JPH0485301A - Amylose particle and its production - Google Patents

Abstract

PURPOSE:To obtain high-quality amylose particles excelling in flow, being difficultly water-absorptive and being useful as the material of foods, pharmaceuticals, cosmetics, etc., by imparting specified properties thereto. CONSTITUTION:Amylose particles wherein substantially spherical particles exist in a state of a separate particle or of an agglomerate composed of a plurality of separate particles, and which have a diameter or major diameter within a range of about 2-10mu, give the pattern of B-starch when analyzed by powder X-ray diffraction, and have a number-average molecular weight of about 4000-7000 when determined by gel permeation chromatography and the weight-average molecular weight to number-average molecular weight ratio of about 1.4-1.7.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to amylose particles and a method for producing the same, and more particularly, the present invention relates to amylose particles having a specific shape, X-ray diffraction pattern, and molecular weight, and cyclodextrin or The present invention relates to a method for producing amylose particles, which comprises applying cyclomaltodextrin glucanotransferase to an aqueous solution containing starch to produce amylose particles insoluble in the aqueous solution, and collecting the amylose particles.

[Prior Art] Various methods for producing amylose are known as shown below.

(1) T, J, 5choc
h) is the Journal of American Chemical Society.
Chemical 5ociety) Vol, 64
．． 2957 (1942), the night coalescence precipitation R method using butanol.

(2) K. H. Meyer (2) 1. Meyer
) etc. are Helveti force, Kimi force, Acta () level
ticaChimica Acta) Vol, 24.
378 (1951), an extraction method using hot water.

(3) Bus etc. are disclosed in Japanese Patent Publication No. 32-8975, U.S. Patent No. 2,822,305 and U.S. Patent No. 2,
Salting out method using magnesium sulfate disclosed in No. 829,990.

(4) A hydrolysis method using debranching enzymes such as pullulanase and isoamylase, which is disclosed by Sanki et al. in Japanese Patent Publication No. 54-21420.

(5) Hans Benderl (Hans Benderl), Force-Bohydrate Research (Carbohydrate Research)
ateResearch) Vol, 65.85-97
(197g), a method of glycosyltransfer reaction to receptors using cyclomaltodextrin glucanotransferase.

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

Although the method (4) has a high yield of 80 W/W% or more from starch, it can only be obtained as a mixture of long-chain amylose and short-chain amylose, and usually the complicated process of combining long-chain amylose and short-chain amylose is difficult. Even though a separation process is required, there is a risk of contamination of unfractionated #* by starch cutting enzymes.

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

[Problems to be solved by the invention] There is a strong need to establish high-quality amylose particles with relatively uniform molecular weights and a method for producing them stably, easily, and at high yields by eliminating the drawbacks of conventional amylose production methods. desired.

[Means for Solving the Problems] The present invention was made to solve the above-mentioned drawbacks, and in particular, by using cyclomaltodextrin glucanotransferase (EC2, 4, 1, 19). , conducted intensive research with the aim of establishing a new amyas particle and its manufacturing method.

As a result, cyclomaltodextrin glucanotransferase (hereinafter abbreviated as CGT-ase) was added to a relatively highly concentrated aqueous solution of cyclodextrin or starch.

) was found to form a precipitate in the aqueous solution by reacting it at a relatively low temperature, and it was discovered that by collecting the precipitate, high-quality amylose particles could be easily produced at a high yield. , completed the invention.

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

(Since insoluble amylose particles are precipitated in the 1-N reaction solution, their separation and recovery are easy, and expensive organic precipitants such as methanol and butanol are not required.)
0 (2) It is also easy to make a high yield vehicle with a yield of 50 W/W% or more from raw materials.

(3) The molecular weight of the produced amylose particles can also be adjusted by changing conditions such as differences in raw materials, origins of CG T-ase, action temperature, action amount, and action time.

The raw material cyclodextrin used in the present invention may be any one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, or a mixture of two or more thereof. Furthermore, as the cyclodextrin, it is optional to use a commercially available product or one produced by acting CGT-ase on starch. At this time, if glucose, low-molecular oligosaccharides, etc. coexist with the cyclodextrin, it is desirable to remove them as much as possible before the cyclodextrin acts.

It is also advantageous to cause CG T-ase and starch debranching enzyme to act on starch to generate cyclodextrin and amylose particles.

In addition, the starch used in the present invention includes potato starch, horsetail starch,
It may be underground starch such as tapioca starch, or above ground starch such as corn starch, wheat starch, rice starch. In order to cause CG T-ase to act on starch, CG T-ase may be added to starch milk and heated to solidify and liquefy the starch.If necessary, starch may be treated with acid or α-amylase in advance. And low DE.

Desirably, CGT-ase after liquefaction to less than DE1
may be applied.

The CGT-ase used in the present invention is a well-known Bacillus stearothermophilus (Bacillus stearothermophilus).
Bacillus circulans
), Bacillus macerans
Bacillus species such as Bacillus cerans, Klebsiella pneumoniae
Enzymes derived from microorganisms belonging to the genus Klebscilla, such as AEL, are used. In particular, thermophilic CG T-ase derived from Bacillus stearothermophilus, which can also be used to heat and gelatinize starch, can be advantageously used. In addition, starch debranching enzymes include pullulanase (EC3, 2, 1,
41) known as Aerobacter
Enzymes derived from microorganisms belonging to the genus Bacillus, Bacillus, etc. are used as isoamylases (EC3, 2, 1, 68).
Known Pseudomonas (Pseudo+5onas)
Genus, Flavobacterium
ium I genus, Cytophaga
Enzymes derived from microorganisms belonging to the genus can be advantageously used. These enzymes do not necessarily need to be purified before use, as long as they can achieve the purpose of the present invention, and culture supernatants or partially purified enzymes thereof are usually used. Moreover, it is also optional to use it after immobilization, if necessary.

More specifically, in the present invention, CG T-ase is added to an aqueous solution containing about 10 to 50 W/W% of cyclodextrin or starch in an amount of 1 to 1 per solid duram.
, 000 units, temperature of approximately 5 to 100° C., and pH of 3 to 9 for an appropriate period of time, amylose particles are deposited in the aqueous solution to form a precipitate.

When using starch milk, for example, CGT-
Add T-ase or CG T-ase and starch debranching enzyme and heat to 70 to 100°C to gelatinize the starch.
After liquefaction, cool to 60°C or less, preferably 5 to 50°C, add CG T-ase as necessary,
The reaction is allowed to proceed for ~500 hours to precipitate amylose particles. The amylose particles can be easily recovered by filtration or centrifugation.

When we investigated the physical and chemical properties of the amylose particles obtained in this way, we found that (1) almost spherical particles were in a single state;
or exists in a plurality of bonded states, and their diameter or major axis is about 2 to 10 μ-; (2) powder x W1 diffraction gives a B-shaped starch pattern; and (3) gel permeation chromatography shows that the number average molecular weight is (Mn) or about 4,00
0 to 7,000 (approximately 2 as the average degree of polymerization of glucose)
5 to 43), the weight average molecular weight (Mw)/number average molecular weight (Mn) is about 1.4 to 1.7, (4) the iodine coloration is bluish-purple to blue, and (5) the β-amylase It was found that the reaction produced a theoretical amount of maltose.

It is also optional to further purify the amylose particles obtained by the reaction using CG T-ase by repeating dissolution and precipitation. For example, amylose particles are heated and dissolved in water, cooled, and precipitated. The mixture is filtered or centrifuged, and dried to collect white powdery amylose particles.

It is also optional to further pulverize or granulate the obtained amylose particles by a known method.

The amylose particles obtained in this manner have good fluidity and low hygroscopicity in powder form, and when dissolved by heating in water, they are easily affected by the action of amylase and easily solidify when cooled.

Utilizing these properties, the amylose particles of the present invention are
For example, jelly sweets, mochi sweets, rice crackers, bread, kutski,
It is used in the food field as tablets, powder, etc., in the pharmaceutical field, cosmetics field, etc. as powder, tablets, paste, etc.
It is widely used in various fields as a mold release agent, anti-adhesion agent, filler, excipient, film material, etc.

Furthermore, since high-quality amylose particles can be stably produced, it can be advantageously used as a substrate for amylase measurement in clinical tests.

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

Experiment 1 Effect of substrate concentration on the production of amylose particles CGT derived from Bacillus stearothermophilus was added to a substrate solution containing α-cyclodextrin dissolved in various concentrations.
-ase (sold by Hayashibara Biochemistry Research Institute) at 50 units per cyclodextrin ram, temperature 30℃, pH
5.5 for 20 hours, the produced amylose particles were collected by centrifugation, and the yield i (v/W%) was determined.

In addition, this amigosu particle is used as TS sold by Tosoh Corporation.
K-GEL G40QOP and G3000P were used in conjunction and subjected to gel permeation chromatography using 5hodex 5TANDARDP-82 sold by Showa Denko Co., Ltd. as a molecular weight standard to determine the number average molecular weight (fan). Result is.

It is shown in Table 1.

From the results shown in Table 1, the production of amylose particles depends on the substrate concentration, and the higher the substrate concentration, the greater the amount of amylose particles produced. there were. The molecular weight of the generated amylose particles is
were almost the same.

Experiment 2 Effect of temperature on the formation of amylose particles CGT-ase derived from Bacillus stearothermophilus was added to a 25v/su% solution of α-cyclodextrin at 50 units per cyclodextrin gram at various temperatures at pH 5.5. After 20 hours of action, amylose particles were collected.

Table 2 shows the measurement results of the yield and molecular weight of the obtained amylose particles.

Table 2 Experiment 3 Comparison of CGTase sources that support the production of amylose particles 25v/so% of α-cyclodextrin and γ-cyclodextrin? CGT-ase derived from Bacillus stearothermophilus and Bacillus macerans in the volume + Pi
50 units per cyclodextrin gram, pH 5.5
, Ar was applied at 14° C. for 20 hours, and the yield, molecular weight, and iodine coloration of the obtained amylose particles were measured. The results are shown in Table 3.

From the results in Table 2, the production of amylose particles is dependent on temperature, and the lower the temperature, the greater the amount of amylose particles produced.
The temperature was preferably below 0°C. The molecular weight of the produced amylose particles was influenced by temperature.

As shown in Table 3, depending on the type of substrate and the origin of the enzyme,
The yield, molecular weight, and iodine coloration of the produced amylose particles are different, and by taking advantage of these differences, the desired amylose particles can be produced! ! It becomes far more possible.

Examples will be described below.

Example 1 CGT-ase derived from Bacillus stearothermophilus was added to a 25% aqueous solution of α-cyclodextrin at a rate of 50 units per cyclodextrin,
The reaction was carried out at pH 5, 5, and 30° C. for 20 hours. The generated amylose particles were collected using a centrifuge. Add this with water 2
The mixture was washed by centrifugation and vacuum dried at 40° C. overnight to obtain amylose particles with a yield of about 84 W/W%.

The physical and chemical properties of this product were investigated and found to be as follows.

(1) Particle size When examined using a scanning electron microscope at a magnification of 2,000 times, as shown in Figure 1, almost spherical particles could be confirmed in a single state or in a multiple bonded state. Their diameter or major axis was approximately 2-10 μI.

(2) Powder X-ray Analysis The control starch and the amylose particles of the present invention were examined by the powder method using an XS diffractometer (manufactured by Rigaku Denki Co., Ltd., trade name: Geigerflex RAD-II B 1CuKα rays). The results are shown in Figure 2. ■ is the A-shaped figure of starch,
(4) is the B-shaped pattern of starch. ■ is an X-ray diffraction pattern of the amylose particles of the present invention, and it was found that it gives the same B-shaped pattern of starch as in (■).

(3) Molecular Weight When the molecular weight (number average molecular weight (Mn) and weight average molecular weight (My)) was examined by gel filtration chromatography, the values were 5, 300 and g, ooo, respectively.

Therefore, its My/Mn is about 1.5, which means that it is a high quality amylose with a narrow molecular weight distribution.

(4) Specific optical rotation [a] 20163° (l = 1, c = 0.9.0.5t NaOH) (5) Infrared absorption spectrum determined by KBr tablet method, and the results are shown in Figure 3. .

(6) Iodine coloring The sample was colored bluish-purple with an iodine-potassium iodide solution, and its maximum absorption wavelength (λl1aXl) was around 57 Onm.

(7) Amylase decomposition Since it is hydrolyzed by sweet potato-derived β-amylase (Seikagaku Corporation, crystal specimen) to produce a theoretical amount of maltose, the present amylose particles are substantially α-1, It is thought to consist of 4 glucoside bonds.

Example 2 70 units of Bacillus macerans-derived CGT-ase per cyclodextrin gram was added to a 25W/W% aqueous solution of 7-cyclodextrin, and the mixture was 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 amylose particles with a high yield of 66%.
Obtained in W/W%.

Number average molecular weight (Mn), weight average molecular weight (My) of this product
are 4 and 700.6,500 respectively, and their My/
Mn was about 1.4.

Other physical and chemical properties of this product were the same as in Example 1.

Example 3 CG T-as derived from Bacillus stearothermophilus was added to a suspension containing 20%/V of β-cyclodextrin.
Add 200 units of e per cyclodextrin ram, add p
After maintaining the temperature at H5.7 and reacting at 50°C for 4 hours, cooling, and reacting at 40°C for 10 hours and 30°C for 10 hours, for a total of 24 hours, the produced amylose particles were treated as Example 1. A similar sample was obtained with a yield of 60i+l/l/%.

Number average molecular weight (Mn), weight average molecular weight (My
) are 5 and 200.7.900 respectively, and their My/
Mn was about 1.5.

Other physical and chemical properties of the ice crystals were the same as in Example 1.

Example 4 20W/W% corn, ::LM milk powder (p) 16. OL
= Adjustment), added 1 unit of thermophilic CG T-ase derived from Bacillus stearothermophilus per solid duram,
Liquefaction was carried out at 90°C for 20 minutes9, and this liquefied liquid was heated to 70°C.
The mixture was cooled to 0.degree. C., 5 units of CG T-ase was added per solid duram, and the reaction was allowed to proceed for 24 hours to produce cyclodextrin. This reaction solution was maintained at 100°C for 20 minutes to
G T-ase was inactivated, then the pH was adjusted to 4.5, glucoamylase was added at 10 units per solid, and 55
Incubate at ℃ for 200 hours. This reaction solution was maintained at 100° C. for 10 minutes to inactivate glucoamylase, filtered, decolorized with activated carbon, and concentrated.

This concentrated solution was subjected to column chromatography packed with a sodium-type strongly acidic cation exchange resin according to the method disclosed in Japanese Patent Publication No. 62-51120 to remove glucose and to remove α-β-17-cyclodextrin. The mixture was obtained in solution with a yield of 25 W/IJ%.

This solution was brought to a concentration of approximately 35 W/W%, 50 units of CG T-ase derived from Bacillus stearothermophilus was added per cyclodextrin gram, the pH was maintained at 5.7, and the mixture was heated at 65°C for 4 hours and then cooled. The mixture was allowed to react at 40° C. for 10 hours and at 30° C. for 10 hours for a total of 24 hours, and the produced amylose particles were collected in the same manner as in Example 1 to obtain a yield of 85 W/W% based on the cyclodextrin mixture.

The number average molecular weight of ice crystals is 104nl, the weight average molecular weight (My
) are 6,600.10,500 respectively, and their My/
)4n was approximately 1.6.

Other physical and chemical properties of ice crystals'i! In other words, it was the same as in Example 1.

Example 5 25W/W% Potato II powdered milk (pH adjusted to 6.0) Thermophilic CG T derived from Nihatillus stearothermophilus
-ase was added at 2 units per solid duram, and at 90 tons, 2 units
Liquefaction was performed for 0 minutes. This liquefied liquid was cooled to 55°C,
P) After adjusting to 15.5, add 100 units of each of CGT-ase1 isoamylase (sold by Hayashibara Biochemical Research Institute Co., Ltd.) per solid duram, let it work for 3 hours, and do not generate cyclodextrin. Cool, 40
℃ for 6 hours and 30t' for 17 hours, a total of 26 hours, and the generated Amyloaf particles were collected in the same manner as in Example 1.
A yield of about 55% starch was obtained.

Number average molecular weight (Mn), weight average molecular weight (Mv) of ice crystals
) are 5, 400.9, 200 respectively, and their My
/Mn was about 1.7.

Other physical and chemical properties of the ice crystals were the same as in Example 1.

Effect of the invention 1 As stated in the main text, the novel amylose particles of the present invention are
They are high-quality particles with a relatively uniform molecular weight, and are easy to separate and recover because they form a precipitate in a reaction solution in which cyclodextrin or starch is reacted with cyclodextrin glucanotransferase.
Organic precipitants such as butanol are not required.

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

Furthermore, by changing the reaction conditions such as differences in raw materials, differences in the origin of CG T-ase, working concentration, and working amount,
It is also very advantageous to be able to adjust the molecular weight of the amylose particles produced.

The new amylase particles obtained in this way have good fluidity and low hygroscopicity in the powder form, and when heated and dissolved in water, they are easily affected by the action of amylase and solidify by cooling. has.

Utilizing these properties, they can be advantageously used as materials for foods, medicines, cosmetics, etc., and therefore have great industrial significance in these industries.

[Brief explanation of drawings]

FIG. 1 is an enlarged photograph (2,000 times magnification) of amylose particles taken by an electron microscope as an example of the present invention. Figure 2 is a powder X-ray diffraction diagram of starch and amylose particles of the present invention, where ■ is the A-shaped pattern of starch, ■ is the B-shaped pattern of starch, and ■ is the X-ray diffraction pattern of the amylose particles of the present invention. It is a diagram. FIG. 3 shows an infrared absorption spectrum of amylose particles as an example of the present invention.

Claims (2)

[Claims] (1) Approximately spherical particles exist in a single state or in a plurality bonded state, and their diameter or major axis is about 2 to 10
Pm, the B-shaped pattern of starch is obtained by powder X-ray diffraction, and the number average molecular weight is approximately 4,000-7,000 by gel permeation chromatography, and the weight average molecular weight/number average molecular weight is approximately 1.4-1. Amylose particles characterized by giving 7. (2) A process for producing amylose particles, which comprises making amylose particles insoluble in the aqueous solution act on an aqueous solution containing cyclodextrin or starch to produce amylose particles that are insoluble in the aqueous solution, and collecting the amylose particles. Production method.