JP3494686B2 - Method for producing isomaltosyl fructoside - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing isomaltosyl fructoside, which is known as a sweetener that suppresses cariogenicity. [0002] Sucrose has a good sweet taste,
Further, since it is extremely soluble in water, it has been widely used as a representative sweetener. However, in recent years, it has been revealed that sucrose is highly cariogenic and causes caries. In other words, sucrose is converted into insoluble glucan by microorganisms in the oral cavity, anaerobic fermentation is performed on the surface of the teeth covered with the glucan, and organic acids such as lactic acid are generated, and this organic acid is converted into tooth enamel. It was clarified that the decay caused caries. For these reasons, a sweetener that can replace sucrose and a sweetener that suppresses the cariogenicity of sucrose are desired. Isomaltosyl fructoside is known as a typical sweetener that suppresses the cariogenicity of sucrose.Since this isomaltosyl fructoside has the effect of suppressing the production of insoluble glucan, it is an alternative to sucrose. As well as having the advantage that it can be used in combination with sucrose. [0003] To date, methods for producing isomaltosyl fructoside include Aspergillus niger (Asp.
ergillus niger) [J. Chem. Soc. , 2064 (195)
7)] and a method using a sucrose transferase of the genus Bacillus (JP-A-4-30796);
And Mucor Javanese
(Patent Science, Vol. 35, No. 2, P93-102 (1988), JP-A-2-128652, JP-A-4-287692). [0004] However, among strains belonging to the genus Aspergillus, only a method for producing isomaltosyl fructoside using a glycosyltransferase of Aspergillus niger has been known so far. I didn't. Furthermore, this method is much more expensive than inexpensive sucrose, and therefore, it has been desired to develop a more productive method for producing isomaltosyl fructoside. The present invention has been made under such circumstances, and an object of the present invention is to provide an efficient method for producing isomaltosyl fructoside. [0005] In order to solve the above-mentioned problems, the present inventors have developed Aspergillus niger (Aspergillus niger) among strains belonging to the genus Aspergillus.
From the viewpoint of producing isomaltosyl fructoside more efficiently than S. niger, strains of the genus Aspergillus other than Aspergillus niger were searched, and their ability to produce isomaltosyl fructoside was investigated. As a result, Aspergillus awamori (Aspergillus awamori)
mori) or Aspergillus phoensis (As
production to glycosyltransferase activity of pergillus phoenicis), when allowed to act on a substrate solution containing sucrose and glucosyl group donor, found that efficiently generated isomalto Sylph lactoside than Aspergillus niger, the present invention Was completed. [0006] That is, the present invention relates to a method for preparing Aspergillus awamori or Aspergillus phoenesis in a substrate solution containing sucrose and a glucosyl group donor.
glucosyltransferase activity produced by L. phoenicis)
The present invention relates to a method for producing isomaltosyl fructoside, which is characterized by allowing an active substance to act. The substrate solution in the present invention is a solution containing sucrose, which becomes a glucosyl group acceptor, and a glucosyl group donor in a glucosyl group transfer reaction by a glycosyltransferase active substance . Glucosyl group donors are oligosaccharides and polysaccharides having a glucosyl group bonded to a disaccharide or higher, such as soluble starch, partially hydrolyzed starch, amylose, maltose, maltotriose, maltotetraose, and maltopentaose. Oose, maltohexaose, and mixtures thereof, and the like. The ratio of sucrose to glucosyl group donor in the substrate solution is desirably about 10: 1 to 1:10. The substrate concentration can be about 5 to 70% by weight, and preferably about 20 to 60% by weight. The pH of the substrate solution can be in the range of 2 to 8, and preferably 3 to 7. The temperature of the substrate solution can be between 5 and 70 ° C.
To 55 ° C. [0007] In the present invention, the glycosyltransferase active substance acting on the substrate solution refers to a glucose residue generated by cleavage of a glucosidic bond of a glucosyl group donor at the 6-position of a sucrose glucose residue. Trisaccharides transferred to carbon and linked by α-glucoside bonds, that is, cells or cell extracts having an action of producing isomaltosyl fructoside, which means cells in a medium in which microorganisms are cultured. And the cells once isolated from the medium according to a conventional method. The term "cell extract" refers to a crushed cell, or a partially purified enzyme-containing substance having the above-mentioned glucosyl transfer activity according to a conventional method. Furthermore, it also means an enzyme having the above-mentioned glucosyltransferase activity, which is purified by a known method, if necessary. [0008] The glycosyltransferase active substance used in the production method is
Aspergillus awamori (Aspergillus)
awamori) or Aspergillus phoenicis. Typical examples thereof include Aspergillus awamori (Asperg)
illus awamori IFO433, Aspergillus phoensis (Aspergillus ph)
oenicis) IFO 6649, IFO 6650, I
FO8833 or ATCC1555. These strains were obtained from the Fermentation Research Institute (Inst
ituteof Fermentation, Osak
a) and the American Type Culture Collection (American Type Culture C)
collections can be freely obtained by anyone. In the present invention, as a medium that can be used, any natural medium or synthetic medium may be used as long as the above-mentioned microorganism can be proliferated by culture. For example, as a carbon source, glucose, molasses, sucrose, starch, starch saccharified solution, cellulose decomposition product, and the like are used. As a nitrogen source, ammonium salts such as ammonia, ammonium sulfate, ammonium nitrate, and phosphorus phosphorus, urea, nitrates and the like are appropriately used. As the inorganic salt, salts such as phosphoric acid, potassium and magnesium, for example, common industrial chemicals such as ammonium phosphate, potassium phosphate, sodium phosphate and magnesium phosphate may be used, and trace elements may be added in addition. In addition, as trace organic nutrients, vitamins, amino acids, nucleic acid related substances, etc.
Although it is not particularly necessary for the growth of the fungus, these may be added, or organic substances such as corn steep liquor, meat extract, yeast extract, peptone, etc. may be added. These media can be used in any form of a liquid medium or a solid medium. Representative medium compositions include, for example, soluble starch 40 g, corn steep liquor 30 g, sodium nitrate 0.5 g, potassium dihydrogen phosphate 0.1 g, magnesium sulfate heptahydrate 0.05 g, potassium chloride 0.05 g.
(Each component is dissolved in distilled water to make 1 L). The cultivation is preferably performed under aerobic conditions such as shaking and aeration and stirring, but can also be performed in a static state. The cultivation temperature can range from 15 ° C. to 35 ° C.
It is desirable to be around 5 ° C to 30 ° C. PH in the culture solution is 3
To 8, and preferably around pH 4 to 6. According to the present invention, Aspergillus awamori (As
pergillus awamori or Aspergillus phoensis (Aspergilluspho)
By reacting the glycosyltransferase activity produced by E. enicis) with a substrate solution containing sucrose and a glucosyl group donor, isomaltosyl fructoside can be efficiently produced. EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. (Example 1) Aspergillus awamori (Aspergillus aw) obtained from the Fermentation Research Institute
amori) IFO433 in 100 ml of natural medium (40 g of soluble starch, 30 g of corn steep liquor, 0.5 g of sodium nitrate, 0.1 g of potassium dihydrogen phosphate,
0.05 g of magnesium sulfate heptahydrate and 0.05 g of potassium chloride dissolved in distilled water to make 1 L).
Shaking culture was performed at 25 ° C. for 2 days in a 0 ml flask.
100 ml of the same culture solution was added with 2 L of the above-mentioned natural medium.
The cells were transferred to an L flask and cultured at 25 ° C. for 2 days. After completion of the culture, the cells were collected by filtration, washed with deionized water, collected again, and the wet cell weight and the dry cell weight were measured. Sucrose 20%, Tetrap-H syrup (purchased from Hayashibara Shoji Co., Ltd.) 6.7% (solid content 5
%) Of 50 M of 0.05 M phosphate buffer (pH 7) was added with 1 g of the dry cells, and reacted at 20 ° C. for 5 hours. After heating the obtained reaction solution at 95 ° C. or higher for 10 minutes, a reaction solution supernatant was obtained by centrifugation. The obtained reaction solution supernatant was analyzed using a high performance liquid chromatography apparatus under the following analysis conditions, and as a result, formation of 0.61 g of isomaltosyl fructoside was confirmed. (Analysis conditions) Column: Amide 80 (4.6 × 25, manufactured by Tosoh Corporation)
0 mm) Eluent: acetonitrile: water = 75: 25 Flow rate: 1.0 ml / min Temperature: 70 ° C. Detector: differential refractometer (Example 2) Aspergillus phoenesis obtained from Fermentation Research Institute (Asperg
(illus phoenicis) IFO6649,
Cell culture was carried out in the same manner as in Example 1, and the wet cell weight and the dry cell weight were measured. Sucrose 2
To 50 ml of 0.05 M phosphate buffer (pH 7) containing 6.7% (solid content: 5%) of 0%, Tetrap-H syrup (purchased from Hayashibara Shoji Co., Ltd.) was added wet cells of 1 g of dry cell weight. And reacted at 20 ° C. for 5 hours. After heating the obtained reaction solution at 95 ° C. or higher for 10 minutes, a reaction solution supernatant was obtained by centrifugation. As a result of analyzing the obtained reaction solution supernatant in the same manner as in Example 1, production of 0.81 g of isomaltosyl fructoside was confirmed. Example 3 Aspergillus phoeniisis (Asperg) obtained from the Fermentation Research Institute, Japan
(illus phoenicis) IFO6650,
Cell culture was carried out in the same manner as in Example 1, and the wet cell weight and the dry cell weight were measured. Sucrose 2
To 50 ml of 0.05 M phosphate buffer (pH 7) containing 6.7% (solid content: 5%) of 0%, Tetrap-H syrup (purchased from Hayashibara Shoji Co., Ltd.) was added wet cells of 1 g of dry cell weight. And reacted at 20 ° C. for 5 hours. After heating the obtained reaction solution at 95 ° C. or higher for 10 minutes, a reaction solution supernatant was obtained by centrifugation. As a result of analyzing the obtained reaction solution supernatant in the same manner as in Example 1, production of 0.90 g of isomaltosyl fructoside was confirmed. Example 4 Aspergillus phoeniisis (Asperg) obtained from the Fermentation Research Institute
(illus phoenicis) IFO 8873,
Cell culture was carried out in the same manner as in Example 1, and the wet cell weight and the dry cell weight were measured. Sucrose 2
To 50 ml of 0.05 M phosphate buffer (pH 7) containing 6.7% (solid content: 5%) of 0%, Tetrap-H syrup (purchased from Hayashibara Shoji Co., Ltd.) was added wet cells of 1 g of dry cell weight. And reacted at 20 ° C. for 5 hours. After heating the obtained reaction solution at 95 ° C. or higher for 10 minutes, a reaction solution supernatant was obtained by centrifugation. The obtained reaction solution supernatant was analyzed in the same manner as in Example 1, and as a result, generation of 0.45 g of isomaltosyl fructoside was observed. Example 5 American Type Culture Collection (American TypeCul)
Aspergillus phoensis (Aspergillus)
(Phenicis) ATCC1555 was cultured in the same manner as in Example 1, and the wet cell weight and the dry cell weight were measured. Sucrose 20%, Tetrap-H syrup (purchased from Hayashibara Shoji Co., Ltd.)
To 50 ml of a 0.05 M phosphate buffer (pH 7) containing 6.7% (solid content: 5%) was added wet cells of 1 g in weight of dry cells, and reacted at 20 ° C. for 5 hours. The obtained reaction solution was 9
After heat treatment at 5 ° C. or more for 10 minutes, a reaction solution supernatant was obtained by centrifugation. The obtained reaction solution supernatant was analyzed in the same manner as in Example 1, and as a result, formation of 0.72 g of isomaltosyl fructoside was observed. (Comparative Example) American Type Culture Collection (American TypeCult)
Aspergillus niger (Aspergillus nig)
er) Cell culture was performed on ATCC9642 in the same manner as in Example 1 to determine the wet cell weight and the dry cell weight. Sucrose 20%, Tetrap-H syrup (purchased from Hayashibara Corporation) 6.7% (solid content 5
%) Of 50 M of 0.05 M phosphate buffer (pH 7) was added with 1 g of the dry cells, and reacted at 20 ° C. for 5 hours. After heating the obtained reaction solution at 95 ° C. or higher for 10 minutes, a reaction solution supernatant was obtained by centrifugation. The obtained reaction solution supernatant was analyzed in the same manner as in Example 1, and as a result, the weight of the produced isomaltsyl fructoside was 0.11 g. As is evident from the results, Aspergillus niger (Aspergillus niger)
In contrast to the method for producing isomaltosyl fructoside using the glycosyltransferase of ger), the production method of the present invention shows extremely excellent productivity. According to the present invention, Aspergillus awamori or Aspergillus phoensis (Aspergill)
us phoenicis) producing glycosyltransferase activity
Things to, by acting on a substrate solution containing sucrose and glucosyl group donor, inhibits representative sweeteners caries induced sucrose isomalto Sylph lactoside efficiently, can be produced.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-3-262494 (JP, A) JP-A-56-51982 (JP, A) JP-A-3-27285 (JP, A) JP-A 64-64 80283 (JP, A) Biochem. Mol. Biol. Int. 1993, Vol. 30, No. 1, p. 107-113 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19/64 JICST file (JOIS) PubMed BIOSIS / WPI (DIALOG)

Claims (1)

(57) [Claims 1] Aspergillus awamori (Aspergillus awamori) is added to a substrate solution containing sucrose and a glucosyl group donor.
gillus awamori or Aspergillus phoensis
A method for producing isomaltosyl fructoside, comprising reacting a glycosyltransferase activity produced by icis).