein-containing aqueous solution of the present invention may be subjected to a low molecular weight component removal treatment for removing at least a part of components having a molecular weight lower than that of the above arabinogalactan protein. Here, the low molecular weight component is not particularly limited as long as it is a component having a molecular weight lower than that of arabinogalactan protein contained in grape sap, and examples thereof include free amino acids, organic acids, saccharides and minerals.

  Moreover, as a method for removing at least a part of the low molecular weight component, dialysis treatment, ultrafiltration treatment, microfiltration treatment, gel filtration, etc. may be mentioned, but it is not limited thereto.

[Method of recovering grape sap containing arabinogalactan protein]
Although the recovery method of sap (conduit fluid) which overflows from the pruned branch of the grape cultivated in open field before the germination period of spring is not particularly limited, the recovery method of arabinogalactan protein-containing grape sap described below is preferable.

  In the early spring, grape sap is spilled out from cut pruned branches, and it is possible to collect it during 2-3 days from sap flow before sprouting, when the vines end their winter dormancy and the roots start to soak up water. It is about a week.

  However, depending on the temperature, rainfall conditions, field topography, soil, and cultivars (eg, Kyoho, Koshu, Kaiji, or Delaware) at that time, the timing and spill status of sap extravasation in each vines (continuous? The quality of the grape sap differs greatly depending on whether it is intermittent or the spillover amount is different.

  For example, during the collection period, grape sap may spill over continuously, but the temperature may fall due to the return of cold in the early spring, the spill may stop, and then it may become warm again and spill over may not necessarily be constant. Volume does not spill over time.

  In addition, since the source of grape sap is the moisture absorbed by the grapes from the soil, minerals and nutrient components stored in the roots, etc., the grape sap that overflows depending on the amount of rainfall and the soil on which the roots grow. Differences in the quantity and quality of

  According to the experience collected in Yamanashi, which is the No.1 grape producing area in Japan, sanding is generally more effective than sanding when it is compared between a well-drained sandy area along the river and a well-watered clayed area Although the spillover start time is early, the spillover period is short and the quantity is small.

  According to the recovery method of the present invention, it is possible to selectively recover grape sap having a high AGP concentration by using the urine sugar test paper, and moreover, it is economical and efficient because it is simple.

  Urine sugar test paper measures the glucose concentration in grape sap, and does not measure AGP concentration, but it is possible to measure AGP concentration in grape sap using the coloration of the urine sugar test paper as an index. So far, there is no known method for measuring the concentration of AGP in grape sap that can be carried out at the site of grape sap collection.

  The inventor considered that if the glucose concentration standard is 50 mg / dL or 100 mg / dL, only grape sap having high AGP concentration and high utility can be recovered. If 50 mg / dL is adopted as the standard, more grape sap can be recovered than when 100 mg / dL is adopted as the standard, and if 100 mg / dL is adopted as the standard, 50 mg / dL is adopted as the standard Higher quality grape sap can be recovered, which contains higher concentrations of AGP than it does.

  The method for recovering arabinogalactan protein-containing grape sap according to the present invention (hereinafter simply referred to as "the recovery method of the present invention") comprises (1) extravasation from the pruned branches of the open-grown grape before the germination period. Collecting grape sap, collecting (2) contacting the grape sap with urine sugar test paper, and (3) recovering or collecting grape sap based on the color of the urine sugar paper And a recovery step of recovering the grape sap when it is determined that it is to be recovered. Below, each process of the collection | recovery method of this invention is demonstrated.

(1) Harvesting step The harvesting step is a step of harvesting a grape sap extravasated from a pruned branch of an open-field grown grape before the germination period.

  The harvest time of the grape sap is within a week, two weeks or three weeks after the extravasation of the grape sap starts from the cut end of the vines cut in the early spring. However, even during this period, if the extravasation of the grape sap is interrupted or stopped, or the grape sap is colored, it is not possible to collect the grape sap.

  The method for collecting the grape sap is not particularly limited as long as it can collect the grape sap extravasated from the pruned branches of the open-field grown grapes before the germination period, but for example, the sap flows in a small container etc. It can be carried out by attaching sap to a pruned area and collecting sap flowing out into a container. A preservative may be added to the container.

  Since collection efficiency of grape sap is high and working quality can be prevented, it is possible to collect sap from various positions at one time because the quality deterioration can be prevented, for example, JP-A-10-295207. It is preferable to use the sap collection device and sap collection method described in 4.

  The grape from which the grape sap is collected may be a plant belonging to the genus Vitis, and its species, variety, etc. are not particularly limited. For example, as grapes, European grapes (V. vinifera, Viniera species), American grapes (V. labrusca, Laburuska species), muscadines (V. rotundifolia, Rotundifolia species), Amur grapes (V. amurensis, Amrensis species) , These hybrids and the like. Further, as varieties of grapes, Kyoho, Koshu, Kaohji, Muscat Berry A, Delaware, Campbell Early, Pione, Neo Muscat, Niagara, Concord, Steuben, Cabernet Sauvignon, Pinot Noir, Merlot, Semillon, There may be mentioned varieties such as Riesling and Chardonnay.

(2) Contacting step The contacting step is a step of contacting grape sap and urine sugar test paper.

  Urine sugar test paper utilizes the color reaction by the glucose oxidase method. Since the color reaction is quantitative, it can be used for the determination of glucose by combining it with a color chart showing the relationship between coloration and glucose concentration.

(3) Sorting Step The sorting step is a step of judging whether or not the grape sap is to be recovered based on the coloration of the urine sugar test paper.

  In the sorting step, first, it is determined whether or not the coloration of the urine sugar test paper is equal to or greater than a predetermined reference.

  Next, it is judged that the grape sap is collected if the coloration of the urine sugar test paper is equal to or greater than a predetermined reference, and it is judged that the grape sap is not collected if it is less than the reference.

  As a standard, it is preferable to set it as 50 mg / dL in glucose concentration, for example.

  The urine sugar test paper is not particularly limited, but preferred is New Uri Ace Ga (manufactured by Terumo Corporation), New Uri Ace BT (manufactured by Terumo Corporation) or Uri Ace Kc (manufactured by Terumo Corporation). It can respond to both the case where the reference value of the glucose concentration is set to 50 mg / dL and the case where it is set to 100 mg / dL.

(4) Recovery step The recovery step is a step of recovering grape sap when it is determined to be recovered in the sorting step.

  In order to prevent the deterioration of the quality due to contamination, deterioration, etc., the collected grape sap is preferably divided into a half-day collected portion, a 1-day collected portion, etc. and stored preferably, and is preferably stored frozen. The temperature at the time of cryopreservation is not particularly limited as long as it can maintain the frozen state of the grape sap, but it is preferably -18 ° C or less, more preferably -20 ° C or less, because chemical growth can be suppressed. -60 ° C or less is more preferable, and -80 ° C or less is still more preferable because it can suppress the progress of

  It is preferable to mix and use the collected grape sap. The mixing makes the quality of grape sap uniform and less variation, which is advantageous in terms of quality assurance and quality control.

  Also, the collected grape sap can be used as a liquid substance by mixing with a preservative or the like as needed, or as a powder after drying.

  In addition, the collected grape sap may be subjected to a heat treatment and a clarification treatment as necessary. As a result, it is possible to more reliably prevent the clouding and browning of the sap. The heat treatment can be carried out by heating grape sap, for example, at 60 to 100 ° C., preferably at 80 to 90 ° C., for 10 minutes or more. Within this range, it is possible to prevent white turbidity and browning, and to prevent deterioration of sugars and amino acids contained in grape sap. The heat treatment can be performed at any time, and may be performed on the collected grape sap, or may be performed after the cosmetic composition containing the grape sap. In addition, the fining treatment can be performed using a known fining agent such as activated carbon, bentonite, silica gel, silica sol, polyvinylpolypyrrolidone (PVPP) or the like. Although any clarifier suppresses the progress of turbidity and discoloration of the lotion containing the grape sap, in particular bentonite is preferable because it suppresses both the turbidity and discoloration of the lotion well.

  Another advantage of the method for collecting grape sap according to the present invention is that by collecting grape sap whose glucose concentration is above the reference value, it is excellent by containing AGP at a high concentration while securing the liquid volume. It is in the point which can obtain the grape sap which has moisture retention.

[Method for producing arabinogalactan aqueous solution]
The method for producing the arabinogalactan protein-containing aqueous solution of the present invention is not particularly limited, but from each step of the method for recovering arabinogalactan-containing grape sap of the present invention described above and the harvested grape sap, Also preferred is a method for producing an arabinogalactan protein-containing aqueous solution, comprising a low molecular weight component removing step of removing at least a part of low molecular weight components.

  Here, the low molecular weight component is not particularly limited as long as it is a component having a molecular weight lower than that of arabinogalactan protein contained in grape sap, and examples thereof include free amino acids, organic acids, saccharides and minerals.

  Moreover, as a method for removing at least a part of the low molecular weight component, dialysis treatment, ultrafiltration treatment, microfiltration treatment, gel filtration, etc. may be mentioned, but it is not limited thereto.

  In the case of dialysis treatment in the dialysis treatment step, the method is not particularly limited, and conventionally known dialysis treatment methods can be used.

  It is preferable that the semipermeable membrane used in the dialysis treatment is one that is permeable to low molecular weight components having a molecular weight of 1000 or less.

  The dialysate used in the dialysis treatment is not particularly limited, but water is preferable from the viewpoint of the removal efficiency of the component that permeates the dialysis membrane, and ion exchange water, distilled water, reverse osmosis water (RO water), etc. are used be able to.

  The temperature in the dialysis treatment is not particularly limited, but 0 ° C. to 25 ° C. is preferable, 0 ° C. to 15 ° C. is more preferable, and 1 ° C. 5 as long as the grape sap (AGP-containing aqueous solution) and the dialysate do not freeze. ° C is more preferred. By dialysis, the purity of the arabinogalactan protein-containing aqueous solution can be further increased, and it becomes more suitable as a raw material of a cosmetic composition and a functional food to be described later. In addition, since the impurities are reduced, it becomes difficult to discolor or rot the AGP-containing aqueous solution.

[Arabinogalactan protein-containing powder]
The arabinogalactan protein-containing powder of the present invention is obtained by drying and pulverizing the above-mentioned arabinogalactan protein-containing aqueous solution.

  By drying and pulverizing the arabinogalactan protein-containing aqueous solution, it is possible to obtain a concentrated powder which can be preserved without addition of a preservative and which can be easily handled.

  The method for producing the arabinogalactan protein-containing powder of the present invention is not particularly limited, and conventionally known drying methods such as air drying, lyophilization and spray drying can be used. The manufacturing method is preferred.

[Method for producing arabinogalactan protein-containing powder]
The method for producing the arabinogalactan protein-containing powder of the present invention is not particularly limited, but each step of the method for producing the arabinogalactan protein-containing aqueous solution described above and the produced arabinogalactan protein-containing aqueous solution are dried, The method for producing an arabinogalactan protein-containing powder, which comprises the step of

  By drying and pulverizing the produced AGP-containing aqueous solution, it is possible to obtain a freeze-dried powder which can be preserved without addition of a preservative and which is easy to handle.

  The drying and powdering methods in the powdering step are not particularly limited, and conventionally known drying and powdering methods can be used, and examples thereof include air drying, lyophilization, and spray drying.

  In the case of drying and pulverization by lyophilization, the first step of lyophilization may be freezing, as long as the AGP-containing aqueous solution can be frozen, and various conventionally known methods can be used.

  The drying process which is the second step of the lyophilization process only needs to be capable of removing water (ice) from the frozen AGP-containing aqueous solution by sublimation, and various conventionally known methods can be used.

  The lyophilization process may be performed using a lyophilization apparatus capable of performing the lyophilization process as a series of processes.

[Cosmetic composition]
The cosmetic composition of the present invention comprises the above-mentioned arabinogalactan protein-containing aqueous solution or the above-mentioned arabinogalactan protein-containing powder.

  Expected effects of AGP include the proliferation of skin cells, the production of hyaluronic acid and type I collagen, and also high water retention ability per se, rough skin, wrinkles, skin elasticity, melanin production suppressing action and pigment Although the effect of improving deposition is also confirmed and has the effect of keeping youthful skin, these effects can be expected as cosmetics or functional foods.

  In addition to the arabinogalactan protein-containing aqueous solution or the arabinogalactan protein-containing powder of the present invention, the cosmetic composition of the present invention may further contain components generally used in cosmetic compositions. Here, the components generally used in cosmetic compositions are not particularly limited, but polyhydric alcohols, lower alcohols, ultraviolet absorbers, ultraviolet scattering agents, preservatives, antioxidants, chelating agents, pH adjusters, A surfactant, a fragrance, a pigment, water and the like can be appropriately blended. These can be used in the range which does not impair the effect of this invention.

  Examples of the polyhydric alcohol include glycerin, propylene glycol, 1,3-butylene glycol and sorbitol. As said lower alcohol, ethanol, 2-propanol, butanol, a benzyl alcohol etc. are mentioned, for example. Examples of the UV absorber include t-butylmethoxydibenzoylmethane (4-tert-butyl-4'-methoxydibenzoylmethane), ethylhexyl paramethoxycinnamate (4-ethylmethoxycinnamic acid), oxybenzone And -3 (2-hydroxy-4-methoxybenzophenone) and the like. Examples of the ultraviolet light scattering agent include titanium oxide and zinc oxide. Examples of the preservative include paraben (p-oxybenzoic acid ester), benzoic acid, sodium benzoate (sodium benzoate), sodium dehydroacetate (sodium dehydroacetate), hinokitiol and the like. Examples of the above-mentioned antioxidant include BHT (dibutyl hydroxytoluene), BHA (butyl hydroxy anisole), propyl gallate, tocopherol and the like.

  The content of the grape sap in the cosmetic composition when producing the cosmetic composition of the present invention can be appropriately changed depending on the use of the cosmetic composition, but in general, 5% by mass or more is preferable in terms of unprepared sap.

  Also, the dosage form of the cosmetic composition is optional, and it may be soluble, emulsifying, powder, dispersion, etc., and the application is also for lotion, milk, cream, face wash, soap, shampoo, bath cosmetics etc. It can be widely used for cosmetics in general.

[Functional food]
The functional food of the present invention comprises the above-mentioned arabinogalactan protein-containing aqueous solution or the above-mentioned arabinogalactan protein-containing powder.

  Expected effects of AGP include the proliferation of skin cells, the production of hyaluronic acid and type I collagen, and also high water retention ability per se, rough skin, wrinkles, skin elasticity, melanin production suppressing action and pigment Although the effect of improving deposition is also confirmed and has the effect of keeping youthful skin, these effects can be expected as cosmetics or functional foods.

  As a functional food, AGP itself is water-soluble, and since it may be relatively stable to heating during processing, the processing method is not particularly limited, and the aspect as a food is a drink beverage or It can be used in various forms according to the purpose of use, such as gel foods and solid foods.

[Arabinogalactan protein]
Arabinogalactan protein (hereinafter sometimes referred to as "AGP") is an important physiological functional component involved in cell adhesion and recognition as well as differentiation and growth of plant cells through signal transmission in plants. It is considered. AGP is a glycoprotein composed of a core protein portion and a sugar chain portion bound to it. The sugar chain portion accounts for 90% or more of the mass of AGP, and is mainly composed of arabinose (hereinafter sometimes referred to as "Ara") and galactose (hereinafter sometimes referred to as "Gal"). Sometimes referred to as "AG" chain.

  AG has a (1 → 3) -β-D-galactan chain as a main chain, to which a side chain composed of (1 → 6) -β-D-galactan or Ara is bonded. It is characterized as having a high degree of branching. Furthermore, in the side chain, rhamnose (hereinafter sometimes referred to as "Rha"), fucose (hereinafter sometimes referred to as "Fuc"), xylose (hereinafter sometimes referred to as "Xyl") and glucose (hereinafter referred to as "Xy") as minor components. Other sugars such as “Glc” may be bound. Also, depending on the plant, not only the sugar composition of the side chain (such as Rha, Fuc, Xyl, Man, Glc, GalA or GlcA), but also the conformation and binding mode differ.

  In addition to the complexity of the structure of such AG, purification and structural analysis is very difficult, and the content in plants is also low. An example in which the entire structure is completely clarified for each AG There is not much.

  In order to identify the moisturizing component in the grape sap, the present inventor made a high molecular weight component of the grape sap extravaginated from the cut end of the pruned branch of the grapevine in early spring, which has a glucose concentration of 500 mg / dL or more. The whole sugar analysis was done including. As a result, it was found that grape sap contains arabinose and galactose at high concentrations.

  In addition, the present inventor predicted that arabinose and galactose are derived from a high molecular weight polysaccharide, and further analyzed the components in grape sap, and obtained high molecular weight components having a molecular weight of 70,000 or more by gel filtration chromatography. .

  When this high molecular weight component was reacted with YARI reagent which binds specifically to AGP to form a precipitate, a reddish brown precipitate was formed, and it was found that this high molecular weight component is AGP.

  Furthermore, when the sugar chain of this AGP was analyzed, it was found to be an arabinogalactan chain having (1 → 3) -β-D-arabinogalactan as a main chain. In addition, it was speculated that this AGP sugar chain is different in sugar structure from the known sugar chains of AGP derived from grape fruits.

  Until now, it has not been known that AGP is contained in grape sap, nor has AGP been obtained from grape sap.

Molecular weight of AGP
The grape sap-derived AGP of the present invention has an average molecular weight of at least 70,000 measured by gel filtration chromatography (hereinafter sometimes referred to as "GPC").
Here, the conditions of GPC are as described below.
Column: Superdex 75 HR 10/30 10 mm × 30 cm (manufactured by GE Healthcare)
Solvent: 50 mM ammonium formate, pH 5.3
Column temperature: Room temperature Detector: Differential refractive index detector RID-10A (manufactured by Shimadzu Corporation)

<Structure of core protein and sugar chain part>
The grape sap-derived AGP of the present invention has a core protein moiety and a carbohydrate moiety, and the carbohydrate moiety (AG) has (1 → 3) -β-D-galactan as the main chain.

  In AG, (1 → 6) -β-D-galactan side chain is attached to O-6 position of (1 → 3) -β-D-galactan in the main chain, and further, O-3 position of side chain It is considered that Ara (1 → is bound to

  Also, the main chain is linked to the hydroxyproline residue of the core protein moiety.

<Reactivity with Yarib's reagent>
The grape sap-derived AGP of the present invention reacts with the Yarib reagent that specifically binds to AGP to form a complex, resulting in a reddish brown precipitate.

  In AGP studies, Yarib's reagent has been used that specifically binds and inhibits its physiological function. It has been unclear for a long time where the Yarib reagent binds to AGP, but in recent years, AGP derived from Japanese radish and AGP derived from acacia have (1 → 3) -β-D-galactan of the main chain of the sugar chain. Was found to bind specifically to

  EXAMPLES The present invention will be more specifically described below with reference to examples, but these do not limit the scope of the present invention.

Example 1
<Identification of moisturizing ingredients contained in grape sap>
"Total sugar analysis of grape sap"
1. Samples Grape sap collected immediately after the start of extravasation from the cut end of a pruned branch of a grape tree was used. The glucose concentration of this grape sap was measured using new Uriace Ga (manufactured by Terumo Corporation), and the color tone was ++ rating (glucose concentration 500 mg / dL or more and less than 2000 mg / dL).

2. Method: 400 mL of grape sap was freeze-dried, 1 mg of the freeze-dried product was transferred to a pressure-proof, heat-resistant screw-cap test tube, 1 mL of 2 M trifluoroacetic acid (TFA) was added, and heated at 121 ° C. for 1 hour. Next, the solvent was removed by dry air while warming at 40 ° C. or lower to remove TFA. After drying, 250 μL of 1 M hydrochloric acid-methanol (HCl-MeOH 5% (w / v), manufactured by GL Science) was added, and the mixture was heated at 80 ° C. for 15 hours. To this was added 100 μL t-butyl alcohol and the solvent was removed by dry air. After adding 0.05 mL of pyridine, 0.1 mL of hexamethyldisilazane, and 0.05 mL of trimethylchlorosilane and stirring, the mixture was heated at 80 ° C. for 20 minutes or more to perform trimethylsilylation (TMS treatment). The TMS agent was removed by dry air at room temperature. After dissolving in 1 mL of hexane, gently stirring, and centrifuging at 3000 rpm at room temperature for 10 minutes, the supernatant was removed using a Pasteur pipette and transferred to a new screw-cap test tube. The solvent was removed by dry air at room temperature. Using a syringe, it was dissolved in about 10 μL of hexane, and gas chromatography analysis was performed using 1 μL of the solution (DBI 30 m × 0.25 mm × 0.2 μm, column condition: 140 ° C. (2 ° C./min)→200) ° C (30 ° C./min)→275° C. (10 minutes), split ratio = 50: 1, gas chromatograph GC-17A (manufactured by Shimadzu Corporation). Furthermore, the following two methods were also used as a method for quantifying sugar in grape sap. Anthrone sulfuric acid method was used for the determination of neutral sugars. That is, 1 mL of 0.2% anthrone solution in which anthrone powder is dissolved with concentrated sulfuric acid is gently poured into 500 μL of sample (containing 20 μg or less of neutral sugar), mixed immediately, and kept in boiling aqueous solution for 5 minutes Quenched on ice. The absorbance at 620 nm of this sample was measured. The metahydroxybiphenyl method was used for the determination of the acidic sugar. That is, 1.2 mL of boric acid solution (4.77 g NaB ₄ O ₇ · ₁₀ H ₂ O per 1iter of conc. H ₂ SO ₄ ) is gently poured into 200 μL of a sample (containing 20 μg or less of acidic sugar) and mixed immediately Then, it was kept in boiling aqueous solution for 5 minutes and then quenched on ice. To this, 20 μL of a 0.15% metahydroxybiphenyl solution was added, immediately mixed, allowed to stand at room temperature for 5 minutes, and then the absorbance at 520 nm of this sample was measured.

3. Results TMS conversion was performed on the sugar component of grape sap by the method as described above, and the sugar composition was investigated by gas chromatography (GC) analysis. As a result, it was found that glucose concentration was most abundant, but other arabinose and galactose were abundant. In addition, the glucose concentration was reduced to about 1/50 compared to immediately after the start of extravasation and one week after the extravasation, and the concentrations of both arabinose and galactose were also reduced to about 1/20 (see below). Glucose in grape sap is known to be present as free sugar from general component analysis, but arabinose and galactose were expected to be derived from high molecular weight polysaccharides.

"Molecular weight measurement of grape sap high molecular weight component"
1. Samples Grape sap collected immediately after the start of extravasation from the cut end of a pruned branch of a grape tree was used. The glucose concentration of this grape sap was measured using new Uriace Ga (manufactured by Terumo Corporation), and the color tone was ++ rating (glucose concentration 500 mg / dL or more and less than 2000 mg / dL).

2. Method 200 mL of grape sap was dialyzed against distilled water at 4 ° C. for 2 days using a dialysis membrane (Spectra / Por Membrane 6 MWCO: 1,000). This treatment removed small molecules having a molecular weight of 1000 or less. Next, the sample was dissolved in ammonium formate, and the molecular weight was measured by gel permeation chromatography (GPC: Gel Permeation Chromatography) (column: Superdex 75 HR 10/30 10 mm × 30 cm (manufactured by GE Life Sciences); solvent: 50 mM Ammonium formate, pH 5.3; room temperature; Detector: Differential Refractive Index Detector RID-10A (manufactured by Shimadzu Corporation).

3. Result As a result of GPC analysis, a peak having an average molecular weight of more than 70,000 was confirmed in the polymer fraction.

Extraction of grape sap-derived AGP
From the above results, it was predicted that a high molecular weight polysaccharide having a molecular weight of more than 70,000 containing arabinose and galactose is arabinogalactan protein (AGP).

1. Samples Grape sap collected immediately after the start of extravasation from the cut end of a pruned branch of a grape tree was used. The glucose concentration of this grape sap was measured using new Uriace Ga (manufactured by Terumo Corporation), and the color tone was ++ rating (glucose concentration 500 mg / dL or more and less than 2000 mg / dL).

2. Method After freeze-drying grape sap, 20 mL of 80% ethanol was added and suspended, and it heated at 80-90 degreeC for 10 minutes. After the solution had cooled to about room temperature, it was centrifuged at 5000 × g and 4 ° C. for 10 minutes, the supernatant was removed, and the precipitate was lyophilized. Add 100 mL of 50 mM CDTA (trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid) per 1 g of precipitate, stir at room temperature for 6 hours, 10 × at 5000 × g, 4 ° C. After centrifugation for a minute, the supernatant was dialyzed into distilled water. In order to fractionate AGP from the resulting solution, Yalib precipitation was performed using Yalib reagent (Biosupplies Australia Pty Ltd) that specifically binds to AGP. AGP and Yarib reagent were mixed at a mass ratio of 1: 2 and dissolved in distilled water, but with 1% sodium chloride (NaCl) aqueous solution, the property of precipitating as an AGP-yarib reagent complex was used. The Yarib reagent was added to the sample at a weight ratio of 1: 2, and a 2% (w / v) aqueous solution of NaCl was added to give a final solution concentration of 1% (w / v). After standing overnight at 4 ° C. and centrifuging (5000 × g, 4 ° C., 20 minutes), the precipitate (AGP-Yarib reagent complex) was suspended in a 1% aqueous solution of NaCl and centrifuged again. In order to remove the insoluble residue, the precipitate (AGP-Yarib reagent complex) is dissolved in distilled water, centrifuged, and the precipitate (insoluble residue) is removed to a final concentration of 1% (w / w). 10% (w / v) NaCl aqueous solution was added to become v). Centrifugation was performed again, the supernatant was removed, and the collected precipitate (AGP-Yarib reagent complex) was dissolved in distilled water. At this time, this operation was repeated until no insoluble residue appeared in the supernatant. Dissolve the final precipitate (AGP-Yarib reagent complex) with distilled water, add sodium dithionite (Na ₂ S ₂ O ₄ ) to a final concentration of 10% (w / v), reddish brown It was left at room temperature until it disappeared. As soon as the color disappeared, it was desalted (eluted with distilled water) with a PD-10 column (manufactured by GE Healthcare), freeze-dried, and used as a yarib precipitation fraction (AGP).

3. Result The grape sap was freeze-dried by the method as described above, and the YARI reagent that specifically binds to AGP was added to the CDTA soluble fraction, resulting in precipitation of a complex reddish brown, so it was in the grape sap Certainly, it turned out that AGP exists.

"Structural analysis of sugar in grape sap high molecular weight fraction"
1. Samples Grape sap collected immediately after the start of extravasation from the cut end of a pruned branch of a grape tree was used. The glucose concentration of this grape sap was measured using new Uriace Ga (manufactured by Terumo Corporation), and the color tone was ++ rating (glucose concentration 500 mg / dL or more and less than 2000 mg / dL). 200 mL of grape sap was dialyzed against distilled water at 4 ° C. for 2 days using a dialysis membrane (Spectra / Por Membrane 6 MWCO: 1,000). This treatment removed small molecules having a molecular weight of 1000 or less. Furthermore, using 2 mg of a freeze-dried product of AGP prepared from the Yarib precipitated fraction by the same method as the above-described AGP extraction method from grape sap, structural analysis of the sugar chain was performed by the partially methylated alditol acetate method.

2. Method (1) Methylation A sample was placed in a screw-cap test tube, 500 μL of DMSO (dimethyl sulfoxide) was added, and the mixture was stirred overnight at room temperature. 100~200μL of ^K + DMSO in a draft ^- was added (potassium methylsulfinyl carba anion) was stirred at room temperature for 2 hours. While the sample was frozen on ice, 150 μ of methyl iodide (MeI) was added in the crucible. The mixture was removed from the ice, returned to room temperature, and stirred for 1 hour (methylation reaction). Next, 500 μL of distilled water was added to stop the methylation reaction. Bubbled with argon (Ar) to remove the MeI. It was then dialyzed against distilled water at 4 ° C. for 1 day.

(2) Acetylation of partially methylated alditol The dialyzed and freeze-dried sample was transferred to a screw test tube, 200 μL of 2 M trifluoroacetic acid (TFA) was added, and the mixture was heated at 121 ° C. for 1 hour. The solvent was removed by dry air at room temperature. 250 μL of 2-propanol were added and the solvent was removed by dry air at room temperature. Next, 200 μL of sodium borohydride (NaBD ₄ ) (NaBD ₄ 10 mg / 1 M NH ₄ OH 1 mL) was added, and the mixture was allowed to react at room temperature for 1 hour (reduction reaction). 50 μL of acetic acid was added to break up excess boron deuterated compounds. 200 μL of acetic acid: methanol (= 1: 9) was added, the solvent was removed by dry air, and this operation was then performed 3 times. 200 μL of methanol was added, the solvent was removed by dry air, and this operation was then performed 3 times. 100 μL of acetic anhydride was added and heated for 3 hours at 121 ° C. (acetylation reaction). The acetylated sample was neutralized by adding a small amount of sodium carbonate (anhydrous). 500 μL of chloroform was added, stirred and centrifuged at 15000 rpm for 5 minutes. The supernatant was removed to leave the organic layer and the solvent was removed by dry air. 200 μL of acetone was added to dissolve and collect the sample in the screw test tube. Transfer to a new screw-cap test tube, remove the solvent with dry air, dissolve in about 10 μL of acetone, and use 1 μL of them for gas chromatography one analysis (SP ^TM 2330 30m × 0.25mm × 0.2μm; column conditions : 170 ° C. (2 minutes) → 170 ° C. (4 ° C./minute)→235° C. (10 minutes); gas chromatograph GC-17A (manufactured by Shimadzu Corporation), and gas chromatography mass spectrometry (GC-MS) were performed. Column conditions: 50 ° C (2 minutes) → 170 ° C (30 ° C / minute) → 235 ° C (20 minutes), gas chromatograph HP HEWLETT PACKARD 5890; mass spectrometry system JEOL JMS -DX 303 HF).

3. Results Structural analysis of the sugars of the high molecular weight polysaccharide contained in the high molecular weight fraction by the above method revealed that there are a large number of 3,6-galactan, which is the binding mode of sugar chains characteristic of AGP, and high molecular weight of grape sap The polysaccharide was suggested to be mostly AGP. In addition, when a sugar chain structural model of AGP was prepared, it was predicted that the sugar chain of 3, 6-galactan had a characteristic feature of AGP, but it was characterized that the 1,2-arabinan chain was long. It was suggested that AGP derived from grape sap is a different substance from AGP derived from grape fruits. The sugar composition shown in Table 2 and the sugar chain structural model (expected) shown in FIG. 1 were obtained as analysis results of the sugar chain binding mode.

<Production of arabinogalactan protein-containing aqueous solution and powder>
1. Samples Grape sap collected immediately after the start of extravasation from the cut end of a pruned branch of a grape tree was used. The glucose concentration of this grape sap was measured using new Uriace Ga (manufactured by Terumo Corporation), and the color tone was ++ rating (glucose concentration 500 mg / dL or more and less than 2000 mg / dL).

2. Method 200 mL of grape sap was dialyzed against distilled water at 4 ° C. for 2 days using a dialysis membrane (Spectra / Por Membrane 6 MWCO: 1,000). This treatment removed small molecules having a molecular weight of 1000 or less. Next, the arabinogalactan protein-containing grape sap from which low molecular weight components were removed was lyophilized.

3. Result: 108 mg of freeze-dried product was obtained. In the case of grape sap, most of it was considered to be AGP because it contains almost no other high molecular weight component. In Non-Patent Document 2 (Tsuruya, 2014), in the case of Japanese radish adult roots, AGP obtained from 1 kg of living tissue is described to be about 50 mg, and in comparison with this, about 1 kg to about 10 times or more in this grape sap. As a result, 500 mg will be obtained, which is a simple and very efficient method of AGP recovery.

<Summary>
From the above results, the inventor of the present invention has found that the polymer fraction of grape sap (conduit fluid) is mainly composed of AGP, and the AGP-containing aqueous solution obtained from the grape sap by the recovery method of the present invention is known. It is considered that AGP is contained at a high concentration as compared with an aqueous solution containing AGP extracted from a plant. In addition, since the total mass of grape sap that can be recovered is large, it is considered that the total mass of AGP obtained from the grape sap is also an amount that can not be considered conventionally.

Example 2
<Measurement of grape sap sugar>
Uri-sugar test paper "New Uriace," which is generally marketed, as a method for measuring glucose sugar concentration in grape sap simply and quickly and at low cost by a farm in the field where grape sap is collected. The glucose concentration of various grape sap spilled in the early spring was measured using "Ga" (made by Terumo).

About sugar test paper It was judged by the color 30 seconds after soaking the test paper in grape sap. The glucose concentration of the test paper and the color tone table are shown in Table 3.

・ Test implementation 15 samples of various grape sap spilled out in early spring from the trees of grape varieties (Koshu, Kyoho, Delaware, Kaiji and Berry A) widely grown in the vineyards of Yamanashi Prefecture are collected and collected (farming site The sugar concentration was analyzed with sugar test paper and the color tone was recorded. After that, the sugar concentration of the same sample was measured by a general analysis method (HPLC method) at a test laboratory.

Test Results Fifteen grape sap solutions were in the range of------+-++ in the color tone table as measured by urine sugar test paper. Moreover, as a result of generally analyzing the sugar concentration of the same sample, it was in the range of 0.2 to 12 g / L (20 to 1200 mg / dL). This is within the effective concentration range (0 to 2000 mg / dL) which can be measured with urine sugar test paper, and it has been found that it can be sufficiently subjected to sugar (glucose) examination of grape sap.

[Example 3]
<Collection of grape sap containing high concentration AGP>
Collecting grape sap
In the vineyard in Yamanashi Prefecture (Japan), three vines (“No. 1”, “No. The grape sap was collected from each of the trees from 200 mL / day for 2 weeks after the extravasation of the grape sap was observed from each of the trees (2) and "No. 3". Hereinafter, the collected grape sap may be referred to as "collected sample". At the time of collection, the glucose concentration of the collected sample was measured and recorded using a sugar test paper (New Uri Ace ^(R) Ga, Terumo Co., Ltd.) according to the method described in "Determination of glucose concentration with sugar test paper". . After collection, each collected sample was immediately stored frozen at -18 ° C without mixing with other collected samples. Table 4 shows the collection date, collection sample number, collection amount and glucose concentration for each collection sample.

<< Method for measuring glucose concentration with urine sugar test paper >>
Urine sugar test paper (New Uri Ace Ga, Terumo Co., Ltd.) is dipped in each collected sample (grape sap) for about 1 second, and after 30 seconds, the color of the urine sugar test drug is compared with the attached color chart to compare each collected sample The glucose concentration in the medium was measured and indicated by a symbol.

The relationship between the color of the color tone table and the glucose concentration is approximately as follows.
Color tone: "yellow", symbol: "-", sugar concentration: below detection limit Color tone: "yellow-green", symbol ... "±", sugar concentration: about 50 mg / dL
Color tone: "green", symbol: "+", sugar concentration: about 100 mg / dL
Color tone: "Slightly dark green", symbol: "++", sugar concentration: about 500 mg / dL
Color tone: "dark green", symbol: "+++", sugar concentration: about 2000 mg / dL

<Preparation of test sample>
Among the collected samples collected from grapevine (No. 1), those whose glucose concentration measurement result by glucose test paper is “±” or more (glucose concentration is about 50 mg / dL or more), that is, from collected sample number X1 The test samples were prepared by selecting up to X7 collected samples and mixing them in equal amounts. Hereinafter, this test sample may be referred to as "test sample A-1".

<Measurement of glucose concentration of test sample>
Using glucose standard solution I (200 mg / dL), glucose standard solution II (500 mg / dL) and distilled water, glucose standard solution (concentration 50 mg / dL, 100 mg / dL, 200 mg / dL, 300 mg / dL, 400 mg / dL) , 500 mg / dL) was prepared. Using these as samples, the absorbance was plotted on the vertical axis and the glucose concentration on the horizontal axis to create a calibration curve. A sample for measurement was prepared by mixing 0.02 mL of a test sample with 3.0 mL of a coloring reagent and warming at 37 ° C. for 5 minutes. The absorbance of the measurement sample at a wavelength of 505 nm was measured using a spectrophotometer (optical path length 10 mm, square cell, measurement temperature 20 ° C.). The glucose concentration of the test sample was determined from the absorbance of the measurement sample and the calibration curve. The glucose concentration of the test sample determined is shown in the "glucose concentration" column of Table 5.

Evaluation of the moisturizing properties of test samples
In order to evaluate the moisturizing effect, 20 panelists performed in vivo water sorption-desorption test (Tagami, H., 7 others, “Water sorption-desorption test of the skin in vivo for functional assessment” of the stratum corneum ", Journal of Investigative Dermatology, 1982, Vol. 78, No. 5, pp. 425-428). In the evaluation method, a fixed amount of each test sample is applied to the upper arm of 20 panelists, and the stratum corneum water content before application and application using the epidermal stratum corneum water content measuring device SKICON-200EX (manufactured by IBS) After 10 minutes, 1 hour after application and 4 hours after application, the stratum corneum water content was measured, and the evaluation was performed based on the number of people who increased the stratum corneum water content compared to before the application. The evaluation results are shown in the column of "moisturizing [person]" in Table 5.

Evaluation of storage stability of test sample
Each test sample was left in a thermostat at 30 ° C., and after 1 day, 3 days and 1 week, the color tone and turbidity were observed visually with the naked eye and evaluated according to the following evaluation criteria. The evaluation results are shown in the "storage stability" column of Table 5.

(Evaluation criteria)
No change in color tone and turbidity ... "○"
Slight yellowing and / or slight clouding ... "△"
Yellowing and / or cloudiness ... "x"
In order to be used as a cosmetic raw material, it must be "Δ" or higher, and in particular, it is preferable to be "○" evaluation in order to use for applications requiring transparency such as a cosmetic water raw material.

[Examples 4, 5]
Among grape sap (collected sample) collected from grapevines (No. 2), those whose glucose concentration measurement result by glucose test paper is “±” or more (glucose concentration is about 50 mg / dL or more), ie, collected The collected samples of sample numbers Y1 to Y8 were selected and mixed in equal amounts to prepare test samples. Hereinafter, this test sample may be referred to as “test sample A-2”. Among grape sap (collected sample) collected from grapevines (No. 3), those whose glucose concentration measurement result by glucose test paper is “±” or more (glucose concentration is about 50 mg / dL or more), ie, collected The test samples were prepared by sorting the collected samples of sample numbers Z1 to Z6 and mixing them in equal amounts. Hereinafter, this test sample may be referred to as “test sample A-3”. For test samples A-2 and A-3, glucose concentration measurement, and evaluation of moisture retention and storage stability were performed in the same manner as in Example 3. Evaluation of storage stability in the column of "glucose concentration [mg / dL]" in Table 5 and in the column of "moisturizing [person]" of Table 5 in the glucose concentration measurement results in the column of "glucose concentration [mg / dL]" The results are shown in the "storage stability" column of Table 5, respectively.

Comparative Examples 1 to 3
Among grape sap (collected sample) collected from grapevines (No. 1), those whose glucose concentration measurement result by sugar paper was "-" (less than measurement limit), that is, collected sample numbers X8 to X14 The test samples were prepared by selecting up to collected samples and mixing them in equal amounts (Comparative Example 1). Hereinafter, this test sample may be referred to as "test sample B-1". Among the grape sap (collected sample) collected from grapevines (No. 2), those whose glucose concentration measurement result by sugar paper was "-" (less than measurement limit), that is, collected sample numbers Y9 to Y14 Test samples were prepared by selecting up to collected samples and mixing them in equal amounts (Comparative Example 2). Hereinafter, this test sample may be referred to as "test sample B-2". Among the grape sap (collected sample) collected from grape trees (No. 3), those whose glucose concentration measurement result by sugar paper was "-" (less than the detection limit), that is, collected sample numbers Z7 to Z14. Test samples were prepared by selecting up to collected samples and mixing them in equal amounts (Comparative Example 3). Hereinafter, this test sample may be referred to as “test sample B-3”. For test samples B-1, B-2 and B-3, measurement of glucose concentration and evaluation of moisture retention and storage stability were performed in the same manner as in Example 3. Evaluation of storage stability in the column of "glucose concentration [mg / dL]" in Table 5 and in the column of "moisturizing [person]" of Table 5 in the glucose concentration measurement results in the column of "glucose concentration [mg / dL]" The results are shown in the "storage stability" column of Table 5, respectively.

Comparative Example 4
Comparative Example 4 is a conventional method for collecting grape sap, that is, an example using grape sap obtained by collecting grape sap over a period of 2 to 3 weeks from the start of extravasation thereof. All the grape sap collected during two weeks from the start of the extravasation of the grape sap was mixed and mixed with another grape tree (No. 4) different from the grape trees (No. 1 to No. 3) The test sample described in the example in Japanese Patent No. 3169067 was prepared by aseptically filtering the grape sap with a cellulose acetate type membrane filter (DISMIC 25CS045AN, manufactured by Advantec) with a pore size of 0.45 μm. Hereinafter, this test sample may be referred to as "test sample C-1". For test sample C-1, measurement of glucose concentration and evaluation of moisturizing property and storage stability were performed in the same manner as in Example 3. Evaluation of storage stability in the column of "glucose concentration [mg / dL]" in Table 5 and in the column of "moisturizing [person]" of Table 5 in the glucose concentration measurement results in the column of "glucose concentration [mg / dL]" The results are shown in the "storage stability" column of Table 5, respectively.

[Comparative Examples 5 to 7]
An aqueous glucose solution with a concentration of 200 mg / dL (comparative example 5, sometimes referred to as "test sample D-1" hereinafter), an aqueous glucose solution with a concentration of 200 mg / dL (comparative example 6, hereinafter referred to as "test sample D-2" And purified water (Comparative Example 7, hereinafter sometimes referred to as "Test Sample D-3") were prepared. For each test sample, the glucose concentration measurement and the moisturizing evaluation were performed in the same manner as in Example 3. The results of glucose concentration measurement are shown in the "glucose concentration [mg / dL]" column of Table 5 and the evaluation results of the moisturizing property are shown in the "moisturizing [person]" column of Table 5.

[Moisturizing property]
As shown in Examples 3 to 5, excellent moisturizing is achieved by collecting grape sap (collected sample) having a glucose concentration measurement result of "±" or more by sugar test paper, ie, a glucose concentration of about 50 mg / dL or more. It was possible to obtain grape sap having test characteristics (Test Sample A-1, Test Sample A-2, Test Sample A-3) (Table 4, Table 5, FIG. 2).

  From the comparison of Examples 3 to 5 and Comparative Examples 1 to 4, it was shown that the higher the glucose concentration of the grape sap, the higher the moisture retention and storage stability of the grape sap (Table 5, FIG. 2).

  Also, from the comparison of Examples 3 to 5 and Comparative Examples 5 and 6, the moisture retaining property of grape sap is not due to glucose alone, but depends on the concentration of AGP containing glucose, that is, AGP in grape sap. It was suggested that the higher the concentration, the higher the moisturizing property.

  Therefore, it was suggested that the glucose concentration of grape sap is a good indicator of the concentration of AGP in grape sap, in other words, the glucose concentration of glucose tree is linked to the concentration of AGP in grape sap.

[Preservation stability]
In addition, since the concentration of the substance capable of causing a browning reaction (aminocarbonyl reaction) increases as the concentration of AGP increases, a decrease in storage stability is expected, but Examples 3 to 5 and Comparative Examples 1 to 1 According to the comparison with No. 4, contrary to the expectation, Examples 3 to 5 in which the glucose concentration is high and the concentration of AGP is high are lower than those in Comparative Examples 1 to 4 in which the concentration of AGP is also low. It showed high storage stability.

[Collection amount of grape sap]
A sufficient amount of grape sap having excellent moisture retention and storage stability can be obtained by collecting grape sap having a glucose concentration measurement result of “±” or more, ie, a glucose concentration of about 50 mg / dL or more, using sugar paper It could be done (Table 4, Table 5, Figure 2).

Claims (7)

A harvesting step of harvesting grape sap extravasated from a pruned branch of an open-field grown grape before the germination period;
A step of contacting the grape sap with a urine sugar test paper utilizing a color reaction by the glucose oxidase method;
A sorting step of collecting grape sap having a glucose concentration of 50 mg / dL or more based on the coloration of the urine sugar test paper and determining that grape sap having a glucose concentration of less than 50 mg / dL is not collected;
And a recovery step of recovering grape sap having a glucose concentration of 50 mg / dL or more
A method for producing an arabinogalactan protein-containing aqueous solution , comprising Each step of the arabinogalactan method for producing protein-containing aqueous solution according to claim 1, and comprising the step of drying the collected grape sap, arabinogalactan protein content producing method of powder. Comprising the steps of arabinogalactan method for producing protein-containing aqueous solution according to claim 1, or comprises the steps of the method of manufacturing the arabinogalactan protein content powder according to claim 2, preparation of a cosmetic composition How . Comprising the steps of arabinogalactan method for producing protein-containing aqueous solution according to claim 1, or comprises the steps of the method of manufacturing the arabinogalactan protein content powder according to claim 2, preparation of functional foods How . A harvesting step of harvesting grape sap extravasated from a pruned branch of an open-field grown grape before the germination period;
A step of contacting the grape sap with a urine sugar test paper utilizing a color reaction by the glucose oxidase method ;
A sorting step of collecting grape sap having a glucose concentration of 50 mg / dL or more based on the color of the urine sugar test paper and determining that grape sap having a glucose concentration of less than 50 mg / dL is not collected;
And a recovery step of recovering grape sap having a glucose concentration of 50 mg / dL or more . Each step of the method for recovering arabinogalactan protein-containing grape sap according to claim 5;
A low molecular weight component removing step of removing at least a part of a component having a molecular weight lower than that of the arabinogalactan protein from the collected grape sap, and a method of producing an aqueous solution containing arabinogalactan protein. Each process of the manufacturing method of the arabinogalactan protein containing aqueous solution of Claim 6;
And drying the arabinogalactan protein-containing aqueous solution obtained by removing low molecular weight components, and pulverizing the aqueous solution containing the arabinogalactan protein.