JP2610452B2 - Method for producing polymer branched dextrin composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a polymer-branched dextrin composition having excellent emulsification stability and coatability using an immobilized cyclodextrin glucanotransferase adsorbed on an ion exchange material sheet. The present invention relates to a method for producing a product, and this product is useful as an emulsifier or a coating agent in the fields of food, medicine, cosmetics, and the like.

[Conventional technology]

Low-DE dextrin (high molecular weight dextrin), which is slightly decomposed by the action of α-amylase on waxy starch, is superior in fragrance retention or emulsion stability when dried compared to dextrin made from starch other than waxy starch. It is well known. However, the emulsification stability of the polymer-branched dextrin is equal to or less than that of natural gums or decomposed products of modified starch.

On the other hand, it has been known for a long time that cyclodextrin glucanotransferase (hereinafter, referred to as CGTase) acts on starch to produce cyclodextrin and has been put to practical use. Moreover, cyclodextrin produced by the action of CGTase has a cyclic structure and thus has an inclusion ability and is excellent in fragrance retention and emulsifying properties. However, cyclodextrin alone does not have sufficient emulsification stability as compared with natural gum or modified starch. Further, since cyclodextrin is expensive, it is not economical to add cyclodextrin to polymer-branched dextrin.

Conventionally, there is a low-purity cyclodextrin,
It contains oligosaccharides of DE20 or higher and low-molecular-weight dextrins. Oligosaccharides other than cyclodextrins and low-molecular-weight dextrins alone do not have fragrance retention or emulsifying properties. Therefore, the fragrance retention and emulsion stability of the low-purity product are mainly caused only by cyclodextrin.

[Problems to be solved by the invention]

The method of producing cyclodextrin by reacting CGTase with a polymer-branched dextrin in a batch reaction requires a long reaction time and requires a high concentration of a substrate to be added to a high-concentration substrate. As a means for solving such a problem, there is a technique called immobilization of an enzyme. Among them, ion exchange resins are often used as carriers for immobilized enzymes because of their ease of immobilization and little damage to the enzymes. But,
If a high-molecular-weight, high-viscosity substrate solution such as low-DE dextrin that is slightly decomposed by the action of α-amylase on waxy starch is used as the substrate solution, it is difficult to obtain a high flow rate due to the resin structure, and the reaction efficiency is also high. Low. Therefore, CGTa
When se is immobilized on a resin, there is a problem that the utilization efficiency of the enzyme does not increase.

[Object of the invention]

Accordingly, an object of the present invention is to provide a polymer-branched dextrin composition having improved emulsifying power of hyperbranched dextrin by further improving the conventional method for immobilizing enzymes, and having excellent emulsification and coating stability. is there.

[Means for solving the problem]

The present inventors have conducted intensive studies and found that high-viscosity polymer-branched dextrin solution can be passed at a high flow rate by using an ion-exchange material sheet as a CGTase immobilization carrier. The present inventors have found that a cyclodextrin-coexisting polymer-branched dextrin composition having a high emulsifying property can be obtained by a synergistic effect of the cyclodextrin produced by the action and the polymer-branched dextrin, thereby completing the present invention.

 Hereinafter, the present invention will be described specifically.

As the CGTase used in the present invention, any known CGTase can be used. For example, an enzyme produced by Bacillus macerans is known as a typical one.

As the ion exchange material sheet used for the immobilization carrier, known ones, for example, DEAE zetaprep, QAE zetaprep, SP zetaprep manufactured by LKB or equivalents thereof can be used. However, when the isoelectric point of the enzyme is lower than the reaction pH, an anion exchange group must be used, and when the isoelectric point is opposite, a cation exchange group must be used. For example, macerance
In CGTase, weakly basic DEAE zetaprep can be used.

The immobilization is performed by passing the enzyme solution through an ion exchange material sheet cartridge. Basically, it is performed in the same manner as that having a normal ion exchange group. That is, after the exchange group is released with an acid or alkali solution, the exchange group is washed and pretreated. A CGTase solution (activity 0.05 to 17 U / ml, preferably
0.5 to 2 U / ml) at a flow rate SV = 1 to 100, preferably SV = 5 to 15
And let it pass. If activity leaks, the effluent may be recycled and immobilized. In the present invention, the enzyme activity is
The amount of enzyme that produces 1 mg of cyclodextrin per minute under the reaction conditions of pH 6.0 and 50 ° C. using 5% concentration of soluble starch as a substrate is expressed as 1 unit (U). Quantification of cyclodextrin is performed by high performance liquid chromatography using an amino column (Ultron NH2 manufactured by Shinwa Chemical Industry Co., Ltd.), the solvent is acetonitrile / water = 65/35, and the detection is performed with a differential refractometer.

In the immobilized enzyme of the present invention, a polymer-branched dextrin composition having a target cyclodextrin content can be produced by selectively combining the amount of the immobilized enzyme, the substrate concentration, and the flow rate. That is, the substrate solution to be passed is
Use waxy corn starch liquefied so that DE = 10 or less. Starch other than waxy corn starch cannot be used because of low emulsion stability. The means for liquefaction may be an acid treatment or an enzyme treatment. At DE10 or higher, the characteristics as a polymer branched dextrin are lost. Substrate concentration is 1 ~
A concentration of 50%, preferably 5-30% is used. As shown in FIG. 1, the higher the substrate concentration, the lower the amount of cyclodextrin produced. The pH and the reaction temperature may be adjusted under the condition that the enzyme used generates cyclodextrin. For example, in the case of macerans CGTase, the reaction is performed at pH 6 and a reaction temperature of 50 ° C.
The flow rate is set at SV = 100 or less. As shown in FIG. 1, the higher the flow rate, the lower the cyclodextrin content. The greater the amount of immobilized enzyme (up to 2200 U per ml of ion exchange material sheet cartridge), the higher the amount of cyclodextrin produced.

The enzyme-immobilized sheet cartridge has been in continuous operation for one week,
Further, the activity is stable even after refrigeration at 4 ° C. for one month.

The emulsion stability of the polymer branched dextrin composition prepared as described above shows a synergistic effect when the cyclodextrin content is 2% or more. But more than 30% or DE10
As described above, since dextrin is reduced in molecular weight, a synergistic effect is lost. In addition, when the cyclodextrin concentration is low, the synergistic effect is greater than when cyclodextrin and polymer-branched dextrin are simply mixed. Further, the emulsion produced by using the composition prepared according to the present invention as an emulsifier has excellent acid and salt resistance.

[Examples] Examples of the present invention will be described below, but the present invention is not limited thereto.

Example 1. Preparation of immobilized enzyme Weakly basic anion exchange material sheet cartridge (LKB
13400 ml of a Macerans CGTase enzyme solution (pH 6.2, activity 0.91 U / ml) was passed through DEAE Zetaprep 250 (manufactured by K.K.) at a flow rate of SV = 8 to adsorb and immobilize the enzyme to obtain the immobilized enzyme of the present invention. The amount of immobilized activity was 48.4 U per ml of cartridge.

Example 2. Method for producing a polymer-branched dextrin composition using an immobilized enzyme 5% of waxy corn starch liquefied liquid of DE5 (FZ-100 manufactured by Hosei Oil Co., Ltd.) was added to the immobilized enzyme obtained in Example 1 and 20%.
% Solution (pH 6.0) was passed at 50 ° C. at different flow rates (SV = 1-100). The amount of generated cyclodextrin was quantified by HPLC to determine the content of cyclodextrin.

As a comparative example, CGTase was immobilized using a weakly basic anion exchange resin Amberlite IRA-93 (manufactured by Organo). 90 ml of the resin was packed in a column having a diameter of 2 cm and a length of 28 cm, and immobilization was carried out in the same manner as in the case of the above sheet except for the flow rate SV = 3.7. The amount of immobilization activity was 98 U per ml of wet resin. The substrate solution was flowed in the same manner as described above, and the generated cyclodextrin was quantified.

The above results are shown in FIG. In both cases of sheet immobilization and resin immobilization, the cyclodextrin production increased as the flow rate decreased. In addition, as the substrate concentration decreased, the amount of cyclodextrin produced increased. However, when compared at the same substrate concentration and flow rate, the immobilized sheet produced more cyclodextrin than the immobilized resin. That is, although the enzyme activity per unit was half in the sheet immobilization as compared to the resin immobilization, the content of the generated cyclodextrin was twice.

Example 3 Stability of Immobilized Enzyme A liquefied liquid of DE5 waxy cons (FZ-100 manufactured by Hosei Oil Co., Ltd.) was applied to a sheet cartridge (immobilized enzyme of the present invention) on which CGTase obtained in Example 1 was immobilized. 20% concentration, pH 6,
The solution was passed at a temperature of 50 ° C. and a flow rate of SV = 2.5 for one week. After storing this in a refrigerator for one month, the solution was passed again under the same conditions. FIG. 2 shows the cyclodextrin formation content (phase solid content%). All were stable at around 10%.

As a comparative example, a batch reaction was carried out by adding 13 U of the enzyme amount to 50% ml of the above substrate solution. 24 hours after the start of the reaction, the cyclodextrin production content reached a plateau at 9.8%.

The amount of the polymer-branched dextrin composition per enzyme activity is higher than that of the batch reaction (0.77 g / U) by immobilizing the sheet (8.7 g / U).
Above) is more than 11 times higher.

Example 4 Amount of Enzyme Immobilized In the same manner as in Example 1, 130 U, 1300 U, and 6500 U were immobilized on DEAE-zetaprep 15 as the enzyme activity. D. as substrate
E.2 Waxy cons liquefied liquid (APDEX manufactured by Hosei Refinery Co., Ltd.)
50) was passed through at 30% concentration, pH 6, 50 ° C. and SV = 4.3. The cyclodextrin content of the resulting polymer-branched dextrin composition was 2.0%, 5.3%, and 10%, respectively. As the amount of immobilized enzyme increased, the cyclodextrin content also increased.

Test Example 1. Flavor Retention To 3 g of a 30% solution of the polymer-branched dextrin composition (a) containing 10% of cyclodextrin produced in Example 4, 3 mg of vanillin, which is a main component of vanilla essence, was added. This solution was freeze-vacuum dried to obtain 0.9 g of powder.

As a comparative example, polymer-branched dextrin of DE2 (b)
A freeze-dried powder was obtained in the same manner using dextrin (c) of DE12 and DE12 instead of (a).

As an evaluation method of the fragrance retention, the amount of residual vanillin was examined. That is, the powder was dissolved in 2.1 g of water, treated with amylase to decompose dextrin, vanillin was extracted with chloroform and quantified by gas chromatography (GC) to determine the amount of residual vanillin. The recovery rate of vanillin by chloroform after the aqueous solutions (a), (b), and (c) before drying were treated with amylase was 100%.

As a result, the residual rates of vanillin were (a), (b),
(C) 80%, 67% and 43%, respectively.

The GC analysis was performed using a column: 3 mm in diameter x 1 m in length, packing material: FFAP 1% Uniport HP 60/80, temperature: injector 20
The measurement was performed using a 0 ° C. column at 150 ° C. and a detection FID.

Test Example 2. Synergistic effect on emulsifying property 15 ml of soybean white squeezed oil and 15 ml of various aqueous solutions shown in Table 1
The resulting solution was placed in a centrifuge tube having a volume of ml, and emulsified at 10,000 rpm for 2 minutes using an Ultra Disposer LK21 manufactured by Yamato Scientific. This centrifuge tube was centrifuged at 5,000 rpm for 10 minutes, and the emulsification stability was evaluated by the ratio of the remaining emulsified layer (the higher the ratio of the emulsified layer, the better the stability).

As shown in Table 1, the synergistic effect of the polymer branched dextrin having a DE of 10 or less and the cyclodextrin was recognized, but the synergistic effect of the DE12 and the cyclodextrin was not recognized.

Test Example 3. Difference from Mixing Polymer branched dextrin (A) containing 10% of cyclodextrin prepared according to the present invention used in Test Example 1, commercially available pure cyclodextrin (C), polymer branched dextrin of DE5 and ( The mixture (B) obtained by mixing C) was emulsified with the composition shown in Table 2 in the same manner as in Test Example 2 to evaluate the stability. If the cyclodextrin concentration of the solution is
%, A synergistic effect of cyclodextrin and branched polymer dextrin was observed. When the cyclodextrin concentration was 1%, no synergistic effect was observed in the mixture (B), but a synergistic effect was observed in the mixture (A) prepared according to the present invention.

Test Example 4. Acid resistance and salt resistance of emulsification The present invention (A) used in Test Example 3 was added to 5% saline solution or acetic acid-added water adjusted to pH 3.5 or pure water at a concentration of 30%.
Sucrose fatty acid ester (HLB6) was dissolved at a concentration of 1%, and xanthan gum (Echo gum manufactured by Dainippon Pharmaceutical Co., Ltd.) was dissolved at a concentration of 0.25%, and emulsification was carried out in the same manner as in Test Example 2. The emulsification stability was evaluated by the ratio of the remaining emulsified layer after centrifugation at 2000 rpm for 1 minute. The product of the present invention was resistant to acid and salt.

[Effect of the Invention] By using the immobilized enzyme of the present invention, a polymer containing cyclodextrin can be used at an efficiency of 11 times or more compared to the batch method, and at a rate of 4 times or more as high as that of a conventional resin immobilized enzyme. A branched dextrin can be produced and is sufficiently practical.

Furthermore, the polymer-branched dextrin composition produced according to the present invention is not only excellent in flavor retention and emulsification stability as compared with conventional branched dextrins and cyclodextrins, but also has an effect of simply mixing both. Is shown.
In addition, it produces more stable emulsification than natural gums and conventional emulsifiers, and is excellent in acid resistance and salt resistance.

[Brief description of the drawings]

FIG. 1 shows the effect of flow rate and substrate concentration on cyclodextrin production. In the figure, ○ indicates the product of the present invention, and △ indicates that the conventional resin-immobilized enzyme was used. The solid line shows the results at a substrate concentration of 5%, and the broken line shows the results at a substrate concentration of 20%. FIG. 2 shows the stability of the enzyme immobilized on the ion-exchange material sheet of the present invention, and shows the change in cyclodextrin content in the polymer branched dextrin composition.

 ────────────────────────────────────────────────── ─── Continuation of the front page Examiner Hiroshi Ueno (56) References JP-A-61-185196 (JP, A) JP-A-63-44886 (JP, A) JP-A-52-25043 (JP, A)

Claims (2)

(57) [Claims] 1. A polymer-branched dextrin composition, comprising contacting an immobilized enzyme having cyclodextrin glucanotransferase adsorbed on an ion exchange material sheet with a liquefied waxy starch having a glucose equivalent (DE) of 10 or less. Manufacturing method. 2. The method for producing a polymer-branched dextrin composition according to claim 1, wherein the composition contains 2 to 30% by weight of cyclodextrin and 98 to 70% by weight of branched dextrin.