JPH03180172A - Production of palatinose and trehalose - Google Patents

Abstract

PURPOSE:To efficiently produce the subject palatinose and trehalose almost without by-production of glucose and fructose as monosaccharides and simultaneously improve the ratio of trehalose production by acting an enzyme produced by an microorganism belonging to Klebsiella genus on sucrose. CONSTITUTION:Palatinose and trehalose are produced from sucrose utilizing an enzyme produced by a microorganism belonging to Klebsiella genus and capable of conversion of sucrose into palatinose and trehalose. As the above mentioned microorganism belonging to Klebsiella genus, Klebsiella planticola MX-1 (Bikokenkinki No. 11123), Klebsiella planticola MX-2 (Bikokenkinki No. 11124) and Klebsiella terrigena ATCC 33257 are used.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing palatinose and trehalulose. More specifically, the present invention relates to microorganisms belonging to the genus Klebsiella, particularly Klebsiella planticola (
Klebsiella planticola ) M
X-1 (Feikoken Bacteria Deposit No. 11123), Klebsiella
Planticola MX-2 (Feikoken Bacteria Deposit No. 11124) and Klebsiella terrigena ATCC33257
The present invention relates to a method for producing palatinose and trehalulose from sucrose using.

(Conventional technology)

Palatinose and trehalulose are currently widely used as nasal sweeteners, and are industrially produced by reacting sucrose with α-glucosyltransferase derived from microorganisms. Conventionally, the microorganism that converts sucrose into palatinose or trehalulose is Protaminoba.
Strains of the genera Cter, 5erratia, and Erwinia are known. For example, German patent specification 1,
No. 049,800 has Protaminobacter
rubrum, 5erratia plastic
A method for converting sucrose into palatinose using enzymes derived from microorganisms, such as Japanese Patent Publication No. 57-10720, describes a method for producing palatinose by culturing Protaminobacter or Serratia in a sucrose solution under aerobic conditions; Tokuko Showa 6
No. 0-9797 describes a method for producing isomaltulose using an immobilized enzyme 5 using microorganisms of the genus Erwinia.

(Problems to be Solved by the Invention) In the production of palatinose using these conventionally known enzymes produced by microorganisms, the production ratio of palatinose and trehalulose is about 6:1 to 10:1.

Palatinose easily crystallizes, so after purifying the reaction solution, it is concentrated and crystallized to produce a product. Trehalulose does not crystallize, so it remains in the final sound after collecting the palatinose crystals, and this is used to further refine and concentrate the liquid product. Palatinose・
It is the main component of syrup. However, in the above enzymatic reaction, in addition to the target products palatinose and trehalulose, monosaccharides such as glucose, fructose, isomaltose, and isomaltose are produced as byproducts. Among these, the production ratio of monosaccharides glucose and fructose is about 5% of the sugar composition in the reaction solution. If these monosaccharides coexist, the product will become colored due to repeated heat treatments such as concentration during the manufacturing process, and a decoloring process will be essential in the product recovery process, resulting in problems with the production efficiency of the desired product and the manufacturing process. was there. Furthermore, when the product obtained by this method is added to foods as a sweetener, palatinose may crystallize in the final product because of its low solubility in water. Therefore, the content is limited, whereas palatinose syrup, which has highly soluble trehalulose as its main ingredient, does not have this problem and is in high demand due to its high quality sweet taste. However, despite this high demand, conventional production methods have only yielded syrups with low trehalulose content. Under the above circumstances, an enzyme-producing bacterium that produces a high trehalulose production ratio and a small amount of monosaccharides has been desired.

(Means for Solving the Problems) The present inventors have been searching for producing bacteria that produce less monosaccharide and have a high production ratio of trehalulose. Klebsiella planticola (K.
MX-1 and MX-2, which belong to the genus Lebsiella planticola, efficiently produce palatinose and trehalulose from sucrose without producing much of the monosaccharides glucose and fructose. being higher than the body;
In addition, Klebsiella terrigena (Klebsiella
It was discovered that ATCC33257 (A. terrigena) also has similar properties, and the present invention was completed. Conventionally, there is no known method for producing palatinose and trehalulose using Klebsiella.

Next, the microorganism Klebsiella plantico of the present invention, -
The results of the identification test for La MX-1 and MX-2 are shown. These two strains were subjected to microscopic observation and 23 identification tests using the API20E kit, etc., and the following results were obtained.

Test items and result items 1.0 NPG test 2, fulginine hydrolase 3, lysine decarboxylase 4, ornithine decarboxylase 5, citric acid utilization testro, hydrogen sulfide production test 7, urease 8, tryptophan deaminase 9, indole 10, VP Test 11, Gelatin Hydrolysis 12, Carbohydrate Utilization Glucose Mannite Inosit Sorbitrhamnose MX-1 10 + Sucrose + + Melibiose + + Amygdalin
＋ ＋Arabinose ＋
+13-oxidase test 14, *Growth at 10°C + +
15-Melezitose availability test This oxidase test uses cytochrome oxidase test filter paper.Growth at 110°C using Nutrient agar.
Inoculate the test strain into medium (Difco) and inoculate for 10 minutes.
Barsik melezitose utilization test cultured in 'C
OW medium to which melezitose was added to a final concentration of 1%, was cultured at 37°C. Observation with an optical microscope revealed that MX-1 and MX-2 had a diameter of 0.5
It is a straight, non-motile, Durham-negative bacillus with a length of 1-1 lm and a length of 1-411 m. Based on these results and the above test results, the taxonomic position of this strain was determined by BERGEY'SMA.
NUAL OF Systematic Bacter
As a result of checking with iology Volume 1,
This strain is a facultative anaerobic Durham-negative bacillus Klebsiel.
It was considered to belong to la planticola. Klebsiella strain Klebs as a control.
iella planticola ATCC3353
1, Klebsiella terrigena ATC
C33257, Klebsiella pnuemon
As a result of conducting a similar test on three strains of iaeIFO13541, MX-1 and MX-2 were found to be Klebsiell.
It was confirmed that the strain matched A. planticola ATCC33531 and was different from the other two bacterial species. However, the present inventors found that the above-mentioned comparative bacterial species Klebsiella p.
lanticola ATCC33531 is not capable of producing palatinose from sucrose, but MX-1 and M
X-2 converts sucrose into palatinose, so Kle
This strain was determined to be a new strain of Klebsiella planticola and was classified as Klebsiella plantico.
la MX-1 and MX-2. These new strains of Klebsiella planticola MX
-1 and MX-2 were deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as "Microorganism Deposit Number: Microorganism Research Institute No. 11123 and No. 11124." Then Kleb
Palatinose,
When searching for production capacity of trehalulose, Klebsi
It has also been found that ella terrigena ATCC 33257 converts sucrose to palatinose.

It has not been previously known that a bacterial species belonging to the genus Klebsiella converts sucrose into palatinose, and the use of a bacterial strain of this genus is an important feature of the present invention.

The method for producing palatinose and trehalulose related to the present invention will be explained below. The enzyme produced by this bacterium that converts sucrose into palatinose and trehalulose is also considered to be α-glucosyltransferase, similar to Protaminobacter, Serracha, and Erwinia, and the presence of sucrose, fructose, and palatinose in the culture medium. It is inducibly produced by the bacteria and exists incidentally in the bacterial cells. Therefore, industrially, after culturing bacterial cells in an enzyme production medium, an immobilized enzyme is produced by immobilizing the bacterial cells as is, and the immobilized enzyme is filled into a bioreactor, and a sucrose solution is continuously passed through this to cause a reaction. Palatinose and trehalulose are produced, and after desalting, purifying and concentrating the reaction solution, the desired product is separated.

Cultivation is usually carried out aerobically using a liquid medium. The medium composition for enzyme production uses sucrose as a carbon source, and the quantitative proportion of sucrose in the medium is 1 to 15% (w/v), particularly preferably 5 to 13% (w/v). use. Yeast extract, peptone, which can be used by microorganisms as a nitrogen source
Malt extract, corn steep liquor, etc. are used, and yeast extract, corn steep liquor, etc. are particularly preferred. As the inorganic salts, salts such as phosphoric acid, magnezium, calcium, potassium, and iron are used. Furthermore, other organic substances and inorganic substances are added to the medium as necessary.

The culture is carried out at a temperature of 15 to 38°C, at which the bacterial cells grow, but is preferably in the range of about 25 to 35°C.

The pH of the medium is 5.0 to 7.0, preferably pH 6.0 to 7.0.
It is adjusted within the range of 7.0. In culture using a normal culture tank, ventilation of about 1/10 to 1 vvm and 30
Stir at ~400 rpm. The culture time is 10-
It takes about 24 hours. After the culture is completed, the culture solution is cooled and the precipitate is collected by centrifugation. Various methods can be used to obtain an immobilized enzyme; for example, the immobilized enzyme is mixed with sodium alginate and then mixed with a calcium chloride solution to form a gel in the form of particles.

This granulated enzyme is further treated with polyethyleneimine and geltaraldehyde to obtain an immobilized enzyme. This is packed into a column, and a sucrose solution having a concentration of 20 to 60% W/W is passed through the column at a temperature of about 25° C. and a pH of 5.5, and the reacted tinose is recovered by centrifugation and used as a product. In addition, difficult-to-crystallize trehalulose is recovered as syrup and used as a product. A highly pure product can be obtained by fractionating this syrup by conventional chromatography using a Ca-type ion exchange resin.

(Efficacy) New strains MX-1, MX-2 and Klebsiella terrigena ATC
Palatinose and trehalulose-producing bacteria of the genus Klebscilla such as C33257 produce about half the amount of monosaccharides fructose and glucose compared to conventional bacteria such as P. Because a clear reaction solution with less coloring due to heating is obtained, the cleaning process can be simplified, and compared to conventional bacteria, the amount of trehalulose produced is large, making it possible to increase the production amount of palatinose syrup. It becomes possible to produce syrup. In addition, since the proportion of palatinose in the sugar composition of the reaction solution is lower than that of conventional bacteria, crystallization of palatinose from the reaction solution can be avoided, making it possible to increase the concentration of the raw material sucrose solution, thereby avoiding microbial contamination during the process. Cost reduction is possible through high-concentration raw material processing.

Example 1 [Enzyme production] Final concentration of sucrose in water: 50 g, C, S, L
30g.

The materials were dissolved to give 5 g of yeast extract, 2 g of disodium phosphate, and 3 g/e of sodium chloride, and the pH was adjusted to 6.5 to 7.0 with a sodium hydroxide solution to prepare a medium. Sterilization conditions are autoclave at 120°C.
The duration was 20 minutes. Pour 50 ml of culture medium into a 500 ml shaking flask, and add K. planticola MX-
Inoculate 1 slant and inoculate 2 at 28°C and 114 rpm.
The cells cultured for 4 hours were used as seed bacteria.

Capacity 3. Culture medium 2e was put into a fermentor of Oe, sterilized and cooled to a temperature of 28°C, and the seed culture was inoculated, and the aeration rate was 1/4 vvm, stirring at 400 rpm to approx.
It was cultured for 2 hours. The humidity during cultivation was 28°C, and the pH was approximately 6.
It was kept at 0. The α-glucosyltransferase activity of the culture solution was 15 U/ml. IU is pH 5.5 2
When reacted at 206C in a 0% sucrose solution, 1 unit (U) was defined as the amount of enzyme at which the initial rate of conversion of sucrose to a product was 1 pmol per minute.

[Immobilization of enzyme 1 The culture solution was cooled to 5°C, centrifuged at 20,000 G for 30 minutes, the supernatant liquid was discarded, and the precipitate was collected.

Precipitation was carried out in a 1:1 (w/w) solution with 4% sodium alginate solution.
) and dropped into a 0.25 M calcium chloride solution from a nozzle with a pore size of 0.5 mm using a dropping device to gel the soil into granular soil. After aging for 2 hours, the soil was washed with water to obtain a granulating enzyme. Next, this granulating enzyme was mixed with 2% polyethyleneimine (PE) adjusted to pH 5.5 by adding hydrochloric acid.
I) Mixed with the solution in a ratio of 1:1 (w/w) and left for 5 minutes, immediately filtered to recover the PEI-absorbed granulated enzyme, followed by cooling to 5 °C. The immobilized enzyme 2
00g was produced. As a result of investigating the characteristics of this immobilized enzyme, the activity was 33 U/g, and a heat resistance test in which 5 g of the immobilized enzyme was immersed for 18 hours in 90 ml of calcium acetate buffer solution at a temperature of 25-50°C showed that it did not exceed 40°C. Activity decreased rapidly. In experiments where the pH was 5.0-6.0 and the reaction temperature was 25-35°C, the higher the temperature, the more palatinose was produced, while the lower the temperature, the more trehalulose was produced, and the more glucose was produced. , the production rate in fructose became smaller.

[Manufacture of palatinose and trehalulose] Above, M
The immobilized enzyme X-1 was packed into a column with an inner diameter of 25 mm and a length of 600 mm, and a sucrose solution with a solid content of 50% (w/v) was passed through it at a temperature of 25°C and a flow rate of 60 ml/h (SV = 0.3). The sugar composition of the reaction solution flowing out from the column was investigated.

Survey results Reaction liquid sugar composition Fructose 1.1% Glucose 1.0 Sucrose
1.3 Palatinose (P)
60.7 Trehalulose (T) 35.8
Others 0.1 total
100% P/T ratio
1.7 The above reaction solution is filtered and passed through a cation exchange resin tower and an anion exchange resin tower for desalting and purification, and then sent to a vacuum crystallizer, and while concentrating at a temperature of 45-60°C, seeds are added and decocted. After sugaring, the white part was placed in a crystallizer while cooling and separated using a centrifuge to obtain palatinose crystals with a purity of 99.5% and a recovery rate of 50%. The sugar composition of the syrup after collecting the palatinose crystals was as shown below.

Syrup Sugar Composition Fructose 2.7% Glucose 2.5 Sucrose
1.3 Palatinose (P)
24.0 Trehalulose (T) 69.3 Others MX as above 0.2
Regarding the production ratio of palatinose and trehalulose in the sugar composition in the reaction solution obtained by -1, the P/T ratio was 1.7, in which trehalulose was abundant, and the production of monosaccharides was also halved. Therefore, the syrup obtained after collecting the palatinose crystals has a lower monosaccharide content of 5.2% compared to conventional palatinose syrup, is almost colored and clear, and has a high trehalulose content.

Example 2 In the same manner as in Example 1, planticola
When MX-2 was used, the sugar composition of the reaction solution flowing out of the column and the syrup was as follows.

Reaction liquid sugar composition Syrup sugar composition Fructose 1.1% Fructose 2.
4% Glucose 1.0 Sucrose 0.5 Palatinose (P) 60.0 Trehalulose (T) 37.3 Others 0.1 Total 100% P/T ratio 1.6 Implemented in Example 3 using terrigena ATCC33257 Using an immobilized enzyme prepared in the same manner as in Example 1, a sucrose solution with a solid content of 45% (w/v) was heated to a temperature of 25°C.
The sugar composition of the reaction solution flowing out of the column when the liquid was passed through the column at a flow rate of 60 m1/h (SV = 0.3) and the sugar composition of the syrup after recovering Palatinose crystals from the reaction solution with a recovery rate of about 70% are as follows. It was as follows.

2.2 0.5 22.0 72.7 0.2 glucose sucrose Palatinose (P) Trehalulose (T) others Reaction liquid sugar composition fructose glucose sucrose Palatinose CP) Trehalulose (T) others total PIT ratio 1.1% 1.0 1.0 76.5 20.0 0.4 100% 3.8 syrup sugar composition fructose glucose sucrose Palatinose (P) Trehalulose (T) others 4.4% 4.0 1.6 24.0 64.7 1.3

Claims (4)

[Claims] (1) Klebsiella planticola MX-1 (FEB Deposit No. 11123) and Klebsiella planticola MX-2 (FEK Deposit No. 11124), which have the ability to produce palatinose and trehalulose. (2) A method for producing palatinose and trehalulose, which is characterized by producing palatinose and trehalulose from sucrose by using oxygen produced by microorganisms belonging to the genus Klebsiella to convert sucrose into palatinose and trehalulose. (3) The microorganism belonging to the genus Klebsiella is Klebsiella planticola MX-1 (Feikoken Bacteria Deposit No. 11123)
, Klebsiella planticola MX-2 (Feikoken Bacteria Deposit No. 11124) and Klebsiella terrigena AT
3. The method of claim 2, which is CC33257. (4) The method according to claim 2, which utilizes an immobilized enzyme.