JP2926434B2 - Amylose particles and method for producing the same - Google Patents

Abstract

Novel amylose granules advantageously used in the fields of food products, pharmaceuticals and cosmetics exist in an approximately globular-shape of amylose granule or in a conjugation form consisting of two or more of the amylose granules linked together, and having about 2.10 mu m in diameter or major axis, B type form of starch on powder X-ray diffraction analysis, the number-average molecular weight of about 4,000-7,000 on gel permeation chromatography, and the weight- average molecular weight per the number-average molecular weight of about 1.4-1.7. The granules may be prepared by allowing cyclomaltodextrin glucanotransferase (EC 2.4.1.19) to act on an aqueous solution containing a cyclodextrin or starch. In examples the enzyme is derived from Bacillus stearothermophilas or Bacillus macerans; other sources are mentioned. <IMAGE>

Description

Description: FIELD OF THE INVENTION The present invention relates to amylose particles and a method for producing the same, and more particularly, to amylose particles having a specific shape, X-ray diffraction pattern, and molecular weight, and cyclodextrin or starch. The present invention relates to a method for producing amylose particles, characterized in that cyclomaltodextrin glucanotransferase is allowed to act on an aqueous solution containing to produce amylose particles insoluble in the aqueous solution, and the amylose particles are collected.

[Prior Art] As a method for producing amylose, various methods are known as follows.

(1) TJ Schoch has published the Journal of American Chemical Society, Vol. 64, 29
57 (1942), a complex precipitation method using butanol.

(2) KHMeyer and others have joined the Helvetica Chimica Act
a) Extraction method using warm water reported in Vol. 24, 378 (1951).

(3) A salting-out method using magnesium sulfate disclosed in Japanese Patent Publication No. 32-8675, U.S. Pat. No. 2,822,305 and U.S. Pat.

(4) A hydrolysis method using a starch debranching enzyme such as pullulanase and isoamylase disclosed by Sugimoto et al. In Japanese Patent Publication No. 54-21420.

(5) Hans Bender, Carbohydrate Research Vol.6
5, 85-97 (1978), a method of transglycosylation to a receptor by cyclomaltodextrin glucanotransferase reported in 1978.

However, in the methods (1), (2) and (3), not only the yield from starch is as low as less than 25 W / W%, but also the quality and yield of amylose depending on the type and lot of raw starch. Is not stable, and there is a risk of contamination with amylopectin.

In the method (4), although the yield from starch is as high as 80 W / W% or more, it can be obtained only with a mixture of long-chain amylose and short-chain amylose. Requires a separation step. Further, there is a possibility that undegraded products may be mixed in with the starch debranching enzyme.

In the method (5), the yield is as low as less than 30 W / W%, although the concentration of amylose produced is low and an organic precipitant such as methanol is required for its recovery.

[Problems to be Solved by the Invention] The disadvantages of the conventional method for producing amylose are solved, and there is a strong probability that high-quality amylose particles having a relatively uniform molecular weight and a method for stably and easily producing them at a high yield. Is desired.

[Means for Solving the Problems] The present invention has been made in order to solve the above-mentioned drawbacks. In particular, the present invention provides a novel amylose particle using cyclomaltodextrin glucanotransferase (EC 2.4.1.19). We studied hard to establish the manufacturing method.

As a result, by applying cyclomaltodextrin / glucanotransferase (hereinafter abbreviated as CGT-ase) to a relatively high concentration aqueous solution of cyclodextrin or starch at a relatively low temperature, new amylose particles are formed in the aqueous solution. It was found that a precipitate was formed, and it was found that high-quality amylose particles could be easily produced in high yield by collecting the precipitate, thereby completing the present invention.

It has also been found that the present invention has the following features.

(1) Since insoluble amylose particles are precipitated in the reaction solution, their separation and recovery are easy, and expensive organic precipitants such as methanol and butanol are not required.

(2) It is easy to increase the yield from raw materials to a high yield of 50 W / W% or more.

(3) Difference in raw materials, difference in origin of CGT-ase, working temperature,
The molecular weight of the produced amylose particles can be adjusted by changing conditions such as the amount of action and the time of action.

The raw material cyclodextrin used in the present invention may be any one or a mixture of two or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. In addition, as the cyclodextrin, a commercially available product or a product produced by allowing CGT-ase to act on starch is optional. At this time, if glucose, low molecular weight oligosaccharides and the like coexist in the cyclodextrin, it is desirable to remove as much as possible to act.

In addition, it is also possible to advantageously carry out the action of CGT-ase and starch debranching enzyme on starch to produce cyclodextrin and amylose particles.

Further, the starch used in the present invention may be an underground starch such as potato starch, sweet potato starch, tapioca starch, or a ground starch such as corn starch, wheat starch, rice starch. In order for CGT-ase to act on starch, CGT must be added to starch milk.
-The starch may be gelatinized and liquefied by adding the ase and heating, and if necessary, liquefying the starch with an acid or α-amylase to a low DE, preferably less than DE1, followed by CGT
-Ase may act.

CGT-ase used in the present invention is a known Bacillus stearothermophilus.
s), Bacillus circulan
s), Bacillus such as Bacillus macerans, Klebsi pneumoniae (Klebsi)
An enzyme derived from a microorganism belonging to the genus Klebsiella such as ella pneumoniae is used. In particular, a thermophilic CGT-ase derived from Bacillus stearothermophilus, which can be used even when heating and gelatinizing starch, can be advantageously used. The starch debranching enzyme is pullulanase (EC 3.2.1.4).
An enzyme derived from a microorganism belonging to the genus Aerobacter or Bacillus known as 1) is known as Pseudomonas (Ps) as an isoamylase (EC3.2.1.68).
eudomonas, Flavobacteriu
m) Enzymes derived from microorganisms belonging to the genus Cytophaga can be advantageously used. These enzymes only need to achieve the object of the present invention and need not necessarily be used after purification. Usually, a culture supernatant or a partially purified enzyme thereof is used. In addition, if necessary, it is optionally used by immobilization.

More specifically, in the present invention, CGT-ase is added to an aqueous solution containing about 10 to 50 W / W% of cyclodextrin or starch by adding 1 to 1,000 units per gram of solid matter, at a temperature of about 5 to 100 ° C., and at pH 3 By acting for an appropriate time under the conditions selected from ９9 to ９9, the amylose particles are precipitated and precipitated in the water-soluble manner. When using starch milk, for example, after adding CGT-ase, or CGT-ase and starch debranching enzyme to starch, heating to a temperature of 70 to 100 ° C. to gelatinize and liquefy the starch The temperature is lowered to 60 ° C. or lower, preferably 5 to 50 ° C., and if necessary, CGT-ase is added, and the reaction is allowed to proceed for 5 to 500 hours to precipitate and form amylose particles. The amylose particles can be easily recovered by filtration or centrifugation.

The physicochemical properties of the amylose particles thus obtained were examined. (1) Almost spherical particles exist in a single state or in a state of a plurality of particles, and their diameter or major axis is about 2 to 10 μm. (2) X-ray powder diffraction gives a B-shaped figure of the starch, and (3) gel permeation chromatography has a number average molecular weight (Mn) of about 4,000 to 7,000.
(Average degree of polymerization of glucose is about 25 to 43), weight average molecular weight (Mw) / number average molecular weight (Mn) is about 1.4 to 1.7, (4) iodine color changes from bluish purple to blue, and (5) It was found that β-amylase produced a theoretical amount of maltose.

The amylose particles obtained by the reaction with CGT-ase may be further purified by a method such as repeated dissolution and precipitation. For example, amylose particles are dissolved in water by heating, cooled, precipitated, filtered or centrifuged, and dried to collect white powdery amylose particles. Also,
The obtained amylose particles may be further pulverized or granulated by a known method.

The amylose particles obtained in this manner have good fluidity and poor hygroscopicity in powder form, and have the property of being easily dissolved by heating in water, easily affected by amylase, and easily solidified by cooling.

Utilizing these properties, the amylose particles of the present invention can be used, for example, in the food field as jelly confectionery, rice cake, rice confectionery, bread, cookies, tablet confectionery, powdering, etc., in the pharmaceutical field as powders, tablets, pastes, etc. It is widely used in various fields such as a cosmetics field, and further as a release agent, an antiadhesive, a bulking agent, an excipient, a film material, and the like.

Further, since high-quality amylose particles can be stably produced, it can be advantageously used for clinical tests as a substrate for amylase measurement and resolution of optical isomers as a filler for liquid chromatography.

 Hereinafter, the present invention will be described in detail using experiments.

Experiment 1 Effect of Substrate Concentration on Formation of Amylose Particles CGT-a derived from Bacillus stearothermophilus was added to a substrate solution in which α-cyclodextrin was dissolved at various concentrations.
se (sold by Hayashibara Biochemical Laboratory) is used for 50 hours per gram of cyclodextrin, allowed to act for 20 hours at a temperature of 30 ° C and a pH of 5.5, and the produced amylose particles are collected by centrifugation to recover the yield. (W / W%) was determined. In addition, these amylose particles were transferred to TSK-GEL G4000PW and G300
Using 0PW connected, gel permeation chromatography using Shodex STANDARDP-82 sold by Showa Denko KK as a molecular weight standard substance was performed to determine the number average molecular weight (Mn). The results are shown in Table 1.

From the results in Table 1, it can be seen that the production of amylose particles depends on the substrate concentration, and the higher the substrate concentration, the more the amount of amylose particles produced, and a high yield of 50 W / W% or more at a concentration of 15 W / W% or more. Was. The molecular weight of the produced amylose particles was almost the same.

Experiment 2 Effect of temperature on formation of amylose particles Bacillus.
Amylose particles were collected by allowing CGT-ase derived from Stearothermophilus to act at 50 units per cyclodextrin ram at pH 5.5 for 20 hours at various temperatures. Table 2 shows the measurement results of the yield and molecular weight of the obtained amylose particles.

From the results in Table 2, the production of amylose particles depends on the temperature, and the lower the temperature, the more the amount of amylose particles produced,
50 ° C. or less was preferred. The molecular weight of the resulting amylose particles was affected by temperature.

Experiment 3 Effect of CGT-ase on the production of amylose particles Comparison of α-cyclodextrin and γ-cyclodextrin
CGT-ase derived from Bacillus stearothermophilus or Bacillus macerans was allowed to act on a 25 W / W% solution at 50 units per cyclodextrin ram at pH 5.5 at a temperature of 40 ° C. for 20 hours, and the resulting amylose particles were collected. The rate, molecular weight and iodine coloration were measured. The results are shown in Table 3.

As shown in Table 3, the yield, molecular weight, and iodine coloration of the produced amylose particles are different depending on the type of the substrate and the origin of the enzyme, and the desired amylose particles can be produced by utilizing these differences. become.

 Hereinafter, examples will be described.

Example 1 CGT-ase derived from Bacillus stearothermophilus was added to an aqueous solution of α-cyclodextrin at 25% w / w at a ratio of 50 units per cyclodextrin rum at pH 5.5, 30.
The reaction was performed at 20 ° C. for 20 hours. The produced amylose particles were collected by a centrifuge. This is centrifuged twice with water, and
Overnight to obtain amylose particles in a yield of about 84 W / W%.

Examination of the physicochemical properties of this product revealed the following.

(1) Particle size When examined with a scanning electron microscope at a magnification of 2,000 times, as shown in FIG. 1, almost spherical particles are in a single state.
Alternatively, it was confirmed in a state of multiple bonds, and their diameter or major axis was about 2 to 10 μm.

(2) X-Ray Powder Analysis Control starch and amylose particles of the present invention were examined by an X-ray diffractometer (manufactured by Rigaku Denki Co., Ltd., trade name: Geigerflex RAD-IIB, using Cukα ray) by a powder method. Result is,
As shown in FIG. Is an A-shaped figure of starch, and is a B-shaped figure of starch. Is an X-ray diffraction pattern of the amylose particles of the present invention, and was found to give the same B-form of starch.

(3) Molecular weight When the molecular weight (number average molecular weight (Mn) and weight average molecular weight (Mw) were examined by gel filtration chromatography, the values were 5,300 and 8,000, respectively. At about 1.5, this means a high quality amylose with a narrow molecular weight distribution.

(4) Specific rotation ▲ [α] ²⁰ _D ▼ 163 ° (l = 1, c = 0.9, 0.5N-NaO
H) (5) Infrared absorption spectrum Measured by the KBr tablet method, and the results are shown in FIG.

(6) Coloration of iodine The iodine-potassium iodide solution exhibited blue-violet color, and its maximum absorption wavelength (λmax) was around 570 nm.

(7) Amylase degradation The amylose particles are substantially hydrolyzed by β-amylase derived from sweet potato (Seikagaku Corporation, crystal standard) to produce the theoretical amount of maltose. It is determined to be composed of glucosidic bonds.

Example 2 CGT-ase derived from Bacillus macerans was added to a 25 W / W% aqueous solution of γ-cyclodextrin in an amount of 70 units per cyclodextrin gram, and allowed to act at pH 5.7 and 40 ° C for 20 hours.
The produced amylose particles were washed and dried in the same manner as in Example 1 to obtain amice particles at a yield of about 66 W / W%.

Number average molecular weight (Mn), weight average molecular weight (Mw) of this product
Were 4,700 and 6,500, respectively, the Mw / Mn was about 1.4, and the specific rotation was ▲ [α] ²⁰ _D ▼ 161 °.

Other physicochemical properties of this product were the same as in Example 1.

Example 3 A CGT-ase derived from Bacillus stearothermophilus was added to a suspension containing β-cyclodextrin at 20% w / w in an amount of 200 units per cyclodextrin, and the pH was maintained at 5.7.
After acting at 50 ° C for 4 hours, cool and at 40 ° C for 10 hours,
The resulting amylose particles were allowed to act for 10 hours at 24 ° C. for a total of 24 hours.

The number average molecular weight (Mn) and the weight average molecular weight (Mw) of this product were 5,200 and 7,900, respectively. The Mw / Mn was about 1.5, and the specific rotation was ▲ [α] ²⁰ _D ▼ 163 °.

Other physicochemical properties of this product were the same as in Example 1.

Example 4 A thermophilic CGT-ase derived from Bacillus stearothermophilus was added to 20 W / W% corn starch milk (adjusted to pH 6.0) at a rate of 1 unit per gram of solid matter, and liquefied at 90 ° C. for 20 minutes. . This liquefied liquid was cooled to 70 ° C., and 5 units of CGT-ase was added per gram of solid substance, and reacted for 24 hours to produce cyclodextrin. This reaction solution was maintained at 100 ° C. for 20 minutes to inactivate CGT-ase, then adjusted to pH 4.5, added with 10 units of glucoamylase per solid, and reacted at 55 ° C. for 20 hours. The reaction solution was maintained at 100 ° C. for 10 minutes to inactivate glucoamylase, filtered, decolorized with activated carbon, and concentrated.

The concentrated solution was subjected to column chromatography packed with a sodium-type strongly acidic cation exchange resin according to the method disclosed in JP-B-62-51120 to remove glucose, and to remove α-, β-, γ- The cyclodextrin mixture was obtained in solution with a yield of about 25 W / W%.

This solution was adjusted to a concentration of about 35 W / W%, and CGT-ase derived from Bacillus stearothermophilus was added in an amount of 50 units per cyclodextrin ram, maintained at pH 5.7, and maintained at 65 ° C for 4 hours.
Then, the mixture was cooled and allowed to act at 40 ° C. for 10 hours and at 30 ° C. for 10 hours, for a total of 24 hours. The resulting amylose particles were collected in the same manner as in Example 1, and the yield per cyclodextrin mixture was about 85 W / W%.
I got it.

The number average molecular weight (Mn) and the weight average molecular weight (Mw) of this product were 6,600 and 10,500, respectively. Its Mw / Mn was about 1.6, and the specific rotation was ▲ [α] ²⁰ _D ▼ 166 °.

Other physicochemical properties of this product were the same as in Example 1.

Example 5 Thermophilic CGT-ase derived from Bacillus stearothermophilus was added to 25 W / W% potato starch milk (adjusted to pH 6.0) at 2 units per gram of solid matter, and liquefaction was carried out at 90 ° C. for 20 minutes. Was.
After cooling this liquefied liquid to 55 ° C and adjusting the pH to 5.5, CGT-
ase, isoamylase (available from Hayashibara Biochemical Laboratory Co., Ltd.)
After acting for 3 hours to produce cyclodextrin,
The mixture was cooled and allowed to act at 40 ° C. for 6 hours and at 30 ° C. for 17 hours for a total of 26 hours, and the produced amylose particles were collected in the same manner as in Example 1 to obtain a yield per starch of about 55 W / W%.

Number average molecular weight (Mn), weight average molecular weight (Mw) of this product
Was 5,400 and 9,200, respectively, the Mw / Mn was about 1.7, and the specific rotation was ▲ [α] ²⁰ _D ▼ 163 °.

Other physicochemical properties of this product were the same as in Example 1.

[Effects of the Invention] As described in the text, the novel amylose particles of the present invention are high-quality particles having relatively uniform molecular weights, and are prepared in a reaction solution obtained by reacting cyclodextrin or starch with cyclodextrin-glucanotransferase. Separation and recovery are easy due to precipitation, and expensive organic precipitants such as methanol and butanol are not required.

It is also easy to increase the yield from raw materials to 50 W / W% or more.

Furthermore, it is also very convenient that the molecular weight of the produced amylose particles can be adjusted by changing the reaction conditions such as the difference in the raw materials, the difference in the origin of the CGT-ase, the action concentration and the action amount.

The novel amylose particles thus obtained have good fluidity and poor hygroscopicity in powder form, and have the property of being easily dissolved by heating in water and easily solidified by amylase. By utilizing these properties and being advantageously used as a material for foods, pharmaceuticals, cosmetics, and the like, the industrial significance in these industries is extremely large.

[Brief description of the drawings]

FIG. 1 is an enlarged photograph (2,000-fold) of an example of the amylose particle structure of the present invention, which is taken by an electron microscope. FIG. 2 is an X-ray powder diffraction pattern of starch and amylose particles of the present invention, wherein A is a form of starch, B is a form of starch B, and is an X-ray diffraction pattern of amylose particles of the present invention. . FIG. 3 is an infrared absorption spectrum of amylose particles as an example of the present invention.

Continuation of the front page (56) References JP-B-54-21420 (JP, B1) JP-B-51-46817 (JP, B1) JP-B-54-28457 (JP, B1) Starch Science, 40 [4] ( 1993) p. 375-381 Carbohydr. Res, 65 [1] (1978) p. 85-97 Carbohydr. Res, 101 [2] (1982) p. 279-285 Carbohydr. Res, 78 [1] (1980) p. 147-162 (58) Field surveyed (Int. Cl. ⁶ , DB name) C08B 30/20 CA (STN)

Claims (3)

(57) [Claims] (1) A substantially spherical particle exists in a single state or a plurality of bonded states, and has a diameter or a long diameter of 2 to 2.
X-ray powder diffraction gave a starch B-shaped figure, and the number average molecular weight was determined by gel permeation chromatography.
4,000 to 7,000, weight average molecular weight / number average molecular weight is 1.4 to
Amylose particles which give 1.7 and consist essentially of α-1,4 glucosidic bonds. 2. An aqueous solution containing 10 to 50% w / w of cyclodextrin and containing substantially no other saccharides is reacted with cyclomaltodextrin / glucanotransferase to form insoluble amylose particles in the aqueous solution. A method for producing amylose particles, comprising obtaining the amylose particles according to claim 1. 3. The method for producing amylose particles according to claim 2, wherein the cyclodextrin is prepared from starch.