JP2666068B2 - Method for producing xylitol by Rhonera - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing xylitol useful as a low-calorie sweetener, and more particularly to a method for producing xylitol from xylose by Rhonera.

[Prior Art] Xylitol is a type of sugar alcohol obtained by reducing xylose, and has recently attracted attention as an effective sweetener for diabetic patients.

However, at present, it is manufactured industrially by the chemical reduction method of xylose, and the manufacturing cost is high, so that the product is considerably expensive.

In addition, production by enzymatic reaction in the reduction of xylose has been attempted (JP-A-61-63291, JP-A-61-881).
No. 5186, Japanese Patent Application Laid-Open No. 62-104588), these inventions involve expensive NAD (P) H, so that NAD (P)
A reproduction system for NAD (P) H is required.

Enzyme reactions that require coenzymes such as NAD (P) H require that coenzymes are significantly more expensive than reaction products, and that a practical separation method from the reaction system has not been established. And has not yet been put to practical use.

In addition, the yeast Pichia quercips
xylose by fermentation using microorganisms such as us) (Japanese Patent Publication No. 48-23038), but the culture time is long,
It is not a good method.

[Object of the present invention] An object of the present invention is to provide a method for producing xylitol which can be easily and inexpensively produced by utilizing microorganisms and which can be industrially profitable. .

[Means for Solving the Problems] In order to achieve the above object, the present inventors conducted research on various microorganisms, and found that microorganisms belonging to the genus Rhonera specifically produce xylitol from xylose. As a result, the method of the present invention has been developed.

Thus, the method for producing xylitol according to the present invention comprises the steps of (1) inoculating a medium containing D-xylose as a main raw material with a microorganism belonging to the genus Rhonera capable of producing xylitol, and culturing it under aerobic conditions to produce xylitol. (2) reacting a liquid containing D-xylose with cells of the genus Rhonera having xylitol-producing ability obtained by culturing, and treating the cells with a solution containing D-xylose; To produce xylitol and collect it.

Examples of the microorganisms belonging to the genus Rhonella used in the present invention include, for example, Rahnella aquatilis (Rahnella aquat).
ilis: JCM-1683).

In the above-mentioned method of the present invention, the concentration of xylose used for xylitol production in the culture method can be used in a wide range of 1 to 20%, but about 10% is appropriate.

In addition, the medium contains other nitrogen compounds necessary for the growth of microorganisms, such as peptone, amino acids, ammonium sulfate,
Any of ammonium chloride and the like may be added. In some cases, inorganic salts such as calcium, magnesium and phosphate may be added.

The medium thus adjusted was inoculated with Rhonea aquatilis, which had been pre-cultured, and 20-37 ° C, preferably 30 ° C.
Under aerobic conditions at a temperature of 1 to 10 days, preferably 2
After shaking culture for ３3 days, xylitol accumulates in the medium.

The xylitol produced by the cells of the microorganism obtained by the culturing and the processed cells can be used in a considerably wide range of xylose concentration of 1 to 40%, preferably xylose concentration. About 20% is appropriate.

The cells used for the reaction are monosaccharides such as xylose, disaccharides such as sucrose, and organic acids such as gluconic acid as carbon sources, and nitrogen compounds necessary for the growth of microorganisms (eg, peptone, amino acids, ammonium sulfate,
Cells grown on a medium containing ammonium chloride or the like can also be used.

As the cell treatment used in the present invention, an extract of the cells cultured by the above method and the cells obtained by the above method and those obtained by immobilizing the cell extract and the cell extract by a usual method such as calcium alginate or carrageenan are used. can do.

The xylitol-producing ability of the microorganism used in the present invention is not yet known, and the Rhoneella aquatilis (Rahnella aquatis) used in the present invention is not known.
atilis: JCM-1683) is the first one.

EXAMPLES Examples of the method of the present invention will be described below, but the method of the present invention is not limited to these examples.

Example 1 D-xylose 10% gluconic acid 3% ammonium sulfate 0.4% yeast extract 0.14% monopotassium phosphate 0.34% disodium phosphate (12-hydrate) 2.7% magnesium sulfate 0.08% Calcium chloride ... 0.06% pH ... 7.0 Put 100 ml of the above medium into a 500 ml Erlenmeyer flask,
Separately cultured Rhonela aquatilis culture
1 ml was inoculated, and shaking culture (160 rpm) was performed at 30 ° C. for 4 days using a rotary shaker.

When xylitol produced was measured by high performance liquid chromatography, 3.4 g of xylitol was produced from 10 g of xylose.

The amount of xylitol was determined by high performance liquid chromatography [pump: L-6000 type, manufactured by Hitachi, Ltd .; detector: SE-31 type, manufactured by Showa Denko; column: Shodex RSpak DC-613; manufactured by Showa Denko; solvent: acetonitrile: water = 70 : 30 (0.8ml flow rate
/ min., column temperature is 60 ℃. ) Was carried out and the peak area was determined.

 The same applies hereinafter.

Example 2 100 ml of the medium used in Example 1 was placed in a 500 ml Erlenmeyer flask, inoculated with 1 ml of a pre-cultured culture of Rhonea aquatilis, and shake-cultured at 30 ° C. for 1 day with a rotary shaker ( 160 rpm).

After completion of the culture, the cells were collected by centrifugation (15000 rpm) using a centrifuge, and the collected cells were washed twice with 50 ml of distilled water and suspended in 50 ml of distilled water.

To this cell suspension was added 50 ml of a 1/5 M phosphate buffer (pH 7.0) containing 20 g of xylose, and the reaction was carried out at 30 ° C for 20 hours.

As a result of measuring the amount of xylitol in the reaction-finished liquid by high performance liquid chromatography, it was 1.7 g.

[Effects] According to the method of the present invention, xylitol is produced and cultured from xylose by using the genus Rhonela. Therefore, the conventional method of chemically reducing xylose and the method of producing a coenzyme by using an expensive enzymatic reaction are expensive. In contrast, xylitol can be easily mass-produced, and xylitol can be produced on an industrially profitable basis.

Claims (2)

(57) [Claims] A medium containing D-xylose as a main raw material,
A method for producing xylitol by the genus Rhonera, comprising inoculating a microorganism belonging to the genus Rhonella having xylitol-producing ability, culturing under aerobic conditions to produce and accumulate xylitol, and collecting the xylitol. 2. A solution containing xylitol obtained by culturing and containing a D-xylose solution.
la) A method for producing xylitol by the genus Rhonela, wherein xylitol is produced by reacting cells of the genus and treated cells.