JPH0445794A - Production of maltopentaose syrup - Google Patents

Abstract

PURPOSE:To obtain in high efficiency the title syrup having polymerization degree higher than that of maltohexaose, with low content of e.g. maltooligosaccharide by acting maltopentaose-productive enzyme followed by alpha-amylase on starch etc. CONSTITUTION:The objective syrup can be obtained by acting (A) maltopentaose-productive enzyme followed by (B) alpha-amylase on (C) at least one substance selected from starch, its compositional fractions and degradation products of starch. The enzyme A has the following characteristics: (1) acting on amylose, soluble starch, potato starch etc. into maltopentaose; (2) stable at 45 deg.C and a pH of 6.5 - 9; (3) optimal temperature at pH 6.5 is 50 - 55 deg.C; (4) deactivated when left to stand at >=55 deg.C for 15min; and (5) inhibited in a solution containing 0.4mM mercuric p-chlorobenzoic acid and 1mM monoiodoacetamide; however, being pref. 40 - 50% in the inhibition rate.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing maltopentaose (hereinafter referred to as G5) starch syrup, and more specifically, a method for producing maltopentaose (hereinafter referred to as G5) starch syrup, and more specifically, a method for producing maltopentaose (hereinafter referred to as G5) starch syrup. G with very low content of malto-oligo or polymer dextrin
5. Concerning a method for producing starch syrup.

[Problems to be solved by conventional techniques and inventions] In recent years,
As research on maltooligosaccharides progresses, enzymes that specifically produce maltooligosaccharides are discovered, and uses for maltooligosaccharides are developed, various proposals have been made regarding methods for producing maltooligosaccharides.

G5 starch syrup is expected to be used not only as a clathrate powdered base material 2 food improver, but also as a raw material for nutritional foods for the sick, infants, and the elderly. This G5 starch syrup has conventionally been produced by treating raw materials such as starch with a 05-generating enzyme. No. 59-44070) is used.

However, G5 starch syrup obtained by this method contains 20% or more of malto-oligosaccharides or polymer dextrins with a degree of polymerization of 06 or higher.

Therefore, ll1i! ! When the temperature is high, the turbidity is also high, which is not desirable as a product.

Therefore, the present inventors conducted research to develop a method for producing G5 starch syrup containing as little as possible malto-oligosaccharides or polymer dextrins having a degree of polymerization of 0.6 or higher.

[Means for Solving the Problems] As a result, it was discovered that the target G5 starch syrup can be obtained by allowing the above-mentioned G5-generating enzyme to act on raw materials such as starch and then acting on α-amylase, and the present invention has been achieved based on this discovery. It was completed.

That is, the present invention is characterized in that a maltopentaose-generating enzyme is applied to starch, a composition fraction of S powder, and a small amount of starch decomposition products, and then α-amylase is applied to the maltopentaose-generating enzyme. The present invention provides a method for producing maltopentaose starch syrup.

In the present invention, various starches can be used as the starch, such as potato, sweet potato, rice, tapioca, corn, waxy corn, sago, and maize. , Those obtained from arbitrary raw materials such as wheat can be used alone or in combination of two or more kinds. Examples of starch composition fractions include amylose and amylopectin. Further, starch decomposition products include, for example, roasted dextrins such as white dextrin, yellow dextrin, and British gum; modified starches such as oxidized starch and low-viscosity modified (by treatment with enzymes, acid 7 mechanical high-speed stirring, etc.) starch; Starch derivatives such as starch ethers and starch esters represented by starch phosphate and starch acetate; physically treated starches such as starch irradiated with radiation or neutron beams, high frequency treatment, or heat treatment; α-starch, etc. I can do it.

These starches may be used alone or in combination of two or more.

Next, as a G5-producing enzyme, for example, Pseudomonas K.
The enzyme produced by O-89,40 (FERM P-7456, see Japanese Patent Publication No. 49955/1983 for the mycological properties of magnolia) can be used. The properties of this enzyme are shown below.

)9 This enzyme acts on amylose, soluble starch, potato starch, sweet potato starch, rice starch, tapioca starch, corn starch, waxy corn starch, sago starch, etc. to produce maltopentaose.

ii) This enzyme is optimally p) 16 to 7 at 45°C and is stable at pH 6.5 to 9.

ij), this enzyme has an optimum temperature of 50 to 50 at pH 6.5.
The temperature is 55°C, and it is deactivated if left at a temperature of 55°C or higher for 15 minutes.

iv) The enzyme is inhibited in 0.4mM mercuric parachlorobenzoate and 1mM monoiodoacetamide solution, but the inhibition rate is 40-50%.

v) The molecular weight of this enzyme is 72,500+2,500 (according to disk gel electrophoresis).

vi), the isoelectric point of this enzyme is pt16. 5 (by ampholine electrophoresis).

In addition, examples of α-amylase include enzymes produced by Bacillus licheniformis (manufactured by Novo Nordisk Bio-Industry, trade name: Termamil).In the present invention, in order to generate G5 more efficiently, Enzymes such as pullulanase can be used together.

The starches used as raw materials for producing C5 starch syrup are liquefied if necessary. For liquefaction, enzyme liquefaction methods using liquefied α-amylase, etc., or acids.

A physical method using an alkali can be applied.

Since the G5 production rate is affected by enzyme concentration, substrate concentration, reaction pH, reaction temperature, etc., optimal conditions should be selected.

G5-generating enzyme is stable in the pH range of 6.5 to 9, and p
By acting at H8-8.5, the G5 production rate increases. Furthermore, the lower the substrate concentration, the higher the G5 production rate, but industrially it is preferably 5 to 20%.

The appropriate reaction temperature is 45 to 50°C, but in the presence of calcium salts, the reaction proceeds at 60°C or lower.The enzyme concentration should be selected so that the reaction is sufficiently carried out, and excessive amounts should be avoided. There is no need to use enzymes.

On the other hand, α-amylase (trade name: Termamil) is generally reacted at a pH of 6 to 6.5, but also works well at a pH of 5 to 8. The action temperature of this enzyme is 95 to 100°C, but in the present invention, 50°C was used.

Therefore, the G5 production reaction according to the present invention has a substrate concentration of 5
~20%, pH 8-8.5. The G5-generating enzyme is allowed to act at a temperature of 50°C, and after inactivation and termination of the reaction at an appropriate stage, α-amylase (trade name: Termamil) is added, p) 16. It is desirable to operate at a temperature of 50°C. In addition, when adding the waikikiri enzyme, there is no restriction on the timing of its addition, and it may be added before or at the same time as the addition of the G5-generating enzyme.

The G5 starch syrup obtained by the present invention contains 50% or more of G5 and less than 0.05 glucose (hereinafter referred to as G1),
Maltose (hereinafter referred to as G2) Maltotriose (
Hereinafter, it will be referred to as G3. ) and maltotetraose (hereinafter referred to as G4) is 40% or more, but the amount of polymeric sugar with a degree of polymerization of 06 or higher is suppressed to 10% or less.

As a result, the properties of G5 starch syrup are improved, and a highly concentrated G5 starch syrup that is clear even at low temperatures is obtained, and its sweetness is slightly increased.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

The C5-generating enzyme used in the Examples was Pseudomonas KO-8940 (FERM P-7456) by the method of Okemoto et al. (^pp1.Microbio1.Biotec
hno1. (1986) 25:137-142), and the culture solution was concentrated by salting out ammonium sulfate and dissolved in a buffer solution to obtain a crude enzyme solution. In addition, the activity of the enzyme was measured by the following method, and the activity was 5210/Mi.

Reduced 2% soluble starch (Merck) 0.5 paper, O,
Add 0.4 d of enzyme solution to 0.4 d of 1M phosphate buffer. Add 1d,
The reaction was carried out at 45° C., and the resulting reducing sugar was measured by the Somogyi-Nerhun method, and the enzyme activity for cleaving a glucoside bond of 1 μm+aol per minute was defined as 1 unit (IU). Also, α
-The activity of amylase (trade name: Termamil) is 37±
0.05℃, calcium concentration 0.0003M, pH 5,
5.26g of starch (manufactured by Merck & Co.) per hour under the conditions of 6.
The enzyme activity that degrades 1 novo α-amylase unit (I
Ntl). The activity of pullulanase was 40±0.
1℃.

p) Is, o, Substrate The amount of enzyme that brings about an increase in reducing power equivalent to 1 μM glucose per minute under the conditions of 0.5% pullulan aqueous solution is 1 pullulanase power unit (PtlN
).

Example 1 18.8 mg of bacterial liquefied α-amylase and 150 sf of water were added to 15 g of corn starch, stirred and liquefied in a boiling water bath, and immediately autoclaved at 120° C. for 15 minutes.
After cooling, the pH was adjusted to 8.4 with an aqueous sodium hydroxide solution, followed by pullulanase a 5 opvN, 1!
:(:, 45 IU of the 5-producing enzyme was added, reacted at 50°C for 9 hours, and then heated at 100°C for 30 minutes to inactivate it. The composition of the sugar produced at this point is shown in Table 1 as Sample 1. show.

Add α-amylase (product name: Termamil 60) to the above reaction solution.
Add 150μf of L) and react at 50°C for 2 hours.
Immediately after the reaction was completed, the pH was adjusted to 4.5 with hydrochloric acid, and the pH was adjusted to 120
The mixture was autoclaved at 0.degree. C. for 15 minutes, and then insoluble matter was removed by diatomaceous earth filtration to obtain C5 starch syrup. The composition of this starch syrup is shown in Table 1 as Sample 2.

Sugar composition (%) GI G2 G3 G4 G5
G6 G6〈 Sashipur 1 Sample 2 1.4 5.3 9.6 7.0 16.1 5.6 7.9 50.5 55.4 4.9 3.2 26.7 6.4 It is clear from the table As shown, the total amount of G6 and 66 or higher polymer moieties was 31.6%, which was reduced to 9.6% by the termamyl reaction. G5 also increased from 50.5% to 55.4%. The sugar composition of the obtained reaction solution was analyzed by high performance liquid chromatography (hereinafter referred to as HPLC). The HPLC analysis conditions are as follows.

Equipment used: LC-4A manufactured by Shimadzu Corporation Column: Aminex Carbohydrate HPX-42A manufactured by Bio-Rad 7.8we (diameter) x 300m Eluent: Distilled water temperature: 80°C Detection: R-Cho Example 2 Waxy corn starch 70g Add 17.5 mg of bacterial liquefied α-amylase and 700 d of water, stir in a boiling water bath, liquefy, immediately autoclave at 120''C for 15 minutes, allow to cool, and add sodium hydroxide aqueous solution to pH 8.45.
Adjusted to. Next, 140 Itl of G5-generating enzyme was added and reacted at 50°C for 5 hours, heated at 100°C for 30 minutes to inactivate, and allowed to cool. The composition of the sugar produced at this point is shown in Table 2 as Sample 3.

Pullulanase 2100PtlN, α-
100μl of amylase (product name: Termamil 60L)
The mixture was added and reacted at 50°C for 5 hours. Immediately after the reaction was completed, the pH was adjusted to 4.5 with hydrochloric acid, and the mixture was autoclaved at 120°C for 15 minutes. Next, insoluble matter was removed by diatomaceous earth filtration to obtain G5 starch syrup. The composition of this starch syrup is shown in Table 2 as Sample 4.

I! Composition (%) GI G2 G3 G4 G5 G6 G
6<Sample 3 0 0.9 1.6
1.829.1 0.7 65.9 sample 4
3.1mM2.4 18.0 8.651.3
2.2 As is clear from Table 4.4, the total amount of polymer moieties of 06 and G6 or higher in the obtained G5 starch syrup is 6.
．． 6%, and low molecular weight sugars mainly containing G5 account for 51.3%.

Example 3 32 grams of bacterial liquefied α-amylase and 400 grams of water were added to 40 grams of waxy corn starch, stirred in a boiling water bath, liquefied, immediately autoclaved at 120°C for 15 minutes, left to cool, and then mixed with 0.05 M) Add 100 d of Lis-HCl buffer,
p) Adjusted to 18.4.

Next, 200 IU of G5-generating enzyme and 120 IU of pullulanase
After adding 0PUN and reacting at 50°C for 4.5 hours, 1
The mixture was heated at 00° C. for 30 minutes to inactivate it, and then allowed to cool. The composition of the cellulose produced at this point is shown in Table 3 as Sample 5, and the HPLC elution curve is shown in FIG.

Add 3N hydrochloric acid to the above reaction solution to adjust the pH to 6, then add 200μ of α-amylase (trade name: Termamil 60L)! After the reaction, the pH was adjusted to 4.5 with hydrochloric acid and autoclaved for 15 minutes at 120°C. Next, insoluble matter was removed by diatomaceous earth filtration to obtain G5 starch syrup. Ta.

The composition of Ni-no-Mizuame is shown in Table 3 as Sample 6.

Moreover, the HPLC elution curve is shown in FIG.

Sugar composition (%) GI G2 G3 G4 [;5 G6
G6<Sample 5 0 6.2 8.4
5.349.7 4.1 26.3 Sample 6
2.812.0 15.9 7.853.4
1.7 As is clear from Table 6.4, the obtained G5 starch syrup contains a total of 8 G6 and 06 or higher polymer moieties.
．． It only contains 1%, and the majority is low-molecular weight, which contains 5334% G5.

[Effects of the Invention] According to the present invention, it is possible to efficiently produce maltopentaose which has a very low content of malto-oligo pulp and polymer dextrin having a degree of polymerization higher than maltohexaose.

The obtained G5 starch syrup is expected to be used not only as a clathrate powder base material 5 food improver, but also as a raw material for nutritional foods for sick people, infants, and the elderly.

[Brief explanation of the drawing]

FIGS. 1 and 2 show HPLC elution curves of the sugar solutions obtained in Examples. In the figure, Gl is glucose and G2 is maltose. G3 is maltotriose, G4 is maltotetraose,
G5 is maltopentaose, G6 is maltohexaose 2, and D is dextrin. Patent Applicant: Shimizu Port Refining Co., Ltd. Director, Food Research Institute, Ministry of Agriculture, Forestry and Fisheries New Technology Corporation Figure Elution Time Figure Elution Time

Claims (2)

[Claims] (1) Maltopentaose, which is produced by treating at least one substance among starch, a compositional fraction of starch, and a decomposition product of starch with a maltopentaose-generating enzyme, and then treating with α-amylase. Method for producing starch syrup. (2) The method according to claim 1, wherein the maltopentaose-generating enzyme has the following properties. i) This enzyme contains amylose, soluble starch, potato starch,
Sweet potato starch, rice starch, tapioca starch, corn starch,
It acts on waxy corn starch, sago starch, etc. to produce maltopentaose. ii) This enzyme has an optimum pH of 6 to 7 at 45°C;
Stable at pH 6.5-9. iii) The optimal temperature for this enzyme is 50 at pH 6.5.
~55°C, and is deactivated if left at a temperature of 55°C or higher for 15 minutes. iv) This enzyme is inhibited in 0.4mM mercuric parachlorobenzoate and 1mM monoiodoacetamide solution, but the inhibition rate is 40-50%. v) The molecular weight of this enzyme is 72,500±2,500 (according to disk gel electrophoresis). vi) The isoelectric point of this enzyme is pH 6.5 (according to ampholine electrophoresis).