JPH0427818B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Field of Application) The present invention relates to a method for producing a syrup containing a large amount of branched oligosaccharide, which is used as a sweetener, particularly a sweetener having an anti-caries effect. (Prior Art) Recently, sweeteners such as sugar have tended to be avoided from the viewpoint of preventing tooth decay, and branched oligosaccharides such as isomaltose have begun to attract attention as an alternative sweetener. However, as a method for industrially and economically mass-producing these branched oligosaccharides has not yet been established, isomaltose and the like are currently only sold as expensive reagents and are not used in foods. has not been reached yet. Conventionally, the method for producing branched oligosaccharides has been to use polymeric polysaccharides such as pullulan and dextran with many α-1,6 bonds as substrates, hydrolyze them with appropriate enzymes or acids, and then separate the molecules. Fractionation methods such as fractional chromatography and ion exchange chromatography have been attempted. However, pullulan, dextran, etc. are very expensive, and this method is not suitable for industrial production. Furthermore, it has been known that in the process of producing glucose, glucoamylase and the like cause a retrosynthesis reaction of glucose as well as a saccharification effect, producing small amounts of so-called branched oligosaccharides such as isomaltose and panose. The amount of branched oligosaccharides produced is usually around 5% of the sugar solid content, but from the perspective of glucose production, the production of branched oligosaccharides is not desirable, and therefore efforts are being made to suppress their formation as much as possible. It has been paid. It is also known that when starch is hydrolyzed with acid at high temperatures, a retrosynthetic reaction occurs and sugars having α-1,6 bonds are produced. However, in this case, the conversion rate to branched oligosaccharides is extremely low, and the conditions are extremely harsh, so there are many products due to side reactions. Therefore, there is almost no possibility that this method can be practically used as a means for producing branched oligosaccharides. (Problems to be Solved by the Invention) The present invention, in view of the usefulness of branched oligosaccharides having an anti-caries effect, in the current situation where an industrial production method for branched oligosaccharides has not yet been established as described above,
The object of the present invention is to provide an industrial method for producing branched oligosaccharides. (Means for Solving the Problems) As a result of repeated studies on the industrial production of branched oligosaccharides, the present inventors have discovered an extremely economical production method based on enzyme transfer reaction. The outline of the present invention is to reduce the branched oligosaccharide content in the carbohydrate solids by allowing β-amylase, starch debranching enzyme, and an enzyme having a sugar transfer action to coexist on a starch liquefied liquid adjusted to DE5 to 15. After producing a branched oligosaccharide in which the isomaltose content is 40% or more and the ratio of isomaltose in the branched oligosaccharide is 50% or more, glucose and other types other than branched oligosaccharides are separated and separated by an appropriate method if necessary. It is to be removed. To obtain syrup with the above branched oligosaccharide composition, β
- There is a method in which amylase (EC3.2.1.2.), a starch debranching enzyme, and an enzyme that has a saccharide transfer effect are used in coexistence. In the present invention, it has been found that glycosyltransferase can be made to act on the starch liquefaction liquid simultaneously with β-amylase, starch debranching enzyme, etc. In conventional research using maltose as a substrate for the transfer reaction, in order to generate a large amount of branched oligosaccharides, it is necessary to generate maltose before adding the transferase. It was thought that However, contrary to this conventional understanding, the present inventors have found that by simultaneously acting on each enzyme in the starch liquefaction solution, starch hydrolysis and transfer reaction proceed in a well-balanced manner, and branched oligosaccharides can be obtained in good yield. I discovered that it can be done. In this case, the reaction conditions include adding β-amylase such as a soybean enzyme preparation (manufactured by Nagase Sangyo Co., Ltd., pure β-amylase, etc.) to a starch liquefied solution with a DE of 5 to 15 per solid content of the substrate. 1.2% (w/w), as a debranching enzyme, for example, commercially available bacterial pullulanase (manufactured by Novo, Promozyme 200L, etc.) is used at 0.3 to 0.8% (w/w) per solid substrate content, an enzyme with glycosyl transfer activity. For example, Aspergillus niger α-glucosidase ((EC3.2.1.20) 0.03~
11U/g・Substrate coexisting, pH4.0~
6.0, it is optimal to react at 50-65℃. The production of branched oligosaccharides occurs from 1 to 1 after the addition of transferase.
It peaks within two days and then gradually decreases. The sugar solution containing branched oligosaccharides obtained in this way has a branched oligosaccharide content of 40% in the carbohydrate solid content.
In addition, the ratio of isomaltose in the branched oligosaccharide is 50% or more, and it can be used as a branched oligosaccharide syrup as it is, or if necessary, it can be further processed to make a highly concentrated branched oligosaccharide syrup. can. The method for this is
Examples include: (1) A method of adding salt to a sugar solution to crystallize and remove glucose-salt double salt crystals. (2) A method in which saccharides other than branched oligosaccharides are precipitated and removed by adding alcohols, acetone, or other organic solvents to the sugar solution. (3) A method of removing sugars other than branched oligosaccharides using molecular fractionation chromatography such as an activated carbon column or gel filtration, or ion exchange chromatography using an ion exchanger. (4) A method of assimilating sugars other than branched oligosaccharides by allowing yeast to act on a sugar solution. (Effects of the Invention) As described above, the method of the present invention provides a practical method for industrially producing syrup with a high content of branched oligosaccharides. The syrup thus obtained containing a high concentration of branched oligosaccharides has a refreshing sweet taste, and has a wide range of uses as a sweetener or flavor improver for various foods. Furthermore, since it has an anti-caries effect, it can be widely used as a sweetener for the purpose of preventing dental caries. In addition, branched oligosaccharides have various types of crystallization inhibiting effects, and can be used as crystallization inhibitors by adding small amounts to sugar solutions such as sugar, glucose, isomerized sugar, and maltose. (Example) Reference example 1 Aspergillus niger α-glucosidase in sugar solution containing 70% maltose (solid content 30%)
0.08 IU/s substrate was added, and the mixture was reacted at pH 5 and 55° C. for 24 hours to obtain a sugar solution containing branched oligosaccharides. Its sugar composition is shown in Table 1. Example 1 33% cornstarch aqueous solution (PH6.3) with heat-resistant α-amylase (Novo, Termamill 60L)
0.08% (w/w) was added and reacted at 105°C for 10 minutes. To this, 0.03% (w/w) of the same enzyme was added and reacted at 95℃ for 3 hours to obtain DE12, and then α-
Add 0.3% β-amylase (manufactured by Nagase Sangyo, No. 150) to the liquefied liquid that has been adjusted to pH 5 by inactivating amylase.
(w/w) and 0.2% pullulanase (Promozyme 200L, manufactured by Novo) were added and reacted at 60°C for 24 hours.
Thereafter, 0.11 U/g of Aspergillus niger α-glucosidase and substrate were added, and the mixture was reacted at 55°C for 24 hours to obtain a branched oligosaccharide syrup. The sugar composition is shown in Table 1. Example 2 β-amylase (manufactured by Nagase Sangyo, No. 150) 1.0% (w/w),
Pullulanase (manufactured by Novo, Pumozyme 200L) 0.7
% (w/w), Aspergillus niger α-glucosidase 0.02 IU/g, substrate added at the same time 60
The reaction was carried out at ℃ for 36 hours to obtain a syrup rich in branched oligosaccharides. The sugar composition is shown in Table 1. Reference Example 2 Aspergillus awamori α-glucosidase was added (0.5 IU/g/substrate) to a sugar solution containing 30% solids, of which 70% was maltose, and the pH was 4.5.
The reaction was carried out at 60°C for 24 hours to obtain a sugar solution containing 43% or more of branched oligosaccharides. The sugar composition is shown in Table 1.

[Table] Reference Example 3 The sugar solution obtained in Example 1 was decolorized and ion-purified with activated carbon, concentrated to 60%, and treated with an ion exchange resin (manufactured by Dowex Co., Ltd., Dowex Co., Ltd.).
88) was used to obtain a syrup containing branched oligosaccharides. Its sugar composition is shown in Table 2.

[Table] Processing conditions Column 50mmφ×1000mmh (resin height 700mm) With jacket 65℃ Packing Dowex 88 (Na type) Loaded sugar amount 30g (dry matter) Flow rate SV=0.1 Reference example 4 Sugar obtained in Example 1 After decolorizing the liquid with activated carbon and ion-purifying it, 15% (opposed portion) of common salt was added and dissolved to concentrate to a concentration of approximately 85%, and a horizontal stirring type crystallizer with a 3-liter jacket was used. Glucose-salt double salt crystals were crystallized under the following conditions, and the crystals were removed using a centrifuge.
The separated solution was purified using an ion exchange resin to remove residual common salt, and a sugar solution with a high content of branched oligosaccharides was obtained. Its sugar composition is shown in Table 3. Product conditions Sugar concentration 84.7% Temperature and time Performed according to the program shown in Figure 1 Seed Glucose-salt crystals with a solid content of 0.5% Stirring 10 rpm

[Table] Reference Example 5 A crystallization prevention test was conducted using the branched oligosaccharide-rich syrup obtained in Reference Example 2. (1) Sample A: Maltose 80% syrup (solid content 75%) B: Maltose 83% syrup mixed with 10% of the sugar solution obtained in Example 4 (solid content 75%) (2) Storage temperature 5. 12, 20℃ and room temperature (winter) (3) Results As shown in Table 4 Table 4 Number of days until crystallization Storage temperature (℃) A B 5 9 40 12 5 20 20 9 23 Room temperature 8 24 (Note) Syrup The day on which 2 to 3 crystals were observed with the naked eye was defined as the day of crystallization. As is clear from Table 4, the number of days until crystallization was longer in the case of the sample to which isomaltose was added under all storage conditions, showing a remarkable effect of suppressing crystallization.

[Brief explanation of drawings]

FIG. 1 is a temperature and time program for crystallization of glucose-salt double salt crystals in Reference Example 4. The horizontal and vertical axes represent crystallization time (hours) and crystallization temperature (°C), respectively.

Claims (1)

[Claims] 1. A starch liquefied solution adjusted to DE5 to 15 is treated with β-amylase, starch debranching enzyme, and an enzyme that has a saccharide transfer action in the coexistence state, and the branched oligosaccharide content in the carbohydrate solid content is 40% or more. , and the ratio of isomaltose in the branched oligosaccharide is 50% or more. A method for producing a syrup containing a large amount of branched oligosaccharide.