JPS6012038B2 - Method for producing starch bran with high maltotriose content - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a starch sugar with a high maltotriose content and a method for producing the same, and more particularly, the present invention relates to a starch sugar having a high maltotriose content and a method for producing the same, and more specifically, a starch sugar having a high maltotriose content in the solid content of 60% or more, preferably 75 to 95%. This invention relates to starch sugar and its manufacturing method.

In recent years, as people's eating habits have improved, consumers' tastes in food have diversified, and there is also a tendency to dislike the hassle of cooking, leading to an increase in the number of foods that are highly processed and not overly sweet. Demand for food is increasing.

In order to respond to these changes in consumer tastes, new processing technologies are being developed one after another in the food industry, but the harsh conditions required for these new processing technologies are Due to the lack of processed materials with favorable properties that can withstand these conditions, there is an urgent need to develop these new food processing materials. Starch sugar is an important processing material that has been frequently used in the food industry, especially the confectionery industry, but all of the products currently in practical use have a high content of glucose and maltose, resulting in sweetness. It has the disadvantage that the sweetness of confectionery made using it is increased more than necessary. In addition, starch sugars with low contents of glucose, maltose, etc. are known to be obtained by partially hydrolyzing starch with acids or enzymes. For example, DE3 starch sugar is commercially available as a partial acid hydrolyzate, but this product contains various kinds of starch hydrolysates, from the monosaccharide glucose to the polymer dextrin. However, it is difficult to control the quality of each lot, and it is difficult to pass through a furnace and has a high viscosity, which makes it difficult to manufacture and handle, and it is difficult to say that it has a sufficiently low sweetness.
On the other hand, starch sugar with DEIO ~ 20 obtained by two-step hydrolysis of starch with Q-amylase has recently been put on the market, but this product has a relatively average molecular weight and a high oligosaccharide content. Moreover, there is little polymer dextrin.
It is a desirable processed material with low sweetness. Furthermore, a starch sugar containing 3 to 5 malto-oligosaccharides as a main component has been announced as a product with further improved properties (Japanese Patent Application Laid-open No. 1983-1992).
3584). However, these starch sugars still contain a large amount of sugars of tetrasaccharide or higher, so they have the disadvantage that they cannot be used for special purposes, such as raw materials for producing processed materials for low-calorie, low-sweetness foods. For these reasons, there has been a problem in that conventional starch sugar alone cannot sufficiently satisfy the diversifying demands for processed materials for confectionery.
The present inventors have conducted various studies to resolve the above-mentioned problems with raw materials in the food industry, and as a result, they have succeeded in producing large amounts of maltotriose using the N-A468 enzyme previously obtained from actinomycetes. Succeeded in obtaining starch sugar that contained in
As a result of further research, the present invention was completed.

The main object of the present invention is to provide starch sugar having a maltotriose content of 60% or more in solid content. Another object of the present invention is that the maltotriose content in the solid content is 75 to 9.
The starch content is 5%. Still another object of the present invention is to provide a method for producing starch sugar having a high maltotriose content. Other objects of the invention will become apparent from the description below. According to the present invention, there is provided a starch sugar having a maltotriose content in the solid content of 60.% or more, preferably 75 to 95%. In other words, the starch sugar provided by the present invention has a maltotriose content in the solid content of 60% or more, preferably 75 to 9
5%, and the content of starch hydrolysates other than maltotriose is 40% or less, preferably 5 to 25%. According to the present invention, starch sugar having the composition as described above is produced by the following method.

That is, it is produced by liquefying a solution or suspension of starch or starch-based carbohydrates as desired, then adding Q-1,6-glucosidase and N-A468 enzyme to the solution and hydrolyzing it under heating. . N-A468 used in the present invention
The enzyme is a novel enzyme found in the culture solution of Leptomyces gricius N-A46 Tsukushi (Choken Bacteria No. 2227), and has the following physical and chemical properties.

Properties of the N-A46 enzyme: When this enzyme acts on a starch-based substrate, a large amount of maltotriose is produced.

Since the sugar in the reaction product is almost exclusively maltotriose, this enzyme is considered to be an amylase that hydrolyzes the substrate into maltotriose units. '2} Substrate specificity Converts maltotriose by acting on other oligosaccharides such as maltotetraose, maltobentaose, maltohexaose, and maltoheptaose in starch substrates such as amylose, amylobectin, glycogen, potato starch, and waxy starch. generate.

It does not act on maltotriose, maltose, 8-cyclodextrin, pullulan, etc. {3' Optimum pH and stable pH The optimal pH is pH 5.0 to 6.0 in an enzyme reaction of 40 qo.
0, and the stable pH range is 3.5 to 7.5 in which 90% or more of residual activity is present after 3702 hours of treatment.

'41 Method for measuring titer Add 0.1 of enzyme and 0.4 of 0.1 molar McIlvene buffer, pH 5.5, on 0.5 of 2% soluble starch and react in 40 CO for 15 minutes.

After the reaction, quencher 1' is added to stop the reaction, and the amount of reducing sugar is measured by the Somogyi-Nelson method using the 1 block.
The amount of reducing sugar is determined as maltotriose from a standard curve prepared separately using a maltotriose sample, and the amount of maltotriose in the reaction solution is calculated. Enzyme titer is 1 per hour
The potency to produce maltotriose in the evening is defined as 1 unit. [5- Range of suitable temperature for action When this enzyme is allowed to act for 15 minutes at the optimum pH using soluble starch as a substrate, the amount of maltotriose produced is 45%.
It reaches its maximum at oC and shows more than 90% of the production amount within the range of 40 o'clock to 50 o'clock.

The suitable temperature range for action is 4000-500C under these conditions.
It is. {6} Conditions for inactivation due to pH and temperature This enzyme was treated with 37001 at a pH of 2.5 or 7.5.
The residual activity is approximately 50%, and the activity is gradually lost at pH below 2 or above pH 8.

Note that the buffers used are acetate buffer and Tris buffer. Further, this enzyme has a residual activity of about 10% when heated at an optimum pH of 55°C for 18 minutes, and is almost completely inactivated at 6000°C. '7} Inhibition, activation and stabilization This enzyme is inhibited by cadmium, silver, etc. at a concentration of 0.01 molar, and is inhibited by calcium, tin, magnesium, lead,
It is hardly affected by nickel, strontium, etc.

No ions have been found that help with activation. {8} Purification method The culture solution obtained by performing liquid culture in a jar fermenter was filtered, and this fermentation solution was used as crude enzyme Z solution for purification.

Ammonium sulfate was added to a crude enzyme solution having an activity of 9.2 units per unit while keeping the temperature between 0 and 50 C under ice.
．． 7. Take the precipitate fraction that occurs when saturation is reached, and divide this into 8.
After dissolving in 00' pure water and extracting the active part, the unremoved Z part is removed by centrifugation or the like.

After desalting by dialysis against pure water for 2 days, insoluble matter is further removed, and enzyme powder is obtained by freeze-drying. CM-Sephadex C-50 column (column volume: 7 x 27.5 c) buffered with 1 hM acetate buffer (pH 6.0)
Enzyme powder is added to the solution, and the active fraction is desorbed by a concentration gradient lacquering method using the same buffer containing 0.9% sodium chloride as the final elution solution.

After dialyzing the active fraction against pure water and the same buffer, a CM-Sephadex C-50 column (column volume: 4.3 x 50
2 on the skin), and the active fraction is eluted using a concentration gradient elution method at a salt concentration of 0 to 0.3M. The obtained active fraction was sufficiently dialyzed against pure water and freeze-dried to obtain an enzyme powder, which was then placed on a Sephadex G-100 column (column volume: 2.6 x 3100 c) buffered with the above buffer. , elute the active fraction with the same buffer. This is dialyzed against pure water and then dried by freeze-drying to obtain a purified enzyme powder. This purified enzyme was determined to be single by disk electrophoresis using polyacrylamide gel, and its specific activity was 5750 (
unit/m9Pmtein).

■ Molecular weight (by SDS electrophoresis) approximately 55,000 Stiffness Mobility (by disk electrophoresis) Using 7.5% polyacrylamide gel, 4-tris-glycine buffer (pH 8.3), current 3 hA/gel,
After lacquering with energization of 6 females, stain with Amidoshvaru and 7
Decolorization was performed with % acetic acid.

Mobility was measured with promphenol blue added at the same time as the sample (
The value was calculated assuming that the mobility of BPB) was 1.0. As a result, BPB was 7.8 degrees, and the enzyme was 3.8 degrees, and the enzyme moved to the anode side, so the mobility was 0.49. The N-A468 enzyme having the above-mentioned properties is
It can be manufactured by the method described in No. for example,
Soy flour 2.0%, dry yeast 0.1%, beptone 0.1%
, ammonium sulfate 0.1%, starch 2.0%, salt 0.
A medium containing 2% potassium dihydrogen phosphate, 0.1% potassium dihydrogen phosphate, and 0.2% calcium carbonate was adjusted to pH 6 and 7 and sterilized, and then Streptomyces gricius N-A4 was added to the medium.
Inoculated with 6 pairs of spores or hyphae, 25oo~30oo
If culture is carried out for 2 to 5 days while maintaining the temperature, the desired enzyme will be produced and accumulated in the culture. The accumulated N-A468 enzyme is isolated and purified from the culture as follows.

For example, solids in the culture solution are removed by furnace filtration or centrifugation, and then a salting-out agent such as ammonium sulfate or an organic solvent such as acetone or ethanol is added to the furnace solution or supernatant liquid to remove solids from the culture solution. Proteins such as enzymes are precipitated to obtain a fraction rich in enzyme proteins. Then, after dialyzing this precipitate, CM-
Purification is performed by chromatography using Cephadex C-50 and gel filtration using Cephadex G-50 to obtain a purified solution of N-A46 enzyme. The above purification operation is desirably carried out at a temperature of 5° C. or lower in order to prevent deactivation of the enzyme. Q-1,6 used in the present invention
- Glucosidase of any origin can be used, but in order to make it act at the same time as or before and after the N-A468 enzyme, the action conditions of the enzyme are set to N-A46
It is preferable that the conditions match or approximate the conditions for the 8 enzymes.

Examples of such Q-1,6-glucosidase include pullulanase produced by Aerobacter aerogenes ATCC 8724 and isomylase produced by Pseudomonas amylodermosa. These Q-1,6-
All glucosidases are known enzymes, and their properties, preparation methods, etc. are described in detail in the following publications. That is, for pullulanase produced by Aerobacter aerogenes, Biochemische Zei$
chrjft), 334, 79-95 (1961) and Japanese Patent Publication No. 45-19299-46-37849, and isoamylase produced by Pseudomonas amylodermosa is described in Japanese Patent Publication No. 45-16788-4.
6-28151.

These enzymes are commercially available from Hayashibara Co., Ltd. and can be easily obtained. According to the invention,
The above-mentioned N-A468 enzyme and -1,6-glucosidase can be used not only as purified enzymes but also as crude enzymes. Starch or starch-based carbohydrates are used as the raw material of the present invention. That is, as the starch, any of underground starches such as potato starch and corn starch, or above ground starches such as corn starch and wheat starch can be used. In addition, as the starch carbohydrate, either one with a high degree of polymerization such as amylovectin, or one with a relatively medium or low degree of polymerization such as an intermediate hydrolyzate obtained by treating starch with acid, enzymes, mechanical liquefaction, etc. may be used. I can do it. In addition, when using a high viscosity polymeric substrate such as starch as a raw material, the starch is liquefied using a conventional method, for example, using Q-amylase, and then N-A4 fin enzyme and Q-1,6-glucosidase are applied. It is preferable. The conditions for saccharification according to the present invention vary depending on the quality of the target product, but the following conditions are usually selected mainly according to the physicochemical properties of the N-A468 enzyme. The pH of the reaction solution used is in the range of 4.5 to 7.2, preferably in the range of pH 5.0 to 6.5. The reaction temperature is suitably in the range of 30 to 53 degrees Celsius, preferably in the range of 40 to 50 degrees Celsius. The amount of enzyme used varies depending on the decomposition rate and reaction time, but the amount of N-A46 enzyme used is 200 to 50 per gram of raw material.
0 mountain units, preferably 500 to 2000 units are used. Further, when pullulanase is used, Q-1,6-glucosidase is used in an amount of 5 to 5 units, preferably 10 to 3 units, per 1 g of raw material, and when isoamylase is used, 100 to 5000 units are used per 1 g of raw material. In this case, 1 unit of pullulanase means 1 unit of pullulanase using pullulan as a substrate at pH 5.0 and 1 minute at 30°C.
It refers to the activity of liberating maltotriose of French M, and the activity unit of isoamylase represents the value measured by the method described in Biohimi, Biofis, Acta, 212, 458-469 (1970). That is, 0.5% of rice starch having 1% solubility, 0.1% of 0.9M acetate buffer, and 0.1% of enzyme solution were reacted at 4030 °C for one hour.
{B} Add 0.5 mm of 0.01M iodine-potassium iodide solution to 0.5 mm of the above reaction solution, let stand at room temperature for 15 minutes, then dilute with water to 12.5 mm, layer length / The absorbance at a wavelength of 61 m is measured.

B Blank is performed in the same way for the inactivated enzyme.

The unit is defined as the enzyme power that changes the absorbance by 0.1 in 60 minutes under the above conditions. The concentration of starch or starch-based carbohydrates used as raw materials varies depending on the decomposition rate according to the purpose of the product, the zero action time according to manufacturing requirements, the amount of enzyme, etc.
5% to 30% is used, preferably 10% to 20%.
% is used.

According to the invention, N-A468 enzyme and Q-1,6-
The time required for saccharification using glucosidase varies considerably depending on the saccharification conditions, but is usually 30 minutes to 4 hours.

After the saccharification reaction is thus completed, the saccharification solution is heated to 90°C or higher to deactivate the enzyme.

After decolorizing with activated carbon, this liquid is desalted by passing it through an ion exchange column of ion exchange resin, such as a Monopet ion exchange tower consisting of 1:2 of Amberlite Melon-12 fertilizer and Amberlite IRA-410. By concentrating in a conventional manner, a starch sugar solution containing maltotriose as a main component can be obtained with a yield of 90% or more based on the raw material. The physical properties of the starch sugar obtained by the present invention vary somewhat depending on the composition of the sugar contained in the product, but for example, the properties of the starch sugar obtained in Examples 1 and 3 are as shown in the table below. be.

*Indirect reducing sugar value is determined by hydrolyzing the sample. Let the reducing power measured be glucose. Directly calculate the reducing sugar value from the value expressed as This is the subtracted value.

The starch sugar obtained by the present invention has an extremely high maltotriose content of 60% or more, but the dextrin content is extremely low, so the viscosity of the liquid is not excessively high, and it is easy to handle. It has favorable properties as a processed material, such as the ability to easily obtain products of consistent quality.

Furthermore, starch sugar with low glucose and maltose content is not only suitable as a low-sweet food processing material and for use in the diet of kidney disease patients, but also can be used to create other special food materials, such as The starch sugar of the present invention is very useful as a food processing material because it can also be used to produce low-sweetness and low-calorie food materials. Next, reference examples and examples will be shown to explain the present invention in more detail and concretely.
The present invention is not limited to these examples. Reference example 1 Preparation of N-A46 enzyme Glucose 0.5%, Fluid starch 1.5%, Soybean flour 2%
Dispense 1 medium containing 0.1% dry yeast, 0.2% calcium carbonate, 0.2% sodium chloride, 0.1% beptone, and 0.1% ammonium sulfate into five 500' Erlenmeyer flasks. After sterilization, Streptomyces grissius N
- A468 strain (Shikoken Bacteria No. 2227) was shielded, and 2
After culturing with shaking at 70°C for 7 hours, the culture solutions were combined and filtered.

The total volume of the furnace liquid was 650', the titer was 12.9 units, and the specific activity was 1.
．． Total activity is 8.388 at 57 units/m9protein
It was a unit. Example 1 Soluble starch 10 female and liquefied type Q
．． -Amylase (Nagase Sangyo Sawmill Neospitase K750
Disperse and suspend 300' of skin/g in 100 ml of water to make an emulsion.

This emulsion was gradually dropped into 300 °C of hot water at 85 to 90 °C, and a liquefaction reaction was carried out for about 3 seconds. During the dropping of the emulsion and the liquefaction reaction, the reaction solution is maintained at about 85 qo while being vigorously stirred. After the reaction, the reaction solution was heat-treated in an autoclave for 120,000 minutes to inactivate the enzyme, cooled to 40 qo, and a buffer solution was added to adjust the pH to 5.5.
shall be. This was added to NA prepared in the same manner as in Reference Example 1.
468 enzyme, 6930 mountain brush rank and pullulanase (manufactured by Hayashibara,
4. Add 1 g of crude enzyme powder (200 blood/g). Feeding time 40
Perform the saccharification reaction. After the saccharification is completed, the enzyme is inactivated by heat treatment at a temperature of 90° C. for 3 hours. After the reaction solution was decolorized with activated carbon, it was desalted with an ion exchanger monobet and concentrated to obtain a colorless and transparent starch sugar solution with a high maltotriose content.
The solid content yield of this product was 96.3%. The product composition of this starch sugar solution was confirmed by gas chromatography, and it was found that glucose was 0.9% and maltose was 6%.
．． 5%, maltotriose 89.6%, and oligosaccharides of maltotetraose or higher were 3.0%. Example 2 10 parts of potato starch and 300 parts of liquefied Q-amylase (Neospitase K750 skin/g manufactured by Nagase Sangyo) were added to 1 part of water.
00' to make an emulsion.

This emulsion was gradually dropped into 300 hot water of 85 to 90 mm, and a liquefaction reaction was carried out for about 30 minutes. During the dropping of the emulsion and during the liquefaction reaction, the reaction liquid performs a strong hawking,
After the reaction, the reaction solution was heated in an autoclave at 12,000 ℃ for 30 minutes to starve the enzyme, cooled to 40 qo, and a buffer solution was added to adjust the pH to 5.
．． Set to 0. This was combined with N prepared in the same manner as in Reference Example 1.
-Add 6930 mountain units of A468 enzyme and 1 g of pullulanase (manufactured by Hayashibara, crude enzyme powder 200 melon/g) for 40 hours for 9 hours.
Carry out the saccharification reaction at noon. After the saccharification is completed, the enzyme is deactivated by heat treatment at a temperature of 990° C. for 3 minutes. The reaction solution was decolorized with activated carbon, desalted with an ion exchange resin Monopet, and concentrated to obtain a colorless and transparent starch sugar solution with a high maltotriose content.

The solid content yield of this product was 95.6%. The product composition of this starch sugar solution was confirmed by gas chromatography, and it was found that glucose: 6.0%, maltose: 1%.
8.1%, maltotriose 79.8%, and oligosaccharides of maltotetraose or higher were 0%. Example 3 Partially enzyme-hydrolyzed starch (average degree of polymerization = 15.5) lk9
was dissolved in water for 10 minutes, and then heat-treated by autoclaving (1
20oo, 3 minutes).

After cooling, the pH was adjusted to 5.5, and 90,000 units of N-A46 mother enzyme and 2 females of brulanase (made by Hayashibara, crude enzyme powder 200 melon/g) were added and kept at 40°C for 4 hours.
Perform 0 minute saccharification reaction. After the saccharification is completed, the enzyme is deactivated by heat treatment at a liquid temperature of 95q0 for 3 minutes. The above reaction solution is purified and concentrated in the same manner as in Example 1 to become a colorless and transparent starch sugar solution with a high maltotriose content.

The solid content yield of this product was 90.1%. In addition, the product composition of this starch sugar solution is gask.

Confirmed by chromatographic method, glucose 2.6%, maltose 10.5%, maltotriose 7%
7.9%, and oligobran containing more than maltotetraose was 9.0%. Example 4 A liquefied solution obtained using soluble starch 1 female in the same manner as in Example 1 is adjusted to pH 4.5 using a buffer solution.

Claims (1)

[Claims] 1 Starch or starch carbohydrates are combined with N-A468 enzyme and α-
A method for producing starch sugar with a high maltotriose content, which comprises hydrolyzing it with 1,6-glucosidase.