JP2006342168A - Method for producing trehalose derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method with which high-quality trehalose derivatives (ethers or esters) as a safe surfactant useful as a food and drink, a cosmetic, a medicine, etc., can inexpensively be produced in good yield. <P>SOLUTION: The method for producing the trehalose derivatives is carried out as follows. An anhydrous trehalose having <3% moisture content measured by the Karl Fischer method is reacted with a reagent having reactivity with a non-anomeric hydroxy group under anhydrous conditions to afford the trehalose derivatives (e.g. octaacetyltrehalose, fatty acid esterified trehaloses, dodecyl etherified trehaloses or sulfuric esterified trehaloses) by an esterification or an etherification reaction. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel method for producing a trehalose derivative, and more particularly to a method for producing a trehalose derivative characterized in that a reactive reagent is reacted with anhydrous trehalose under anhydrous conditions.

  Recently, acid esters and alkyl ethers of trehalose such as fatty acid esters and sulfuric acid esters are in the spotlight. That is, in Non-Patent Document 1, it is reported that stearic acid ester, palmitic acid ester and myristic acid ester of trehalose markedly suppressed the growth of malignant tumor in vivo and in vitro, and that these derivatives are promising as antitumor agents. Suggests. For example, as seen in Patent Document 1, sulfate ester has a remarkable effect of keeping moisture in the skin in a well-balanced and moisturizing manner, and has already been used in some cosmetics as an excellent moisturizer and skin beautifier. ing. Further, ethers with alkyl having 8 to 25 carbon atoms are known to be useful as safe and highly active surfactants.

  As can be seen in Non-Patent Document 2 and Non-Patent Document 3, trehalose derivatives are usually prepared by reacting trehalose hydrated crystals with a reactive reagent under anhydrous conditions, resulting in high-quality trehalose derivatives with good yield. Whether or not it can be obtained depends on whether or not water can be completely removed from the reaction system. However, even if the trehalose hydrous crystal is solid, it usually contains about 10% (w / w) of water, and if it is used as it is, good results cannot be obtained. For this reason, until now, prior to the reaction, attempts have been made to dry to an anhydrous state with a drying agent such as phosphorus pentoxide, but it has not been easy to remove the water contained as crystal water. Therefore, it has been extremely difficult to prepare a high-quality trehalose derivative at a high yield and at a low cost.

JP-A-4-290808 Nishikawa et al., “Journal of the Chemical Society of Japan”, No. 10, pp. 661 to 1,666 (1982) CK Lee, Developments in Food Carbohydrate, 1980, published by Applied Science Publishers, pages 1-89 K. Yoshimoto et al., “Chemical and Pharmaceutical Bulletin”, Vol. 30, No. 4, pp. 1,169 to 1,174 (1982)

  In view of such circumstances, an object of the present invention is to provide a method capable of producing a high-quality trehalose derivative with good yield and at low cost.

  This invention solves the said subject by the manufacturing method of the trehalose derivative characterized by making a reactive reagent react with anhydrous trehalose under anhydrous conditions.

  Anhydrous trehalose used as a substrate in the present invention is substantially free of moisture. As a result, according to the production method of the present invention, anhydrous trehalose is dried very easily, or in some cases without drying, even if it is subjected to the reaction as it is, a high-quality trehalose derivative is produced with high yield.

  Hereinafter, the present invention will be described based on experimental examples, examples and the like. Trehalose derivatives referred to in the present invention are esters, ethers, halides, nitrogen-containing derivatives obtained by reacting a reactive reagent with trehalose under anhydrous conditions. And all trehalose derivatives including sulfur-containing derivatives. Accordingly, the reactive reagent in the present invention means acids, bases, alcohols, ketones, halogens, amines and reactive derivatives thereof that react with trehalose under anhydrous conditions to give such derivatives. As a result, the present invention provides the desired trehalose derivative quality and / or yield, for example, by reducing the reactivity of the reactive reagent, causing an undesirable side reaction or inhibiting the main reaction. Therefore, the present invention can be applied to general chemical reactions that lower the production cost and increase the production cost.

  The anhydrous trehalose referred to in the present invention usually means crystalline or amorphous anhydrous trehalose containing substantially no moisture and having a moisture content of less than 3% (w / w) by the Karl Fischer method. These anhydrous trehalose can be used in this invention as well, but crystalline anhydrous trehalose is generally useful because it generally has a high trehalose content and is relatively inexpensive. Although depending on the type of reaction and the use of the derivative, in general, the higher the trehalose content in anhydrous trehalose, the better, and usually 70% or more, preferably 80% or more per solid content is used.

  Among such anhydrous trehalose, amorphous anhydrous trehalose is obtained by dissolving trehalose in a small amount of water and lyophilizing the aqueous solution as it is, or by spray drying or the like, at a temperature exceeding the melting point of trehalose-containing crystals, usually 100 It can be obtained by drying at a temperature exceeding ℃. On the other hand, crystalline anhydrous trehalose has a water content of less than 10% (w / w), preferably a sugar composition containing 60% or more of trehalose per solid content, as disclosed in, for example, JP-A-6-170221. Is a syrup that exceeds 2.0% (w / w) and does not exceed 9.5% (w / w), and crystal anhydrous trehalose as a seed crystal is 0.01 to 20% per solid content. In addition, crystallization is carried out while maintaining the temperature at 40 to 140 ° C., and crystalline anhydrous trehalose is collected from the obtained mass kit, or dried as a mass kit to obtain a solid product. The anhydrous trehalose thus obtained is usually substantially free of moisture with a moisture content of less than 3% (w / w).

  There are no particular restrictions on the origin and origin of trehalose, which is a raw material for anhydrous trehalose. Even if trehalose is obtained from yeast cells, it is obtained by allowing maltose to act on a complex enzyme system comprising maltose phosphorylase and trehalose phosphorylase, or maltose. -It may be obtained by converting maltose directly into trehalose with trehalose converting enzyme or by enzymatic saccharification of a partially hydrolyzed starch. However, from the economical viewpoint of producing a high-quality trehalose derivative at a low cost, those obtained by the third or fourth method are desirable.

  To prepare trehalose from starch, first, the degree of glucose polymerization such as maltotriose, maltotetraose, maltopentaose, maltohexaose obtained by gelatinizing and liquefying starch with acid and / or α-amylase Japanese Patent Application No. 5-349216 (Japanese Patent Laid-Open No. 7-143876) and Japanese Patent Application No. 6-90705 (Japanese Patent Laid-Open No. 7-322883) are used for reducing a reduced starch partial hydrolyzate comprising three or more maltooligosaccharides. No. 6-166611 (Japanese Patent Application Laid-Open No. 8-66188) or Japanese Patent Application No. 6-190183 (Japanese Patent Application Laid-Open No. 8-84586). To convert maltooligosaccharide to a non-reducing carbohydrate having a trehalose structure at the end. The non-reducing carbohydrates are disclosed in Japanese Patent Application No. 6-59834 (Japanese Patent Application Laid-Open No. 7-298880), Japanese Patent Application No. 6-79291 (Japanese Patent Application Laid-Open No. 7-213283), The trehalose releasing enzyme disclosed in Japanese Patent No. 166126 (Japanese Patent Laid-Open No. 8-66187) or Japanese Patent Application No. 6-190180 (Japanese Patent Laid-Open No. 8-336388) is allowed to act, and trehalose is converted from the non-reducing carbohydrate. Liberate. At this time, the non-reducing saccharide-forming enzyme and trehalose-releasing enzyme may be reacted simultaneously or sequentially, and if both enzymes are used together with starch debranching enzymes such as isoamylase and pullulanase, the product The trehalose content is further improved. On the other hand, in order to directly convert maltose into trehalose, maltose or a sugar composition containing maltose is disclosed in, for example, Japanese Patent Application No. 6-144092 (Japanese Patent Application Laid-Open No. 7-170977) and Japanese Patent Application No. 6-156399. (Japanese Patent Application Laid-Open No. 8-263), Japanese Patent Application No. 6-187901 (Japanese Patent Application Laid-Open No. 9-9986) or Japanese Patent Application No. 6-260984 (Japanese Patent Application Laid-Open No. 8-149980). Maltose / trehalose converting enzyme may be allowed to act. These specifications disclose a method for producing trehalose using maltose trehalose converting enzyme, and any of them can be advantageously applied to the preparation of anhydrous trehalose used in the present invention. When higher purity trehalose is required, column chromatography using a salt type strongly acidic cation exchange resin in a fixed bed system, moving bed system or simulated moving bed system is applied to the product thus obtained. A fraction containing high trehalose may be collected. The products and fractions thus obtained contain at least 70% trehalose per solid content and are suitable as raw materials for anhydrous trehalose.

  In order to react a reactive reagent with anhydrous trehalose under anhydrous conditions, a conventional method in this field can be adopted. For example, CK Lee “Developments in Food Carbohydrate”, 1980, published by Applied Science Publishers, 1st to 89th, K. Yoshimoto et al. “Chemical and Pharmaceutical Bulletin, Volume 30, No. 4, pp. 1,169 to 1,174 (1982) and “Carbohydrates as Organic Raw Materials”, 1991, published by VCH Any of these reactions can be applied to anhydrous trehalose in the present invention, and can be appropriately selected according to the desired trehalose derivative.

  Outlined about a typical method for producing a trehalose derivative, a carboxylic acid ester with acetic acid or benzoic acid can be obtained by reacting an acid anhydride or acid halide corresponding to anhydrous trehalose in a basic organic solvent such as pyridine. be able to. In order to produce a sulfate ester, a complex of sulfur trioxide and dimethyl sulfoxide or pyridine may be reacted in an inert gas or rare gas stream. Fatty acid esters with lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, etc. can be obtained by a condensation reaction in the presence of a basic catalyst or by reacting with a corresponding fatty acid halide. . Ethers such as methyl ether, benzyl ether, trityl ether, methylsilyl ether, dodecyl ether are reacted with an excess of the corresponding alcohol in anhydrous trehalose under an acid catalyst or with a corresponding alkyl halide under a basic catalyst. Can be obtained.

  Depending on the application, the reactant containing the trehalose derivative thus obtained is usually freed from unreacted reactive reagents and / or solvents by, for example, filtration, extraction, liquid separation, fractional precipitation, dialysis, distillation, etc. After that, it is used as it is. When a higher purity trehalose derivative is required, for example, in this field for purifying sugars or sugar derivatives such as thin layer chromatography, column chromatography, ion exchange chromatography, gas chromatography, distillation, crystallization, etc. Conventional methods may be applied, and these purification methods are applied in combination as necessary. As is well known, trehalose provides eight reactive functional groups in a normal substitution reaction mainly composed of non-anomeric hydroxyl groups. This means that compositions containing trehalose derivatives with different degrees of substitution in various proportions can be produced depending on the type and conditions of the reaction. Such a composition is usually used as it is, but if necessary, one or more of the above purification methods may be applied and only the desired components may be isolated prior to use.

  The trehalose derivative obtained by the production method of the present invention has a wide range of uses in various fields such as the food industry, the cosmetic industry and the pharmaceutical industry. Fatty acid esters and alkyl ethers are highly active and are useful as safe surfactants for foods, cosmetics, pharmaceuticals, and the like. Depending on the fatty acids, they are expected to be used as antitumor agents. Sulfate ester can be advantageously used in cosmetics as an excellent moisturizing agent and skin beautifying agent, and halide is useful as an intermediate for synthesizing various derivatives.

  Next, the effect of this invention is demonstrated based on an experiment example.

<Experimental Example 1 Preparation of Enzyme>

<Experimental Example 1-1 Preparation of Non-reducing Carbohydrate Generating Enzyme>
In a 500 ml Erlenmeyer flask, 2.0% (w / v) maltose, 0.5% (w / v) peptone, 0.1% (w / v) yeast extract, 0.1% (w / v) hydrogen phosphate 100 ml of a liquid medium (pH 7.0) containing disodium and 0.1% (w / v) sodium dihydrogen phosphate was taken and sterilized by autoclaving at 120 ° C. for 20 minutes. After cooling, the liquid medium in the Erlenmeyer flask was inoculated with Rhizobium species M-11 (FERM BP-4130) and seed-cultured at 27 ° C. for 24 hours under rotary shaking. Thereafter, 20 l of a liquid medium having the same composition as described above was placed in a 30 l jar fermenter, sterilized, and inoculated with 1% (v / v) of the seed culture solution prepared above, while maintaining the pH at 6 to 8, The culture was aerated and stirred at 24 ° C for 24 hours.

  Next, about 18 liters of the culture prepared above is taken in an ultra-high pressure microbial cell disruption apparatus, and after disrupting the microbial cells, ammonium sulfate is added to about 16 liters of supernatant collected by centrifugation so as to be 20% saturated. After standing for 1 hour, the precipitate was removed by centrifugation. Ammonium sulfate was added to the obtained supernatant so as to be 60% saturation, and the mixture was allowed to stand at 4 ° C. for 24 hours. The precipitate was collected by centrifugation and dissolved in a minimum amount of 10 mM phosphate buffer (pH 7.0). After dialyzing against 10 mM phosphate buffer (pH 7.0) for 24 hours, insoluble matters were removed by centrifugation. The newly obtained supernatant was loaded onto a Tosoh ion exchange chromatography column “DEAE-Toyopearl” that had been equilibrated in advance with 10 mM phosphate buffer (pH 7.0) and increased from 0 M to 0.5 M. Under a concentration gradient of sodium chloride, 10 mM phosphate buffer (pH 7.0) was passed through the column. A fraction having enzyme activity was collected from the eluate, dialyzed against 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate for 10 hours, and then insoluble matters were removed by centrifugation. Thereafter, the supernatant was loaded onto a column for hydrophobic chromatography “Butyl Toyopearl” manufactured by Tosoh that had been equilibrated in advance with 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate, and the ammonium sulfate descending from 2 M to 0 M was loaded. Under a concentration gradient, 50 mM phosphate buffer (pH 7.0) was passed through the column. A fraction having enzyme activity is collected from the eluate, loaded on a column “Toyopearl HW-55” for gel filtration column chromatography manufactured by Tosoh that has been equilibrated in advance with 50 mM phosphate buffer (pH 7.0), and applied to the column. A 50 mM phosphate buffer solution (pH 7.0) was passed through, and a fraction having enzyme activity was collected from the eluate. The specific activity of the non-reducing saccharide-forming enzyme thus purified was about 195 units / mg protein, and the yield was about 220 units per liter of culture.

  In the present invention, the activity of the non-reducing saccharide-forming enzyme is measured by the following method and displayed as an activity value (unit). Specifically, 4 ml of 50 mM phosphate buffer (pH 7.0) containing 1.25% (w / v) maltopentaose was added, 1 ml of enzyme solution was added thereto, and the mixture was incubated at 40 ° C. for 60 minutes for reaction. The reaction is stopped by heating at 100 ° C. for 10 minutes. After diluting the reaction solution 10 times with distilled water, the reducing power is measured by the Somogi-Nelson method. As a control, the enzyme previously inactivated by heating at 100 ° C. for 10 minutes is treated in the same manner as described above. One unit of non-reducing saccharide-forming enzyme is defined as the amount of enzyme that reduces the reducing power corresponding to 1 μmol of maltopentaose per minute under the above conditions.

<Experimental Example 1-2 Preparation of Trehalose Free Enzyme>
In a 500 ml Erlenmeyer flask, Matsutani Chemical's partial starch hydrolyzate “Paindex # 4” is 2.0% (w / v), peptone is 0.5% (w / v), and yeast extract is 0.1%. (W / v), 100 ml of a liquid medium (pH 7.0) containing 0.1% (w / v) disodium hydrogen phosphate and 0.1% (w / v) sodium dihydrogen phosphate was taken at 120 ° C. And sterilized by autoclaving for 20 minutes. After cooling, Rhizobium species M-11 (FERM
BP-4130) was inoculated and seeded at 27 ° C. for 24 hours under rotary shaking. Thereafter, 20 l of a liquid medium having the same composition as described above was placed in a 30 l jar fermenter, sterilized, inoculated with 1% (v / v) of the seed culture solution prepared above, and maintained at pH 6 to 8 at 30 ° C. The culture was aerated and stirred for 24 hours.

  About 18 l of the culture thus prepared was crushed in the same manner as in Experimental Example 1-1, and the crushed product was purified. As a result, about 650 units of trehalose-free enzyme with a specific activity of about 240 units / mg protein was obtained per liter of culture. It was.

  In the present invention, the activity of trehalose-free enzyme is measured by the following method and displayed as an activity value (unit). That is, 4 ml of 50 mM phosphate buffer (pH 7.0) containing 1.25% (w / v) α-maltotriosyltrehalose was added, 1 ml of enzyme solution was added thereto, and the mixture was incubated at 40 ° C. for 30 minutes for reaction. . Then, 1 ml of the reaction solution is taken and added to 2 ml of the copper foil of somogi to stop the reaction, and then the reducing power is measured by the somogi-Nelson method. As a control, the enzyme previously inactivated by heating at 100 ° C. for 10 minutes is treated in the same manner as described above. One unit of trehalose-free enzyme is defined as the amount of enzyme that increases the reducing power corresponding to 1 μmol of glucose per minute under the above conditions.

<Experimental Example 2 Preparation of Trehalose>

<Experimental Example 2-1 Preparation of Trehalose Hydrous Crystal>
Suspend 1 part by weight of potato starch in 10 parts by weight of water, add bacterial liquefied α-amylase according to a conventional method, heat to 90 ° C, gelatinize and liquefy to DE 0.5, then immediately heat to 130 ° C. The enzyme reaction was stopped. The obtained starch liquefaction liquid was rapidly cooled to 45 ° C., and then 1 unit of non-reducing saccharide-forming enzyme prepared in Experimental Example 1-1 and trehalose-free enzyme prepared in Experimental Example 1-2 per 1 g of starch solid content. One unit, 200 units of an isoamylase agent manufactured by Hayashibara Biochemical Laboratories was added and saccharified for 48 hours while maintaining the pH at around 6.0 to obtain a reaction product containing 80.5% trehalose per solid content. This reaction product is decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, concentrated to 75% (w / w), taken into an auxiliary crystal can, heated to 50 ° C., and then seeded with trehalose-containing hydrate crystals. The powder was added at 1% per solid and cooled to 30 ° C. over 24 hours with gentle stirring. The thus obtained mass kit containing the trehalose hydrous crystals was honeyed with a basket-type centrifuge, and the collected crystals were washed by spraying a small amount of water to obtain trehalose hydrous crystals containing 99.0% trehalose per solid content. A yield of 47% per raw starch solids was obtained.

<Experimental Example 2-2 Preparation of crystalline anhydrous trehalose>
A portion of the trehalose hydrous crystals prepared in Experimental Example 2-1 is taken, dissolved in a small amount of water by heating, transferred to an evaporation kettle, and boiled under reduced pressure to give a syrup having a water content of 9.5% (w / w). It was. Take this syrup in a crystallizer, add 1% of crystalline anhydrous trehalose powder as a seed crystal per solid, and crystallize for 5 minutes at 100 ° C. with stirring, then dispense the mass kit into a plastic vat. And aged at 70 ° C. for 3 hours. Thereafter, the mass kit solidified into a block shape is taken out from the vat, pulverized by a conventional method, fluid dried, and about 90% of crystalline anhydrous trehalose powder with a water content of about 1% (w / w) per solid content The yield was obtained.

<Experimental Example 2-3 Preparation of Amorphous Anhydrous Trehalose>
A portion of the trehalose hydrated crystals prepared in Experimental Example 2-1 was taken, dissolved in water to a concentration of about 40% (w / w), freeze-dried, and pulverized to obtain a water content of about 2% (w / W) amorphous anhydrous trehalose powder was obtained with a yield of almost 100% per raw material solids.

<Experimental Example 3 Preparation of Octaacetyl Trehalose>
Take 65 g of dry pyridine containing 50 g of acetic anhydride in a reaction vessel, cool to 0 ° C., and then add any of the trehalose hydrous crystals, crystalline anhydrous trehalose or amorphous anhydrous trehalose prepared in Experimental Examples 2-1 to 2-3. 3 g was added and dissolved gently by stirring at the same temperature. The solution was allowed to stand at room temperature for 18 hours to react, and then the reaction product was poured into ice water, allowed to stand for a while, then separated by decanting, and the organic solvent layer was collected and concentrated. The concentrate was analyzed by a usual method using gas chromatography, and the amount of octaacetyl trehalose produced by the reaction was quantified and the colored state was visually observed. The results are shown in Table 1.

（表中、「＋＋」は、肉眼観察により着色が著しかったことを、「±」は、肉眼観察により着色が少なかったことを示す。）

(In the table, “++” indicates that coloring was significant by visual observation, and “±” indicates that coloring was small by visual observation.)

  The results shown in Table 1 show that, when trehalose hydrous crystals are used as a substrate, only 60% of the theoretically significant octaacetyl trelohath is obtained. However, when crystalline or amorphous anhydrous trehalose is used as a substrate, the coloring is low. It shows that high-quality octaacetyl trehalose is produced with almost theoretical values. This confirms that the yield and quality of trehalose derivatives are significantly improved when the production method of the present invention using anhydrous trehalose as a substrate is used as compared with the case of using trehalose hydrous crystals.

  Examples of the present invention will be described below.

<Linoleic acid ester>
10 g of amorphous anhydrous trehalose obtained by the method of Experimental Example 2-3 and 200 ml of anhydrous pyridine were placed in a reaction vessel, and 4 g of thiazolythione-linoleic acid amide dissolved in 5 ml of anhydrous pyridine was added under an argon stream. 85 mg of 60% (w / w) oily sodium hydride was added and reacted at room temperature for 2 hours. After 1.5 ml of saturated aqueous ammonium chloride solution was added to the reaction product, pyridine was distilled off under reduced pressure, and 8.5 g of residue was obtained. Obtained. When this was purified by silica gel chromatography, 5.3 g of trehalose linoleic acid ester having an average substitution degree of 1.4 was obtained.

  The tasteless, odorless and highly active product can be advantageously used in foods, cosmetics, pharmaceuticals and the like as a safe nonionic surfactant. As a control, when the trehalose hydrous crystal obtained by the method of Experimental Example 2-1 was reacted in the same manner, only 2.3 g of colored trehalose linoleate was obtained.

<Myristic acid ester>
220 g of crystalline anhydrous trehalose obtained by the method of Experimental Example 2-2 was dissolved in 800 ml of N, N′-dimethylformamide, 60 g of myristic acid methyl ester and 4 g of calcium carbonate were added, and the mixture was stirred under reduced pressure of 100 to 200 mmHg. The reaction was performed at 85 to 95 ° C. for 24 hours. Thereafter, the solvent was distilled off from the reaction product under reduced pressure, the residue was immersed twice in 300 ml of acetone, the leachate was concentrated, and the viscous oil obtained by washing with benzene and petroleum ether was again immersed in 300 ml of acetone. The leachate was ice-cooled, and the precipitate was collected and dried. As a result, 310 g of trehalose myristic acid ester having an average substitution degree of 1.7 was obtained.

  The tasteless, odorless and highly active product can be advantageously used in foods, cosmetics, pharmaceuticals and the like as a safe nonionic surfactant. In addition, this product has an action of suppressing the growth of malignant tumors in and outside the living body and is also useful as an active ingredient of pharmaceuticals. As a control, when the trehalose hydrous crystals obtained by the method of Experimental Example 2-1 were reacted in the same manner, only 90 g of highly colored trehalose myristic acid ester was obtained.

<Dodecyl ether>
390 g of n-dodecanol was placed in a reaction vessel, heated to 125 ° C., 1 g of p-toluenesulfonic acid was added as a catalyst, and the inside of the vessel was depressurized to 5 to 10 mmHg. Separately, 100 g of amorphous anhydrous trehalose obtained by the method of Experimental Example 2-3 was suspended in 130 g of n-dodecanol and added dropwise to the reaction vessel at a rate of 2.3 g / min for 100 minutes to react. . Thereafter, the reaction product was neutralized with a saturated aqueous sodium carbonate solution, and unreacted alcohol was distilled off. As a result, about 140 g of a composition containing 79.9% trehalose dodecyl ether per solid content was obtained.

  This highly active product can be advantageously used as a safe surfactant in general detergents including laundry detergents, kitchen detergents and shampoos. As a control, when the trehalose hydrous crystals obtained by the method of Experimental Example 2-1 were reacted in the same manner, only about 80 g of a remarkable colored composition containing 27.5% trehalose dodecyl ether was obtained.

<Sulfate ester>
1 part by weight of amorphous anhydrous trehalose obtained by the method of Experimental Example 2-3 was placed in a reaction vessel, and 5 parts by weight of a sulfur trioxide-dimethylformamide complex separately prepared according to a conventional method was added dropwise under a nitrogen stream. The reaction was allowed to proceed for 4 hours and then at 70 ° C. for an additional hour. 5N sodium hydroxide was added in an appropriate amount to neutralize, 5 volumes of methyl alcohol was added, and after standing for a while, the precipitate was collected by suction filtration. As a result, trehalose sulfate having an average substitution degree of 7.7 was about 95%. Yield.

  This high-quality product can be advantageously blended and used in general cosmetics as a moisturizer and skin beautifier. As a control, the trehalose hydrous crystals obtained by the method of Experimental Example 2-1 were reacted in the same manner. As a result, a highly colored trehalose sulfate ester having an average substitution degree of 6.5 was obtained in a yield of about 63%. There wasn't.

<Sulfate ester>

<Example 5-1 Preparation of maltose / trehalose converting enzyme>
In a 500 ml flask, 2.0% (w / v) glucose, 0.5% (w / v) polypeptone, 0.1% (w / v) yeast extract, 0.1% (w / v) dihydrogen phosphate Liquid medium (pH 7) consisting of potassium, 0.06% (w / v) sodium dihydrogen phosphate, 0.05% (w / v) magnesium sulfate heptahydrate, 0.5% (w / v) calcium carbonate and water .2) 100ml each, sterilized by heating at 115 ° C for 30 minutes, and after cooling, Pimerobacter species R48 (FERM
BP-4315) was inoculated and seeded at 27 ° C. and 200 rpm for 24 hours. Then, 20 liters of fresh liquid medium with the same composition as above was taken into a 30 liter jar fermenter, sterilized in the same manner, cooled to 27 ° C., and inoculated with 1% (v / v) of the seed culture solution obtained above. The culture medium was aerated and stirred at 27 ° C. for 40 hours while maintaining the pH of the medium at 6.0 to 8.0.

  The culture is centrifuged, and the resulting bacterial cells (wet weight of about 0.5 kg) are suspended in 10 mM phosphate buffer (pH 7.0), pulverized by a conventional method, and centrifuged to obtain a crude enzyme solution of about 4. 5 l was obtained. Ammonium sulfate was added to the crude enzyme solution so as to become 30% saturation, and the mixture was allowed to stand at 4 ° C. for 4 hours for salting out, and then centrifuged to collect the supernatant. Ammonium sulfate was added to the supernatant to 80% saturation, and the mixture was allowed to stand overnight at 4 ° C., and the precipitate was collected by centrifugation, dissolved in a small amount of 10 mM phosphate buffer (pH 7.0), and 10 mM phosphate buffer. Dialyzed against the solution (pH 7.0) for 24 hours. The supernatant collected by centrifuging the dialyzed internal solution was loaded onto a Tosoh ion exchange chromatography column “DEAE Toyopearl” previously equilibrated with 10 mM phosphate buffer (pH 7.0). A 10 mM phosphate buffer (pH 7.0) was passed through the column under a sodium chloride concentration gradient rising to 4M. A fraction having enzyme activity was collected from the eluate, dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate for 10 hours, and centrifuged to collect a supernatant. The supernatant was loaded onto Tosoh's hydrophobic chromatography column “Butyl Toyopearl” that had been equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the concentration of ammonium sulfate decreased from 1 M to 0 M. Under a gradient, 10 mM phosphate buffer (pH 7.0) was passed through the column. A fraction having enzyme activity is collected from the eluate and loaded onto a Pharmacia ion exchange chromatography column “Mono Q HR5 / 5” which has been equilibrated in advance with 10 mM phosphate buffer (pH 7.0). A 10 mM phosphate buffer (pH 7.0) was passed through the column under a sodium chloride concentration gradient rising to 0.5 M, and a fraction having enzyme activity was collected from the eluate. The specific activity of the maltose / trehalose converting enzyme thus purified was about 17 units / mg protein, and the yield was about 46 units per culture 11.

  In the present invention, the activity of maltose / trehalose converting enzyme is measured by the following method and displayed as an activity value (unit). That is, 1 ml of 10 mM phosphate buffer (pH 7.0) containing 20% (w / v) maltose was added, 1 ml of an enzyme solution diluted to an appropriate concentration was added, and the mixture was incubated at 25 ° C. for 60 minutes for reaction. The reaction is stopped by heating at 0 ° C. for 10 minutes. The reaction product was diluted 11-fold with 50 mM phosphate buffer (pH 7.5), 0.4 ml of the diluted reaction product was taken, 0.1 ml of a solution containing 1 unit / ml of trehalase was added thereto, and the solution was added at 45 ° C. for 120 minutes. After incubation, the amount of glucose in the reaction is quantified by the glucose oxidase method. At the same time, a system using an enzyme solution deactivated by heating at 100 ° C. for 10 minutes is provided and treated in the same manner as above to serve as a control. The amount of trehalose produced by the reaction is estimated from the glucose amount thus determined. One unit of maltose / trehalose converting enzyme is defined as the amount of enzyme that produces 1 μmol of trehalose per minute under the above conditions.

<Example 5-2 Preparation of crystalline anhydrous trehalose>
Onion starch is suspended in water to a concentration of 15% (w / w), adjusted to pH 5.5, and liquefied α-amylase agent “Spitase HS” manufactured by Nagase Seikagaku Kogyo Co., Ltd., 2 units per gram of starch solids In addition, the starch was gelatinized and liquefied by heating to 90 ° C. with stirring. The starch liquefaction solution was autoclaved at 120 ° C. for 20 minutes to inactivate the enzyme, rapidly cooled to 55 ° C., adjusted to pH 5.0, and then the isoamylase agent and Nagase Biochemistry manufactured by Hayashibara Biochemical Laboratories per gram of starch solid content. 300 or 20 units of industrial α-amylase agent was added and reacted for 24 hours to obtain a reaction product containing 92% maltose per solid content. The reaction product was heated at 100 ° C. for 20 minutes to inactivate the enzyme, adjusted to 20 ° C. and pH 7.0, and then maltose / trehalose converting enzyme prepared in Example 5-1 was added to 1.5 per gram of starch solids. Units were added and reacted for 72 hours to obtain a reaction product containing 71% trehalose per solid content. The reaction product is heated at 95 ° C. for 10 minutes to inactivate the enzyme, inactivate the enzyme, cool, and purify in the same manner as in Experimental Example 2-1, up to a water content of 9.5% (w / w). Concentrated. Take the concentrate in an auxiliary crystal machine, add 1% of crystalline anhydrous trehalose powder as a seed crystal per solid content, and under stirring, promote the auxiliary crystal at 110 ° C. for 10 minutes, and then dispense the mass kit into a plastic vat. The mixture was left to stand at 3 ° C. for 3 hours for aging. Thereafter, the mass kit solidified into a block shape is taken out from the vat, pulverized by a conventional method, fluidized and dried to obtain a crystalline anhydrous trehalose powder having a water content of about 2% (w / w) at about 95 per starch solid content. % Yield.

<Example 5-3 Preparation of sulfate ester>
When 100 g of crystalline anhydrous trehalose prepared in Example 5-2 was taken and sulfated by the method of Example 4, about 240 g of a composition containing trehalose sulfate having an average substitution degree of about 8 was obtained.

  This high-quality product can be advantageously blended and used in general cosmetics as a moisturizer and skin beautifier.

  As described above, the present invention can produce a high-quality trehalose derivative that could not be easily obtained by a conventional method in high yield by reacting a reactive reagent with anhydrous trehalose under anhydrous conditions. It is something to squeeze. Unlike trehalose hydrous crystals, anhydrous trehalose has no water of crystallization, which can significantly shorten the drying process before the reaction or even omit it in some cases, greatly reducing the production cost of trehalose derivatives. Will be able to. The trehalose derivative obtained by the method of the present invention can be used in the production or synthesis of foods, cosmetics, pharmaceuticals, detergents, chemicals as surfactants, moisturizers, skin beautifiers, antitumor agents, synthetic intermediates, etc. Has a variety of uses.

Claims (1)

Reagents having reactivity with non-anomeric hydroxyl groups under anhydrous conditions are reacted with anhydrous trehalose having a water content of less than 3% (w / w) measured by the Karl Fischer method, and subjected to esterification or etherification. And a method for producing a trehalose derivative selected from esters selected from carboxylic acid esters and fatty acid esters, or methyl ether, benzyl ether, trityl ether, methylsilyl ether, and dodecyl ether.