JP6505813B2 - A fibronectin-binding soybean-derived water-soluble polysaccharide - Google Patents

Description

  The present invention relates to a method for enhancing the binding to fibronectin for a water-soluble polysaccharide derived from soybean. The present invention also relates to a soybean-derived water-soluble polysaccharide having enhanced binding to fibronectin.

  In recent years, it has been clarified that intake of dietary fiber causes changes in the shape and cell division of the small intestine, suggesting the existence of a mechanism by which intestinal epithelial cells recognize dietary fiber and directly interact. With regard to pectin, which is a kind of water-soluble dietary fiber, it has been reported that the length of small intestinal villi and the depth of crypts increase when it is caused to act on the intestinal tract (Non-patent Documents 1 and 2) It has been reported that prune pectin, which is a kind of fibronectin, specifically binds to an extracellular matrix protein called fibronectin present on the surface of the small intestine (Non-patent Documents 2 and 3).

  Pectin has conventionally been reported to have various physiological actions such as suppressing the increase in blood cholesterol and suppressing the onset of colon cancer, but the above findings suggest that such as water soluble dietary fiber Physiological actions are not only secondary actions such as physicochemical actions that have been proposed up to now, but also fermentation actions by intestinal microbes, but both act directly on intestinal tissue cells and the cells respond to them. It suggests that an interaction exists. In particular, from the report on prune pectin, it is considered that the morphological change of small intestine villi by pectin is caused as a result of the specific binding of pectin to fibronectin present on the surface of the small intestine.

Chun W., Digestion. 1989; 42 (1): 22-9 Gifu University Applied Biosciences Applied Life Science Course Food Life Science Course Food Ingredient Functional Chemistry Laboratory Website (Pectin Project) http://work.tomioyabe.net/web/research/pectin.html Akari Honda, Atsuko Tomita, Atsushi Nakamura, Yoshitaka Kanamaru, Koji Kato, Yuichiro Mori, Hiroki Yamamoto, Takao Igami, Tomio Yabe: Identification of human small intestine-derived proteins that recognize pectin

  From the above-mentioned conventional findings, it is speculated that pectin binds to fibronectin on the surface of the small intestine to cause morphological change of small intestinal villi. It is preferable to enhance the binding of pectin to fibronectin in order to use pectin at a lower intake to cause morphological change of small intestinal villi efficiently and exert beneficial physiological actions.

  Therefore, an object of the present invention is to provide a method for enhancing the binding property to fibronectin for a soybean-derived water-soluble polysaccharide such as soybean pectin. Another object of the present invention is to provide a soybean-derived water-soluble polysaccharide having enhanced binding to fibronectin.

  The present inventors have repeatedly studied to solve the above-mentioned problems, and it has been found that polysaccharides (Shoyu Polysaccharides) contained in soybean pectin and soy sauce belonging to a water-soluble polysaccharide derived from soybean are sodium and potassium It has been found that the binding to fibronectin is enhanced by coexistence with an alkali metal ion such as E. coli or an alkaline earth metal ion such as calcium or magnesium.

  The present invention has been completed based on such findings and comprises the following embodiments.

(I) A soybean-derived water-soluble polysaccharide (I-1) having enhanced binding to fibronectin (I-1) at least one metal selected from the group consisting of alkali metals and alkaline earth metals in a total amount of 6 to 65 mass A water-soluble polysaccharide-containing composition derived from soybean, comprising
(I-2) The water-soluble polysaccharide-containing composition derived from soybean according to (I-1), wherein the water-soluble polysaccharide derived from soybean is soybean pectin or soy sauce polysaccharide (Shoyu Polysaccharides).
(I-3) The water-soluble soybean-derived water described in (I-1) or (I-2), wherein the alkali metal and the alkaline earth metal are at least one selected from potassium, sodium, calcium and magnesium Polysaccharide containing composition.

(II) An oral or enteral composition (II-1) containing a water-soluble polysaccharide-containing composition derived from soybean (II-1) (I-1) to (I-3) An oral or enteral composition comprising a saccharide-containing composition.
(II-2) The oral or enteral composition according to (II-1), wherein the oral or enteral composition is any of food, drink, medicine or quasi drug.

(III) A method for enhancing the binding property of a water-soluble polysaccharide derived from soybean to fibronectin (III-1) A water-soluble polysaccharide derived from soybean is at least one selected from the group consisting of an alkali metal ion and an alkaline earth metal ion A method for enhancing the binding ability of a soybean-derived water-soluble polysaccharide to fibronectin, which comprises coexistence of species ions.
(III-2) 6 to 65% by mass of a water-soluble polysaccharide derived from soybean and at least one ion selected from the group consisting of an alkali metal ion and an alkaline earth metal ion The method for enhancing binding described in (III-1), which is
(III-3) The method for enhancing the bondability according to (III-1) or (III-2), wherein the water-soluble polysaccharide derived from soybean is soybean pectin or soy sauce polysaccharide (Shoyu Polysaccharides).

(IV) Method of Using Soy-Derived Water-Soluble Polysaccharide (IV-1) Percentage of Potassium and / or Alkaline Earth Metal Contained in a Composition Containing a Soy-Derived Water-Soluble Polysaccharide That is Soy Pectin or Soy Sauce Polysaccharide 5 so they become 6-65 weight% on a dry solid basis, the soybean-derived water-soluble polysaccharide in potassium and / or outer added amount per 100 mass% final composition an alkaline earth metal on a dry solid basis by adding in a proportion of 60% by weight, to generate or enhance the avidity for full Iburonekuchin water-soluble polysaccharide in the composition, the use of the water-soluble polysaccharide.
(IV-2) before Kia alkaline earth metal painter calcium, and is at least one selected from magnesium, (IV-1) Use according.

  The water-soluble polysaccharide derived from soybean containing alkali metal and / or alkaline earth metal of the present invention is compared with the water-soluble polysaccharide derived from soybean not containing alkali metal and / or alkaline earth metal. Binding to fibronectin, which is known to be present on the surface of the small intestine, is significantly enhanced. Therefore, by orally (or orally) or orally taking (administering) the water-soluble polysaccharide-containing composition of the present invention derived from soybean, the water-soluble polysaccharide derived from soybean can be efficiently bound to fibronectin to make small intestinal villi Morphological changes can be produced, through which it is possible to exert beneficial physiological actions.

In the experimental example 2 (2), the effect of calcium on the binding to fibronectin is shown in Biacore for soybean pectin (FIG. 1 (A)) and soy sauce polysaccharide (FIG. 1 (B)) subjected to neutralization treatment by various methods. It shows the results of investigation using the system. The RU value at a point 120 seconds after the addition (0 s) of the test sample (analyte) (120 s) and the RU value at a point 240 s (240 s) (120 s after the dissociation start) are shown. In Fig. 1 (A) and (B), the horizontal legends are "Control: 1 mg of soybean pectin solution dissolved in 1 mL / mL concentration", "PBS + Ca ²⁺ : calcium ion as neutralization treatment" in order from the left The sample which dialyzed by using PBS containing as a dialysis outer solution "distilled water: The sample which dialyzed by using distilled water as a dialysis outer liquid as neutralization process is shown. The results of examining the influence of the calcium ion concentration (0.01 to 0.1 M) on the binding to fibronectin in soybean pectin in Experiment Example 3 are shown using the Biacore system. The RU value at a point 120 seconds after the addition (0 s) of the test sample (analyte) (120 s) and the RU value at a point 240 s (240 s) (120 s after the dissociation start) are shown. In Experimental Example 4, soybean pectin was dissolved in (A) aqueous potassium chloride solution and (B) aqueous magnesium chloride solution so as to be 10 mg / ml, and various concentrations (a: 1 mg / mL, b: 0.5 mg / mL) The results of measuring the binding (RU value) to fibronectin over time with a Biacore system after dilution to c: 0.25 mg / mL, d: 0.17 mg / mL, e: 0.125 mg / mL) are shown. In Experimental Example 5 (1), the results of measuring the influence of calcium ions on the binding of acid-treated soy sauce polysaccharide to fibronectin over time using a Biacore system are shown. In the figure, samples a, b and c were prepared by dissolving soy sauce polysaccharide in hydrochloric acid containing 0.2% NaCl so as to be 10 mg / mL, dialysing with PBS containing calcium ion, and dialysing with distilled water. The figure shows the time-dependent change of RU value measured as an analyte after dilution of each to various concentrations (a: 1 mg / mL, b: 0.5 mg / mL, c: 0.3 mg / mL). In Experimental Example 5 (3), after dissolving each of the soy sauce polysaccharide in an aqueous solution of calcium chloride (FIG. 5 (A)) and an aqueous solution of potassium chloride (FIG. 5 (B)) to 10 mg / mL, The results show that the binding to fibronectin was measured over time using the Biacore system after dialysis against. In the figure, a, b, c, d and e are prepared by dissolving soy sauce polysaccharide in 0.2% NaCl / 0.1 M CaCl ₂ (or 0.2% NaCl / 0.1 M KCl) solution and then dialyzing with distilled water to obtain various concentrations. The time course of RU value measured as the sample diluted in (a: 1 mg / mL, b: 0.5 mg / mL, c: 0.25 mg / mL, d: 0.17 mg / mL, e: 0.125 mg / mL) as the analyte Indicates a change. Each concentration of (A) CaCl ₂ solution, (B) KCl solution, and (C) MgCl ₂ solution (all: a: 10 mM, b: 5 mM, c: 2.5 mM) is subjected to the Biacore system, respectively. The results of measuring the binding strength to fibronectin over time are shown.

(I) Soy-Derived Water-Soluble Polysaccharide Having Enhanced Binding to Fibronectin (1) Soy-Derived Water-Soluble Polysaccharide The soybean-derived water-soluble polysaccharide targeted by the present invention is a water-soluble soybean-derived water-soluble polysaccharide Polysaccharides of

  With soybean origin, the raw material should just be soybean. Specifically, not only polysaccharides obtained from raw soybeans themselves, but polysaccharides obtained from various processed soybean products such as heat-treated soybeans, defatted soybeans, soybean hulls, soybean meal, soybean meal, tofu, and the like included.

  Water solubility means the property of dissolving in water. The water solubility can be evaluated according to the regulations of the Japanese Pharmacopoeia, and specifically, 1 g of polysaccharides derived from the target soybean is put in 30 mL of distilled water, and it is 5 at 25 ± 5 ° C. It can be evaluated as "water soluble" if it dissolves within 30 minutes when shaken vigorously for 30 seconds each minute. The term "dissolving" means that distilled water to which soybean-derived polysaccharides have been added is clear without detecting insoluble matter, or very little even when insoluble matter is observed.

  The polysaccharide derived from soybean is a polysaccharide having a molecular weight of 2,000 or more, which contains a galacturonic acid having a carboxyl group. As a content rate of galacturonic acid, 10 mass% or more, Preferably 15 mass% or more, More preferably, 20 mass% or more can be mentioned.

  Specific examples of such a water-soluble polysaccharide derived from soybean include soybean pectin and soy sauce polysaccharide.

  Here, soybean pectin refers to pectin derived from soybean. Not only soybean pectin but also “pectin” is generally a repeating unit of a disaccharide consisting of polygalacturonic acid consisting of polygalacturonic acid to which galacturonic acid is bonded to α- (1 → 4), galacturonic acid and rhamnose alternately A complex polysaccharide in which the side chain consisting of arabinose and galactose is linked to the C4 position of rhamnose residue and has a broad molecular weight distribution. It consists of three major structural regions of rhamnogalacturonan-II, which is a complex polysaccharide region with a molecular weight of about 5000, in which four different side chains are linked, and homogalacturonan accounts for the majority. The carboxyl group of galacturonic acid is methylated or acetylated at each ratio.

  Soy pectin is a water-soluble pectin obtained from soybeans or processed soybeans, and is usually composed of three components having molecular weights of 55,000, 25,000 and 5,000. It is characterized by a low content of galacturonic acid (10-30%) compared to pectin derived from citrus fruits (Reference: "All of soybeans" Keisuke Kitamura et al., Published by Science Forum, Inc. P450, published on February 18, 2010 ).

  It is preferable that the method for producing soybean pectin includes the step of heating a material obtained from soybean or soybean hulls. Here, "heating" includes, for example, moist heat heating such as boiling and boiling after adding water to soybean so as to swell, and dry heat heating such as roasting.

  As a production method, for example, a method of adding an extraction solvent to a raw material (soybean or soybean hulls etc.), steaming or simmering after boiling or boiling, extracting the filtrate or simmerant; And stirred, and solid-liquid separation to obtain the filtrate. In addition, by-products generated during cooking and processing of soybeans (soybean broth) and by-products generated in the process before fermenting soybeans in the process of producing soy sauce (soybean soup) are used as extraction objects You can also. In addition, as an extraction solvent, water can be mentioned suitably here. The water may contain a chelating agent. When the extraction solvent contains a chelating agent, it is useful because the recovery of soybean pectin from soybean can be improved. As a chelating agent, hexametaphosphoric acid, citric acid, and salts thereof can be exemplified. The use concentration of the chelating agent in the extraction solvent is not limited, but can usually be 0.25 to 2% by mass, preferably 0.5 to 2% by mass. Although a more detailed production method is illustrated in the examples, without being limited thereto, commercially available soybean pectin can also be used.

The sauce polysaccharide (S hoyu P oly s accharides), although polysaccharides derived soybean solubilized undergo degradation by Aspergillus enzyme, not completely decomposed, remained at about 1% concentration in soy sauce It is a water soluble polysaccharide. Molecular weight is more than 2,000 and contains 10-20% of galacturonic acid (Reference: "Anti-allergic activity and immunoregulatory function of soy sauce polysaccharide SPS" Hiroaki Matsushita, Makio Furubayashi, Ryoichi Ikuyama, Kenji Yamamoto, Research and Technology for Soy Sauce Vol. 33, No. 2, 2007). The production method is described in detail in the examples, but is not limited thereto, and commercially available soy sauce polysaccharides can also be used.

(2) Alkali Metals and Alkaline Earth Metals As alkali metals, sodium and potassium can be mentioned, and as alkaline earth metals, calcium and magnesium can be mentioned.

  Suitable metals may be either alkali metals or alkaline earth metals, but are preferably alkaline earth metals, and particularly preferred is calcium.

  Any of these may be used alone or in combination of two or more. The embodiment to be combined is not particularly limited, but an embodiment in which an alkali metal and an alkaline earth metal are used in combination is preferable, and specifically, a combination of sodium and calcium can be exemplified.

(3) Soy water-soluble acidic polysaccharide containing an alkali metal and / or alkaline earth metal The present invention is a water-soluble polysaccharide derived from soybean characterized by containing the above-mentioned alkali metal and / or alkaline earth metal It is. In other words, the present invention is a composition containing an alkali metal and / or an alkaline earth metal in addition to a water-soluble polysaccharide derived from soybean (in the present invention, a composition containing a water-soluble polysaccharide derived from soybean Also referred to as "object"). From another angle, in other words, it is a composition in which an alkali metal and / or an alkaline earth metal is externally added to a water-soluble polysaccharide derived from soybean.

  The ratio of alkali metal and / or alkaline earth metal contained in the water-soluble polysaccharide-containing composition derived from soybean is such that the amount of alkali metal and / or alkaline earth metal inherent in the water-soluble polysaccharide derived from soybean The ratio is not particularly limited as long as it is a ratio by which the water-soluble polysaccharide derived from soybean itself is enhanced in the binding property to fibronectin due to the presence thereof. As the proportion of alkali metal and / or alkaline earth metal added (externally added) to the water-soluble polysaccharide derived from soybean, generally, with regard to alkali metal, 100% by mass of the water-soluble polysaccharide containing composition of alkali metal externally added There can be mentioned a range in which the concentration thereof is 1 to 60% by mass in terms of dry matter. Preferably it is 5-50 mass%, More preferably, it is 10-40 mass%. With respect to the alkaline earth metal, a range in which the proportion in 100% by mass of the externally added alkaline earth metal water-soluble polysaccharide-containing composition is 1 to 60% by mass in dry matter can be mentioned. Preferably it is 5-50 mass%, More preferably, it is 10-40 mass%.

  In view of the ratio of the alkali metal and / or the alkaline earth metal inherent in the water-soluble polysaccharide derived from soybean, the alkali metal contained in the water-soluble polysaccharide-containing composition derived from soybean is equivalent to the dry matter Usually, from the range of 6 to 65% by mass, the alkaline earth metal can be selected and set in the range of 6 to 65% by mass in terms of dry matter. The amount of alkali metal is preferably 10 to 55% by mass, and more preferably 15 to 45% by mass. The content of the alkaline earth metal is preferably 10 to 55% by mass, and more preferably 15 to 45% by mass.

  The water-soluble polysaccharide-containing composition derived from soybean of the present invention contains, for example, an alkali metal salt and / or an alkaline earth metal salt desired for a water-soluble polysaccharide derived from soybean, and contains an alkali metal and / or an alkaline earth metal. It can manufacture by adding and mixing so that a ratio may become a ratio mentioned above. Here, specific examples of the alkali metal salt include chloride of sodium, chloride of potassium; carbonate of sodium and carbonate of potassium; hydrogencarbonate of sodium and hydrogencarbonate of potassium . Preferred are sodium chloride and potassium chloride, more preferably potassium chloride. Further, as the alkaline earth metal salt, specifically, chloride of calcium, chloride of magnesium; carbonate of calcium, and carbonate of magnesium; hydrogencarbonate of calcium, and hydrogencarbonate of magnesium may be mentioned. it can. Preferred are calcium chloride and magnesium chloride, more preferably calcium chloride.

  The soybean-derived water-soluble polysaccharide-containing composition of the present invention can also be produced by dissolving a soybean-derived water-soluble polysaccharide in an aqueous solution containing an alkali metal ion and / or an alkaline earth metal ion.

  The form of the water-soluble polysaccharide-containing composition derived from soybean is not particularly limited, and may have a solid form such as tablet, pill, powder (powder), granule, and capsule. It may have a semi-solid form such as gel-like liquid (jelly-like) or cream-like or a liquid such as an emulsion or solution.

The water-soluble polysaccharide-containing composition derived from soybean thus prepared is characterized in that the binding property to fibronectin is enhanced as compared with the binding property of the water-soluble polysaccharide derived from soybean alone. The binding property to fibronectin can be determined by using a Biacore system, using a soybean-derived water-soluble polysaccharide-containing composition as an analyte and fibronectin as a ligand, as described in the examples below (binding property) It can be determined by measuring Specifically, the binding rate constant (Ka) and the dissociation rate constant (Kd) can be calculated from the curve of the sensorgram, and the affinity of the two can be determined as the dissociation constant (K _D ) from the ratio. Incidentally, K _D of more smaller bonding force indicates greater.

For example, when the water-soluble polysaccharide derived from soybean is a soy sauce polysaccharide having a molecular weight of 25,000, it has no binding ability to fibronectin itself, but an alkali metal salt and / or an alkaline earth metal salt is added (externally added) As a result, binding ability to fibronectin is generated. The dissociation constant of such a soybean-derived water-soluble polysaccharide-containing composition containing an alkali metal and / or an alkaline earth metal is K _D = 2.30 × 10 ⁻⁶ M.

(II) Oral or enteral composition containing a water-soluble polysaccharide-containing composition derived from soybean As described above, the water-soluble polysaccharide-containing composition derived from soybean has enhanced binding to fibronectin. It features. By orally or enterally taking (administering) a water-soluble polysaccharide-containing composition derived from soybean, the water-soluble polysaccharide derived from soybean taken into the body binds to fibronectin present on the surface of the small intestine, and indirectly It is thought that various physiological effects are expressed.

  Therefore, the water-soluble polysaccharide-containing composition derived from soybean of the present invention is preferably taken orally or enterally into the body so as to pass through the small intestine, and therefore oral administration (intake) or enteral administration It is prepared to have a form of (intake).

  Examples of the oral composition can include foods and drinks, oral medicines, and oral quasi-drugs, and the ratio and form thereof can be appropriately set according to the purpose.

  As the enteral composition, there can be mentioned food and drink for gastrostomy, enteral medicine, or enteral drug quasi-drug, and the proportion and form thereof can be appropriately set according to each purpose.

(III) Method of enhancing the binding of soybean-derived water-soluble polysaccharide to fibronectin The present invention also provides a method of enhancing the binding of soybean-derived water-soluble polysaccharide to fibronectin.

  The method can be carried out by causing a water-soluble polysaccharide derived from soybean to coexist with at least one ion selected from the group consisting of an alkali metal ion and an alkaline earth metal ion. The soybean-derived water-soluble polysaccharides targeted here are as described in the section (I), and the description is similarly incorporated in the section (III) including preferred embodiments. Further, as alkali metal ions, sodium ions and potassium ions can be mentioned, and as alkaline earth metal ions, calcium ions and magnesium ions can be mentioned. The alkali metal ion is preferably potassium ion, and the alkaline earth metal ion is preferably calcium ion. In addition, these can be used combining 1 type, or 2 or more types arbitrarily, and the aspect which combines alkali metal ion (Na ion, K ion) as an aspect of a combination, alkaline-earth metal ion (Ca ion) And Mg ions) and an embodiment in which alkali metal ions (Na ions, K ions) and alkaline earth metal ions (Ca ions, Mg ions) are combined. As a combination aspect in the case of combining an alkali metal ion and an alkaline earth metal ion, a combination of a sodium ion and a calcium ion can be suitably exemplified although it is not limited.

  As a ratio of the alkali metal ion and / or the alkaline earth metal ion to be made to coexist with the water-soluble polysaccharide derived from soybean, a ratio in which the concentration of the ion is 1 to 60% by mass can be mentioned.

  As a method of coexisting a water-soluble polysaccharide derived from soybean with an alkali metal ion and / or an alkaline earth metal ion, it is characterized by containing a water-soluble polysaccharide derived from soybean and an alkali metal and / or an alkaline earth metal. The method of melt | dissolving the water-soluble polysaccharide containing composition derived from soybean of this invention mentioned above of this invention in the solution of pH 2-9 can be mentioned. The term "dissolution" as used herein refers to the water-soluble polysaccharide-derived composition derived from soybean of the present invention as long as a coexistence state of the water-soluble polysaccharide derived from soybean and an alkali metal ion and / or an alkaline earth metal ion is formed. The substance is not limited to complete dissolution, and includes partial dissolution. For example, a semi-solid or liquid composition in which the water-soluble polysaccharide-containing composition derived from soybean of the present invention is partially or completely dissolved in a solvent such as water is a water-soluble polysaccharide derived from soybean and an alkali metal ion It can be said that it is in the state of coexistence with the alkaline earth metal ion. Further, by dissolving (including partially dissolving) the soybean-derived water-soluble polysaccharide-containing composition of the present invention in solid form in a solvent such as water, the soybean-derived water-soluble polysaccharide and alkali metal ion and / or A state in which it coexists with an alkaline earth metal ion can be formed. Furthermore, by taking the water-soluble polysaccharide-containing composition derived from soybean of the present invention together with a drink (solution) such as water if necessary, the water-soluble polysaccharide derived from soybean and the alkali metal ion and / or Or it will be in the state of coexistence with the alkaline earth metal ion.

  The water-soluble polysaccharide derived from soybean which has been treated in this manner and coexists with the alkali metal ion and / or the alkaline earth metal ion is a soybean derived from the state which does not coexist with the alkali metal ion and / or the alkaline earth metal ion Compared with the water-soluble polysaccharides of the present invention, the binding to fibronectin is enhanced, and it can be bound with stronger affinity to fibronectin present on the surface of the small intestine in vivo.

The presence or absence of increased binding to fibronectin can be determined by using a Biacore system, using a soybean-derived water-soluble polysaccharide as an analyte and using fibronectin as a ligand, an alkali metal ion and / or an alkali, as described in the examples below. It can be determined by measuring the interaction (binding property) of the two in the presence of the earth metal ion. Specifically, the binding rate constant (Ka) and the dissociation rate constant (Kd) can be calculated from the curve of the sensorgram, and the affinity of the two can be determined as the dissociation constant (K _D ) from the ratio.

  Hereinafter, the configuration and effects of the present invention will be described in more detail based on examples and experimental examples. However, the present invention is not limited to these examples and the like.

(I) Experimental Material, and Preparation of the Same Soy pectin and soy sauce polysaccharide were used as test samples in the test examples described later. The methods for preparing these test samples are described below.

(I-1) Soy Pectin 100 g of soybean seed coat was heat-extracted (at 95 ° C., 3 hours) with 1000 mL of a 1.5% aqueous citric acid solution, and pressed and centrifuged. An equal amount of ethanol was added to the obtained centrifugation supernatant, and the mixture was centrifuged, and then the precipitate was recovered and freeze-dried to obtain 5 g of freeze-dried soybean pectin. The molecular weight measured by the method described in the following experimental method (II) was about 500,000.

(I-2) Soy Sauce Polysaccharide Add 1000 mL of ethanol to 500 mL of soy sauce containing soybean and wheat as main raw materials, mix well after leaving to stand overnight, remove the supernatant by decantation, and thoroughly wash with 1500 mL of 66% ethanol. did. The formed precipitate was collected by decantation and centrifugation, ethanol was scattered for about 2 hours at room temperature, and then lyophilized to obtain 25 g of a lyophilizate. Dissolve 20 g of this dried product in about 100 mL of water, and use a dialysis tube (average pore size: 25 Å, molecular weight cut off (MWCO): 12,000 to 16,000, size: 24, width: 32 mm, diameter: 20 mm, film thickness: 30 μm: seamless It was packed in a cellulose tube (Wako) and dialyzed in running water overnight. Two volumes of ethanol was added to the obtained dialyzate and mixed, followed by centrifugation, and the resulting precipitate was freeze-dried to obtain 2.6 g of a freeze-dried product of soy sauce polysaccharide. The molecular weight measured by the method described in the following experimental method (II) was about 25,000.

(I-3) Fibronectin The dimeric fibronectin consisting of two monomer components of about 2500 amino acid residues is linked by a disulfide bond at the C-terminus, and it is the first of the three modules constituting this It has been found that the C-terminal two-third peptide (molecular weight about 7,000) of the type III module is involved in the binding to pectin. Therefore, this peptide is purchased from Abcam Co., Ltd. (Abcam), and amine coupling is performed on the sensor chip of Biacore system (Biacore J (GE Healthcare Co., Ltd.)) in which dextran having a carboxymethyl group introduced is immobilized on a gold film. It was immobilized by the method.

(II) Experimental method
(II-1) Measurement of Molecular Weight The molecular weight of the test sample was measured using gel filtration chromatography (HPLC) under the following conditions. Specifically, glucose (Mw. 180), dextran T10 (Mw. 10500), dextran T20 (Mw. 20400), dextran T40 (Mw. 43500), dextran T70 (Mw. 70000), the molecular weights of which are known as standard products. A calibration curve is created by the same HPLC using dextran T110 (Mw. 105000), dextran T500 (Mw. 487000), and dextran T 2000 (Mw. 2000000), and the molecular weight of the test sample is determined based on the calibration curve. The In addition, Shodex SUGAR KS-805 was used as a column filler for measurement of the molecular weight of soybean pectin and soy sauce polysaccharide.

[HPLC conditions]
Stationary phase: Filler Shodex SUGAR KS-805 (manufactured by Showa Denko KK),
Column inner diameter and length: 8.0 x 300 (mm)
Mobile phase: Distilled water flow rate: 0.5 mL / min
Column temperature: 45 ° C
Detection method: Using a differential refractometer JASCO 830-RI detector.

(II-2) Measurement of the binding ability to fibronectin The evaluation of the interaction (binding ability) of each test sample with fibronectin described above was performed using the Biacore system (Biacore J (GE Healthcare)). Immobilization of fibronectin as a ligand on a sensor chip was carried out by flowing a sample solution containing each test sample (analyte) through the sample flow channel, where fibronectin is N-hydroxysuccinimide (NHS, GE Healthcare) and carbodiimidohydrochloride. A mixture of equal amounts of (EDC, GE Healthcare) is added to the sensor chip CM5 (GE Healthcare) for 6 minutes to NHS activate the carboxy group on the sensor chip surface, and immediately after using 10 mM acetate buffer ( pH 4.5) fibronectin fragment III ₁ -C concentration of 80 [mu] g / mL diluted in (molecular weight of about 7,000, Abcam) solution were coupled by the addition of 6 minutes . Thereafter, 1 M ethanolamine solution (pH 8.5) was added 6 min, and blocking the activated NHS groups remaining on the chip surface was prepared fibronectin immobilized sensor chip.

Experimental Example 1 Evaluation of Binding Ability of Each Test Sample to Fibronectin Each test sample (soybean pectin, soy sauce polysaccharide) was dissolved in distilled water (10 mg / mL), and interaction with fibronectin was performed using the Biacore system under the following conditions ((1) Connectivity) was evaluated.

[Measurement conditions of Biacore]
Running buffer: HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20), pH 7.4
Velocity: Medium
Temperature: 25 ° C
Inject Time: 120 seconds.

As a result, for soybean pectin, binding to fibronectin was confirmed, but for soy sauce polysaccharide, binding was not confirmed. From this, the dissociation constant (K _D ) of soybean pectin was calculated to be 1.13 × 10 ⁻⁷ M.

Experimental Example 2 Binding Ability of Test Sample after Acid Treatment to Fibronectin (1) Using soybean pectin in which binding to fibronectin was found in Experimental Example 1 and soy sauce polysaccharide in which binding was not confirmed, after oral ingestion An experiment was conducted that mimics the internal environment. Specifically, first, soya pectin and soy sauce polysaccharide are dissolved in artificial gastric juice (hydrochloric acid containing 0.2% NaCl, pH 2) so as to be 10 mg / mL, and then using Mini Dialysis Kit (manufactured by GE Healthcare) Then, dialysis was performed against PBS containing calcium (0.1 M) to neutralize. Thus, test samples (soybean pectin and soy sauce polysaccharides) prepared through treatment simulating acid treatment with gastric juice and neutralization treatment with duodenum (a soy pectin and soy sauce polysaccharide) in various concentrations (a: 1 mg / mL, b: 0.5 mg / mL) , C: 0.25 mg / mL, d: 0.17 mg / mL, e: 0.125 mg / mL) after dilution with Biacore running buffer HBS-EP, subject to the Biacore system under the same conditions as in Experimental Example 1, and bind to fibronectin The sex was measured.

  The results are shown in FIG. 1 ((A) soy pectin, (B) soy sauce polysaccharide). In FIG. 1, control means a sample of soybean pectin (A) and soy sauce polysaccharide (B) itself dissolved at a concentration of 1 mg / mL. In addition, in FIG. 1, 120 S and 240 S, respectively after 120 seconds (120 s) and 240 seconds (240 s) after addition start (0 s) of a test sample (analyte) to Biacore system (after dissociation start) The result (RU value) at 120 s) is shown. After 120 seconds (120 s), RU simply indicates the amount of interaction substance present in the flow path of the Biacore system (see reference experiment described later), and the binding amount to the ligand fibronectin after 240 seconds (240 s) It can be evaluated by RU at time point (the same applies to the following experiments).

  As shown in FIG. 1, not only soybean pectin but also soy sauce polysaccharide which did not show binding to fibronectin in Experimental Example 1 showed binding to fibronectin after 240 seconds (240 s) . When acid treatment was performed with sodium chloride-free hydrochloric acid (pH 2) and dialyzed with calcium-free PBS as a neutralization treatment, none of the test samples showed binding to fibronectin (Results) Not shown). From this, it was thought that the presence of sodium and calcium is important for the binding of the soybean-derived water-soluble polysaccharide to fibronectin.

(2) Influence of calcium on the binding ability to fibronectin Instead of the dialysis external fluid (PBS containing calcium (0.1 M)) used in the neutralization treatment performed in (1) above, using distilled water (1 The dialysis was performed in the same manner as in.

  Using the test sample after neutralization treatment thus prepared, the binding to fibronectin was measured using the Biacore system as in (1). The results are shown in FIG. 1 (A: soybean pectin, B: soy sauce polysaccharide).

As can be seen from this result, after acid treatment, the test sample dialyzed with PBS containing calcium (0.1 M) rather than the test sample neutralized by dialysis with calcium-free distilled water (in FIG. ^The fact that the binding to fibronectin is much stronger than in the case of “ ²⁺ ” indicates that the size of the binding of soybean pectin to fibronectin may be greatly affected by the concentration of coexistent calcium.

Experimental Example 3 Effect of Calcium on Binding to Fibronectin In order to confirm the influence of calcium ion on the binding of soybean water soluble polysaccharide to fibronectin, different concentrations of CaCl ₂ solution (0.01 M, 0.05 M, 0.1, M) Soy pectin (10 mg / mL) was dissolved in each and the binding strength to fibronectin was measured using Biacore system.

  The results are shown in FIG.

  As shown by “240S” in FIG. 2, it was confirmed that all soybean pectins have increased binding to fibronectin in a calcium concentration-dependent manner. From this result, it was found that the binding ability (binding ability) of soybean pectin, which is a water-soluble polysaccharide derived from soybean, to fibronectin is enhanced in the presence of calcium ion.

Experimental Example 4 Effects of Alkali Metals and Alkaline Earth Metals on the Bonding Strength of Soy Pectin to Fibronectin Soy pectin is dissolved in 10 M aqueous solution of 0.1 M KCl and 0.1 M MgCl ₂ so as to have various concentrations. After dilution to (a: 1 mg / mL, b: 0.5 mg / mL, c: 0.25 mg / mL, d: 0.17 mg / mL, e: 0.125 mg / mL), the Biacore system was used under the same conditions as in Experimental Example 1. Then, the binding strength to fibronectin was measured. The binding strengths to fibronectin of soybean pectin dissolved in KCl aqueous solution and MgCl ₂ aqueous solution are shown in FIG. 3 (A) and (B), respectively. From this result, it was confirmed that the binding amount of soya pectin to fibronectin is increased in the presence of potassium or magnesium.

Experiment 5 Effect of calcium on binding of soy sauce polysaccharide to fibronectin
(1) Effect of calcium on the binding of acid-treated soy sauce polysaccharide and fibronectin The soy sauce polysaccharide that has become bound to fibronectin by acid treatment is reacted with calcium and then dialyzed with distilled water as an external solution. The binding ability to fibronectin when calcium was removed was examined.

  Specifically, after dissolving soy sauce polysaccharide in hydrochloric acid (pH 2) containing 0.2% NaCl so as to be 10 mg / mL, dialysis was performed using PBS containing calcium as an external solution. The external solution was further replaced with distilled water, and dialysis was continued to remove salts in the solution. After diluting this sample to various concentrations (a: 1 mg / mL, b: 0.5 mg / mL, c: 0.3 mg / mL), the binding to fibronectin as an analyte was examined with a Biacore system. The results are shown in FIG.

  As shown in FIG. 4, soy sauce polysaccharide showed concentration-dependent binding to fibronectin, suggesting that once calcium is incorporated into soy sauce polysaccharide, it is likely not to disappear even by dialysis It was done.

(2) Examination of the need for acid treatment for soy sauce polysaccharides So far, if soy sauce polysaccharides have only been dissolved in distilled water, binding with fibronectin is not observed, but when treated with an acid solution containing 0.2% NaCl, Acid treatment has been considered a necessary operation for binding as it appears to be associated with fibronectin. However, considering that calcium ions greatly affect the binding to fibronectin, the presence of calcium may indicate binding even without acid treatment.

Therefore, prepare a sample solution in which soy sauce polysaccharide is dissolved in 0.1 M CaCl ₂ aqueous solution containing 0.2% NaCl at a concentration of 10 mg / mL, dilute to various concentrations, and then bind to fibronectin with the Biacore system. Examined. As a result, all of them strongly bound to fibronectin, and the dissociation constant was 2.297 × 10 ⁻⁶ M.

  From this, it was suggested that acid treatment is not an essential treatment, but the presence of calcium ion is important in order for soybean-derived water-soluble polysaccharides such as soy sauce polysaccharides to bind strongly to fibronectin.

(3) Effect of soy sauce polysaccharide on fibronectin binding ability in removal of calcium by dialysis 0.1 M CaCl ₂ solution containing 0.2% NaCl (0.2% NaCl / 0.1 M CaCl ₂ ), and 0.2% NaCl Soy oil polysaccharide is dissolved in 0.1 M KCl solution (0.2% NaCl / 0.1 M KCl) to a concentration of 10 mg / mL and dialyzed against distilled water to obtain various concentrations (a: 1 mg) After dilution to / mL, b: 0.5 mg / mL, c: 0.25 mg / mL, d: 0.17 mg / mL, e: 0.125 mg / mL), the binding strength to fibronectin was examined with the Biacore system. The results are shown in FIG. FIG. 5 (A), the preprocessing 0.2% NaCl / 0.1 M sauce polysaccharide dissolved in CaCl ₂ As a result, FIG. 5 (B), soy sauce multi dissolved in 0.2% NaCl / 0.1 M KCl as a pretreatment The results for saccharides are shown respectively. As shown in FIG. 5, the soy sauce polysaccharide was found to bind to fibronectin both in the case of dissolution after dissolution in 0.1 M CaCl ₂ solution and in the case of dissolution after dissolution in 0.1 M KCl solution. The constants were 1.51 × 10 ⁻⁴ M and 9.62 × 10 ⁻⁵ M, respectively.

  From these results, it was shown that both calcium ion and potassium ion were incorporated into the soy sauce polysaccharide molecule and involved in the binding to fibronectin.

Reference experiment Influence of minerals in the solvent on the binding of fibronectin In order to examine how the minerals contained in the test sample affect the measurement of the binding strength to fibronectin, various concentrations of CaCl ₂ solution, KCl solution And MgCl ₂ solutions (all: a: 10 mM, b: 5 mM, c: 0.25 mM) were respectively applied to the Biacore system to examine their binding ability to fibronectin. The results of measuring the binding strength to fibronectin with respect to CaCl ₂ solution, KCl solution, and MgCl ₂ solution over time are shown in FIG. 6 (A), (B) and (C), respectively.

  From the results in FIG. 6 (B), it was confirmed that potassium ion and chloride ion hardly act on fibronectin. From the results of FIGS. 6 (A) and 6 (C), it was confirmed that calcium ion and magnesium ion act on fibronectin from the start of addition (0 s) to 120 seconds, and the binding thereto increases in a concentration-dependent manner However, it was confirmed that no binding was observed after dissociation (after 120 seconds from the start of addition (0 s)). From this, the measurement results at the time after dissociation (240 s) shown in the above-mentioned experimental example are not the metal ion (calcium ion, potassium ion, magnesium ion) contained in the solution, but soy pectin or soy polysaccharide itself It can be said that it shows the binding ability to fibronectin.

  The water-soluble polysaccharide derived from soybean can be used for the above-mentioned application in the form of a water-soluble polysaccharide salt derived from soybean coexisting with an alkali metal and / or an alkaline earth metal, and also an alkali metal ion and / or an alkaline earth Since it has such a property that its binding ability to fibronectin is enhanced in the presence of metal ions, it can be used as a carrier for affinity chromatography to fibronectin. Specifically, since the water-soluble polysaccharide derived from soybean can control the binding strength with fibronectin in accordance with the concentration of the alkali metal ion and / or the alkaline earth metal ion to be coexisted, it is possible to control the binding ability with fibronectin. By changing the ion concentration, it is possible to bind or detach fibronectin to the carrier.

Claims (2)

The soybean such that the proportion of potassium and / or alkaline earth metal contained in the composition containing a water-soluble polysaccharide derived from soybean which is soybean pectin or soy sauce polysaccharide is 6 to 65% by mass in dry matter conversion. The external addition amount of potassium and / or alkaline earth metal per 100% by mass of the final composition is 5 to 60% by mass in terms of dry matter to the water-soluble polysaccharide derived from or enhance cause avidity for full Iburonekuchin the water-soluble polysaccharide, the use of the water-soluble polysaccharide. Using pre Kia alkaline earth metal painter calcium, and is at least one selected from magnesium, according to claim 1.