JPH0591890A - Production of trehalose - Google Patents

Abstract

PURPOSE:To easily obtain a large amount of trehalose in a short time by heating and drying microbial cell containing trehalose and extracting the denaturated cell with water or an organic solvent. CONSTITUTION:A trehalose-containing microbial cell (preferably cell of yeast of the genus Saccharomyces) is denaturated by heating and/or drying. The denaturated cell is extracted with water or an organic solvent (preferably ethanol) and trehalose is separated from the extracted liquid. The above heat- treatment is preferably carried out at 80-100 deg.C for 2hr and the drying is preferably performed at 90-110 deg.C for 5hr.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing trehalose, which is expected to be widely used in the fields of foods and pharmaceuticals as a raw material for sweeteners, preservatives, humectants, and anticancer agents.

[0002]

BACKGROUND OF THE INVENTION Trehalose is produced by microorganisms,
It is also produced by an enzymatic method or the like, but the production amount is high because the yield is small. Therefore, generally, a method of obtaining from baker's yeast containing a large amount of trehalose is used. LC Stewart et al., J. Am. Chem. Soc., 20.
Pages 59 to 2061 and 1950 describe a method of extracting squeezed yeast with 95% alcohol to obtain trehalose. Further, Agricultural Vol. 27, No. 7, pp. 412 to 419 (Showa 28), a method for obtaining trehalose by boiling and extracting baker's yeast with 90% ethanol is described.

In the extraction of trehalose with an aqueous ethanol solution, when the concentration of ethanol is low, a large amount of protein in the cells is also extracted, and the process of recovery and purification of trehalose after extraction becomes complicated. Although it is necessary to use high-concentration ethanol in order to suppress the extraction of protein, the extraction rate becomes very low when high-concentration ethanol is used to extract trehalose from living cells.

Further, in the extraction of trehalose from live cells, when the extraction temperature is low, the amount of trehalose extracted decreases due to decomposition of trehalose by the enzyme trehalase existing in the cells, and this tendency is particularly low. Remarkable when extracting with alcohol or water. For this reason, high-temperature extraction must be performed when extracting from live cells.

Further, it takes a relatively long time to extract trehalose from living cells.

[0006]

Therefore, the present invention is intended to solve the above-mentioned various problems.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned various problems, the present inventors have solved the above-mentioned various problems by denaturing the cells by heating and / or drying. It was found that the present invention was completed. Therefore, in the present invention, the bacterium containing trehalose is subjected to a denaturing treatment by heating and / or drying, and then trehalose is extracted with water or an aqueous solution of an organic solvent, and trehalose is collected from the extract. The present invention provides a method for producing trehalose, which is characterized by the above.

[0008]

DETAILED DESCRIPTION In the present invention, microbial cells of any microorganism containing trehalose, for example, microbial cells of the genus Rhizoctonia, the genus Scleronium, the genus Arthrobacter, the genus Nocardia, etc. can be used. Upper yeast cells are preferred, and particularly Saccharomyces ( Sacchar
Oomyces ) yeast cells are preferred. As the yeast cells, those that have been particularly cultured for the production of trehalose can be used, but yeasts produced for other purposes such as baker's yeast, yeasts that are a by-product in the production of beer, etc. can also be used. ..

In the present invention, the cells are heated and / or dried to inactivate trehalase, which is a trehalose-degrading enzyme, denature and coagulate proteins, and destroy cell walls. Therefore, any heating or drying method that achieves these objects can be used. For example, the heat treatment may be performed on a wet cell, a cell suspension or the like at 70 ° C to 110 ° C, preferably 80 ° C to 100 ° C.
For example, the treatment can be performed at 95 ° C. for 0.5 to 12 hours, preferably about 2 hours while suppressing the evaporation of water. In addition, the drying treatment is performed on the cells at 70 ° C to 110 ° C.
C., preferably 90.degree. C. to 110.degree. C., for example, 100.degree. C. for 2 to 12 hours, preferably about 5 hours, while performing the treatment while evaporating water. Although commercially available yeast for bread making is a dry cell,
Since it was produced under specific conditions that kill or damage the cells to a minimum, it does not correspond to the dried cells of the present invention unless it is subjected to additional heating or drying treatment.

The extraction solvent used in the method of the present invention is water or an aqueous solution of an organic solvent. As the organic solvent in this case,
Water-miscible organic solvents such as methanol, ethanol,
Acetone or the like can be used, but ethanol is preferable from various viewpoints such as nontoxicity, protein aggregating power, trehalose extraction power, lipid extraction power and the like. The preferred concentration of the organic solvent varies depending on the trehalose-dissolving power of the organic solvent, the protein cohesion, and the like.

As for ethanol, for example, as shown in FIG. 1, when extraction is carried out for 1 hour at the boiling point of the extraction solvent, it can be efficiently extracted in the range of 0% (water) to approximately 80%. This is in contrast to that in the live yeast, when the ethanol concentration is 60% or more, the extraction rate sharply decreases as the concentration increases. As shown in Figure 2,
When extraction is performed at the boiling point of the extraction solvent for 1 hour, the amount of protein extracted (dissolved) sharply decreases as the ethanol concentration increases. Therefore, the ethanol concentration is 50% to 90%,
Preferably, trehalose is sufficiently extracted by extraction with an aqueous ethanol solution of 60% to 80%, for example, about 65 to 70%, and the amount of protein dissolved in the extract is small. It can be collected easily.

[0012] For example, the treated yeast of the present invention is extracted at the boiling temperature thereof with an aqueous ethanol solution having the above-mentioned concentration, and the extract is subjected to filtration, centrifugation, etc. to obtain yeast cells,
After removing coagulated proteins and the like, trehalose is obtained in the form of crude crystals by cooling the obtained filtrate or supernatant. The extraction time of 15 minutes or more is sufficient, and the upper limit of the extraction time is not particularly critical. However, even if the extraction time is lengthened, the extraction rate does not improve, so in practice 15 minutes to 1
Time is appropriate. FIG. 3 shows the results of measuring the amount of trehalose extracted with time by performing extraction at the boiling point using water as the extraction solvent. As is clear from this figure, the amount of trehalose extracted by the live yeast for the extraction treatment for 30 minutes is very small, and extraction for at least 1 hour is required. The final extraction amount is also considerably lower than that of the treated yeast of the present invention.

Next, the extraction temperature is in the range of 35 ° C. to the boiling point of the solvent, and sufficient extraction can be performed at any temperature. FIG. 4 shows the results of measuring the amount of trehalose extracted when water was used as the extraction solvent and the extraction treatment was carried out at various temperatures for 1 hour. There is no difference in the amount of trehalose extracted in the yeast treated with the present invention between 35 ° C and 100 ° C, but in the live yeast, it is approximately 30 ° C-60 ° C.
Very little amount of trehalose extracted between ° C. One of the reasons for this is considered to be that trehalose contained in living cells is decomposed during the extraction.
At 70 ° C to 100 ° C, a considerable amount of trehalose is extracted from live cells due to inactivation of trehalase, etc.,
It is considerably smaller than the amount of trehalose from the cells that have been treated according to the present invention.

FIG. 5 shows the same experiment as in FIG.
This is the result obtained by using a 0% aqueous solution. Although the amount of trehalose extracted from live yeast at low temperature is not as low as when water is used, it is considerably smaller than the amount extracted from yeast treated with the present invention. In addition, the amount of trehalose extracted from yeast treated with the present invention in the entire temperature range is always higher than that from live yeast.

Considering the above results, the preferred embodiment of the method for producing trehalose of the present invention is as follows. In the case of heat-treated yeast, a high-concentration ethanol aqueous solution is added to the treated yeast, and the yeast is heated to room temperature or heated for 10 to 2
Stir for 0 minutes and extract. Although the concentration of the aqueous ethanol solution to be added varies depending on the amount of water in the treated yeast, it is preferable that the concentration of ethanol after stirring be 65% or more in consideration of the water in the yeast. This is because deproteinization is performed and the solubility of trehalose during crystallization is further reduced. After extraction, the suspension is filtered and the filtrate is cooled to obtain crude crystals.

For the yeast which has been dried, an aqueous ethanol solution is added to the treated yeast and stirred for about 20 minutes. The suspension is filtered and the filtrate is cooled to give crude crystals. When the trehalose concentration in the filtrate is low, the filtrate is concentrated and then crystallized.

[0017]

As a result of the above, according to the method of the present invention, (1) the extraction amount of trehalose is always large, and (2) the extraction rate is high, as compared with the extraction method using live cells, so that the extraction time is short. (3) The extraction temperature is not limited, so the extraction temperature can be freely set as needed, and (4) the amount of trehalose extracted is high even at high alcohol concentrations where proteins coagulate. From this, a trehalose extract containing a small amount of protein was obtained, and trehalose was extremely easily recovered from this extract, and (5) trehalose extraction ratio was low. Uses less amount of.

[0018]

EXAMPLES Next, the present invention will be described more specifically by way of experimental examples and examples. The following general method was used in the following experimental examples. Baker's yeast manufactured by Oriental Yeast Co., Ltd. was used as the live yeast. The yeast drying and / or heat treatment was as described below. The extraction was carried out by suspending yeast having a wet weight of 30 g in a solvent of 150 ml. Solid-liquid separation after the extraction treatment was performed by filtration using a 0.22 μm Menglan filter. High-performance liquid chromatography (CR-4A manufactured by Shimadzu Corporation; column: Bi
Aminex HPX-87C manufactured by o-Rad), and the protein was quantified by a dye binding method (Coomassie Burilliant).
Blue G-250). The extraction rate of trehalose was calculated by setting the amount of trehalose extracted from raw baker's yeast extracted with water at 100 ° C. for 1 hour to 1.0.

Experimental Example 1. Examination of heating and drying conditions The heat treatment was performed on raw baker's yeast under non-drying conditions, that is, in a state of being sealed with a film, at various temperatures between room temperature and 100 ° C for various times between 0 and 12 hours. The drying treatment is carried out by drying raw baker's yeast at various temperatures between room temperature and 100 ° C. under dry conditions (open to the atmosphere).
It was carried out for various times of 12 hours. The evaluation of the heating / drying treatment results was carried out by the amount of trehalose extracted after treating the treated yeast with pure water as an extractant at 100 ° C. for 1 hour.

As a result, the treatment temperature of drying and heating has a great influence on the extraction rate, and in the treatment for 12 hours,
Almost no trehalose was extracted at around 45 ° C. However, the extraction rate increased as the temperature increased at 45 ° C or higher, and became almost constant at 70 to 80 ° C or higher. Next, the processing time was examined. In the heat-treated yeast at 100 ° C., the extraction rate gradually increased until the treatment for 2 hours, but after that, it was almost constant. Therefore, the heat treatment for 2 hours is sufficient. In the drying treatment at 100 ° C., the extraction rate increased as the drying rate (expressed by the ratio of the reduced mass after drying the live yeast for 105 hours at 105 ° C. for 5 hours to the reduced mass after drying for a predetermined time) increased. , Drying rate 10
It became constant after 5 hours when it became 0%. Therefore, a heating time of 5 hours is sufficient. From the results described below, as the heat-treated yeast, live yeast heated at 100 ° C. for 12 hours was used, and as dry yeast, live yeast was heated at 100 ° C.
The dried product was used for 12 hours.

Experimental Example 2. Examination of ethanol concentration of extractant
(FIGS. 1 and 2) The results of extracting trehalose from live yeast and each treated yeast using various concentrations of aqueous ethanol solutions at the boiling point of each solvent are shown in FIG. The extraction rate of trehalose was most dependent on the ethanol concentration in the case of extraction from raw baker's yeast, and the curve showed a peak at around 40 vol%. At higher concentrations, the extraction rate dropped sharply. On the other hand, dry,
Alternatively, in the yeast which was heated, the dependence was observed, though not as remarkable as in the case of the live yeast. However, it was found that the trehalose extraction rate was higher in the treated yeast than in the live yeast at any ethanol concentration, and the extraction rate was increased by heating or drying.

Further, the relationship between the ethanol concentration and the protein residual rate after solid-liquid separation was investigated. After treating live yeast with boiling point for 1 hour in ethanol extract, 4 ℃, 6000rpm
It is centrifuged in FIG. 2 and the relative amount of protein in the supernatant is shown in FIG. As is clear from this figure, when the alcohol concentration in the extract exceeds 60%,
The amount of residual protein was significantly reduced.

Experimental Example 3. Examination of extraction time (Fig. 3) The extraction rates from each yeast were compared. FIG. 3 shows the change over time in the extraction rate. As is clear from this figure, the extraction from the heat-treated or dried yeast was significantly faster than the extraction from the untreated yeast, and it was found that the extraction for about 15 to 20 minutes was sufficient.

Experimental Example 4. Examination of extraction temperature (Fig. 4 and Fig.
5) The effect of extraction temperature on the extraction from each yeast was examined.
The extraction result with pure water is shown in FIG. 4, and the extraction with 50 vol% ethanol aqueous solution is shown in FIG. In extraction with pure water, the extraction rate from raw baker's yeast was significantly affected by temperature. In addition, the effect was slightly observed in the ethanol aqueous solution. However, it was found that the dried and heat-treated yeast had no effect at all, and extraction at room temperature was also possible.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentration of ethanol in the extraction solvent and the relative amount of trehalose extracted when the extraction time is 1 hour and the extraction temperature is the boiling point of the extraction solvent.

[Fig. 2] Fig. 2 is a graph showing the amount of protein in the filtered extract when live yeast was extracted with boiling point for 1 hour with extractants having various alcohol concentrations.

FIG. 3 is a graph showing the relationship between the extraction time and the relative amount of trehalose extracted when water is used as the extractant and extraction is performed at the boiling point.

FIG. 4 is a graph showing the relationship between the extraction temperature and the relative amount of trehalose extracted when water is used as the extractant for 1 hour.

FIG. 5 is a graph showing the relationship between the extraction temperature and the amount of trehalose extracted when an aqueous solution of 50% ethanol was used as an extractant for extraction for 1 hour.

Claims (1)

[Claims] 1. A bacterium containing trehalose is subjected to a denaturation treatment by heating and / or drying, and then trehalose is extracted with water or an aqueous solution of an organic solvent, and trehalose is collected from the extract. A method for producing trehalose, comprising: