JPH06311891A - Production of trehalose - Google Patents

Abstract

PURPOSE:To provide an industrial economical process for the production of trehalose expected to be usable in a wide field as a raw material for foods, pharmaceuticals, cosmetics, etc. CONSTITUTION:Trehalose is produced by culturing a microbial strain belonging to coryneform bacteria capable of producing trehalose. The cultivation is carried out by using sucrose as a carbon source for sugar replenishment or adding a beta-lactam antibiotic substance during cultivation and continuing the cultivation using sucrose as a carbon source for sugar replenishment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a trehalose obtained by culturing a microorganism belonging to a coryneform bacterium having an ability to produce trehalose to produce trehalose, using sucrose as a carbon source for supplement sugar. Manufacturing method.

[0002]

BACKGROUND OF THE INVENTION Trehalose is a disaccharide (α-D-glucopyranosyl-α-D-glucopyranoside) widely distributed in natural products such as plants, insects, yeasts, molds and seaweeds. Conventionally, three types of extraction methods, enzyme methods, and culture methods are known to obtain trehalose.

As the extraction method, a method of extracting from yeast cells (Nagaka, 27 , 12 (1953)), (J. Am. C
he. Soc. 72 , 2059 (1950)), but the amount of trehalose produced is not sufficient because the amount of trehalose is limited by the amount of microbial cells and the trehalose content in the cells. As an enzymatic method, maltose is used as a substrate and maltose phosphorylase and trehalose phosphorylase are used to produce the enzyme (Japanese Patent Laid-Open No. 58-21669).
No. 5) is known, but it is difficult to adjust the enzyme and it requires a cost.

As a culture method, Arthrobacter parafineus (Arthro), which is a hydrocarbon-assimilating bacterium, is used.
vector / paraffinine) KY4303
A hydrocarbon n-alkane, specifically 10
% By n-paraffin as a carbon source
gric. Biol. Chem. 33 (2), 190,
1969), a method of culturing a microorganism belonging to the genus Nocardia (Japanese Patent Laid-Open No. 50-154485), a method of culturing a microorganism belonging to the genus Rhizoctonia and the genus Sclerotium (Japanese Patent Laid-Open No. 130084), and the like. There is. However, in the production of trehalose by Arthrobacter parafineus KY4303,
The n-alkane used as a carbon source has a problem in safety for foods and pharmaceuticals. In addition, the microorganisms belonging to the genus Nocardia have a very low trehalose-producing ability. Furthermore, the production strains belonging to the genus Rhizoctonia and the genus Sclerotium are plant pathogens, and the culture time is about 1
It takes a week and a long time. Furthermore, the production bacteria of the genus Nocardia, Rhizoctonia, Sclerotium, etc. all accumulate trehalose in the microbial cells, so complicated procedures such as extraction from the microbial cells are required to isolate trehalose. Or require large-scale equipment and a lot of energy.

Therefore, in reality, a method for inexpensively producing a large amount of trehalose has not yet been established.

[0006]

DISCLOSURE OF THE INVENTION The present invention is intended to provide a method for industrially advantageously producing trehalose by culturing a microorganism capable of producing trehalose.

[0007]

Means for Solving the Problems As a result of earnest studies on a method for producing trehalose, the present inventors have found that when trehalose is produced by culturing a microorganism belonging to a coryneform bacterium capable of producing trehalose, trehalose is produced. It was found that by using sucrose as a carbon source, trehalose is secreted outside the cells and the trehalose-producing ability is improved. Furthermore, in this culture, it was found that the trehalose production was further improved by adding the β-lactam antibiotic during the culture and adding sucrose as a supplement sugar.

The present invention has been completed based on the above findings, and when culturing a microorganism belonging to a coryneform bacterium having an ability to produce trehalose to produce trehalose, sucrose is used as a carbon source for supplement sugar. A method for producing trehalose, which comprises culturing using, preferably adding β-lactam antibiotic in the culture,
A method for producing trehalose, which comprises culturing using sucrose as a carbon source for supplement sugar.

First, as microorganisms belonging to the coryneform bacterium having trehalose-producing ability used in the present invention, strains belonging to the genera Corynebacterium, Brevibacterium, Microbacterium, Micrococcus, and mutants thereof , Which has the trehalose-producing ability. For example, Corynebacterium meracecola (Cor
ynebacterium / melassecola)
285 strain (ATCC17965), Brevibacterium d.
ivaricatum, NRRL B-2312), Microbacterium ammoniaphilum (Microb
bacterium, ammoniaphilum, AT
CC 15354), Micrococcus glutamicus, A
TCC 14619) and the like.

The medium used for culturing the above-mentioned microorganism is a medium containing an assimilable carbon source, an ordinary nitrogen source, an inorganic salt and a trace amount of nutrients. As the assimilable carbon source in the initial culture medium of the main culture, for example, glucose, fructose, maltose, sucrose, acetic acid, molasses, molasses, etc. can be used alone or in combination. Molasses or molasses that is inexpensive and contains nutrients such as biotin is preferable as the sucrose concentration is 1 to 10%, preferably 5 to
It is used by adding 7% so that when the sugar concentration in this starting medium becomes 10% or more, the osmotic pressure of the sugar inhibits the culture of the microorganism to be used.

As the nitrogen source of the medium, ammonia, ammonium sulfate, ammonium salt, ammonium nitrate, urea, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and the like can be used alone or in combination. As the inorganic salt, phosphoric acid, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate or the like is used, and for example, 5 μg /
Nutrients such as L or more biotin and 100 μg / L or more thiamine or other various vitamins may be added to the medium.

Culturing is carried out under aerobic conditions such as aeration and shaking, and the pH during cultivation is 5 to 10, preferably around 7, and the adjustment is carried out by adding acid or alkali.
The culturing temperature is 20 to 40 ° C., the culturing period is 12 to 60 hours,
It is preferably 24 to 40 hours. The term "complementary sugar" in the present invention means to add a carbon source continuously or intermittently after the middle stage of the logarithmic growth phase, and to use sucrose as a carbon source for complementing sugar. The amount of sucrose used for supplementing sugar is such that the residual sugar amount of sucrose in the culture solution maintains a concentration of 1 to 3%.

Further, in the addition of the β-lactam antibiotic in the present invention, the amount of bacterial cells is calculated from the OD value using a spectrophotometer as the timing of the addition, and if the logarithmic growth phase is based on this OD value. good. 5 to 5 after the start of culture
It is advisable to add it in the middle phase of the 10th hour logarithmic growth phase, and the addition amount is 0.1 to 100 μg / ml, preferably 0.5 to 10 μg / ml as the final concentration. Examples of β-lactam antibiotics used in this manner include penicillin-based compounds such as penicillin G, penicillin V, and ampicillin, and cephalosporin-based compounds such as cephalexin and cephaloridine, which are appropriately used as soluble salts. I can. Addition of β-lactam antibiotics inhibits cell wall synthesis of the microorganism used,
Trehalose is secreted outside the cells, and trehalose accumulates in the medium at a high concentration.

The trehalose thus produced outside the cells can be separated and purified by a known method. As a method of separation and purification, after removing an ionic substance through an ion exchange column, the trehalose is adsorbed by passing through an activated carbon (granular carbon) column. Trehalose is eluted from this column with a suitable solvent such as methanol, and the eluate is concentrated. Purified trehalose can be obtained by adding ethanol to the obtained residue and allowing it to stand at a low temperature.

[0015]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples shown here. The quantification of trehalose was carried out under the following conditions:
It was performed by a high performance liquid chromatography method.

[High Performance Liquid Chromatography (HPL
C) Measurement conditions] Equipment used: LC-800 system (JASCO) Column: TSK-GEL AMIDE80 (Tosoh) Flow rate: 0.8 ml / min Mobile phase: 65% acetonitrile Detector: Differential refractometer Retention time: Trehalose: 10 .9 minutes

[0017]

Examples 1-2, Comparative Examples 1-4 Seed medium (molasses molasses
(Added as 4% sucrose), MgSO_Four・ 7
H ₂O 0.05%, H₃PO_Four0.15%, urea
0.8%, pH adjusted to 7.0 before sterilization with ammonia water
Dispense 100 ml into a 500 ml Erlenmeyer flask
And sterilize at 120 ° C for 20 minutes, then
Mu Melasecola 285 strain (ATCC 17965)
The cells were inoculated and cultured with shaking at 32 ° C. for 16 hours.

Next, a starting medium for main culture (waste molasses (added so as to be 7% as sucrose), MgSO ₄ .7H ₂ O)
0.05%, H ₃ PO ₄ 0.15%, urea 1%, pH adjusted to 5.6 with NaOH before sterilization) 500 ml
Was placed in a 1 L jar and sterilized at 120 ° C. for 20 minutes, and then 30 ml of the above preculture was added to rotate at 1500 rpm.
Incubation was carried out at 34 ° C. with an rpm and aeration rate of 0.5 L / min. The pH during the culture was maintained at around 7.3 with aqueous ammonia.
As the defoaming agent, a polyalkylene glycol type was used.

Penicillin G (potassium salt) was added 8 hours after the start of culture so that the final concentration was 1 μg / ml. Further, the sucrose supplement sugar was added at an interval of 2 hours from the 8th hour after the start of the culture so that the sucrose concentration of the residual sugar was 1 to 3%. The sucrose concentration of residual sugar was measured using a biosensor BF-2 manufactured by KS System Co., Ltd. Table 1 shows the trehalose concentration per culture solution at 20 and 30 hours of culture.
It was shown to.

[0020]

[Table 1]

In Example 1, sucrose was used as a carbon source for saccharose, in Example 2, penicillin G was used in combination with Example 1, and in Comparative Example 1, glucose was used instead of sucrose in Example 1. When used in an amount, Comparative Example 2 used together with Penicillin G in Comparative Example 1, Comparative Example 3 used the same amount of fructose instead of sucrose in Example 1, and Comparative Example 4 used Penicillin G in Comparative Example 3. This is the case when both are used together.

As is clear from the results shown in Table 1, sucrose was the best as the carbon source for supplemental sugar, and the productivity of trehalose was the best, and the addition of penicillin G to the trehalose during the culturing further increased trehalose. Production has increased significantly. When sucrose was used as a carbon source for supplementing sugar and penicillin G was added at the time of culturing, the trehalose production amount at 30 hours of culture reached 3.2 g / dl, which was 4 times as high as fructose when glucose was used. The production amount was as high as 10 times as high as that of the conventional case.

[0023]

Example 3 Strains used are Brevibacterium dibaricatum (NRRL B-2312), Microbacterium ammoniaphilum (ATCC 15354), and Micrococcus glutamicus (ATCC 14619).
Other than the above, the seed medium, the culturing method, and the main culturing were performed in the same manner as in Examples 1 and 2.

The results are shown in Table 2 below. Even when culturing a microorganism belonging to each coryneform bacterium and having the ability to produce trehalose, trehalose was isolated from the outside of the cell by using sucrose as a carbon source for supplement sugar. It was revealed that trehalose was secreted in an even more significant amount by the addition of penicillin G while being produced in E. coli.

[0025]

[Table 2]

[0026]

INDUSTRIAL APPLICABILITY The present invention provides a method for industrially advantageously producing trehalose by culturing a microorganism capable of producing trehalose. Using a microorganism belonging to a coryneform bacterium as a producing bacterium, By using sucrose as a carbon source for sugar, and more preferably by adding a β-lactam antibiotic during the culture, a large amount of trehalose of 2 to 10 times or more is secreted outside the cells, and trehalose is industrially advantageous. Can be manufactured.

Claims (4)

[Claims] 1. A method for producing trehalose, which comprises culturing a microorganism belonging to a coryneform bacterium having an ability to produce trehalose to produce trehalose, using sucrose as a carbon source for supplement sugar. . 2. The method for producing trehalose according to claim 1, wherein the added amount of sucrose as a carbon source for supplement sugar is maintained at 1 to 3% as the residual sugar amount of sucrose. 3. The method for producing trehalose according to claim 1, wherein the β-lactam antibiotic is added in the culture, and the culture is performed using sucrose as a carbon source for supplement sugar. 4. The method for producing trehalose according to claim 3, wherein the β-lactam antibiotic is added in the logarithmic growth phase, and the added concentration is 0.1 to 100 μg / ml.