JPS6313679B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing oligosaccharides, particularly malto-oligosaccharides of maltotetraose (G ₄ ) or higher, which do not contain low-molecular-weight oligosaccharides of maltotriose (G ₃ ) or lower. More specifically, after inducing yeast to accumulate α-glucosidase activity that decomposes low-molecular oligosaccharides below maltotriose (G ₃ ), the immobilized yeast cells obtained by immobilization treatment are Maltotriose (G ₃ ), maltose (G ₂ ), and glucose (G ₁ ) in the sugar solution are added to the sugar solution containing oligosaccharides and allowed to act.
The present invention relates to a method for assimilating maltotetraose (G ₄ ) and producing a maltooligosaccharide solution consisting of maltotetraose (G 4 ) or more. Conventionally, many methods have been attempted for the preparation of oligosaccharides. For example, separation methods using paper chromatography, cellulose column chromatography, carbon column chromatography, polyacrylamide gel column chromatography, etc. have been attempted. However, in the preparation of oligosaccharides by these chromatographic separations, the loading amount of the sample is small, and in order to separate a large amount, the scale of the separation apparatus is large, which poses many problems. Recently, a method has been proposed for producing oligosaccharides in which both yeast of the genus Satucharomyces and maltase are added to decompose (assimilate) oligosaccharides of maltotetraose (G ₄ ) or less (Tokuko Sho).
56-12437). The above method uses maltase derived from yeast in combination with yeast cells to promote the ability of yeast cells to assimilate low-molecular-weight oligosaccharides,
This is an excellent method for producing oligosaccharides that does not substantially contain low-molecular-weight oligosaccharides of maltotetraose or lower, but in order to use the yeast cells as they are, it is necessary to remove the cells from the sugar solution or remove them from the cells. Not only is it difficult to remove and purify impurities such as eluted dyes, but also a retrosynthetic reaction is likely to occur due to coexisting maltase, resulting in the production of various oligosaccharides with unnecessary branched structures. Furthermore, when commercially available yeast cells alone are used to assimilate low-molecular-weight oligosaccharides, a large amount of cells are required due to their low assimilation ability, and a long reaction time is required. On the other hand, recently, maltotriose (G ₃ ), maltotetraose (G ₄ ), maltopentaose (G ₅ ) and maltohexaose (G ₆ ) have been shown to act on starch.
New enzymes that produce each amylase have been discovered one after another, and the method using these amylases is considered to be the most efficient method for industrial production of oligosaccharides (Starch Science Vol. 28, No. 2, P92 ，
1981). However, although the oligosaccharide-producing enzymes mentioned above act on starch or amylose to produce the respective oligosaccharides as main components, they also decompose the products themselves, so the final products are glucose (G ₁ ), The production of low-molecular-weight oligosaccharides such as maltose (G ₂ ) and maltotriose (G ₃ ) is unavoidable, and therefore, the removal of these low-molecular-weight oligosaccharides is essential for the production of high-purity malto-oligosaccharides. . As described above, in the conventional methods, it has been difficult to produce various desired oligosaccharides with high purity, efficiently, and economically. Therefore, an object of the present invention is to easily, efficiently, and economically advantageously produce high-purity maltooligosaccharides useful as pharmaceuticals, foods, or substrates for amylase analysis. In order to achieve the above object, the present inventors conducted extensive research and found that yeast cells of the genus Satucharomyces were grown in a nutrient medium containing maltose and/or maltotriose as a carbon source to an extent that the yeast did not substantially proliferate. Immobilization obtained by culturing for a period of time to induce accumulation of α-glucosidase having the ability to decompose low-molecular-weight oligosaccharides of maltotriose (G ₃ ) or less in the yeast cells, and then performing immobilization treatment on the α-glucosidase. The optimum pH and optimum temperature of yeast cells are improved compared to untreated live yeast cells, and when these yeast cells are added to an oligosaccharide-containing sugar solution and allowed to act, maltotriose ( G ₃ ) The present invention was completed by discovering that highly pure maltooligosaccharides containing almost no low molecular weight oligosaccharides as shown below can be efficiently produced. That is, the present invention cultivates yeast cells of the genus Satucharomyces aerobically in a nutrient medium containing maltose and/or maltotriose as a carbon source, and induces and accumulates α-glucosidase activity in the yeast cells. After that, by adding the immobilized yeast cells obtained by immobilization to a sugar solution containing oligosaccharides, maltotriose (G ₃ ) and maltose (G ₂ ) in the sugar solution are added. and a method for assimilating glucose (G ₁ ) to produce malto-oligosaccharides of higher purity than maltotetraose (G ₄ ). The present invention will be explained in detail below. The yeast cells used in the present invention are Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces italicus ((Saccharomyces
Any yeast of the genus Saccharomyces such as Saccharomyces italicus or Saccharomyces uvarum can be used, but in the present invention, these yeast cells are used as a carbon source in a medium containing maltose and/or maltotriose. By culturing aerobically at ℃ for about 5 to 15 hours, the α-glucosidase activity that decomposes low-molecular-weight oligosaccharides below maltotriose (G ₃ ) is increased, and the resulting yeast cells are immobilized. That is required. Immobilization of yeast cells may be carried out by known methods, for example, by embedding yeast cells in an entrapping agent such as sodium alginate, gelatin, agar, carrageenan, or acrylamide gel, or by immobilizing yeast cells during embedding or This can be carried out by further crosslinking treatment using a crosslinking agent such as glutaraldehyde after embedding. For example, when using sodium alginate, yeast should be added to 2.5
% sodium alginate solution at a ratio of 1:1 and mix thoroughly, add about 2% sodium alginate solution while stirring.
Approximately 2-3 mm by dropping into _CaCl2 solution
spherical immobilized yeast can be obtained. Next, in the present invention, the immobilized yeast cells are added to a sugar solution containing oligosaccharides and allowed to act.
Here, the sugar solution containing oligosaccharides is obtained by gelatinizing or slightly liquefying any of starch, amylose, or amylopectin, and then allowing various oligosaccharide-producing enzymes to act thereon. Oligosaccharide-producing enzymes include maltotetraose (G ₄ )-producing enzyme produced by Pseudomonas stuzeri, maltopentaose (G ₅ )-producing enzyme produced by Bacillus licheniformis, and Aerobacter aerogenes. (Aerobacter aerogenes)
The maltohexaose (G ₆ )-producing enzyme produced by the above may be appropriately selected and used depending on the type of oligosaccharide of interest. According to the present invention, when the immobilized yeast cells are added to an oligosaccharide-containing sugar solution and allowed to act, the α-glucosidase activity accumulated in the yeast cells causes low molecular weight oligosaccharides of maltotriose (G ₃ ) or less. is decomposed and assimilated to obtain malto-oligosaccharides of maltotetraose (G ₄ ) or higher with high purity. Furthermore, since the yeast cells are immobilized, the desired oligosaccharide can be efficiently produced without requiring an operation to remove free yeast cells. Hereinafter, the present invention will be specifically explained with reference to Examples. The materials and analysis methods used in the examples are as follows. (1) Analysis method Quantitative analysis of oligosaccharides was performed using the method of Kainuma et al. (Journal of chromatography, 212.126-
131.1981) by high performance liquid chromatography (HPLC). Further, qualitative analysis was conducted by a high temperature (60°C) raising method using a developing agent of n-butanol/pyridine/water = 6/4/4. After development, it was treated with glucoamylase and color detection was performed by the alkaline silver nitrate method. (2) Yeast Yeast is commercially available baker's yeast (Oriental Yeast Kogyo Co., Ltd.)
After aerobically shaking the following tissue culture medium (manufactured by Pressed Yeast), centrifuged cells were collected. Thiamine hydrochloride 20mg Pyridoxine hydrochloride 20mg Nicotinic acid 20mg MgSO ₄・7H ₂ O 10g (NH ₄ ) ₂ SO ₄ 10g CO (NH ₂ ) ₂ 20g Maltose 75g Maltotriose 75g 1/10M phosphate buffer (PH6) 500ml A composition of 100 g or more of yeast was shaken for 5 hours using a rotary shaker (30°C, 200 rpm) using a No. 5 Erlenmeyer flask. Under these conditions, the added yeast cells did not substantially proliferate. As a result of comparing the assimilation rate of low-molecular-weight oligosaccharides below maltotriose in the yeast cells prepared by the above method with that of the yeast before treatment, the assimilation rate (especially maltose, maltotriose, It can be seen that the results have been significantly improved.

[Table] (3) Method for producing immobilized yeast The yeast prepared in (2) was collected by centrifugation, and 2.5
% sodium alginate solution at a ratio of 1:1 and mix thoroughly, add about 2% sodium alginate solution while stirring.
A spherical immobilized yeast of approximately 2 mm to 3 mm was obtained by dropping into CaCl ₂ solution. Next, it was filtered through Toyo Roshi (No. 5B) and used for testing. As a result of comparing the above-mentioned immobilized yeast with the yeast prepared in (2) in its ability to assimilate low-molecular-weight oligosaccharides below maltotriose, as shown in Table 2 below, it has almost the same ability to assimilate low-molecular-weight oligosaccharides below maltotriose. Almost no adverse effects due to the immobilization procedure were observed.

[Table] Furthermore, as is clear from Figures 1 and 2, this immobilized yeast is stable over a wider pH range than live yeast, has excellent heat resistance, and can withstand industrial use. It is something. Example 1 10 g of short-chain amylose (manufactured by Hayashibara Biochemistry Research Institute, average degree of polymerization () = 23) was dissolved in 200 ml of hot water, and the method of Robyt et al. (Archives.Biochem.
Biophys., 145 , 105, 1971) maltotetraose-generating enzyme (1 IU/g substrate) was
6.7, Glucose 3.1% after acting at 50℃ for 48 hours,
An oligosaccharide-containing sugar solution with a sugar composition of 7.5% maltose, 12.3% maltotriose, and 77.1% maltotetraose was prepared. Next, 5 g of the immobilized yeast obtained in (3) above (yeast amount
2.5 g) was added thereto, and the mixture was reacted with stirring at 45° C. and pH 5.5 for 20 hours. For comparison, 2.5 g, 5 g, and 10 g of the commercially available baker's yeast used in (2) above were added to the oligosaccharide-containing sugar solution.
and 20 g of each were added and reacted at 40° C. and pH 4.5 with stirring for 20 hours. The sugar compositions of the products of the present invention and comparative products thus obtained are shown in Table 3 below.

[Table] Furthermore, as a result of using the immobilized yeast obtained in the present invention 10 times under the same conditions as above and examining the sugar composition of the obtained product, as shown in Table 4 below,
It was found that the yeast could be stably used for a long period of time with almost no deactivation. As is clear from the above results, the immobilized yeast according to the present invention is not only stable under temperature and pH conditions and excellent in the ability to assimilate low-molecular oligosaccharides below maltotriose, but also can be repeatedly reused. . Therefore, by using this, highly pure oligosaccharides can be economically advantageously produced.

[Table] Example 2 1 kg of glutinous corn starch was dissolved in about 20℃ of hot water, and the pH was adjusted to 7.0 with calcium hydroxide. Product name Termamill 120L, 132.4KNu/g) with 0.4KNu/g starch and 1.5U/g of the debranching enzyme pullulanase (1500U/g) produced by Aerobacter aerogenes.
g starch was added and reacted at 55℃ for 30 hours, glucose 4%, maltose 16%, maltotriose 23
%, maltotetraose 11%, maltopentaose 40%, maltohexaose 3%, and other maltooligosaccharides 3%. Next, 300 g of the immobilized yeast obtained in (3) was added to the above sugar solution.
was added and reacted with stirring at 45°C and pH 5.5 for 50 hours to form an oligosaccharide solution consisting of 19.3% maltotetraose, 70.2% maltopentaose, 5.3% maltohexaose, and 5.2% other polymeric oligosaccharides. I got it. Example 3 5 kg of potato starch was dissolved in about 100 ml of hot water, and the pH was adjusted to 6.5 with calcium hydroxide. 20 U/g starch and 1.5 U/g starch of pullulanase, a debranching enzyme produced by Aerobacter aerogenes, were added to the starch.
After reacting at ℃ for 20 hours, purification such as activated carbon decolorization and ion exchange treatment was performed using conventional methods to obtain 3% glucose, 17% maltose, and 19% maltotriose.
%, maltotetraose 10%, maltopentaose 16%, maltohexaose 27%, and other maltooligosaccharides 8%. Next, after adjusting the pH of the sugar solution to 5.5, 15 kg of immobilized yeast prepared in (3) was filled in advance and heated at 40°C.
16% maltotetraose, 26% maltopentaose, 44% maltohexaose, and 13 other maltooligosaccharides.
An oligosaccharide solution consisting of % was obtained.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the assimilation status of glucose (G ₁ ), maltose (G ₂ ), and maltotriose (G ₃ ) due to changes in pH of the immobilized yeast according to the present invention and commercially available live yeast. In the figure, ●---● indicates assimilation of _G1 to _G3 by immobilized yeast, and Γ---Γ indicates assimilation of _G2 by commercially available live yeast. FIG. 2 is a graph showing the state of assimilation of glucose, maltose, and maltotriose according to temperature changes in the immobilized yeast according to the present invention and the commercially available live yeast. In the figure, ●---● indicates assimilation of _G1 to _G3 by immobilized yeast, and Γ---Γ indicates assimilation of _G2 by commercially available live yeast.

Claims (1)

[Claims] 1 Yeast cells of the genus Satucharomyces are cultured aerobically in a nutrient medium containing maltose and/or maltotriose as a carbon source, and α-glucosidase activity is induced and accumulated in the yeast cells. By adding the immobilized yeast cells obtained through the immobilization treatment to a sugar solution containing oligosaccharides, low-molecular oligosaccharides below maltotriose (G ₃ ) in the sugar solution are assimilated and maltotetra A method for producing high-purity maltooligosaccharide, which is characterized by obtaining a maltooligosaccharide solution having a concentration of ose (G ₄ ) or more.