JP4892727B2 - Enzymatic cross-linking reaction of macromolecular tannins - Google Patents

Description

  The present invention relates to a method for producing a highly viscous solution, gel, and resin by an enzymatic crosslinking reaction of polymer tannin.

  Tannin is a natural product extracted from plant trunks, skins, leaves, fruits, etc., and is an environmentally friendly substance generally used as a tanning agent. Tannin includes pyrogallol-based hydrolyzable tannin and catechol-based condensed tannin. Hydrolyzed tannins have been isolated and identified from many plant materials such as traditional Chinese medicine, helping to be relatively small molecules, and most basic research has been done using only this type of tannin. On the other hand, condensed tannin has not been clearly identified. In 1989, Weinges named proanthocyanidins a substance that produces anthocyanidins when a colorless plant extract is heated with acid. A substance composed of a plurality of flavonoid units having a constituent component of flavan-3-ols was defined as proanthocyanidins. It has been clarified that the substance of many fruit components conventionally called condensed tannins is a proanthocyanidin polymer, and many studies on condensed tannins (proanthocyanidin polymers) have been made.

  The plant used as the raw material of the tannin can be supplied by afforestation or the like, and the afforestation cycle is said to be a 10-year cycle in the case of, for example, Black Watle in South Africa. Therefore, the tannin can be supplied permanently without burdening the global environment like logging of southern timber, and it is also necessary to consider the depletion of resources such as synthetic resin produced from petroleum products. Absent.

  High molecular weight tannin has properties such as antioxidation and active oxygen removal action of natural polyphenol, binding to proteins, binding to heavy metals, binding to alkaloids and aldehydes, antibacterial, antioxidant and deodorant properties It is expected to be used as a material for an antiviral, anti-mite gel, and an adsorption resin for proteins, heavy metals and alkaloids.

  The present inventor has already filed a patent application for a technique for cross-linking with boric acid, phosphoric acid, calcium salt, etc. to create a gel of high molecular tannin (see, for example, Patent Document 1).

  On the other hand, it is known that high molecular tannins and enzymes (proteins) are generally strongly bound to form a precipitate, so that many enzymes are inactivated and do not act (for example, see Non-Patent Document 1).

International Publication No. 2006/088551 Pamphlet Agriculture and Horticulture, Vol. 75, No. 1 (2000), pp. 3-13, Chemistry and utilization of fruit tannins and related compounds [1]

  An object of the present invention is to impart new physical properties to the polymer tannin without using chemicals and to effectively use the polymer tannin as a product friendly to humans and the environment.

  As a result of repeated studies in view of the above problems, the present inventor has succeeded in increasing the viscosity, gelation, and resination of the polymer tannin, thereby completing the present invention.

That is, the gist of the present invention is as follows.
(1) A method for producing a highly viscous solution, gel or resin, comprising treating a polymer tannin aqueous solution with hydrogen peroxide and peroxidase.
(2) The method according to (1), wherein the polymer tannin is condensed tannin.
(3) The method according to (2) above, wherein the condensed tannin is kakitannin.
(4) The method according to any one of (1) to (3) above, wherein 24-48 mM hydrogen peroxide is used per mg of peroxidase.
(5) A highly viscous solution obtained by the method according to any one of (1) to (4).
(6) A gel obtained by the method according to any one of (1) to (4).
(7) A resin obtained by the method according to any one of (1) to (4).

  According to the present invention, a highly viscous solution, gel, and resin that make use of the characteristics of polymer tannin can be prepared under mild conditions.

Hereinafter, the present invention will be described in detail.
Tannins are natural products extracted from plant trunks, skins, leaves, fruits, etc., and are environmentally friendly substances. Tannin includes pyrogallol-based hydrolyzable tannin and catechol-based condensed tannin.

  The polymer tannin used in the present invention is a polyphenol polycondensate extracted from plant trunk, skin, leaves, fruits, etc. with hot water, alcohol, etc., exhibits astringency, strongly binds to many proteins and precipitates. In general, the molecular weight is about 2,000 to about 1,000,000, preferably 10,000 or more. In the present invention, any of hydrolyzed and catechol-based condensed tannins may be used, but catechol-based condensed tannins are preferred. Examples of the high molecular tannin include high molecular tannin contained in heartwood and bark such as quebracho tannin, mimosa tannin and wattle tannin; high molecular tannin contained in immature fruits such as banana, apple and oyster; carob beans, grapes, etc. High-molecular-weight tannin contained in unripe pods and seeds.

  In general, polyphenols such as catechin and the like contained in green tea and black tea called “tannin” are low in molecular weight, have almost no skin tanning action, weakly bind to proteins, have a molecular weight of 290 catechins, and even large ones from 400 500, which is different from the polymeric tannin used in the present invention.

  The tannin is usually dried and used as a powder after extraction from the raw material. In the following examples, the powdered tannin dissolved in water was used as the tannin aqueous solution, but the extract from the raw material may be used as it is as the tannin aqueous solution.

  The raw materials for high molecular tannin (for example, persimmon astringent for oyster tannin) contain organic acids, amino acids, etc. in addition to high molecular tannin. May be.

  In the present invention, the polymer tannin concentration in the polymer tannin aqueous solution is usually 0.5 to 10% by weight, preferably 1 to 4% by weight, more preferably 1.5 to 3% by weight, and the pH is usually 4%. -10, preferably 5-9, more preferably 5.5-7. If necessary, the pH may be adjusted with a buffer solution, an acid such as dilute hydrochloric acid, sulfuric acid, acetic acid or citric acid, or a base such as dilute sodium hydroxide, potassium hydroxide, sodium carbonate or ammonia.

  In the present invention, a highly viscous solution, gel or resin can be produced by treating a polymer tannin aqueous solution with hydrogen peroxide and peroxidase.

The peroxidase used in the present invention is an oxidoreductase that catalyzes the reaction of AH ₂ + H ₂ O ₂ → 2H ₂ O + A (AH ₂ is an electron donor), and is derived from animals, plants, microorganisms, or genetic engineering means. Can be used. In the present invention, peroxidase catalyzes a reaction that oxidizes the phenolic hydroxyl group of polymer tannin using hydrogen peroxide as an oxidizing agent. In a general reaction involving low molecular weight polyphenols, phenolic hydroxyl groups are oxidized by peroxidase to form quinones, and this highly reactive quinone reacts with the surrounding benzene ring and phenolic hydroxyl groups. In high molecular weight tannins, it is speculated that the same reaction occurs mainly, but the details are under investigation. The concentration of peroxidase in the reaction solution is usually 0.01 to 100 mg, preferably 0.1 to 10 mg, per 1 ml of the reaction solution.

  Hydrogen peroxide is usually used as a 0.01-1M aqueous solution. The concentration of hydrogen peroxide in the reaction solution is usually 15 to 100 mM, preferably 20 to 40 mM, per mg of peroxidase.

  The treatment temperature is usually 0 to 70 ° C., preferably 10 to 70 ° C., more preferably 20 to 40 ° C., the optimum temperature is around 30 ° C., and the treatment time is usually 30 seconds to 48 hours, preferably 30 minutes to 24 hours.

  The reaction may be either a stationary reaction or a stirring reaction, but the stationary reaction is preferred in terms of yield.

  By adjusting the concentration of the polymer tannin in the aqueous solution and the degree of crosslinking, a highly viscous solution, gel, or resin of the polymer tannin can be appropriately produced according to the purpose.

  Normally, when the molecules of the polymer tannin in the aqueous polymer tannin solution of several percent or less are lightly crosslinked, the molecular size is further increased, so that the viscosity is increased. In the present invention, the high-viscosity solution refers to a solution that remains as an aqueous solution in a state where gelation or precipitation does not occur.

  It is close to the state of a highly viscous solution, and when the polymer tannins are moderately cross-linked, the whole solution solidifies and maintains its original volume, such as tofu, konjac and agar. The state in which the value remains high is called gel. Cross-linking is a trigger, and it is considered that hydrogen bonds, hydrophobic bonds, ionic interactions, van der Waals interactions, etc. work between molecules and solidify.

  When the concentration of the high molecular tannin is high and the degree of cross-linking between molecules is high, a substance having a low water content and high density is produced. In the present invention, this is called a resin. In many cases, as the crosslinking reaction proceeds, the solubility in water decreases, so water separates and precipitates.

  Various additives can be used for this invention in the range which does not impair the effect of this invention as needed.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, the scope of the present invention is not limited to these Examples.

  In the following Examples, purified oyster tannin powder obtained from Maruzen Pharmaceutical Co., Ltd. (Onomichi, Hiroshima Prefecture) was used as oyster tannin, and horseradish peroxidase was used as peroxidase.

(Example 1) Change in amount of reaction product depending on the amount of hydrogen peroxide Using a purified cachytannin solution, a 0.36M aqueous hydrogen peroxide solution and peroxidase, the reaction was carried out according to the following conditions to produce a reaction product (the resulting precipitate). Alternatively, the amount of dried gel) was measured. The results are shown in FIG.
(A) Tannin concentration: final concentration 4%
(B) Amount of hydrogen peroxide: The final concentration was changed from 0 mM to 96 mM.
(C) Peroxidase amount: 1 mg / 2 ml
(D) Reaction solution: total volume 2 ml
(E) pH: adjusted to 5.8 using a phosphate buffer.

(Example 2) Change in amount of reaction product depending on the amount of peroxidase The same treatment as in Example 1 was conducted except that the final concentration of hydrogen peroxide was 36 mM and the amount of peroxidase was changed from 0.25 mg / 2 ml to 2 mg / 2 ml. did.
The results are shown in FIG.

(Example 3) Change in amount of reaction product due to tannin concentration The same as Example 1 except that the final concentration of hydrogen peroxide was 36 mM and the tannin concentration was changed from 1.25% to 10.0%. Processed.
The results are shown in FIG.

(Example 4) Comparison of reaction product amount by turbidity measurement The following four types of samples having different peroxidase amounts were used in the same manner as in Example 2 to perform gelation or resinification of polymer tannin. The results of following the initial reaction with increasing turbidity are shown in FIG. After filtering the reaction solution, the precipitate or gel on the filter paper was dried, and the dry weight was measured to compare the amounts of reaction products in the following samples 1 to 4.
Sample 1: Peroxidase amount 2mg / 2ml
Sample 2: Peroxidase amount 1 mg / 2 ml
Sample 3: Peroxidase amount 0.5 mg / 2 ml
Sample 4: Peroxidase amount 0.25 mg / 2 ml

(Example 5) Standing reaction and stirring reaction Using a purified oyster tannin solution, 0.36M hydrogen peroxide aqueous solution and peroxidase, the reaction was performed according to the following conditions, and peroxidase was added to the substrate of tannin and hydrogen peroxide. When starting the reaction, the amount of the reaction product when the reaction solution was allowed to react while gently stirring using a rotator and when the reaction solution was allowed to stand without doing anything was compared. The results are shown in FIG.
(A) Tannin concentration: final concentration 1.6%
(B) Amount of hydrogen peroxide: final concentration of 36 mM
(C) Peroxidase amount: 0.5 mg / 3 ml to 2 mg / 3 ml.
(D) Reaction solution: total volume 3 ml
(E) pH: adjusted to 5.6 using 50 mM phosphate buffer.
(F) Reaction temperature: 30 ° C
(G) Reaction time: 1 day

(Example 6) Change in amount of reaction product depending on the amount of hydrogen peroxide Using a purified cachitannin solution, 0.18M aqueous hydrogen peroxide solution and peroxidase, the reaction was carried out according to the following conditions to produce a reaction product (the resulting precipitate). Alternatively, the amount of dried gel) was measured. The results are shown in FIG. In the photograph on the upper right of FIG. 6, the final concentration of the hydrogen peroxide amount is 0 mM (No. 1), 6 mM (No. 2), 12 mM (No. 3), 24 mM (No. 4), 36 mM (No. 5) in order from the left. The state of the reaction product at 48 mM (No. 6) is shown.
(A) Tannin concentration: final concentration 1.6%
(B) Amount of hydrogen peroxide: The final concentration was changed from 0 mM to 50 mM.
(C) Peroxidase amount: 1 mg / 3 ml
(D) Reaction solution: total volume 3 ml
(E) pH: adjusted to 5.6 using 50 mM phosphate buffer.
(F) Reaction temperature: 30 ° C
(G) Reaction time: 1 day

(Example 7) Effect of pH Using a purified oyster tannin solution, 0.18M aqueous hydrogen peroxide solution and peroxidase, the reaction was carried out according to the following conditions, and the reaction product (the resulting precipitate or dried gel) was obtained. The amount was measured. The results are shown in FIG. The photograph in the upper right of FIG. 7 shows the state of the reaction product when the top (number 5) is pH 6 and the bottom (number 6) is pH 7. Gel formation is observed.
(A) Tannin concentration: final concentration 1.6%
(B) Amount of hydrogen peroxide: final concentration 24 mM
(C) Peroxidase amount: 1 mg / 3 ml
(D) Reaction solution: total volume 3 ml
(E) pH: changed to 2-9.
(F) Reaction temperature: 30 ° C
(G) Reaction time: 1 day

(Example 8) Effect of reaction temperature Using a purified oyster tannin solution, 0.18M aqueous hydrogen peroxide solution and peroxidase, the reaction was carried out under the following conditions, and a reaction product (the resulting precipitate or dried gel) The amount of was measured. The results are shown in FIG. The upper right photograph in FIG. 8 shows the state of the reaction product when the reaction temperature is 40 ° C. on the top and the reaction temperature is 30 ° C. on the bottom.
(A) Tannin concentration: final concentration 1.6%
(B) Amount of hydrogen peroxide: final concentration 24 mM
(C) Peroxidase amount: 1 mg / 3 ml
(D) Reaction solution: total volume 3 ml
(E) pH: adjusted to 6.7 using 50 mM phosphate buffer.
(F) Reaction temperature: changed from 30 ° C to 70 ° C.
(G) Reaction time: 1 day

(Example 9) Temperature stability of peroxidase reaction Using a purified cachytannin solution, 0.18M aqueous hydrogen peroxide solution and peroxidase, after peroxidase alone was incubated at each temperature for 1 hour according to the following conditions, Substrates (cachytannin and hydrogen peroxide) were added, and the reaction was carried out at 30 ° C. for 1 day, and the amount of the reaction product (the resulting precipitate or dried gel) was measured. The results are shown in FIG. The photograph in the upper right of FIG. 9 shows the state of the reaction product when the pretreatment temperatures are 40 ° C., 50 ° C., 60 ° C., 70 ° C., and 80 ° C. in order from the left.
(A) Tannin concentration: final concentration 1.6%
(B) Amount of hydrogen peroxide: final concentration 24 mM
(C) Peroxidase amount: 1 mg / 3 ml
(D) Reaction solution: total volume 3 ml
(E) pH: adjusted to 5.8 using 50 mM phosphate buffer.
(F) Pretreatment temperature: changed from 40 ° C to 80 ° C.
(G) Reaction temperature: 30 ° C
(H) Reaction time: 1 day

  The highly viscous solution obtained by the method of the present invention can be used as a paint for paper or wood, or as a dye for yarn or cloth, and can be superior in durability to the case of using a conventional aqueous tannin solution. . In addition, it is expected to be waterproof, insect-proof, bacteriostatic, wear-resistant, deodorizing effect, etc., and can be used as a coating agent for various containers.

  The gel obtained by the method of the present invention can be used as an antibacterial, antioxidant, deodorant, antiviral, and anti-mite gel.

  The resin obtained by the method of the present invention can be used as an adsorption resin for proteins, heavy metals, and alkaloids.

It is a figure which shows the change of the amount of reaction products by the amount of hydrogen peroxide. It is a figure which shows the change of the amount of reaction products by the amount of peroxidase. It is a figure which shows the change of the amount of reaction products by a tannin density | concentration. It is a figure which shows the result of having tracked the initial reaction by the increase in turbidity when performing gelatinization or resinification of high molecular tannin using four types of samples with different amounts of peroxidase. It is a figure which shows the result of having compared the amount of reaction products of stationary reaction and stirring reaction. It is a figure which shows the change of the amount of reaction products by the amount of hydrogen peroxide. It is a figure which shows the influence of pH with respect to a crosslinking reaction. It is a figure which shows the influence of reaction temperature with respect to a crosslinking reaction. It is a figure which shows the temperature stability of a peroxidase enzyme.

Claims (4)

  A method for producing a highly viscous solution, gel or resin, comprising treating a polymer tannin aqueous solution with hydrogen peroxide and peroxidase.   The method according to claim 1, wherein the high molecular weight tannin is condensed tannin.   The method according to claim 2, wherein the condensed tannin is kakitannin.   The method according to any one of claims 1 to 3, wherein 24-48 mM hydrogen peroxide is used per mg of peroxidase.

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