JP3089503B2 - Method for producing sweetener containing isopanose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to isopanoses (6-0-
alpha-maltosyl - glucose) and 6 ² -0-alpha-maltosyl - a process for producing a sweetener containing branched maltotriose and branched maltotetraose represented by maltose.

[0002]

Isomaltose containing alpha-1,6-glucosidic bonds of the Prior Art and Problems which the present invention is to solve In recent years maltooligosaccharides panose, isopanose, isomaltotriose, and 6 ² -0-alpha-maltosyl So-called branched oligosaccharides, such as maltose, have been found to be growth factors for bifidobacteria, a useful intestinal bacterium, and saccharide mixtures containing some of these have been used as sweeteners.

[0003] In this production method, starch syrup containing maltose in a larger amount than starch is first produced, followed by A
α-glucosidase produced by molds such as S. spargillus niger is allowed to act, and α-glucosidase is produced by the enzyme.
Generally, branched oligosaccharides are produced by a 1,6-glucosyl transfer reaction. In this case, panose accumulates in the early stage of the reaction, and isomaltose and isomalttriose in the late stage. However, a large amount of glucose is produced as a by-product due to the nature of the reaction, and the total content of the target isomaltose and panose is only about 40%. Meanwhile isopanose is branched oligo trisaccharide, 6 ² -0-α
-Maltosyl-maltose is a branched oligotetrasaccharide,
These are also known to be carbohydrates that show growth of bifidobacteria in the intestine. However, the production of isopanose
This not means other than hydrolysis with Isopururanaze the most expensive polysaccharides pullulan up, also 6 ² -O-
Even with α-maltosyl-maltose, pullulan was hydrolyzed with acid and then fractionated, so that it could not be produced as a food material.

[0004]

An enzyme which can be used acts on pullulan to cleave only its α-1,4-glucosidic bond or both α-1,4- and α-1,6-glucosidic bonds. Any substance belonging to the so-called neopullulanases that mainly produce panose may be used. For example, Bacillus stearothermo
philus TRS40 (Microorganism Deposit No. 9609)
Neopluranase (Journal of Ba)
cterology, 170, 1554, 19
Published in 1988 ), Thermoactinom
α-amylase from Yces vulgaris (A
gricultural and Biologicala
l Chemistry, Vol. 42, p. 1681, 1
978), Bacillus ste
aulothermophilus KP1064 pullulan hydrolase (Applied Microbio
logy and Biotechnology, 21st
Volume, page 20, published in 1985), and Ba
cteroides thetaiotaomicro
n 95-1 neopluranase (Journal o)
f Bacteriology, Vol. 173, No. 296
2 pages, published in 1991), and the like.

As the substrate, any of saccharides containing only α-1,4- or α-1,4- and α-1,6-glucosidic bonds such as starch, pullulan, dextrin, starch syrup, and various maltooligosaccharides can be used. it can. Therefore, starch liquefied with bacterial liquefied α-amylase, and further added with bacterial glycated (hereinafter referred to as BSA) or mold α
Dextrins degraded with amylase are also effective substrates.

[0006] Since this reaction mainly comprises a transfer reaction and a condensation reaction, the reaction proceeds at a higher concentration. Table 1 shows the production ratio of isopanose when the substrate concentration was changed. As described above, the reaction is preferably performed at a substrate concentration of 5% by weight or more (in the present application,% by weight is expressed as%), preferably 20% or more. The reaction conditions such as pH and temperature may be within the working range of the enzyme. In addition, since this reaction is a transfer effect on oligosaccharides, it proceeds without inconvenience even if the enzyme is immobilized. For this reason, even if various immobilized carriers such as chitosan are used to prepare and act on an immobilized enzyme, it can be used efficiently. The saccharified solution thus obtained can be decolorized with activated carbon and purified with an ion-exchange resin, if necessary, to give a product.

Table 1 Substrate Concentration and Isopanose Production Ratio 基質 Substrate Concentration (% ) 15 10 20 30 割 合 Proportion of Isopanose (% ) <2 12.2 17.2 21.3 22.4 ────────────────────────────────── Substrate : Liquefied corn starch liquid (DE10) Saccharification conditions: 58 ° C, pH 6.0, 40 hours, neopullulanase 10u / g substrate

[0008]

The present inventors have recently proposed a thermophilic bacterium Bacillus.
Study on neopluranase produced by stearothermophilus TRS40. This enzyme degrades pullulan to produce mainly panose. Revealed that there is a nature to do. As a result of further studies, it has been found that the present enzyme exhibits remarkable α-1,4- and α-1,6-glycosylation and condensation when acted on a high concentration of substrate.

In general, it is known that α-amylase reacts with α-1,4-glycosylation reaction when it acts on a high-concentration substrate. Α-glucosidase is used for α-1,4- and α-
It is known to carry out a 1,6-glycosylation reaction. However, the sugar transfer reaction and the condensation reaction of neopluranase are not known at all, and the reaction specificity is completely different from these enzymes. That is, neopluranase differs from α-amylase in that it undergoes α-1,6-glycosylation. In addition, the transglycosylation reaction of neopurulanase is mainly maltosyl transfer, whereas that of α-glucosidase is glucosyl transfer. For the above reasons, it can be said that the glycosyltransfer reaction of neopluranase is extremely novel.

When neoplulanase is allowed to act on maltotriose having a concentration of about 1% or less, only glucose and maltose are produced. On the other hand, a small amount of ¹⁴ C glucose is added to 10% maltotriose as in Experiment 1, for example. When neoplulanase is added and allowed to react, maltotriose, isopanose, and 6-0-α-maltotriosyl-glucose, which show radioactivity at the reducing end, are generated in the initial stage of the reaction. Among them, maltotriose is a substance in which maltotriose as a substrate is decomposed and the maltose moiety is transferred to radioactive glucose by α-1,4-bond (α-1,4-maltosyl group transfer), and isopanose is that the maltose moiety is radioactive. It has been transferred to glucose by an α-1,6-bond (α-1,6-maltosyl group transfer). 6-0-α
-Maltotriosyl-glucose is a product of maltotriose condensed with radioactive glucose at an α-1,6-bond (condensation reaction). That is, the present enzyme is not a mere hydrolase, and causes a transfer reaction and a condensation reaction when acted on a high concentration of a substrate. Then, an oligosaccharide containing an α-1,4-linkage and an α-1,6-linkage is generated as a generated sugar. Furthermore, since the present enzyme has a higher ability to cleave α-1,4-linkage than α-1,6-linkage, branched oligosaccharides containing α-1,6-linkage accumulate later in the reaction.

Experiment 1 After dissolving 2.5 μg of ¹⁴ C glucose in 0.1 ml of a 10% maltotriose solution, neoplulanase (1 U / m
l) 0.1 ml was added and reacted at 40 ° C. and pH 6.0, spotted on a filter paper over time, and butanol / pyridine / water (6 /
4/3) was developed four times with a solvent, and an autoradiogram was prepared in close contact with the X-ray film. The result was as shown in FIG. The structure of the produced radioactive oligosaccharide was confirmed by Rf value and digestion experiments of various enzymes. The method for measuring the activity of neopluranase is as follows. 200% of 2% pullulan
Add an equal volume of enzyme solution to the sodium phosphate buffer,
And react for 30 minutes. The amount of reducing sugars generated is quantified by the dinitrosalicylic acid method. Under these conditions, 1 μmo per minute
The amount of the enzyme that produces reducing sugars corresponding to 1 glucose is 1 U
And

[0012]

When neoplulanase is allowed to act on 10% maltotriose, maltopentaose is produced as a sugar, and B5 is a branched oligosaccharide (hereinafter, the branched sugar is represented by B, and the pentaose is represented by B5). perhaps 6 ³ -0-alpha-maltosyl - maltotriose generated. Further, in the latter stage of the reaction, the transglycosylation reaction, the overall reaction, and the hydrolysis are repeated, and finally the isopanose (6-0-
alpha-maltosyl - glucose) and 6 ² -0-alpha-maltosyl - generating a branch oligosaccharides mainly maltose.

Among them, the structure of isopanose was confirmed by Rf value by paper chromatography, digestion experiment with glucoamylase and neopurulanase, and methylation analysis. Meanwhile 6 ² -0-alpha-maltosyl - maltose performs pullulanase, digestion experiments with glucoamylase together with standards, it could be identified from the result.

[0015]

According to the present invention, maltooligosaccharides, dextrins,
By making neoplulanases act on starch etc.,
Previously it could not mass-produced while health effectiveness is expected isopanose (6-0-alpha-maltosyl - glucose) and became 6 ² -0-alpha-maltosyl that maltose may be easily prepared sweetener containing a large amount of Was.

[0016]

【Example】

Example 1 Neopurulanase and BSA (bacterial saccharified α-amylase) were added to a 30% cornstarch liquefied liquid (DE10) solution.
And a system to which only BSA was added at 58 ° C., respectively.
The reaction was carried out at pH 6.0 for 48 hours. The reaction products were analyzed by high performance liquid chromatography, and the proportions of various oligosaccharides in the products were quantified, as shown in Table 2. Among them, G1, G2, G3, and ≧ G4 represent linear oligosaccharides having a polymerization degree of glucose, maltose, maltotriose, and maltotetraose or more, respectively.
2, B3, and B4 respectively isomaltose, isopanose, and 6 ² -0-alpha-maltosyl - shows the maltose. Table 2 Ratio (%) of each oligosaccharide in the reaction product Ｇ G1 G2 G3 ≧ G4 B2 B3 B4 ────────────────────────────────── neopurulanase + BSA 25.7 13.4 0 0 6.7 30.4 23.8 BSA 29.8 26.0 14.1 30.1 0 0 0 ────────────────────────────────── That is, when only BSA is activated G1, G2, G3,
And when a linear oligosaccharide having a degree of polymerization of 4 or more is produced, and in the presence of neopurulanase, all of G3 and a part of the linear oligosaccharide having a degree of polymerization of 4 and G2 disappear, and the desired B2, B3, and Give B4.

Example 2 BSA (bacterial saccharified α-amylase) was added to a 30% dextrin (Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) solution and reacted at 58 ° C. and pH 6.0 for 24 hours. . Next, neopurulanase was added to this system, and further reacted for 24 hours under the same conditions. The reaction products were analyzed by high performance liquid chromatography, and the proportions of various oligosaccharides in the products were quantified, as shown in Table 3. Table 3 Ratio (%) of each oligosaccharide in the reaction product {G1 G2 G3 B2 B3 B4} ─────── 24.3 15.0 1.4 15.5 30.3 13.4 ──────────────────

Example 3 750 U of neopluranase was added to 100 ml of a 30% dextrin (Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) solution.
Was added and the reaction was carried out at 50 ° C. for 24 hours. The ratio of each oligosaccharide in the reaction product was as shown in Table 4. Table 4 Ratio (%) of each oligosaccharide in the reaction product {G1 G2 G3 B2 B3 B4} ─────── 22.0 27.9 2.6 5.6 25.4 16.5 ──────────────────

Example 4 1 ml of neopurulanase (50 U / ml) was added to 1 g of chitopearl (manufactured by Fuji Boseki Co., Ltd.) and gently stirred for 1.5 hours to prepare an immobilized enzyme. To this, 100 ml of a maltooligosaccharide solution mainly composed of maltotriose (10
%), And the resulting saccharide was subjected to a shaking reaction at 50 ° C. for 16 hours, and the resulting saccharide was analyzed by high performance liquid chromatography to show the saccharide composition shown in Table 5. Table 5 Ratio (%) of each oligosaccharide in the reaction product {G1 G2 G3 B2 B3 B4} ─────── 21.0 19.8 1.4 9.4 25.8 22.6 ──────────────────

[Brief description of the drawings]

FIG. 1 is a diagram of analysis of a reaction product by paper chromatography and autoradiogram.

Continuation of the front page (56) References JP-A-61-181354 (JP, A) JP-A-4-108356 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A23L 1 / 236 C12P 19/14 C12P 19/16 JICST file (JOIS) JAFIC file (JOIS)

Claims (4)

(57) [Claims] 1. The method according to claim 1, wherein only α-1,4- or α-1,4-
And a method for producing a sweetener containing isopanose, wherein neoplulanase is allowed to act on a saccharide containing an α-1,6-glucosidic bond. 2. Isopanose characterized by allowing neopluranase to act on α-1,4-only or a saccharide containing α-1,4- and α-1,6-glucosidic bonds having a carbohydrate concentration of 5% or more. A method for producing a sweetener comprising: 3. The method according to claim 1, wherein only α-1,4- or α-1,4-
And a method for producing a sweetener containing isopanose, wherein α-amylase and neopurulanase are allowed to act on a saccharide containing an α-1,6-glucoside bond. 4. The method according to claim 1, wherein only α-1,4- or α-1,4-
And a method for producing a sweetener containing isopanose, which comprises reacting a saccharide containing an α-1,6-glucosidic bond with α-amylase and then reacting neopullulase.