JPH06269289A - Method for isolating and purifying trehalose by crystallization with water - Google Patents

Abstract

PURPOSE:To obtain a method for simply providing a crystal of trehalose.dihydrate in high purity from an aqueous solution containing the trehalose. CONSTITUTION:This method for purifying trehalose is characterized by depositing a crystal of trehalose.dihydrate from an aqueous solution of the trehalose without using an organic solvent and separating the resultant crystal.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing trehalose. Trehalose is expected to be used as a cell activity retainer, a cold-resistant agent, an antifreeze agent, etc. in the fields of pharmaceuticals and foods.

[0002]

2. Description of the Related Art Conventionally, as a method for producing trehalose,
Known methods include a method of extracting from natural products such as dried yeast, a method of converting maltose, sucrose and the like into an enzyme to convert it into trehalose, a method of culturing a microorganism to produce and accumulate trehalose.

As a method for separating trehalose, there are a method for extracting trehalose from dried yeast and the like, a method for producing and purifying trehalose by an enzymatic method, and a method for producing and purifying trehalose by culturing a microorganism. In the case of extraction from dry yeast,
A method in which ether and alcohol are extracted, and crystallization is repeated using alcohol and acetone (Science, 61 (1587), 57
0, 1925), the yeast was treated with NH ₂ SO ₄ , Hg of heavy metals,
After precipitation with Fe, a method in which a large amount of 95% alcohol is added to the filtrate for crystallization (Science, 82 (2131), 422, 1935)
, Alcohol extracted from yeast, ion exchange resin (Ambe
rlite IR-100, IR-4B), and then crystallization by adding a large amount of 95% alcohol after removing ionic substances (J. Am. Chem. Soc., 72, 2059, 1950). Has been done.

On the other hand, as for the enzymatic method, a method is known in which maltose is treated with maltose phosphorylase and trehalose phosphorylase to produce trehalose, the precipitate is removed from the enzyme reaction solution, and then purified with an anion exchange resin (Japanese Patent Laid-Open No. 2000-242242). 58-216695). The sugar solution purified by anion-exchange resin treatment is passed through a boric acid-type anion-exchange resin to adsorb trehalose and eluted with potassium borate solution, and the resulting trehalose fraction is treated with a cation-type ion-exchange resin. Then, after concentration, lower alcohol is added and distilled to remove boric acid, and alcohol crystallization is repeated to obtain trehalose crystals. Further, glucosyl transferase immobilized on sucrose is caused to act to generate palatinose, and trehalose, which is a by-product, is present in the remaining sugar solution after crystallization and separation. This sugar solution is first chromatographed on a mixed bed of sulfite type and hydrogen sulfite type strong basic anion exchange resin, and then chromatographed on Ca type strong acidic cation exchange resin to separate trehalose. It is known (Japanese Patent Laid-Open No. 4-131090).

Further, in the case of a method for culturing microorganisms to produce trehalose, the culture solution of trehalose-producing bacteria of the genus Nocardia is sterilized and treated with methanol to remove trehalose from the filtrate. A method is known (Japanese Patent Laid-Open No. 50-154485). In this method, the trehalose crystals are obtained by treating the filtrate twice with an anion exchange resin and a cation exchange resin respectively, performing gel filtration, adsorbing and eluting on an activated carbon column and repeating ethanol crystallization. There is also known a method in which cells obtained by culturing a bacterium such as Rhizoctonia or Sclerotium are pulverized and extracted with an aqueous solution of trichloroacetic acid, and trehalose is separated from the extract (JP-A-3-130084). . The extract may first be treated with chloroform and ether to remove lipids and trichloroacetic acid, passed through an ion exchange resin and then evaporated to dryness. This may be dissolved in acetonitrile or the like to separate trehalose by silica gel chromatography. It is shown. After culturing, the bacterial cells were separated, the supernatant was concentrated and then chromatographed with Bio-Gel P-2.
A method of purifying by o-Gel P-2 chromatography and concentrating and drying this trehalose fraction to obtain a dried product of trehalose is also known (Agric. Biol. Chem., 52).
(3), 867-868, 1988).

As described above, the crystallization of trehalose is generally carried out by the alcohol crystallization method, but the trehalose dihydrate crystals obtained by the alcohol crystallization method are fine and have a low purity. .

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems and provide a method for easily obtaining highly pure trehalose dihydrate crystals from an aqueous solution containing trehalose.

[0008]

Means for Solving the Problems The present invention provides a trehalose crystallization method that achieves the above-mentioned object, in which an aqueous trehalose solution is concentrated and cooled to precipitate trehalose dihydrate crystals by crystallization. By separating this, this purpose has been achieved.

The origin of the aqueous trehalose solution is not questioned, and various trehalose aqueous solutions, maltose, sucrose, etc. in the intermediate step of obtaining a trehalose purified product from an extract of natural products such as dried yeast and Iwahiba are enzymatically treated to give trehalose. Various trehalose aqueous solutions in the intermediate step of obtaining the trehalose purified product in the changing method and various trehalose aqueous solutions in the intermediate step of obtaining the trehalose purified product in the method of culturing a microorganism to generate and accumulate trehalose can be used.

The above-mentioned method of culturing microorganisms to produce and accumulate trehalose may be known microorganisms of the genus Nocardia, Rhizoctonia or Sclerotium, or may be newly developed by the present applicant. That is, Brevibacterium genus, Corynebacterium genus, which is conventionally known as a bacterium that produces L-glutamic acid and various amino acids,
It may be an aqueous solution derived from a fermentation broth obtained by culturing a microorganism belonging to the genus Microbacterium or the genus Arthrobacter in a liquid medium containing sucrose or maltose as a main carbon source. As the microorganisms belonging to these genera, any strain can be used as long as it is a microorganism capable of producing trehalose, but specific examples include the following strains.

Brevibacterium lactofermentum ATCC 13869 Brevibacterium flavum ATCC 14067 Brevibacterium tibaricatum ATCC 21642 Corynebacterium glutamicum ATCC 13032 Corynebacterium acetoacidophilum ATCC
13870 Corynebacterium Lilium ATCC 15990 Arthrobacter citreus ATCC 11624 Arthrobacter sulfureis ATCC 15170 Microbacterium ammoniaphilum ATCC 1
5354 Furthermore, mutant strains having improved trehalose-producing ability by subjecting these strains to mutation treatment and the like can also be used.

The culture is preferably carried out under aerobic conditions, the culture temperature is controlled at 20 to 45 ° C., and the pH of the culture solution is controlled at 5.0 to 10.0. Alkaline substances, as well as urea, calcium carbonate,
Ammonia gas or the like can be used.

If necessary, the above-mentioned fermentation liquid can be separated into bacterial cells in advance by a known centrifugation method or filtration method.

The trehalose aqueous solution to which the method of the present invention is applied is an intermediate step solution in each of the above methods, and is usually a step of crystallizing a crude crystal or a purified crystal. These aqueous solutions are obtained from extracts of natural products, enzyme reaction solutions or fermentation solutions, preferably sterilized fermentation solutions, and various purifications are carried out at that time. This may be applied by various known means such as chromatography, but the present inventor is particularly effective in the ultrafiltration membrane treatment and the ion exchange resin treatment in which an anion exchange resin and a cation exchange resin are combined. The ultrafiltration membrane treatment and the ion exchange resin treatment are particularly effective for the fermentation broth obtained by using the above-mentioned L-glutamic acid- and various amino acid-producing bacteria.

The ultrafiltration membrane is a polymer, specifically a molecular weight of about
Use one that can remove 1000 or more, for example 2000 or more. In addition to cellulose-based materials such as acetyl cellulose, a wide variety of materials such as polyamide, polyester, polyacrylonitrile, polyvinyl alcohol, polycarbonate, polyethylene, and inorganic materials are known as the material of the membrane, and any type can be used. The type of the membrane includes a hollow fiber type and a flat membrane type, but is not particularly limited.

Fermentation liquor and the like contain a considerable amount of inorganic salts and other various ionic substances, which are deposited during crystallization. Therefore, trehalose should be desalted before crystallization. It is preferable to set. An ion exchange membrane or the like may be used as the desalting means, but a method of combining a free cation exchange resin and an anion exchange resin is convenient. The cation exchange resin and the anion exchange resin can be used regardless of their types as long as they have a desalting ability. The desalting treatment with the ion exchange resin may be performed before or after the ultrafiltration membrane treatment.

Prior to crystallization, the solution is preferably decolorized with activated carbon or the like. To perform crystallization, first concentrate the aqueous trehalose solution to a trehalose concentration of 70 to 75 g / dl at a liquid temperature of 40 to 80 ° C, inoculate finely ground trehalose dihydrate crystals, and wait for crystallization. . Then, further concentrate to a trehalose concentration of 85-93g / dl, 20-5 ° C.
Gradually cool to a certain degree to grow crystals. 30 minutes at this concentration
It may be left for about 2 hours to eliminate supersaturation, and after solid-liquid separation, the crystals may be washed with a small amount of water. As a result, it is possible to obtain highly pure trehalose having a purity of 99% or more.

[0018]

In the conventional alcohol crystallization, the granularity was small and a very good solubility (transparency of the aqueous solution) could not be obtained. However, the crystal obtained by the method of the present invention is not only high in purity but also very good in solubility. is there. Trehalose has a very high solubility, and even if it was concentrated by the conventional method, it became a candy and could not be crystallized. In the method of the present invention, crystals were precipitated by preliminarily filtering the aqueous solution used for crystallization with an ultrafiltration membrane. It is considered that this is because the crystallization inhibitor was removed by the ultrafiltration membrane.

Next, the present invention will be described in more detail with reference to examples. In addition, quantification of trehalose was carried out by high performance liquid chromatography (column used: Yamamura Chemical Laboratory Co., Ltd.,
PA-03-S-5). All amounts of trehalose are expressed as the amount of dihydrate.

[0020]

【Example】

Example 1 Glucose 4%, Urea 0.5%, KH ₂ PO _{4
0.1%, MgSO 4 · 7H 2} O 0.04%, thiamine hydrochloride 3
00 μg / l, biotin 300 μg / l, soybean degradation concentrate (as total nitrogen) 0.1%, FeSO ₄ .7H ₂ O 0.001%,
A liquid medium consisting of 0.001% MnSO ₄ .4H ₂ O (pH 6.5) was prepared, and 20 ml of each was dispensed into a 500 ml shake flask and heat sterilized at 110 ° C. for 10 minutes. One platinum loop of Corynebacterium lilium ATCC 15990, which had been cultivated in broth agar slant medium for 48 hours at 31.5 ° C, was inoculated into this medium and cultivated at 31.5 ° C for 24 hours in a reciprocating shaker to prepare a seed culture solution. did.

Sucrose 15%, KH ₂ PO ₄ 0.1%, M
_{gSO 4 · 7H 2 O 0.1%} , thiamine hydrochloride 300 [mu] g /
l, Biotin 300 [mu] g / l, Soy decomposition concentrate (as total _{nitrogen) 0.05%, FeSO 4 · 7H} 2 O 0.001%, MnSO 4
・ Dispense a liquid medium containing 2% of an equal mixture of potassium chloride and ammonium chloride into a medium (pH 7.3) consisting of 0.001% of 4H ₂ O into a 1 liter jar fermenter, and store at 120 ° C, 2
After heat sterilization for 0 minutes, inoculate 15 ml of the above seed culture solution at 31.5 ° C,
Culture was started at an aeration rate of 1/2 vvm and a stirring speed of 700 rpm. During the culture, the pH was controlled to 7.3 with ammonia gas. After the start of culturing, the turbidity of the 26-fold dilution of the culture at 562 nm
When it reached 0.60, polyoxysorbitan monopalmitate was added to a concentration of 0.4% to continue the culture, and the culture was terminated when the sucrose in the culture solution was consumed.

Fermentation broth containing 400 g of trehalose (calculated as trehalose dihydrate) thus obtained (protein concentration)
The bacterial cells were removed from 10 l (2.0%) with a centrifuge (pH 7.8). Next, cation exchange resin SKlB H type 10 l column (manufactured by Mitsubishi Kasei Co., Ltd.) and anion exchange resin WA30 OH type 20 l
Columns (manufactured by Mitsubishi Kasei Co., Ltd.) were arranged in series, and the sterilized broth was passed through the column at a flow rate of 10 l / hr to obtain 50 l of a desalted solution. The desalted solution was permeated using an ultrafiltration membrane SIP-0013 (molecular weight cutoff 3000, manufactured by Asahi Kasei Co., Ltd.) to obtain 60 l of a permeate (protein concentration 0.01%). Next, the permeated liquid was pre-concentrated until the trehalose concentration reached about 30 g / dl, to give about 900 ml of pre-concentrated liquid. 30 g of powdered activated carbon in the pre-concentrated liquid
Was added and decolorized under stirring at 60 ° C for 2 hours, and then decolorized filtrate
I got 1000 ml. The decolorized filtrate was further concentrated under reduced pressure with a rotary evaporator to a trehalose concentration of about 75 g / dl. About 2 g of trehalose dihydrate crystals finely pulverized under stirring conditions was added thereto, and after about 30 minutes, the mixture was cooled to 5 ° C at a cooling rate of 5 ° C / h. The resulting trehalose dihydrate crystals were separated by a centrifuge and dried in a vacuum dryer at 40 ° C for 15
Dried for hours. Thus, 178 g of trehalose dihydrate crystal (purity 99.5%, columnar crystal) was obtained. The solubility analysis value (transmittance of trehalose dihydrate 7 g / dl aqueous solution at a wavelength of 400 nm and cell length of 10 mm) was as high as 99.8%, and the crystal grain size was large and uniform as shown in FIG.

Example 2 5 kg of trehalose dihydrate crude crystal obtained by alcohol crystallization was dissolved in water to give trehalose 40 g / dl. To this, 500 g of powdered activated carbon was added, and after decolorization under stirring at 60 ° C for 2 hours, decolorization filtrate
15 l was obtained. The decolorized filtrate was concentrated under reduced pressure to give a trehalose concentration of about 75 g / dl. To this, about 20 g of seed crystals of trehalose dihydrate pulverized under stirring conditions were added,
After 30 minutes, it was cooled to 5 ° C at a cooling rate of 5 ° C / h. The crystals obtained were separated with a centrifuge and then dried with a vacuum dryer at 40 ° C.
After drying for 15 hours, trehalose dihydrate crystals (purity 99.6
%, Columnar crystals) 2.7 kg was obtained. The analytical values of the obtained crystals are shown below.

[0024]

[Table 1]

As described above, the solubility analysis value (trehalose.
The dihydrate 7 g / dl aqueous solution had a very high value of 99.9% at a wavelength of 400 nm and a cell length of 10 mm, and the crystal grain size was uniform.

Comparative Example 1 Trehalose obtained in Example 1
2.0 liter of fermentation broth (protein concentration 2.0%) containing 80 g (trehalose dihydrate equivalent) was added to the ultrafiltration membrane SIP-0013.
(Fraction molecular weight of 3000, manufactured by Asahi Kasei Co., Ltd.) was used for permeation to obtain 5 l of a permeated liquid (protein concentration 0.01%). Next, cation exchange resin SKlB H type 2l column (manufactured by Mitsubishi Kasei Co., Ltd.)
And anion exchange resin WA30 OH type 5 l column (manufactured by Mitsubishi Kasei Co., Ltd.) are arranged in series, and the sterilization broth is flown onto the column at a flow rate of
The solution was passed through at 1 / Hr to obtain 10 1 of desalted solution. Then
The permeate was pre-concentrated to a trehalose concentration of about 30 g / dl to give a pre-concentrate of about 180 ml. 5 g of powdered activated carbon was added to the pre-concentrated liquid, and the mixture was decolorized under stirring at 60 ° C. for 2 hours to obtain 200 ml of a decolorized filtrate. The decolorized filtrate was concentrated to 35 g / dl of trehalose to obtain 180 ml of a concentrated liquid.

Next, 80% ethyl alcohol was placed in a crystallization container.
50 ml was kept at 40 ° C. and left to stir. To this, 180 ml of the trehalose concentrate and 720 ml of 100% ethyl alcohol.
Were simultaneously added at a rate of 50 ml / h and 200 ml / h in proportion to the liquid volume. About 30 minutes later, as soon as the liquid became cloudy, about 0.5 g of finely ground seed crystals of trehalose dihydrate was added, and both liquids were further added simultaneously, and the addition was completed in 3.6 hours. Then, it was cooled to 5 ° C at a cooling rate of 5 ° C / h.

The obtained crystals were separated by a centrifuge and dried in a vacuum dryer at 40 ° C. for 15 hours, and then highly pure trehalose.
75 g of dihydrate crystals (purity 99.2%, columnar crystals) were obtained. Soluble analysis value (wavelength of trehalose dihydrate 7 g / dl aqueous solution
The transmittance at 400 nm and cell length of 10 mm) was as low as 94.4%, and the crystal morphology was fine and inferior in uniformity as compared with Example 1 as shown in FIG.

[0029]

According to the method of the present invention, highly pure trehalose crystals can be easily obtained.

[Brief description of drawings]

FIG. 1 Trehalose-2 obtained in an example of the present invention
It is a drawing substitute photograph which shows the crystal structure of a hydrate crystal.

FIG. 2 is a drawing-substituting photograph showing a crystal structure of a crystal obtained by an alcohol crystallization method which is a comparative example of the present invention.

Claims (1)

[Claims] 1. A method for purifying trehalose, which comprises concentrating and cooling an aqueous solution of trehalose to precipitate trehalose dihydrate crystals by crystallizing and separating the crystals.