JP5993674B2 - Method for producing powder containing α, α-trehalose dihydrate crystal - Google Patents

Description

  The present invention relates to a method for producing a powder containing α, α-trehalose dihydrate crystals, and more specifically, α for producing high purity α, α-trehalose dihydrate crystals-containing powder from starch in a consistent process. , Α-trehalose dihydrate crystal-containing powder, and α, α-trehalose dihydrate crystal-containing powder obtained by the production method.

  Various methods are conventionally known as a method for producing a powder containing dihydrate crystals of α, α-trehalose (hereinafter abbreviated as “trehalose” in the present specification). For example, in Patent Document 1, a starch debranching enzyme is allowed to act on liquefied starch together with β-amylase or β-amylase, and then a maltose / trehalose converting enzyme is allowed to act to obtain a trehalose-containing sugar solution, which is appropriately purified. Subsequently, a method for producing trehalose dihydrate crystal-containing powder by crystallizing trehalose is disclosed, and Patent Document 2 discloses an α-glycosyl trehalose-producing enzyme (also known as “non-non-functional”) together with starch debranching enzyme in liquefied starch. Reducing saccharide-forming enzyme ”) and trehalose-releasing enzyme, and further glucoamylase to obtain a trehalose-containing sugar solution, which is appropriately purified, and then crystallized trehalose to obtain a powder containing trehalose dihydrate crystals. A method of manufacturing is disclosed.

  In Patent Documents 3 and 4, in the production method disclosed in Patent Document 2, starch debranching enzyme and cyclomaltodextrin glucanotransferase (hereinafter abbreviated as “CGTase”) are used in combination. A method for producing a powder containing trehalose dihydrate crystals in which the trehalose content in the trehalose-containing sugar solution is increased by acting an α-glycosyl trehalose producing enzyme and a trehalose-releasing enzyme is disclosed. Furthermore, in Patent Documents 5 and 6 and Non-Patent Documents 1 and 2, liquefied starch is added with an α-glycosyl trehalose producing enzyme derived from a microorganism belonging to the genus Sulfolobus, or an α-glycosyl trehalose producing enzyme and trehalose releasing enzyme. There is disclosed a method for obtaining a trehalose-containing sugar solution by acting, and a method for producing a trehalose-bihydrate crystal-containing powder by appropriately purifying the trehalose-containing sugar solution and then crystallizing trehalose.

  Among these known production methods, in the case of the combination of the enzymes disclosed in Patent Documents 3 and 4, the liquefied starch is used as a raw material, and only the enzyme reaction is performed without performing a fractionation step by column chromatography. A trehalose-containing sugar solution having a trehalose content exceeding 80% by mass in terms of anhydride can be easily prepared, and the composition of the trehalose-containing sugar solution thus prepared is mostly glucose except for trehalose. Trehalose has the advantage of good crystallinity. Therefore, in the case of the production methods disclosed in Patent Documents 3 and 4, relatively high purity is obtained by a honey method in which trehalose dihydrate crystals are crystallized from the trehalose-containing sugar solution and the crystals are collected by centrifugation. The trehalose dihydrate crystal-containing powder can be produced with good yield.

  In particular, in the production methods disclosed in Patent Documents 3 and 4, when an enzyme derived from a microorganism belonging to the genus Arthrobacter is used as the α-glycosyl trehalose producing enzyme and the trehalose releasing enzyme, the genus Arthrobacter is used. Since the microorganisms belonging to the above group grow relatively fast and the productivity of the enzyme is high, it is extremely advantageous for producing trehalose dihydrate crystal-containing powder on an industrial scale. For this reason, the present applicant uses an enzyme derived from a microorganism belonging to the genus Arthrobacter as an α-glycosyl trehalose-producing enzyme and a trehalose-releasing enzyme, and is highly purified by the production methods disclosed in Patent Documents 3 and 4. Manufactures powder containing trehalose dihydrate crystals (trade name “Treha”, sold by Hayashibara Co., Ltd., trehalose purity: 98.0% by mass or more, hereinafter referred to as “food grade trehalose dihydrate crystals containing powder”). However, they are mainly sold as food materials and cosmetic materials. However, according to the present production method, the trehalose content in the trehalose-containing sugar solution obtained by the enzyme reaction is currently about 85% by mass in terms of anhydride even when various enzyme reaction conditions are optimized. The actual condition is that the yield to starch does not reach 40% by mass.

  On the other hand, the methods using α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from Sulfolobus microorganisms disclosed in Patent Documents 5 and 6 and Non-Patent Documents 1 and 2 have relatively high temperatures because the enzymes have heat resistance. Although there is an advantage that there is less concern about contamination of the reaction mixture during the enzyme reaction, there are problems such as poor growth of microorganisms and low enzyme productivity, and the industrial scale of trehalose. It was not suitable for production. Patent Documents 7 and 8 disclose a recombinant enzyme of α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from a Sulfolobus microorganism, respectively, and the problem of enzyme productivity by using the recombinant enzyme is as follows. Eliminate. However, in the method for producing trehalose using an enzyme derived from the genus Sulfolobus, as described in Patent Documents 6 and 8, Non-Patent Documents 1 and 2, etc., it can be obtained by enzymatic reaction even when a starch debranching enzyme is used in combination. The trehalose content in the resulting trehalose-containing sugar solution is as low as about 75 to 81% by mass in terms of anhydride, and even when optimized by adding CGTase, it remains at about 82% by mass in terms of anhydride. It was.

  On the other hand, in Non-patent Documents 3 and 4, the genes for thermostable α-glycosyl trehalose-producing enzyme, thermostable trehalose-releasing enzyme and thermostable isoamylase derived from Sulfolobus solfataricus are expressed in Escherichia coli, respectively. When combined with a recombinant enzyme prepared by adding a mutant enzyme prepared by introducing a site-specific mutation to the soluble starch and acting on soluble starch, the trehalose content is about 87% by mass in terms of anhydride. It is disclosed that a sugar solution is obtained.

  However, the soluble starch used as a raw material in Non-Patent Documents 3 and 4 is a very special and expensive raw material produced by removing the amorphous part in starch granules by acid treatment of starch. Yes, even if an enzyme reaction solution having an increased trehalose content is obtained, it is impossible to use soluble starch as a raw material for industrial production of trehalose dihydrate crystal-containing powder. In addition, when the recombinant enzyme or mutant enzyme disclosed in Non-Patent Documents 3 and 4 is allowed to act on liquefied starch used in production on an industrial scale rather than soluble starch, it can be obtained by enzymatic reaction. As a matter of course, the trehalose content in the resulting trehalose-containing sugar solution is lower than about 87% by mass and stops at about 85% by mass, and the yield of starch containing trehalose dihydrate crystals is higher than that of the current production method. I can not hope to improve.

  Incidentally, if the trehalose content in the trehalose-containing sugar solution is simply increased to 86.0% by mass or more, a column fraction using column chromatography is applied to the trehalose-containing sugar solution to collect a trehalose-rich fraction. Can be considered. However, if column fractionation is performed, the production cost increases as the number of steps increases, and loss of trehalose contained in fractions other than the fraction collected as a trehalose-rich fraction inevitably occurs. Even if a trehalose-containing sugar solution having a trehalose content of 86.0% by mass in terms of anhydride is obtained by column fractionation, a trehalose-bihydrate crystal crystallized from such a sugar solution is collected, Even if the hydrated crystal-containing powder is produced, it is inevitable that the yield with respect to starch is greatly reduced.

  In addition, if the yield to starch is simply increased, the mass kit containing the crystallized crystals is taken out into a container and crystallized and solidified in place of the honey method in which the crystallized crystals are collected by centrifugation. It is also possible to adopt a so-called whole sugar system in which the powder is pulverized or the mass kit is spray-dried to obtain a powder. However, in the case of the all-sugar method, together with the crystallized trehalose, together with the contaminants specific to the production method such as glucose contained in the mass kit, the resulting powder containing trehalose dihydrate crystals is obtained. The trehalose content therein is not higher than the trehalose content in the mass kit, and there is an inconvenience that a high-purity trehalose dihydrate crystal-containing powder cannot be obtained.

  Starch is currently a relatively abundant, inexpensive and readily available raw material, but it is not an inexhaustible material, and the total amount of starch produced by humans on earth per year is There is a limit. On the other hand, starch is widely used, and in addition to conventional industrial use, food use, feed use, or food raw materials, in recent years, due to the rise in demand for clean energy, new bioethanol, etc. It is also used as a fuel raw material. Under such circumstances, it is extremely important from the viewpoint of effective utilization of limited resources to improve the yield of the product, that is, the powder containing trehalose dihydrate crystals, with respect to starch.

JP-A-7-170977 JP-A-7-213283 JP-A-8-73504 JP 2000-228980 A JP-A-8-66188 JP-A-8-66187 JP-A-8-84586 JP-A-8-336388

Kazuhisa Mukai et al., Starch, 1997, 49, 26-30. Kazuo Kobayashi et al., Journal of Fermentation and Bioengineering, 1997, 83, 3, 296-298. Fan et al., Journal of Agricultural and Food Chemistry, 2007, 55, 5588-5594. Fan et al., Journal of Agricultural and Food Chemistry, 2008, 56, 5628-5633.

  The present invention has been made for the purpose of eliminating the inconvenience in the conventional method for producing a powder containing trehalose dihydrate crystals, and further increasing the yield of the starch containing trehalose dihydrate crystals while maintaining the purity of trehalose. A method for producing trehalose dihydrate crystal-containing powder that enables high-purity trehalose dihydrate crystal-containing powder to be produced with high yield in a consistent process using starch as a raw material, and the production method thereof It is an object to provide a novel trehalose dihydrate crystal-containing powder.

  In order to solve the above problems, the present inventors have conducted various studies and trials and errors on the combination of a plurality of enzymes used in the production methods disclosed in Patent Documents 5 and 6, and as a result, α-glycosyl trehalose production. When a thermostable enzyme derived from a microorganism belonging to the genus Sulfolobus is used as the enzyme and trehalose-releasing enzyme, the Geobacillus stearothermophilus Tc-91 strain used so far as CGTase used together with these enzymes By using CGTase derived from the genus Paenibacillus instead of CFERase derived from (FERM BP-11273), trehalose production reaction proceeds more efficiently, and after the glucoamylase treatment step The trehalose content of Reharosu containing sugar solution, without a fractionation step by column chromatography, and found that it is possible to improve to a level in excess of 86.0 wt% on a dry solid basis. Then, the trehalose-containing sugar solution thus obtained is decolored, desalted and concentrated according to a conventional method to crystallize trehalose dihydrate crystals, and the resulting crystals are collected by centrifugation, and this is aged. It was found that by drying, a high-purity trehalose dihydrate crystal-containing powder containing 98.0% by mass or more of trehalose in terms of anhydride could be produced with a higher starch yield than conventional, and the present invention was completed. .

  That is, according to the present invention, α-glycosyl trehalose producing enzyme and trehalose releasing enzyme are allowed to act on liquefied starch together with starch debranching enzyme and CGTase, and then glucoamylase is allowed to act to obtain an α, α-trehalose-containing sugar solution. A step of crystallizing α, α-trehalose dihydrate crystals from the sugar solution, and a step of collecting the crystallized trehalose dihydrate crystals by centrifugation, aging and drying the crystals, A method for producing a powder containing trehalose, wherein in the step of obtaining a trehalose-containing sugar solution, an α-glycosyl trehalose-producing enzyme and a trehalose-free enzyme derived from a microorganism belonging to the genus Sulfolobus and a CGTase derived from a Paenibacillus microorganism are separated by column chromatography. Without going through the painting process Provided is a method for producing an α, α-trehalose dihydrate crystal-containing powder containing 98.0% by mass or more of trehalose in terms of anhydride, wherein the trehalose content is more than 86.0% in terms of anhydride. This solves the above problem.

  According to the present inventors, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention described above is not only the trehalose purity but also has good fluidity. Compared to powders containing water-containing crystals, these powders are comparable to conventional powders containing trehalose dihydrate crystals containing food grades, and can be used in a wide range of fields as food materials, cosmetic materials, etc. .

  Examples of the genus Sulfolobus, which is a source of α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme, include, for example, Sulfolobus acid caldarius (Sulfolobus) disclosed in Patent Documents 5 to 8, Non-Patent Documents 1 to 4, and the like. acidocaldarius) and Sulfolobus solfataricus.

  For the reasons described above, it is difficult to prepare a large amount of α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from Sulfolobus spp., And in reality, recombinant enzymes are used as these enzymes. Is preferred. Furthermore, a mutant enzyme prepared by introducing a mutation into a gene of these enzymes by a conventional method and then expressing it in an appropriate host can also be used.

  As CGTase derived from the microorganism belonging to the genus Paenibacillus used in the production method of the present invention, a natural enzyme derived from the microorganism or a recombinant enzyme prepared by cloning a CGTase gene possessed by the microorganism and expressing it in an appropriate host. Furthermore, a mutant enzyme prepared by introducing a mutation into the CGTase gene by a conventional method and then expressing the same can also be used.

  As a result of further trial and error, the present inventors, as a result of crystallizing trehalose dihydrate crystals from the trehalose-containing sugar solution containing more than 86.0% by mass of trehalose in terms of anhydride, If the pseudo-controlled cooling method is applied, compared to the case where trehalose dihydrate crystals are crystallized by the natural cooling method in which the temperature reduction of the trehalose-containing sugar solution is naturally left, the yield of trehalose dihydrate crystals-containing powder is reduced. It has been found that the rate can be further increased. That is, the present invention provides a method for producing trehalose dihydrate crystal-containing powder, wherein in the production method of the present invention, the step of crystallizing the trehalose dihydrate crystal is performed by a controlled cooling method or a pseudo controlled cooling method. To solve the above problems.

  Note that the reason why the starch yield is improved when the controlled cooling method or the pseudo controlled cooling method is applied is not clear, but according to the controlled cooling method or the pseudo controlled cooling method, in the initial stage of crystallization, rapid cooling due to cooling is performed. Suppressing the increase in supersaturation and the formation of secondary crystal nuclei, generating a large number of minute crystal nuclei of almost the same size, and rapidly cooling in the latter stage of crystallization with a large number of minute crystal nuclei aligned As a result, a large number of crystal nuclei of uniform size can be grown at the same time, so that a mass kit containing crystals with a small particle size and a uniform particle size can be obtained, and the crystals can be easily collected by centrifugation. The crystal collected with a small amount of water can be washed, so it is presumed that the loss of trehalose during washing is reduced.

  In addition, the present inventors have surprisingly found that the trehalose dihydrate crystal-containing powder produced by applying the controlled cooling method or the pseudo controlled cooling method during crystallization of the trehalose dihydrate crystal as described above, The present inventors have found that it is superior in terms of being hard to solidify as compared with a powder containing trehalose dihydrate crystals produced by a natural cooling method or a conventional food grade powder containing trehalose dihydrate crystals. And, it was confirmed that such excellent physical properties were brought about by the difference in trehalose purity and the crystallinity of trehalose dihydrate crystal in the powder containing trehalose dihydrate crystal. The present invention was completed.

  That is, the present invention is a trehalose dihydrate crystal-containing powder obtained by the production method of the present invention to which the controlled cooling method or the pseudo controlled cooling method is applied when crystallizing trehalose dihydrate crystal, and is trehalose in terms of anhydride. Of trehalose dihydrate containing 99.0% by mass to 99.5% by mass and having a crystallinity of 90.0% or more and 95.0% or less by calculation based on the powder X-ray diffraction profile. The above-mentioned problems are solved by providing a water-containing crystal-containing powder.

  Incidentally, the crystallinity of trehalose dihydrate crystals containing 99.0% to 99.5% by mass of trehalose in terms of anhydride and calculated based on the powder X-ray diffraction profile is 90.0% or more and 95.95%. Trehalose dihydrate crystal-containing powder that is 0% or less, according to the present inventors confirmed that the trehalose content is about the same or slightly higher than the conventional food-grade trehalose dihydrate crystal-containing powder, This is a novel powder that has a crystallinity of trehalose dihydrate crystals significantly higher than that of food-grade trehalose dihydrate crystals-containing powders, and is distinguished from conventional food-grade trehalose dihydrate crystal-containing powders.

  The reason why the trehalose dihydrate crystal-containing powder that is difficult to solidify by applying the controlled cooling method or the pseudo controlled cooling method is not clear, but according to the controlled cooling method or the pseudo controlled cooling method, As described above, it is possible to obtain a mass kit containing crystals with a small particle size and a uniform particle size, and it is assumed that the purity of trehalose in the obtained powder and the crystallinity of trehalose dihydrated crystals are increasing. Is done. This is because the crystallinity of trehalose dihydrate crystals in the trehalose dihydrate crystal-containing powder of the present invention obtained by applying the controlled cooling method or the pseudo controlled cooling method is a powder obtained by natural cooling, This is also supported by the fact that the food grade trehalose dihydrate crystal-containing powders are significantly higher in crystallinity than the trehalose dihydrate crystals.

  According to the production method of the present invention, α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from Sulfolobus microorganisms and CGTase derived from Paenibacillus microorganisms are used in a consistent process using starch as a raw material. The hydrous crystal-containing powder can be produced with a high starch yield. Therefore, the outstanding advantage that it contributes to the effective utilization of the starch resource which is a raw material is brought about. In particular, when crystallizing trehalose dihydrate crystals from trehalose-containing sugar solution, when applying controlled cooling method or pseudo-controlled cooling method, the yield of starch containing trehalose dihydrate crystals is improved. There is an advantage that you can. In addition, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention to which the controlled cooling method or the pseudo controlled cooling method is applied has trehalose purity and trehalose two-phase powder compared to the conventional food grade trehalose dihydrate crystal-containing powder. It is a powder excellent in that the degree of crystallinity of the water-containing crystal is high and it is difficult to solidify.

It is an example of the powder X-ray diffraction pattern by the characteristic X-ray of the powder containing a trehalose dihydrate crystal substantially consisting of a trehalose dihydrate crystal. It is an example of the powder X-ray diffraction pattern by the characteristic X-ray of the trehalose powder which consists of a substantially amorphous part. It is an example of the powder X-ray-diffraction pattern by the synchrotron radiation light of the powder containing a trehalose dihydrate crystal substantially consisting of a trehalose dihydrate crystal. It is an example of the powder X-ray diffraction pattern by the synchrotron radiation light of the trehalose powder which consists of a substantially amorphous part. It is a figure which shows various cooling patterns.

1. Definition of Terms In this specification, the following terms have the following meanings.

<Yield to starch>
The term “yield to starch” as used herein refers to the percentage of the mass of the trehalose dihydrate-containing crystal-containing powder obtained per unit mass of the raw material starch in terms of anhydride, expressed as a percentage (%). It is what. In this specification, starch is used as a raw material, and an enzyme is allowed to act on it to produce trehalose, and the resulting trehalose is crystallized, collected, aged, and dried in a series of consistent steps. Since it is premised on producing powder, the term “yield to starch” as used in the present specification refers to the first crystallization from a trehalose-containing sugar solution obtained by allowing an enzyme to act on starch. This means the yield of the trehalose dihydrated crystal-containing powder to be produced with respect to starch. The sugar solution remaining after collecting the crystallized crystals and the nectar separated from the mass kit are returned to the sugar solution and crystallized again. It does not include trehalose dihydrate crystal-containing powder produced from the so-called second crystal. Incidentally, when crystallization is performed by adding a seed crystal, the amount of the seed crystal in the calculation of the starch yield is included in the amount of the trehalose dihydrate crystal-containing powder obtained throughout this specification.

<CGTase activity>
In this specification, “CGTase activity” is defined as follows. That is, 0.2 ml of an enzyme solution appropriately diluted is added to 5 ml of a substrate aqueous solution containing 0.3% (w / v) soluble starch, 20 mM acetate buffer (pH 5.5), and 1 mM calcium chloride, and 40 ml of the substrate solution is added. While maintaining the temperature at 0 ° C., 0.5 ml of the substrate solution was sampled at 0 minutes and 10 minutes of reaction, and immediately added to 15 ml of 0.02N sulfuric acid solution to stop the reaction, and then 0.1N iodine was added to each sulfuric acid solution. Each 0.2 ml of the solution is added to cause coloration, and after 10 minutes, the absorbance at a wavelength of 660 nm is measured with an absorptiometer, and calculated as the amylolytic activity by the following formula [1]. One unit of CGTase activity is defined as the amount of enzyme that completely eliminates the iodine coloration of 15 mg of starch in the solution under such measurement conditions.

Formula [1]:

<Controlled cooling method>
The term “controlled cooling method” as used herein refers to a method of crystallizing crystals by “controlled cooling”. The working time set as the crystallization process is “τ”, and the liquid temperature at the start of crystallization is set. Assuming that “T ₀ ”, the target liquid temperature at the end of crystallization is “T _f ”, and the liquid temperature at time “t” is “T”, the liquid temperature T at time t is generally expressed by the following equation [2]. The cooling method represented.

Formula [2]:

If the controlled cooling method is expressed more specifically (schematically) using a graph in which the working time set as the crystallization step is the horizontal axis and the liquid temperature at the time of crystallization is the vertical axis, the symbol a in FIG. As shown in As shown by symbol a in FIG. 5, according to the controlled cooling method, the liquid temperature gradually decreases in the initial stage of crystallization with a high liquid temperature, and the liquid temperature rapidly increases in the latter stage of crystallization in which the liquid temperature has decreased to some extent. The liquid temperature “T _m ” at the time of t = τ / 2, that is, the intermediate point of the crystallization process, is at least T _m > [(T ₀ −T _f ) / 2 + T _f ]. (That is, the temperature change at the midpoint of the crystallization process is less than 50% of the total temperature change). In this change pattern of the liquid temperature with respect to time, the controlled cooling method includes linear cooling in which the liquid temperature decreases linearly over time τ from T ₀ to T _f (symbol b in FIG. 5), and crystals with a high liquid temperature. In the initial stage of crystallization, the liquid temperature decreases exponentially rapidly, and the liquid temperature gradually decreases as the crystallization is later in the crystallization period (reference c in FIG. 5). Are clearly distinguished. In order to change the liquid temperature T as a function of the time t represented by the above equation [2], for example, a commercially available general-purpose program constant temperature circulator for crystallization system may be used.

  When such a controlled cooling method is applied in the crystallization process, the liquid temperature is gradually cooled in the initial stage of crystallization after the addition of trehalose seed crystals. The rise and the formation of secondary crystal nuclei are suppressed, and crystals having the added seed crystals as crystal nuclei can be preferentially grown. On the other hand, in the latter stage of crystallization where crystals with the added seed crystals as crystal nuclei are gathered, the liquid crystals are rapidly cooled to grow the gathered crystals all at once. For example, there is an advantage that a mass kit including crystals with a small grain size and a uniform grain size can be obtained. The “controlled cooling method” is described in detail, for example, in “Tokuaki Kubota,“ Easy-to-understand batch crystallization ”, Separation Technology Association, issued April 30, 2010, pages 32 to 47”.

<Pseudo controlled cooling method>
The “pseudo controlled cooling method” as used in this specification is a cooling method that simulates the control cooling method literally described above, and the liquid temperature T is strictly changed according to the above equation [2] with respect to time t. The crystal nuclei are mostly at the time of working time t = τ / 2 (intermediate point of the crystallization process), although it depends on the trehalose purity, concentration, supersaturation, amount of seed crystals, etc. in the trehalose-containing solution used for crystallization. Since it is desirable that the temperature changes, the amount of change (T ₀ -T _m ) in the liquid temperature T at the time t = τ / 2 is preferably 5% or more and less than 50% of the total temperature change (T ₀ -T _f ). Means a cooling method in which the liquid temperature T is decreased continuously or stepwise with respect to the time t so as to maintain the range of 10% or more and less than 30%. The liquid temperature T is changed over time so that the change amount (T ₀ -T _m ) of the liquid temperature T at the time t = τ / 2 is 5% or more and less than 50% of the total temperature change amount (T ₀ -T _f ). In the case where the liquid temperature is decreased continuously or stepwise, the liquid temperature T gradually decreases with respect to time t in the initial stage of crystallization with a high liquid temperature, and the latter stage of the crystallization in which the liquid temperature has decreased to some extent. In this case, the liquid temperature T rapidly decreases with respect to the time t. As a result, although there is a case where the above-described controlled cooling method may not be achieved, there is a mass kit containing crystals with a small grain size and a uniform grain size. The obtained advantage is almost the same as that of the controlled cooling method.

Specifically, for example, the working time τ is divided into at least two, preferably three or more sections, and in the initial section of the crystallization process, the temperature gradient in cooling is made gentle (the cooling rate is slow). The temperature gradient is increased (the cooling rate is increased) from the initial period to the middle period to the latter period, and the change amount (T ₀ -T _m ) of the liquid temperature T at the time point t = τ / 2 is the total temperature change. What is necessary is just to reduce the liquid temperature T continuously or stepwise with respect to time t so that it may be 5% or more and less than 50% of the amount (T ₀ -T _f ), desirably 10% or more and less than 30%. When the change amount (T ₀ -T _m ) of the liquid temperature T at the time t = τ / 2 is 50% or more of the total temperature change amount (T ₀ -T _f ), the cooling rate at the initial stage of crystallization Is too early, and there is a risk that secondary crystal nuclei may be formed due to a sudden increase in supersaturation due to cooling. If it is less than 5%, the cooling rate at the initial stage of crystallization is too slow and A mass kit containing crystals with a small grain size and in any case a crystallisation with a small grain size. It becomes difficult to get.

In order to perform the above-described controlled cooling method, it is necessary to change the liquid temperature T as a function of the time t expressed by the formula [2], and an apparatus or a crystallization can capable of controlling the liquid temperature with a set program. However, according to the pseudo control cooling method, the change amount (T ₀ -T _m ) of the liquid temperature T at the time point t = τ / 2 is 5% of the total temperature change amount (T ₀ -T _f ). The liquid temperature T may be decreased continuously or stepwise with respect to time t so that the liquid temperature T is less than 50%, preferably 10% or more and less than 30%. Even if there is no equipment to control precisely, there is an advantage that it can be carried out relatively easily.

<Crystallinity>
As used herein, “crystallinity of trehalose dihydrate crystals” means a numerical value defined by the following formula [3].

Formula [3]:

In the equation [3], the powder X-ray diffraction profile that is the basis for obtaining the analytical values H ₁₀₀ , H ₀ , and Hs can be measured by a powder X-ray diffractometer equipped with a reflective or transmissive optical system. it can. The powder X-ray diffraction profile includes the diffraction angle and the diffraction intensity for the trehalose dihydrate crystal contained in the test sample or standard sample. As a method for determining the analytical value for the crystallinity from the powder X-ray diffraction profile, Examples include the Hermans method and the Funk method. Of these analysis methods, the Hermans method is preferred in terms of simplicity and accuracy. Today, these analysis methods are all made into computer software, so it is convenient to use a powder X-ray diffractometer equipped with an analysis device equipped with any of such computer software.

Further, as the “trehalose dihydrate crystal-containing powder standard sample substantially consisting of trehalose dihydrate crystals” for obtaining the analysis value H ₁₀₀ , the purity of trehalose is 99.9% by mass or more (hereinafter, unless otherwise specified, In this specification, mass% is abbreviated as “%.” However,% given to the crystallinity referred to in this specification is not limited to this.) In the pattern, a diffraction peak peculiar to trehalose dihydrate crystals and substantially consisting of trehalose dihydrate crystals is used. As such powder or single crystal, a powder containing trehalose dihydrate crystals (trade name “trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by the applicant as an analytical reagent) Company Hayashibara Sales), or a trehalose dihydrate crystal-containing powder or a single crystal of trehalose dihydrate crystal obtained by recrystallizing it. Incidentally, the analysis value H ₁₀₀ when the powder X-ray diffraction profile of the trehalose dihydrate crystal-containing powder standard sample substantially consisting of trehalose dihydrate crystals is analyzed by computer software by the Hermans method is usually 50.6. It is about 50.9%.

On the other hand, as the “trehalose-containing powder standard sample consisting essentially of an amorphous part” for obtaining the analysis value H ₀ , a trehalose purity of 99.9% or more and a substance consisting essentially of an amorphous part is used. . As such a powder, for example, the above-mentioned standard sample for obtaining the analytical value H ₁₀₀ is dissolved in an appropriate amount of purified water, concentrated, freeze-dried, and further, the moisture content determined by the Karl Fischer method is 2.0. The powder obtained by vacuum-drying until it becomes% or less is mentioned. Experience has shown that a powder consisting essentially of an amorphous part can be obtained when such a treatment is applied. However, generally, even a powder consisting essentially of an amorphous part is subjected to a powder X-ray diffractometer, and when the obtained powder X-ray diffraction profile is analyzed by the Hermans method, the Funk method, etc., each of these analysis methods is executed. A part of the scattered light derived from the amorphous part is calculated due to the algorithm of the computer software, and the analysis value is not necessarily 0%. Incidentally, the analysis value H ₀ when the powder X-ray diffraction profile of the trehalose-containing powder standard sample consisting essentially of an amorphous part is analyzed with computer software by the Hermans method is usually 8.5 to 8.7. %.

<Average crystallite size>
In general, it is considered that one powder particle in a crystal-containing powder is composed of a plurality of single crystals, that is, a plurality of crystallites. The crystallite size (crystallite diameter) in the crystal-containing powder is considered to be reflected in the characteristics of the crystal-containing powder. The “average crystallite diameter of trehalose dihydrate crystal” (hereinafter simply referred to as “average crystallite diameter”) as used herein is obtained by subjecting a powder containing trehalose dihydrate crystal to powder X-ray diffraction analysis. Among the diffraction peaks detected in the powder X-ray diffraction pattern, five diffraction peaks, that is, in a relatively low-angle region where the influence on the diffraction peak width due to the nonuniform strain of the crystallite is considered to be small Well separated from other diffraction peaks, diffraction angle (2θ) 13.7 ° (Miller index (hkl): 101), 17.5 ° (Miller index: 220), 21.1 ° (Miller index: 221) , 23.9 ° (Miller index: 231) and 25.9 ° (Miller index: 150) diffraction peaks (see symbols a to e in FIG. 1) are selected, and the respective half widths and diffraction angles are used. , Silicon as standard (US National After correcting based on the measured values when using the NIST and the standard sample for X-ray diffraction (“Si640d”), it is calculated by “Scherrer's formula” shown in the following formula [4]. It means the average value of the crystallite diameter.

Formula [4]:

  Since a general powder X-ray diffractometer is equipped with computer software for calculating the crystallite size, as long as the powder containing trehalose dihydrate crystal is available, the average of trehalose dihydrate crystals in the crystal-containing powder is obtained. The crystallite diameter can be measured relatively easily. Prior to powder X-ray diffraction analysis, the test sample is ground with a mortar, sieved with a 53 μm sieve, and powder that has passed through the sieve is used.

<Reducing power>
The “reducing power of the whole powder” as used herein refers to the amount of reducing sugar and total amount based on D-glucose conversion using D-glucose as a standard substance and the Sommoji-Nelson method and the Anthrone-sulfuric acid method widely used in the field. The amount of sugar can be obtained, and the percentage (%) of the amount of reducing sugar relative to the total amount of sugar contained in the powder can be obtained by calculating using the following formula [5].

Formula [5]:

<Particle size distribution>
In this specification, the particle size distribution of the powder is determined as follows. That is, after accurately weighing metal mesh sieves (made by Iida Seisakusho Co., Ltd.) having openings of 425, 300, 212, 150, 106, 75 and 53 μm in accordance with Japanese Industrial Standards (JIS Z 8801-1) In this order, they are mounted on a low-tap sieve shaker (trade name “R-1” manufactured by Tanaka Chemical Machinery Co., Ltd.), and then a fixed amount of the sample weighed is placed on the top sieve (opening) 425 μm), shake for 15 minutes with the sieves overlapped, then weigh each sieve again accurately, and subtract the mass before placing the sample from its mass to capture by each sieve. Determine the mass of the collected powder. Thereafter, the percentage (%) of the mass of the powder having each particle size collected by each sieve to the mass of the sample placed on the sieve is calculated and expressed as a particle size distribution.

2. Method for Producing Trehalose Dihydrate Crystal-Containing Powder of the Present Invention The production method of the present invention basically includes the following steps (1) to (6):
(1) A trehalose producing step of producing trehalose by reacting a liquefied starch solution with α-glycosyl trehalose producing enzyme and trehalose releasing enzyme derived from a microorganism belonging to the genus Sulfolobus together with starch debranching enzyme and CGTase derived from the genus Paenibacillus;
(2) A glucoamylase treatment step in which glucoamylase is allowed to act on the reaction solution containing trehalose obtained by the trehalose production step;
(3) A purification and concentration step of filtering, decolorizing, desalting and concentrating the reaction solution containing trehalose;
(4) a step of adding trehalose dihydrate crystals to a concentrated solution containing trehalose to crystallize trehalose dihydrate crystals;
(5) A step of collecting trehalose dihydrate crystals by centrifugation from the mass kit obtained in the crystallization step;
(6) A step of aging, drying and crushing the collected trehalose dihydrate crystal as necessary.

  Hereinafter, the steps (1) to (6) will be sequentially described.

<Step (1) (Trehalose production step)>
In this process, liquefied starch as a raw material is allowed to act on α-glycosyl trehalose-producing enzyme derived from Sulfolobus microorganisms and trehalose-releasing enzyme derived from Sulfolobus microorganisms together with starch debranching enzyme and CGTase derived from Paenibacillus microorganisms. This is a step of producing trehalose.

  α-Glycosyl trehalose-producing enzyme is an enzyme that acts on liquefied starch to produce α-glycosyl trehalose having a trehalose structure at the end of the molecule, while trehalose-releasing enzyme acts on α-glycosyl trehalose to release trehalose. It is an enzyme. Therefore, trehalose can be produced efficiently by allowing α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme to act on liquefied starch obtained by gelatinizing and liquefying starch together with starch debranching enzyme.

  Further, examples of the microorganism belonging to the genus Sulfolobus which is a source of the α-glycosyl trehalose producing enzyme and the trehalose releasing enzyme used in the production method of the present invention include, for example, Sulfolobus Acidocardarius and Sulfolobus solfataricus are mentioned, More specifically, Sulfolobus acidocardarius ATCC33909 and Sulfolobus solfataricus ATCC35091 are mentioned.

  In the production method of the present invention, a natural enzyme can be used as an α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from a microorganism belonging to the genus Sulfolobus, but a large amount of the natural enzyme is prepared for the reasons described above. Since it is difficult to use, it is preferable to use the recombinant enzymes disclosed in Patent Documents 7 and 8 as these enzymes. Furthermore, mutant enzymes obtained by introducing site-specific mutations into these enzymes can be advantageously used as long as the object of the present invention is not impaired.

  As the “recombinant α-glycosyl trehalose producing enzyme” or “recombinant trehalose releasing enzyme” as used in the present invention, the α-glycosyl trehalose producing enzyme gene or the trehalose releasing enzyme gene of the aforementioned Sulfolobus microorganism is cloned, for example, A recombinant α-glycosyl trehalose producing enzyme or a recombinant trehalose releasing enzyme obtained by expressing in an appropriate host microorganism such as Escherichia coli or Bacillus subtilis can be preferably used. For example, the α-glycosyl trehalose producing enzyme and the trehalose releasing enzyme derived from the aforementioned Sulfolobus acidocardarius ATCC 33909 strain are respectively represented by SEQ ID NOs: 1 and 2 in the sequence listing as disclosed in Patent Documents 7 and 8, respectively. It has the amino acid sequence shown. That is, the recombinant α-glycosyl trehalose producing enzyme and the recombinant trehalose releasing enzyme having the amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing are the same as the respective natural enzymes in the production method of the present invention. Can be used.

  The “mutant enzyme of α-glycosyl trehalose-producing enzyme” referred to in the present invention is an α-glycosyl trehalose-producing enzyme obtained by applying a genetic engineering technique to the gene encoding the α-glycosyl trehalose-producing enzyme derived from the genus Sulfolobus sp. As long as the substrate specificity and enzyme activity are not substantially changed, a mutant enzyme into which a deletion, substitution or insertion mutation of one or more amino acid residues in the amino acid sequence is introduced can be suitably used. . The amino acid sequence is not particularly limited as long as the substrate specificity and enzyme activity as an α-glycosyl trehalose producing enzyme are substantially maintained, but the amino acid sequence of the α-glycosyl trehalose producing enzyme derived from the genus Sulfolobus. For example, using the region conserved above, for example, the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing, the 85th aspartic acid residue to the 90th histidine residue (Asp85 to His90). ) From the 224th glycine residue to the 232nd glycine residue (Gly224 to Gly232), from the 250th lysine residue to the 258th leucine residue (250 Lys to Leu258), from the 438th alanine residue to the 446th Phenylalanine residue (Ala438-Phe446) It is desirable to avoid mutagenesis in this region. On the other hand, Non-patent Document 3 describes the activity of the wild-type enzyme obtained by substituting the 405th phenylalanine residue of the α-glycosyl trehalose-producing enzyme derived from Sulfolobus solfatalicas with another amino acid residue from 29 to 57. % Mutant enzymes are disclosed. This amino acid residue corresponds to the amino acid sequence of the α-glycosyl trehalose-producing enzyme derived from Sulfolobus acidocardarius, that is, the 388th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. If mutations are introduced in the vicinity of these amino acid residues of an α-glycosyl trehalose-producing enzyme, there is a possibility that an enzyme with improved properties can be obtained without significantly affecting substrate specificity and enzyme activity. .

  In the present invention, “a mutant enzyme of trehalose-releasing enzyme” refers to the substrate specificity as a trehalose-releasing enzyme by applying a genetic engineering technique to a gene encoding a trehalose-releasing enzyme derived from a microorganism belonging to the genus Sulfolobus as described above. In addition, a mutant enzyme into which a deletion, substitution, or insertion mutation of one or more amino acid residues in the amino acid sequence is introduced can be suitably used within a range that does not substantially change the enzyme activity. The mutation introduction site on the amino acid sequence is not particularly limited as long as the substrate specificity and enzyme activity as trehalose-releasing enzyme are substantially retained.

  In the production method of the present invention, isoamylase or pullulanase widely used in the art can be used as the starch debranching enzyme. Commercially available enzyme agents or those isolated from microorganisms may be used. As isoamylases, for example, those derived from Pseudomonas amyloderamosa and Myroides odoratatus are well known, and in particular, an isoamylase derived from Pseudomonas amyloderamosa (manufactured by Hayashibara Co., Ltd.). Is preferred. Examples of the pullulanase agent include pullulanase derived from Klebsiella pneumoniae (sales by Hayashibara Co., Ltd.), pullulanase derived from Bacillus amylopullulyticus (trade name “Promozyme”, Japan ) And the like.

  The role of CGTase in the trehalose production step is to disproportionate that CGTase catalyzes malto-oligosaccharide having a degree of glucose polymerization of 4 or less, which is inevitably produced in the course of trehalose production reaction by α-glycosyl trehalose producing enzyme and trehalose releasing enzyme. By converting to maltooligosaccharide having a glucose polymerization degree of 5 or more by a reaction (linear sugar intermolecular transfer reaction, Disproportionation), the trehalose production reaction is further advanced to further increase the trehalose content in the reaction solution. .

  CGTase has been isolated from various microorganisms, and its action and physicochemical properties have been clarified (“Industrial Glucose Enzyme Handbook”, edited by Kodansha Scientific, published by Kodansha, 28 to 32 pages (1999)). In addition, for some of the above CGTases, the genes have been cloned, and the amino acid sequences have been determined from the base sequences of the genes. The CGTase amino acid sequences are common to the enzymes classified into the α-amylase family. It is also known that there are four storage areas that are supposed to exist. Furthermore, the three-dimensional structure of CGTase protein derived from Geobacillus stearothermophilus has already been clarified by X-ray crystal structure analysis. The three catalytic residues of CGTase, namely SEQ ID NO: 4 in the sequence listing, The 225th aspartic acid residue (D225), the 253rd glutamic acid residue (E253), and the 324th aspartic acid residue (D324) in the amino acid sequence shown are also known ("Industrial Carbohydrate Enzyme Handbook") Edited by Kodansha Scientific, published by Kodansha, pages 56 to 63 (1999)).

  In the production method of the present invention, natural or recombinant CGTase derived from a microorganism belonging to the genus Paenibacillus or a mutant enzyme thereof is preferably used as CGTase. Examples of the “natural CGTase derived from a microorganism belonging to the genus Paenibacillus” in the present invention include CGTase derived from known strains such as Paenibacillus illinoisensis, Paenibacillus publi, Paenibacillus amylolyticus, and Paenibacillus isolated from nature. CGTase derived from a genus microorganism can be used. More specifically, Paenibacillus illinoisensis NBRC15959 strain, Paenibacillus illinoisensis NBRC15379 strain, Paenibacillus publi NBRC13638 strain, and Paenibacillus amylolyticus NBRC15957 strain, For example, CGTase derived from a high enzyme-producing mutant strain obtained by introducing a mutation by, for example, ultraviolet irradiation or mutation treatment with a chemical substance can be more suitably used. Further, as CGTase derived from a microorganism belonging to the genus Paenibacillus, for example, CENase derived from Paenibacillus sp. (Trade name “alkali CD amylase”, manufactured by Nagase ChemteX Corporation) can also be used. Incidentally, CGTase conventionally used in the production of food-grade trehalose dihydrate crystal-containing powder is CGTase derived from Geobacillus stearothermophilus.

Moreover, as CGTase used in the present invention, CGTase having partial amino acid sequences shown in the following (a) to (d) is preferably used.
_{(A) Gly-Ser-X} 1 -Ala-Ser-Asp;
(B) Lys-Thr-Ser-Ala-Val-Asn-Asn;
(C) Lys-Met-Pro-Ser-Phe-Ser-Lys;
(D) Val-Asn-Ser -Asn-X 2 -Tyr;
(However, X ₁ means Ala or Ser, and X ₂ means Ala or Thr.)
The partial amino acid sequence is a characteristic amino acid sequence peculiar to CGTase derived from Paenibacillus sp.

  In addition, the “recombinant CGTase derived from the genus Paenibacillus” referred to in the present invention is a group obtained by cloning the CGTase gene of the genus Paenibacillus and expressing it in an appropriate host microorganism such as Escherichia coli or Bacillus subtilis. The replaceable CGTase can be preferably used. In addition, CGTase derived from the aforementioned Paenibacillus illinoisensis NBRC15379 strain, Paenibacillus publi NBRC13638 strain, and Paenibacillus amylolyticus NBRC15957 strain, the applicant has independently cloned the CGTase gene from these microorganisms. When the sequence was determined, it was found to have the amino acid sequences shown by SEQ ID Nos. 1, 2 and 3 in the sequence listing. The recombinant CGTase obtained by expressing the CGTase gene of the above-mentioned Paenibacillus microorganism in Escherichia coli, that is, the recombinant CGTase having the amino acid sequence represented by SEQ ID NO: 1, 2, or 3 in the sequence listing is the production method of the present invention. Can be used similarly.

  The “mutant enzyme of CGTase derived from the genus Paenibacillus” in the present invention (hereinafter abbreviated as “mutant CGTase”) applies a genetic engineering technique to the gene encoding CGTase derived from the genus Paenibacillus. Thus, a mutant CGTase into which a deletion, substitution, or insertion mutation of one or more amino acid residues in the amino acid sequence is introduced as long as the substrate specificity and enzyme activity as CGTase are not substantially changed is preferably used. be able to. The mutation introduction site on the amino acid sequence is not particularly limited as long as the substrate specificity and enzyme activity as CGTase are substantially retained, but common to the enzyme group belonging to the α-amylase family existing on the amino acid sequence of CGTase. Thus, it is desirable to avoid the introduction of mutations into the above-mentioned partial amino acid sequences (a) to (d), which are peculiar to the four regions conserved and CGTase derived from Paenibacillus microorganisms.

  As a more specific example of “natural or recombinant CGTase derived from a microorganism belonging to the genus Paenibacillus or a mutant enzyme thereof” that can be suitably used in the production method of the present invention, SEQ ID NO: 3 in the aforementioned sequence listing SEQ ID NO: 14 or 15 in the sequence listing prepared by introducing a site-specific mutation into CGTase having the amino acid sequence shown by 4 and 5, respectively, or CGTase gene of Paenibacillus illinoisensis NBRC15379 strain described later in Examples. And a mutant CGTase having an amino acid sequence represented by

  The raw material starch used in the production of trehalose may be ground starch such as corn starch, rice starch and wheat starch, or underground starch such as potato starch, sweet potato starch and tapioca starch. Alternatively, it may be a partially decomposed starch obtained by partial decomposition with amylase. The raw starch is usually suspended in water to form starch milk having a concentration of about 10 to 50%, and gelatinized and liquefied by heating in the presence of heat-resistant α-amylase. The degree of liquefaction of the liquefied starch is usually adjusted to less than 10 and more specifically less than 5 as dextrose equivalent (DE).

  In the production method of the present invention, the liquefied starch (pH about 4 to 10) as a raw material is preferably used as a substrate, and the above-mentioned α-glycosyl trehalose-producing enzyme derived from a microorganism belonging to the genus Sulfolobus is added per gram of the substrate solid. 1 to 20 units, 5 to 50 units of trehalose-releasing enzyme derived from the above-mentioned Sulfolobus sp. Microorganism, 100 to 2,000 units of starch debranching enzyme, and 1 to 50 CGTase derived from the above-mentioned Paenibacillus sp. A unit is added and the reaction is carried out at a temperature range where the used enzyme is not inactivated, usually 30 to 70 ° C. for 10 to 100 hours. The trehalose content of the reaction solution at the end of the reaction is usually around 86% in terms of anhydride.

<Step (2) (Glucoamylase treatment step)>
In this step, a glucoamylase agent is further allowed to act on the reaction solution obtained in the trehalose production step (1) to increase the trehalose content in terms of anhydride. That is, the reaction solution obtained by the trehalose production step contains saccharides such as D-glucose, maltose, malto-oligosaccharide having a degree of glucose polymerization of 3 or more, α-glucosyl trehalose, and α-maltosyl trehalose together with trehalose. Therefore, by causing glucoamylase to act on this reaction solution, maltose and maltooligosaccharide having a glucose polymerization degree of 3 or more are decomposed into D-glucose, and α-glycosyl such as α-glucosyl trehalose and α-maltosyl trehalose. By decomposing trehalose into D-glucose and trehalose, the trehalose purity in the reaction solution, that is, the trehalose content in terms of anhydride can be increased.

  As the glucoamylase used in the production method of the present invention, maltose and maltooligosaccharide having a glucose polymerization degree of 3 or more can be decomposed to D-glucose, and α-glycosyl trehalose such as α-glucosyl trehalose and α-maltosyl trehalose. As long as it can be decomposed into D-glucose and trehalose, there are no particular restrictions on its origin or origin. Commercially available glucoamylase agents such as Amano Enzyme Co., Ltd., trade name “Gluczyme AF6”, Nagase ChemteX Co., Ltd., trade name “GlucoTeam” and the like can be suitably used.

  In addition, the trehalose content in the reaction solution after glucoamylase treatment, that is, the trehalose-containing sugar solution is usually more than 86.0%, preferably 87.0% or more in terms of anhydride. Incidentally, in the case of using CGTase derived from Geobacillus stearothermophilus instead of CGTase derived from the genus Paenibacillus, the trehalose content in the trehalose-containing sugar solution after glucoamylase treatment is about 83.0% in terms of anhydride. It will not stop and will not be over 86.0%.

<Step (3) (Purification and Concentration Step)>
In this step, the trehalose-containing sugar solution whose trehalose content in terms of anhydride has been increased after finishing the glucoamylase treatment is subjected to filtration, centrifugation, etc., by an ordinary method to remove insoluble matters, and decolorized with activated carbon. This is a step of desalting with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type) and concentrating to a concentration suitable for crystallization. The trehalose content in terms of anhydride in the reaction solution has already been increased to 86.0% or more by the glucoamylase treatment step of (2). Therefore, in the purification and concentration step of (3), it is determined by column chromatography. A step of further increasing the trehalose content such as a fractionation step is not necessary.

<Step (4) (crystallization step)>
This step is a step of crystallizing trehalose dihydrate crystals in the presence of seed crystals of trehalose dihydrate crystals from the trehalose-containing sugar solution obtained through the steps (1) to (3). That is, the sugar liquid in which the trehalose content in terms of anhydride is increased to a predetermined level is usually adjusted after adjusting the supersaturation degree for trehalose to be in the range of 1.05 to 1.50, in other words, The trehalose concentration is adjusted to about 60 to 85% and the liquid temperature is adjusted to about 40 to 80 ° C., and then transferred to an auxiliary crystal can. Then, the seed crystals of trehalose dihydrate crystals are added to the concentrated sugar solution volume in the auxiliary crystal can. Usually, 0.1 to 5% (w / v), more specifically 0.5 to 2% (w / v) is contained, and the liquid temperature is 5 to 5 over 3 to 48 hours with gentle stirring. Crystallization of trehalose dihydrate crystals is promoted by natural cooling to 60 ° C. When seed crystals of trehalose dihydrate crystals already exist in the auxiliary crystal can etc., it is not necessary to add the seed crystals of trehalose dihydrate crystals. In terms of work efficiency, crystallization of trehalose dihydrate crystals from the concentrate is usually performed in the presence of seed crystals.

In the crystallization step, a controlled cooling method or a pseudo controlled cooling method can be advantageously used instead of the natural cooling method. When crystallization is performed by the controlled cooling method or the pseudo controlled cooling method, the trehalose-containing sugar solution adjusted to a predetermined temperature through the step (3) is transferred to an auxiliary crystal can, and then the trehalose dihydrate crystal The seed crystal is usually contained in an amount of 0.1 to 5% (w / v), more specifically 0.5 to 2% (w / v) with respect to the volume of the concentrated sugar solution in the auxiliary crystal can. By controlling the cooling while stirring, the liquid temperature is gradually decreased at the initial stage of the crystallization process, and the liquid temperature is rapidly decreased at the latter stage of the cooling process to promote crystallization. Although the time required for crystallization varies depending on the amount of seed crystals of trehalose dihydrate crystals added, for example, in the case of the pseudo controlled cooling method, the total cooling time is divided into at least two, preferably three or more sections, Within each section, the temperature is decreased substantially linearly with respect to time, and the change amount (T ₀ -T _m ) of the liquid temperature T at the time when the working time t = τ / 2 (intermediate point of the crystallization process) The liquid temperature T is continuously or stepwise with respect to the time t so as to maintain a range of 5% or more and less than 50% of the total temperature change amount (T ₀ -T _f ), desirably 10% or more and less than 30%. It is good to reduce. For example, when the liquid temperature is cooled from 60 ° C. to 20 ° C. over 10 hours to crystallize the crystal, the cooling time is divided into two sections of 6 hours and 4 hours, and the liquid temperature is changed from 60 ° C. to 50 ° C. Cool to 6 hours and then cool from 50 ° C to 20 ° C over 4 hours, or divide the cooling time into two sections of 7 hours and 3 hours, and the liquid temperature from 60 ° C to 45 ° C It is preferable to cool for 7 hours, and then cool from 45 ° C. to 20 ° C. for 3 hours, and more preferably, the cooling time is divided into three intervals of 4 hours, 3 hours and 3 hours. Then, the liquid temperature is cooled from 60 ° C. to 55 ° C. over 4 hours. In the next section, the liquid temperature is cooled from 55 ° C. to 50 ° C. over 3 hours. It is preferable to cool to 20 ° C.

  Thus, in the case of the controlled cooling method or the pseudo-controlled cooling method, compared with the crystallization method in which natural cooling is performed without performing temperature control, trehalose dihydrated crystal microcrystals are less likely to be formed, and crystals having a uniform particle size are produced. The resulting mass kit can be obtained, and as a result, the yield of trehalose dihydrate crystal-containing powder can be further increased compared to the case of using the natural cooling method. In addition, as will be described later, the obtained trehalose dihydrated crystal powder is more in terms of crystallinity of trehalose dihydrated crystal, which is an important index of trehalose purity and solidification than the powder obtained by the natural cooling method. It has the feature of being high. In addition, in the case of the controlled cooling method or the pseudo controlled cooling method, there is an advantage that a powder having a more uniform particle size distribution can be obtained as compared with a powder obtained by a crystallization method in which natural cooling is performed.

<Step (5) (collecting step)>
This step is a step of collecting trehalose dihydrate crystals by centrifugation from the mass kit obtained in the crystallization step (4) according to a conventional honey method. The collected trehalose dihydrate crystal is washed by spraying (showing) a small amount of purified water in order to remove amorphous nectar adhering to the surface. The amount of purified water used for washing the crystals is usually preferably 3% to 10% with respect to the weight of the mass kit before centrifugation. That is, if the amount of purified water used for washing is less than 3%, washing is not performed sufficiently, amorphous nectar remains, and the desired trehalose purity may not be obtained. On the other hand, if the amount of purified water used for washing exceeds 10%, the amount of trehalose dihydrate crystals dissolved and removed by washing increases and the starch yield may decrease.

<Step (6) (Aging and drying step)>
This step is a step in which the collected trehalose dihydrate crystal is kept in a predetermined temperature and humidity atmosphere for a certain period of time, and the crystal is aged and dried with hot air to obtain a trehalose dihydrate crystal-containing powder. There are no particular limitations on the product temperature of the crystal, the relative humidity of the atmosphere, and the holding time in the aging and drying steps, as long as the desired powder can be obtained. The relative humidity of the atmosphere is preferably maintained at 55 ° C. and 60 to 90%, and the aging and drying time is preferably about 5 to 24 hours. The powder that has undergone the aging and drying steps is then naturally cooled to room temperature. Further, forced cooling to a product temperature of about room temperature by blowing clean air of about room temperature can be advantageously performed. The obtained crystal powder is used as it is or after being pulverized as necessary.

  According to the method for producing a powder containing trehalose dihydrate crystals of the present invention, a trehalose-containing sugar solution having a high trehalose content of more than 86.0% in terms of anhydride can be obtained by enzymatic reaction. A process is unnecessary, there is no loss of trehalose due to fractionation, and a powder containing trehalose dihydrate crystals can be obtained at a high starch yield. In addition, instead of a whole sugar system that crystallizes, solidifies or spray-drys the entire mass kit including crystallized crystals, a honey-type system that removes nectar containing impurities by centrifuging the crystallized crystals is used. Thus, the trehalose content in the obtained trehalose dihydrate crystal-containing powder can be easily increased to 98.0% or more, and a high-purity trehalose dihydrate crystal-containing powder can be produced.

  The thus-produced trehalose dihydrate crystal-containing powder is almost equivalent to conventional food-grade trehalose-containing powder in physical properties such as solidification during storage when crystallization is performed by natural cooling. It is a powder containing trehalose dihydrate crystals containing particles that normally have a particle size of 53 μm or more and less than 425 μm occupying 70% or more of the whole powder, and particles having a particle size of 53 μm or more and less than 300 μm of 50% or more . In addition, when crystallization is performed by the controlled cooling method or the pseudo controlled cooling method, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention is significantly more than the conventional food-grade trehalose-containing powder. Trehalose dihydrate crystals that are hard-to-consolidate and usually have particles with a particle size of 53 μm or more and less than 425 μm occupy 80% or more of the whole powder and particles with a particle size of 53 μm or more and less than 300 μm of 60% or more of the whole powder Contains powder. In addition, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention usually has a reducing power of 0.5% or less of the whole powder obtained by the above formula [6], and is incorporated into foods and pharmaceuticals. However, it is an excellent powder with no fear of discoloration due to browning.

  Therefore, the powder produced by the production method of the present invention can be used as it is or after adjusting the particle size as appropriate, as a powdery food material, cosmetic material, quasi-drug material, or pharmaceutical material. . In particular, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention that applies the controlled cooling method during crystallization is a powder that is significantly harder to set than the conventional food-grade trehalose-containing powder, as described above. It can be said that this is a completely new powder containing trehalose dihydrate crystals that has not been known so far. The powder is a single or a plurality of other powdered foods in each of the fields of food production, cosmetic production, quasi-drug production, and pharmaceutical production using a production plant designed on the premise of handling powder raw materials. It has an excellent advantage that it can be contained safely in materials, cosmetic materials, quasi-drug materials, and pharmaceutical materials.

  Hereinafter, the method for producing the trehalose dihydrate crystal-containing powder of the present invention will be specifically described by experiments.

<Experiment 1: Effect of CGTase origin on trehalose content in enzyme reaction solution>
Reacted α-glycosyl trehalose-producing enzyme derived from Sulfolobus microorganisms and recombinant trehalose-releasing enzyme derived from Sulfolobus microorganisms together with starch debranching enzyme and CGTase on liquefied starch, followed by action of glucoamylase In order to examine how the origin of CGTase used affects the trehalose content in the sugar solution obtained by the enzyme reaction in the enzyme reaction system in which trehalose is generated by the enzyme reaction to be performed.

<Experiment 1-1: Preparation of Recombinant α-Glycosyl Trehalose-Generating Enzyme and Recombinant Trehalose-Free Enzyme Derived from Sulfolobus spp.>
Recombinant DNA containing an α-glycosyl trehalose synthase gene derived from Sulfolobus acidocardarius ATCC33909 by the method described in Example A-2 (b) of Patent Document 7 (Japanese Patent Laid-Open No. 8-84586), Recombinant Escherichia coli ST36 retaining pST36 was cultured, and recombinant α-glycosyl trehalose-producing enzyme was purified by the method of Experimental Example 1 of the same specification to obtain about 3,800 ml of purified enzyme solution containing about 210 units per ml. . In addition, by the method described in Example A-2 (b) of Patent Document 8 (Japanese Patent Application Laid-Open No. 8-336388), a recombinant DNA containing a trehalose-free enzyme gene derived from Sulfolobus acidocardarius ATCC33909 strain, pSU19 The retained recombinant Escherichia coli SU19 was cultured, and the recombinant α-glycosyl trehalose producing enzyme was purified by the method of Experimental Example 1 of the same specification to obtain about 2,050 ml of a purified enzyme solution containing about 2,900 units per ml. . The activities of α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme were measured according to the methods disclosed in Patent Document 5 (JP-A-8-66188) and 6 (JP-A-8-66187).

<Experiment 1-2: CGTase derived from various microorganisms>
The following CGTases were used as CGTases derived from various microorganisms. That is, as CGTase derived from Geobacillus stearothermophilus, CGTase derived from Geobacillus stearothermophilus Tc-91 strain (FERM BP-11273) (manufactured by Hayashibara Co., Ltd.), Bacillus macerans (Bacillus macerans) The CGTase derived from the market is CGTase (trade name “Contiszyme”, sold by Amano Enzyme Co., Ltd.), and the CGTase derived from Thermoanaerobacterium thermosulfurigenes is the commercially available CGTase (trade name “Tolzame”). ”, Novozymes Japan Co., Ltd.).

  Moreover, the following CGTase was prepared as CGTase derived from Paenibacillus sp. That is, Paenibacillus illinoisensis NBRC15959 strain, Paenibacillus illinoisensis NBRC15379 strain, Paenibacillus publi NBRC13638 strain, Paenibacillus amylolyticus NBRC15957 strain, dextrin 2%, potassium hydrogen chloride 0.50, Each suspension supernatant obtained by culturing at 27 ° C. for 3 days in a liquid medium containing 0.05%, magnesium sulfate 0.025% and calcium carbonate 0.5%, and centrifuging the culture broth was prepared according to a conventional method. The crude enzyme solution of CGTase derived from each microorganism was obtained by analysis and dialysis. The obtained CGTase crude enzyme solution was subjected to anion exchange column chromatography using DEAE-Toyopearl 650S gel (manufactured by Tosoh Corporation) and hydrophobic column chromatography using butyl-Toyopearl 650M gel (manufactured by Tosoh Corporation), respectively. And purified partially to prepare partially purified CGTase. In addition, the activity of CGTase derived from each strain was measured according to the above-described method and calculated using the formula [1].

<Experiment 1-3: Trehalose production reaction>
Corn starch was suspended in water to a concentration of 30%, and 0.1% calcium carbonate was added to this suspension. After adjusting the pH of the suspension to 6.0, 0.2% heat-resistant α-amylase agent (trade name “Termamyl 60L”, sold by Novozymes Japan KK) per starch solid was added, and 95 ° C. For 15 minutes to gelatinize and liquefy the starch. The resulting liquefied starch solution was autoclaved at 120 ° C. for 30 minutes, then cooled to 57 ° C., adjusted to pH 5.5, and maintained at the same temperature, while maintaining the same temperature, 10 units of recombinant type per gram of starch solids. α-glycosyl trehalose producing enzyme, 40 units of recombinant trehalose releasing enzyme, 1,500 units of isoamylase agent (manufactured by Hayashibara Co., Ltd.), and CGTase described in Experiment 1-2 or Experiment 1-2 15 units of CGTase was added and reacted for 48 hours. The obtained reaction product was heated at 97 ° C. for 30 minutes to inactivate each enzyme, then adjusted to pH 4.5, and 10 units of glucoamylase agent (trade name “Glucoteam # 20000” per gram of starch solids. ”, Manufactured by Nagase ChemteX Corporation) and reacted for 24 hours. The reaction solution thus obtained was heated at 95 ° C. for 10 minutes to inactivate the enzyme and subjected to the measurement of trehalose content in the reaction solution described below. In addition, the reaction liquid obtained by performing an enzyme reaction on the same conditions except not adding CGTase was used as the control.

<Experiment 1-4: Measurement of trehalose content in reaction solution>
The reaction solutions obtained in Experiment 1-3 were designated as reaction solutions 1 to 8 shown in Table 1, and the trehalose content was determined as follows. That is, each of the reaction solutions 1 to 8 was made into 1% solution with purified water, filtered through a 0.45 μm membrane filter, subjected to HPLC analysis under the following conditions, and the reaction solution was determined from the area of the peak appearing in the chromatogram by the differential refractometer. The trehalose content of was calculated and converted to anhydride. The results are shown in Table 1. The trehalose content in the reaction solution shown in Table 1 is a value obtained with good reproducibility within a range of slight variations even when trehalose production reaction and glucoamylase treatment are repeated 5 times under the same conditions for each CGTase. It is.
・ Analysis conditions HPLC apparatus: “LC-10AD” (manufactured by Shimadzu Corporation)
Degasser: “DGU-12AM” (manufactured by Shimadzu Corporation)
Column: “MCI GEL CK04SS” (Mitsubishi Chemical Corporation)
Sample injection volume: 20 μl
Eluent: Purified water Flow rate: 0.4 ml / min Temperature: 85 ° C
Differential refractometer: “RID-10A” (manufactured by Shimadzu Corporation)
Data processor: “Chromatopack C-R7A” (manufactured by Shimadzu Corporation)

  As shown in Table 1, in the combination of the recombinant α-glycosyl trehalose producing enzyme, the recombinant trehalose releasing enzyme, and the starch debranching enzyme (isoamylase) (reaction solution 1, control), the reaction solution after the glucoamylase treatment The content of trehalose is only 77.1%. However, when CGTase derived from Geobacillus stearothermophilus conventionally used for the production of trehalose is added, the content increases to 82.5% (reaction solution 2). Increased trehalose yield was observed. On the other hand, when CGTase derived from Bacillus macerans was used (reaction liquid 3), 81.2%, and when CGTase derived from Thermoanaerobacterium thermosulfrigenes was used (reaction liquid 4), 80. The value was 6%, which was lower than that in the case of using CGTase derived from Geobacillus stearothermophilus Tc-91 strain conventionally used for the production of trehalose (reaction solution 2).

  On the other hand, when CGTase derived from a microorganism belonging to the genus Paenibacillus was used (reaction liquids 5 to 8), the trehalose content after glucoamylase treatment exceeded 86.0% in terms of anhydride. It increased by about 4% compared with the case of using CGTase derived from Geobacillus stearothermophilus Tc-91 strain used for the production of trehalose (reaction solution 2). In particular, when each CGTase derived from Paenibacillus illinoisensis NBRC15959 (reaction solution 5) and Paenibacillus illinoisensis NBRC15379 (reaction solution 6) is used as CGTase, the trehalose content is 86.0 to 87. It was found to rise to 0.0%. In this experiment, it was found that CGTase derived from Paenibacillus illinois cis was most preferable.

<Experiment 2: Trehalose purity, starch yield, and physical properties of trehalose dihydrate crystal-containing powders produced from sugar solutions with different trehalose contents>

<Experiment 2-1: Preparation of test sample>
<Test samples 1 to 8>
Reaction liquids 1 to 8 having different trehalose contents obtained in Experiment 1 were purified by decolorization using activated carbon and desalting using ion exchange resin, and concentrated to a solid concentration of about 60%. Corresponding to each of 8 to 8, trehalose-containing sugar solutions 1 to 8 (containing 77.1 to 86.8% trehalose in terms of anhydride) were obtained.

  Each of the trehalose-containing sugar solutions 1 to 8 is concentrated under reduced pressure to a solid concentration of about 85%, taken into an auxiliary crystal can, and about 1% (w / v) trehalose dihydrate containing each sugar solution. Crystals were added as seed crystals, and the mixture was stirred and naturally cooled from 60 ° C. to 20 ° C. over about 10 hours to prepare a mass kit in which trehalose dihydrate crystals were crystallized. The trehalose dihydrate crystals are collected from the mass kit by a basket-type centrifuge by a conventional method, and the collected trehalose dihydrate crystals are washed with 8% deionized water with respect to the weight of the mass kit, and are heated at 40 ° C. for 8 hours. After ripening and drying, it was forcedly cooled by blowing clean air at 25 ° C. for 30 minutes and pulverized to obtain a powder containing trehalose dihydrate crystals. Trehalose dihydrate crystal-containing powders obtained from trehalose-containing sugar solutions 1 to 8 were used as test samples 1 to 8, respectively.

<Test sample 9>
As a test sample 9, a food-grade trehalose dihydrate crystal-containing powder (trade name “Treha”, lot number: 9I131, Hayashibara Sales Co., Ltd.) was used.

<Experiment 2-2: Trehalose purity, starch yield, and caking property of test samples 1 to 9>
<Trehalose purity>
The trehalose purity of test samples 1 to 9 was determined by the same HPLC method as in Experiment 1-3. The results are shown in Table 2.

<Yield to starch>
The starch yields of the test samples 1 to 8 prepared above were calculated in terms of anhydride of the raw material starch from the mass of the enzyme reaction solution used for the preparation of each test sample and the concentration (30%) when the raw material starch was charged. The weight of the test samples 1 to 8 obtained by this value was calculated, and the weight in terms of anhydride was divided. The results are shown in Table 2.

<Caking test>
For each of the test samples 1 to 9, the following experiment was performed for the purpose of examining the caking property of each powder. Specifically, 1 gram of each of the test samples 1 to 9 was weighed, and a polypropylene cylindrical tube with a cap of 14 ml with a hemispherical inner bottom (each sold by Becton Dickinson, trade name “Falcon Tube 2059”) with a diameter of 1. 7 cm, height 10 cm), and the tube is placed in a 50 ° C. incubator (trade name “CI-410”, sold by Advantech Toyo Co., Ltd.) with the tube upright on a test tube stand for 24 hours. The tube was taken out of the incubator, removed from the tube, and the tube was slowly turned over to remove the test sample on a black plastic plate, and the state of the taken test sample was visually observed. .

  The presence or absence of consolidation is identified as “solidified” (+) when the test sample is still clearly a hemisphere at the bottom of the tube even on a flat plate (+). The case where it was possible to be “solidified slightly” (±), and the case where the test sample collapsed and the shape of the inner bottom of the tube was not maintained was determined as “no solidification” (−). The results are shown in the column “Caking” in Table 2.

  As shown in Table 2, the trehalose content in terms of anhydride in the trehalose dihydrate crystal-containing powders of the test samples 1 to 8, that is, the trehalose purity exceeds 98.0%, and the conventional food grade trehalose dihydrate crystals It was a high-purity trehalose dihydrate-containing crystal-containing powder in the same manner as the test sample 9 which was a contained powder. However, with regard to the starch yield, in the test samples 2 to 4 using CGTase other than CGTase derived from the Paenibacillus genus, the starch yield was 31% at most, whereas that of Paenibacillus In the test samples 5 to 8 using CGTase derived from microorganisms, the yield with respect to starch reached 39 to 41%, and differences due to the origin of CGTase used were observed. Moreover, when the results in Table 1 and Table 2 are compared, the trehalose dihydrate crystal-containing powder prepared from the enzyme reaction solution having a high trehalose content in the enzyme reaction solution after the glucoamylase treatment tends to have a higher starch yield. A correlation was observed between the trehalose content in the enzyme reaction solution and the yield with respect to starch.

  From these results, when CGTase derived from a microorganism belonging to the genus Paenibacillus is used as CGTase (test samples 5 to 8), the trehalose content in the enzyme reaction solution after the glucoamylase treatment exceeds 86.0%. Further, it was found that the yield of starch with respect to the trehalose dihydrate crystal-containing powder was also increased to about 40%. In addition, when using CGTase derived from a microorganism belonging to the genus Paenibacillus, the improvement in starch yield (starch yield of test samples 5 to 8) is compared with the conventionally used Geobacillus stearothermophilus Tc-91. Compared to the case of using CGTase derived from the strain (test sample 2), it was improved by 8 to 10%, and a remarkable effect was recognized.

  On the other hand, regarding the caking property which is an important physical property in handling as a powder, the test sample 1 produced from the trehalose-containing sugar solution 1 not using CGTase, and CGTase are derived from Thermoanaerobacterium thermosulfurigenes The test sample 4 produced using CGTase was determined to be “consolidated (+)” in the caking test, whereas test samples 2, 3 and 4 produced using other CGTases were used. Nos. 5 to 8 are determined to be “slightly consolidated” (±) in the caking test as in the case of food-grade trehalose dihydrate crystal-containing powders (test sample 9) that have been commercially available. It was. As a result, the powder containing trehalose dihydrate crystals (test samples 5 to 8) produced by the production method of the present invention using CGTase derived from a microorganism belonging to the genus Paenibacillus is a food-grade trehalose dihydrate crystals that have been commercially available. Compared to the contained powder (test sample 9), it is a powder that is not inferior in caking property, and it is a food-grade powder that is commercially available as a powdery food material, cosmetic material, quasi-drug material, or pharmaceutical material. It shows that the powder can be used in the same manner as the powder containing trehalose dihydrate crystals.

<Experiment 3: Effect of pseudo controlled cooling during crystallization on trehalose purity, starch yield, and caking properties of trehalose dihydrate crystal-containing powder>
In this experiment, when crystallizing trehalose dihydrate crystals from trehalose-containing sugar solutions 1 to 8 prepared in Experiment 2-1, a pseudo-controlled cooling method was applied to prepare trehalose dihydrate crystal-containing powder. The effects of the powder on trehalose purity, starch yield, and caking properties were investigated.

<Experiment 3-1: Preparation of test sample>
Concentrate each of the trehalose-containing sugar solutions 1 to 8 with different trehalose contents in terms of anhydride prepared in Experiment 2-1 to a solid concentration of about 85% under reduced pressure, take them into a crystal can, Except that it was auxiliary crystallized by adding about 1% (w / v) trehalose dihydrate crystal as a seed crystal and stirring the mixture while stirring for about 10 hours from 60 ° C to 20 ° C. In the same manner as in 2, a mass kit in which trehalose dihydrate crystals were crystallized was prepared. In addition, the pseudo control cooling is divided into three sections of 4 hours, 3 hours and 3 hours, and the liquid temperature is changed from 60 ° C. to 55 ° C. over 4 hours in the first section. In the last section, the liquid temperature is lowered from 55 ° C to 50 ° C, and in the last section, the liquid temperature is lowered from 50 ° C to 20 ° C over 3 hours. Went by. From the obtained mass kit, trehalose dihydrated crystals were collected by a conventional method using a basket-type centrifuge, and the collected trehalose dihydrated crystals were washed with 8% deionized water based on the weight of the mass kit at 40 ° C. After aging and drying for 8 hours, clean air at 25 ° C. was blown for 30 minutes to forcibly cool and pulverize to obtain a powder containing trehalose dihydrate crystals. Trehalose dihydrate crystal-containing powders obtained from each of the trehalose-containing sugar solutions 1 to 8 by pseudo controlled cooling were used as test samples 1c to 8c, respectively.

<Experiment 3-2: Trehalose purity, starch yield, and caking property of test samples 1c to 8c>
<Trehalose purity>
The trehalose purity of test samples 1c to 8c was determined by the same HPLC method as in Experiment 1-3. The results are shown in Table 3.

<Yield to starch>
The starch yields for the test samples 1c to 8c were calculated by the same method as in Experiment 2-2. The results are shown in Table 3.

<Caking test>

  The caking properties of the test samples 1c to 8c were evaluated by the same caking property test as in Experiment 2-2. The results are shown in Table 3.

  As shown in Table 3, the trehalose purity of the test samples 1c to 8c prepared by applying the pseudo controlled cooling method in the crystallization process was in the range of 98.7 to 99.5%. When this result was compared with the trehalose purity of the test samples 1 to 8 obtained by crystallization by the natural cooling method of Experiment 2 (in the column of “trehalose purity” in Table 2), all of the test samples 1c to 8c were trehalose. The purity was increased by 0.3 to 0.9%. This result shows that the trehalose purity of the powder can be increased by applying pseudo controlled cooling in the crystallization process.

  The test samples 1c to 8c had a starch yield of 31 to 44%, and this result was crystallized by the natural cooling method of Experiment 2 and the test samples 1 to 8 had a starch yield (Table 2). In comparison with the column “Yield to starch” in FIG. 5, the test samples 1c to 8c all had an improvement in starch yield of about 3 to 4%. This result means that when the pseudo controlled cooling method is applied at the time of crystallization, the yield with respect to starch is increased as compared with the case of crystallization by the natural cooling method. It is not clear why the trehalose dihydrate-containing powder obtained by applying the pseudo controlled cooling method increases the starch yield even though there is no change in trehalose content in the trehalose-containing sugar solution used for crystallization. However, as described above, according to the pseudo controlled cooling method, crystals with few crystallites and a uniform particle size can be obtained. Therefore, when collecting crystals from a mass kit by centrifugation and when washing the collected crystals with water, It is presumed that the loss of trehalose is reduced.

  Further, the test samples 1c to 8c were subjected to the same caking test as in Experiment 2-2 to examine the caking properties of the powder. As shown in Table 3, all of the test samples 1c to 4c were “slightly”. It is determined as “solidified” (±), and all of the test samples 5c to 8c are collapsed when taken out on a flat plate and do not maintain the shape of the bottom of the tube, and are determined as “no consolidated” (−). It was. These results show that when the pseudo controlled cooling method is applied during crystallization, the solidification property of the obtained powder tends to be improved compared to the case of crystallization by the natural cooling method. Show. Among them, the conventional food-grade trehalose dihydrate crystal-containing powder (test sample 9) was determined to be “somewhat consolidated” (±) (see Table 2) in the caking test, whereas the trehalose content was 86 The trehalose dihydrated crystal-containing powders (test samples 5c to 8c) obtained by crystallization by applying a pseudo-controlled cooling method from 5 to 8 trehalose-containing sugar liquids 5 to 8 having a relatively high value of “No solidification” The fact that it was determined as (-) is that the conventional food-grade trehalose dihydrate crystals are contained by crystallizing from a trehalose-containing sugar solution having a trehalose content exceeding 86% and applying a pseudo controlled cooling method. It shows that it is possible to produce trehalose dihydrate crystal-containing powders that are significantly less consolidated than powders and have excellent powder properties.

  From the above results, by applying the pseudo controlled cooling method in the crystallization process, the trehalose dihydrated crystal-containing powder having a higher trehalose purity compared to the case of crystallization by the natural cooling method has a higher yield to starch. It was found that can be manufactured. In addition, trehalose dihydrate crystal-containing powder produced by crystallization from a sugar solution having a trehalose content exceeding 86.0% by a pseudo controlled cooling method is a conventional food grade trehalose dihydrate produced by a natural cooling method. It was found that the water-containing crystal-containing powder is a more excellent powder in that it does not solidify even under conditions judged to be “somewhat solidified” (±) and maintains fluidity as a powder.

<Experiment 4: Effect of crystallinity and average crystallite size on difference in caking properties of powder>
In Experiment 3, trehalose dihydrate crystal-containing powder test samples 5c to 8c prepared by applying a pseudo-controlled cooling method from a sugar solution having a relatively high trehalose content of more than 86% are higher in trehalose purity than other test samples. Despite not much difference, it had excellent powder characteristics that it was difficult to consolidate. For the purpose of elucidating the reason, in this experiment, for the test samples 1 to 8 obtained in the experiment 2 and the test samples 1c to 8c obtained in the experiment 3, the crystallinity and average crystal of the powdered trehalose dihydrate crystal The child diameter was measured. As a control, the test sample 9 was examined in the same manner.

<Experiment 4-1: Preparation of standard sample used for measurement of crystallinity>
<Standard sample A>
As test sample A, a standard sample substantially composed of trehalose dihydrate crystals is recrystallized from reagent-grade trehalose dihydrate crystals-containing powder (trade name “trehalose 999”, code number: TH224, purity 99.9% or more). It was prepared by letting. That is, 1,840 g of the reagent-grade trehalose dihydrate crystal-containing powder was heated and dissolved in 1,000 g of purified water, and the dissolved solution was placed in a constant temperature chamber at 20 ° C. and allowed to recrystallize overnight. The trehalose dihydrate crystals crystallized by recrystallization were collected by a conventional method using a basket type centrifuge and dried at 40 ° C. for 8 hours to obtain about 950 g of trehalose dihydrate crystals. This was designated as test sample A. When the trehalose purity of test sample A was measured by the HPLC method described in Experiment 1, it was 100%.

<Standard sample B>
As test sample B, a standard sample consisting essentially of an amorphous part was prepared by the following procedure. That is, the test sample A was dissolved in an appropriate amount of purified water, freeze-dried over 3 days, and then vacuum-dried overnight at 40 ° C. or lower to obtain a powder consisting essentially of an amorphous part. This was designated as test sample B. When the trehalose purity of test sample B was measured by the HPLC method described in Experiment 1, it was 100%. In addition, it was 2.0% when the moisture content of the test sample B was measured by the Karl Fischer method.

<Experiment 4-2: Crystallinity of Test Samples A and B, Test Samples 1 to 9, and Test Samples 1c to 8c>
<Crystallinity>
The crystallinity of the trehalose dihydrate crystals in test samples A and B, test samples 1 to 9, and test samples 1c to 8c was determined as follows. That is, using a commercially available reflected X-ray powder diffractometer (Spectris Co., Ltd., trade name “X'Pert PRO MPD”), a CuKα ray (X-ray tube) that is a characteristic X-ray emitted from a Cu counter cathode. Based on a powder X-ray diffraction profile with a current of 40 mA, an X-ray tube voltage of 45 kV, and a wavelength of 1.5405 angstroms), test samples A and B, test samples were used using dedicated analysis computer software installed in the powder X-ray diffractometer. Analytical values of crystallinity by Hermans method were determined for each of 1 to 9 and test samples 1c to 8c. Prior to analysis of crystallinity by the Hermans method, software is set so that a baseline determined to be optimal can be obtained while taking into account the overlap of peaks, diffraction intensity, scattering intensity, etc. in each powder X-ray diffraction pattern The adjusted granularity and bending factor were adjusted to appropriate levels. As for the Hermans method, P.H. Harmans and A. Weidinger, “Journal of Applied Physics”, Vol. 19, 491-506 (1948), and P.H. Harmans and A. Weidinger, "Journal of Polymer Science", Volume 4, pages 135-144 (1949).

The analysis value H ₁₀₀ is the analysis value of crystallinity for the test sample A, the analysis value H ₀ is the analysis value of crystallinity for the test sample B, and the analysis value of crystallinity for each test sample is Hs. The degree of crystallinity was determined by substituting into the formula [3]. Incidentally, the analysis value (H ₁₀₀ ) of the crystallinity by the Hermans method for the test sample A and the same analysis value (H ₀ ) for the test sample B were 50.69% and 8.59%, respectively. The results are shown in Table 4. In addition, about test sample A and B, the powder X-ray-diffraction pattern was shown in FIG.1 and FIG.2, respectively.

  As shown in FIG. 1, in the powder X-ray diffraction pattern of test sample A, a diffraction peak peculiar to trehalose dihydrate crystals appears clearly and sharply in the diffraction angle (2θ) range of 5 to 50 °, and is amorphous. There was no halo specific to the part. On the other hand, as seen in FIG. 2, in the powder X-ray diffraction pattern of test sample B, unlike the powder X-ray diffraction pattern of FIG. 1, a halo peculiar to the amorphous part appears prominently as a bulge of the baseline. However, no diffraction peak peculiar to the dihydrate crystal or anhydrous crystal of trehalose was observed.

<Experiment 4-3: Powder X-ray diffraction of test samples A and B by synchrotron radiation>
In this experiment, for the purpose of further confirming that the test samples A and B are suitable as samples for determining the analytical values H ₁₀₀ and H ₀ , these standard samples are synchrotron radiation (hereinafter referred to as “ "X-ray source") was used as an X-ray source and subjected to a powder X-ray diffraction of a transmitted light system capable of detecting weak diffraction and scattering signals. The measurement conditions were as follows.

<Measurement conditions>
Powder X-ray diffractometer: High-speed powder X-ray diffractometer (Sold by Kozu Seiki,
Model number “PDS-16”), Debye-Shera mode,
Camera length: 497.2mm
X-ray source: Synchrotron radiation from a deflecting electromagnet (Hyogo Prefecture Beamline (BL08B2))
Measurement wavelength: 1.2394 mm (10.00 keV)
Measurement intensity: 10 ⁹ photons / second Measurement angle: 3 to 38 °
Exposure time: 600 seconds Image shooting: Imaging plate (trade name “Image”, manufactured by FUJIFILM Corporation)
Plate BAS-2040
Image reader: Image analyzer (Fujifilm, Bioimage
Dianalyzer BAS-2500 ”)

  The measurement was performed using the “Hyogo Prefectural Beam Line (BL08B2)” provided in the large synchrotron radiation facility “SPring-8” (1-1-1 Koyo Sayo-cho, Sayo-gun, Hyogo).

  Prior to powder X-ray diffraction measurement, the test samples A and B were ground with a mortar and then screened with a 53 μm sieve, and the powder that passed through the sieve was subjected to X-ray crystal diffraction capillary (trade name “Mark” Tube ", No. 14 (0.6 mm in diameter, made by Lindeman Glass)) was uniformly filled so that the filling length was about 30 mm. Next, the capillary is cut at the end of sample filling, and the opening is sealed with an adhesive, and then the capillary is fixed to the sample mount with clay, so that the longitudinal direction of the capillary is perpendicular to the optical axis of the powder X-ray diffractometer. The sample mount was attached to a powder X-ray diffractometer. In order to eliminate the influence on the powder X-ray diffraction profile by the orientation of the trehalose dihydrate crystal, the sample mount was rotated at a constant speed of 2 times / second during the measurement.

  In the process of analyzing the powder X-ray diffraction profiles obtained for the test samples A and B and creating a powder X-ray diffraction pattern, the powder X-ray diffraction profiles are obtained from each powder X-ray diffraction profile according to a conventional method in order to increase measurement accuracy. The background signal from the line diffractometer was removed. The powder X-ray diffraction patterns for the test samples A and B thus obtained are shown in FIGS. 3 and 4, respectively.

  As shown in FIG. 3, the powder X-ray diffraction pattern of test sample A by powder X-ray diffraction using synchrotron radiation shows that the diffraction peak characteristic of trehalose dihydrate crystals is in the range of diffraction angle (2θ) of 3 to 38 °. Appeared clearly and sharply. Comparing FIG. 3 and FIG. 1, the wavelength of the emitted light (1.2394Å) is different from the wavelength of the characteristic X-ray (1.5405 、). Although there is a difference in that each diffraction peak appears at the diffraction angle (2θ), the diffraction patterns in FIGS. Further, although the intensity of each diffraction peak in FIG. 3 is nearly 50 times stronger than the intensity of the diffraction peak in FIG. 1, the half width of each diffraction peak is clearly narrower than that in FIG. . Further, in the powder X-ray diffraction pattern of FIG. 3, no halo peculiar to the amorphous part was observed as in FIG. 4 described later. This indicates that the crystallinity of the trehalose dihydrate crystal in the test sample A is extremely high, indicating that the test sample A is substantially composed of trehalose dihydrate crystal.

  On the other hand, as shown in FIG. 4, in the powder X-ray diffraction pattern for the test sample B by powder X-ray diffraction using synchrotron radiation, a halo peculiar to the amorphous part appears prominently as a bulge of the baseline, No diffraction peaks peculiar to trehalose dihydrate crystals were observed. This indicates that the test sample B is substantially composed of an amorphous part.

The above results obtained using synchrotron radiation as an X-ray source indicate that the test samples A and B are suitable as samples for determining the analysis value H ₁₀₀ and the analysis value H ₀ in Equation [3]. That it is.

<Experiment 4-4: Average Crystallite Diameter of Test Sample A, Test Samples 1 to 9 and Test Samples 1c to 8c>
The crystallite diameter can be calculated from the half-value width and diffraction angle (2θ) of each diffraction peak in the powder X-ray diffraction pattern. The present inventors consider that the average value of crystallite diameters (average crystallite diameter) calculated from a plurality of diffraction peaks can be a parameter that defines the physical properties of the crystal-containing powder, and testing the trehalose dihydrate crystal-containing powder. The average crystallite size was determined for the sample.

  When the crystallinity was determined for test sample A, test samples 1 to 9, and test samples 1c to 8c, excluding test sample B, which is an amorphous powder and does not show a diffraction peak in the powder X-ray diffraction pattern Each average crystallite diameter was further determined using each powder X-ray diffraction pattern. Comparison that the average crystallite diameter has little influence on the five diffraction peaks in each powder X-ray diffraction pattern of the powder containing trehalose dihydrate crystal, that is, the diffraction peak width caused by the nonuniform strain of the crystallite Diffraction angle (2θ) 13.7 ° (Miller index (hkl): 101), 17.5 ° (Miller index: 220), 21.1 ° (which is well separated from other diffraction peaks) Miller indices: 221), 23.9 ° (Miller indices: 231) and 25.9 degrees (Miller indices: 150) diffraction peaks (symbols a to e shown in FIG. 1) are selected, and the half width of each is selected. And diffraction angle (2θ), using the computer software for analysis attached to the powder X-ray diffractometer (“X'pert Highscore Plus”) After correcting based on the measured value when using silicon (NIST National Standard Institute of Technology (NIST), standard sample for X-ray diffraction (“Si640d”)), the crystallite diameter is calculated based on the formula [4]. The average value of 5 points was obtained and the results are shown in Table 4.

  In Table 4, the results of trehalose purity and powder caking test were reprinted from Tables 2 and 3 for Test Samples 1 to 9 and Test Samples 1c to 8c, respectively. Moreover, also about the test samples A and B used as the standard sample for crystallinity measurement, each was used for the same caking property test as Experiment 2-2 and Experiment 3-2, and the caking property was evaluated. The results are also shown in Table 4.

In crystallinity measurement, analysis values _{H 100} test sample A (trehalose purity 100.0% crystallinity 100.0%) which was a standard sample to determine the average crystallite size of was 3,910Å . Further, as shown in Table 4, the test sample A was determined to be “no consolidation” (−) in the caking test. On the other hand, the test sample B (trehalose purity 100.0%, crystallinity 0.0%) as a standard sample for determining the analysis value H ₀ was taken out from the tube onto a flat plate in the caking test. However, the hemisphere of the inner bottom of the tube was clearly maintained, and it was judged as “consolidated” (+). It should be noted that the hemispherical shape of the inner bottom of the tube held by the test sample B taken out on the flat plate was such that it did not collapse as long as the plate was lightly vibrated. On the other hand, the test sample 9, which is a food-grade trehalose dihydrate crystal-containing powder that has been commercially available, had a trehalose purity of 99.0% and a crystallinity of 85.4%.

  As shown in the column of “crystallinity” in Table 4, the crystallinity of the test samples 1 to 8 obtained by crystallization by the natural cooling method in the crystallization step is in the range of 75.3 to 87.8%. Moreover, the crystallinity of the test samples 1c to 8c prepared by applying the pseudo controlled cooling method in the crystallization step was in the range of 86.2 to 94.8%. When comparing the crystallinity of the test samples 1 to 8 and the test samples 1c to 8c from the viewpoint of the difference in the crystallization method, the test samples 1c to 8c obtained by the pseudo controlled cooling method vary among the samples. The crystallinity was found to be 3.2 to 10.9% higher than the test samples 1 to 8 obtained by the natural cooling method.

  Further, the results shown in Table 4 indicate that the degree of crystallinity is correlated with the caking property of the powder. That is, as shown in Table 4, the test sample A and the test samples 5c to 8c having a crystallinity of 90% or more were “no consolidation” (−), whereas the crystallinity was 85 The test samples 5 to 9 and the test samples 1c to 4c that are not less than 90% and less than 90% are all “slightly consolidated” (±), and the test sample B and the test sample having a crystallinity of less than 85% 1 to 4 were all “consolidated” (+). This indicates that the degree of crystallinity can be a powerful indicator for defining a powder containing trehalose dihydrate crystals that are difficult to solidify.

  Furthermore, this result shows that in the production of trehalose dihydrate crystal-containing powder, if the trehalose content in the reaction solution is increased to more than 86.0%, and the pseudo controlled cooling method is applied in the subsequent crystallization step, the obtained powder As a result, the crystallinity degree of the trehalose dihydrate crystal in the water is 90% or more, and as a result, the trehalose dihydrate crystal-containing powder that is harder to set than the conventional food-grade trehalose dihydrate crystal-containing powder Tells you that you can get.

  On the other hand, as shown in the column of “average crystallite diameter” in Table 4, the average crystallite diameters of test samples 1 to 8 obtained by crystallization by the natural cooling method in the crystallization step are 2,050 to 2,740 mm. In addition, the average crystallite diameters of the test samples 1c to 8c prepared by applying the pseudo controlled cooling method in the crystallization process were in the range of 2,680 to 3,410cm. When comparing the test samples 1c to 8c and the test samples 1 to 8 with respect to the average crystallite diameter, the test samples 1c to 8c obtained by the pseudo controlled cooling method were obtained by the natural cooling method although there were variations among the samples. It was found that the average crystallite diameter was increased by 220 to 740 mm more than the test samples 1 to 8. From this result, it can be said that the application of the pseudo controlled cooling method in the crystallization process of the trehalose dihydrate crystal is an excellent method for obtaining the trehalose dihydrate crystal-containing powder having a large average crystallite diameter.

  Further, in Test Samples 1 to 8 and Test Samples 1c to 8c, it was recognized that the value of the average crystallite diameter was larger as the crystallinity of trehalose purity and trehalose dihydrate crystals in the powder was higher. This tendency is that the test sample 1 having a trehalose purity of 100.0% and a crystallinity of 100.0% has an average crystallite size of 3,910 mm and is a food-grade trehalose dihydrate crystal-containing powder. When the average crystallite size of No. 9 was 2,590 mm, the average crystallite size in the trehalose dihydrate crystal-containing powder shows a certain correlation with trehalose purity and crystallinity.

  Furthermore, the results shown in Table 4 indicate that the average crystallite size is also correlated with the powder solidification. That is, as shown in Table 4, the test sample A and the test samples 5c to 8c having an average crystallite diameter of 3,120 mm or more were “no consolidation” (−), whereas the average crystallite Test samples 5 to 9 and test samples 1c to 4c having a diameter in the range of 2,620 mm or more and less than 2,900 mm are all “slightly consolidated” (±) and have an average crystallite diameter of 2,600 mm. The test sample B and the test samples 1 to 4 that were less than “all” were “consolidated” (+). This indicates that the average crystallite size as well as the degree of crystallinity can be an effective index for defining a powder containing trehalose dihydrate crystals that are difficult to consolidate.

<Experiment 5: Powder characteristics of test sample (storability, solubility in water)>
In order to further clarify the properties of the test samples 1 to 9 and the test samples 1c to 8c as powders, a storage stability test and a water solubility test were performed.

<Experiment 5-1: Preservability test>
To confirm that the caking test conducted in Experiment 2-2, Experiment 3-2, etc. is a valid test for evaluating the caking property of trehalose dihydrate crystal-containing powder during actual storage. The test samples A and B obtained by the method of Experiment 4-1, the test samples 1 to 9 obtained by Experiment 2, and the test samples 1c to 8c obtained by Experiment 3 A storability test was conducted on the assumption that the hydrated crystal-containing powder was actually stored, the environment and the period.

That is, 150 g of each of test samples A and B, test samples 1 to 9 and test samples 1c to 8c were taken and separately separated into polyethylene bags (trade name “Unipack F-4”, manufactured by Production Nippon Co., Ltd., 17 cm × 3 polyethylene bags were collected for each test sample, and the polyethylene bags were collected in a state where the air was removed. Then, a weight of 13.2kg such load is 648kg pieces area 1 m ² per each polyethylene bag, placed as load is applied to the entire top surface on each polyethylene bag, in this state, the hot and humid Stored for 60 days in an avoiding environment. Incidentally, food grade trehalose two hydrous crystalline powder containing products are usually in Packing bags input of 20kg filling, where it is stored in a warehouse or the like in a state where the stacked about 10 stages, migraine area 1 m ² per 648kg of polyethylene bags Is equivalent to the load applied to the product in the lowermost stage in the state where the ten stages are stacked. After storage for 60 days, each test sample is taken out from the polyethylene bag, passed through a sieve with an opening of 425 μm, and the mass of the powder that has passed through and the powder that has not passed through is measured. The mass ratio (%) was determined and the average value of the three bags tested for each test sample was taken to determine the presence or absence of caking in the powder after storage for 60 days. The solidification of the powder is “no consolidation” (−) when the particle size of 425 μm or more is less than 30% of the whole powder, and “consolidation” when the particle is 30% or more of the whole powder (+ ). In addition, when the ratio of particles of 425 μm or more in the powder exceeds 30%, generally, the dissolution of the powder, mixing with other powdered compositions, kneading, and the like are hindered. Therefore, the determination criterion is set to 30%. The results are shown in Table 5.

<Experiment 5-2: Water solubility test>
Each test sample was weighed in an amount of 0.25 g, and placed in a polypropylene cylindrical tube with a cap having a hemispherical inner 14-ml lid (available from Becton Dickinson, trade name “Falcon Tube 2059”). 5 ml of deionized water was added to the tube, heated in a constant temperature water bath at 50 ° C. for 30 minutes, then turned down twice, further held at 50 ° C. for 15 minutes, and the solubility at that time was examined visually. The results are shown in Table 5. The results are shown in Table 5. The results are shown in Table 5.

  As shown in the column of “Preservability” in Table 5, the crystallinity of trehalose was 90.0% in a preservability test in which each test sample was stored for 60 days in an environment avoiding high temperature and high humidity while applying a load. Test samples 1 to 9 and test samples 1c to 4c having an average crystallite diameter of 2,850 mm or less were determined to be “consolidated” (+), while the crystallinity was 90.4% or more Test sample A and test samples 5c to 8c having a mean crystallite size of 95.0% or less and an average crystallite size of 3,120 mm or more were determined to be “no consolidation” (−). This result indicates that the test sample determined to be “solidified” (+) or “slightly consolidated” (±) in the caking test conducted in Experiment 2-2, Experiment 3-2, etc. Is determined to be “consolidated” (+), whereas in the solidification test conducted in Experiment 2-2, Experiment 3-2, etc., the test sample was determined to be “not consolidated” (−) Indicates that it is similarly determined as “no consolidation” (−) in this storage test. This fact is valid as a test for evaluating the caking property of the powder containing trehalose dihydrate crystals in the actual storage environment performed in Experiment 2-2, Experiment 3-2 and the like. It is shown that.

  In addition, as shown in the column of “Solubility in water” in Table 5, in the solubility test in water, the test sample A having a crystallinity of 100% and an average crystallite diameter of 3,910 と is said to be “poor”. In contrast, the test samples 1 to 9 and the test samples 1c to 8c having a crystallinity of 94.8% or less and an average crystallite diameter of 3,410 mm or less were determined to have good solubility. It was done. This result shows that in the powder containing trehalose dihydrate crystals, the crystallinity and the average crystallite diameter are increased to the level of the test sample A, in other words, to the level of the reagent grade trehalose dihydrate crystals-containing powder. This indicates that a problem different from the caking property occurs, that is, the solubility becomes worse.

<Experiment 6: Partial amino acid sequence common to CGTase suitable for production of trehalose>
For the purpose of characterizing CGTase more suitable for the production of trehalose, the above-mentioned CENase derived from the genus Paenibacillus excellent in the effect of increasing the trehalose content in the enzyme reaction solution, that is, Paenibacillus illinoisensis NBRC15379 strain, Paenibacillus publi NBRC13638 strain, -The amino acid sequence of CGTase (SEQ ID Nos. 1, 2 and 3 in the sequence listing) derived from amylolyticus NBRC15957 and the effect of increasing trehalose content in the enzyme reaction solution is weak compared to CGTase derived from Paenibacillus sp.・ Sterothermophilus Tc-91 strain, CGTase amino acid derived from Bacillus mazellans, Thermoanaerobacter thermosulfrigenes The acid sequences (SEQ ID NOs: 4, 5 and 6 in the sequence listing) were compared. The amino acid sequences represented by SEQ ID NOs: 1 to 3 in the sequence listing used for the comparison of the amino acid sequences are all Paenibacillus illinoissis NBRC15379 strain, Paenibacillus publi NBRC13638 strain, and Paenibacillus amylolyticus NBRC15957 strain, respectively. The amino acid sequence encoded by the base sequence determined by the applicant's original cloning and determination of the CGTase gene was used. In addition, the amino acid sequences shown in SEQ ID NOs: 4 and 5 in the sequence listing were determined by the applicant and originally disclosed in Japanese Patent Application Laid-Open No. 61-135581 by the same applicant as the present application, Geobacillus stearothermophilus ( It is an amino acid sequence of CGTase derived from the Bacillus stearothermophilus) Tc-91 strain and Bacillus mazellans in the old classification. Incidentally, the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing is not the same as the CGTase derived from Bacillus mazelans (trade name “Contiszyme”, sold by Amano Enzyme Co., Ltd.) used in Experiment 1, but the same CGTase derived from Bacillus mazelans. It was substituted because it was an amino acid. As the amino acid sequence of CGTase derived from Thermoanaerobacter thermosulfrigenes, accession No. is listed in the gene database “GenBank”. The one registered as 35484 was used.

In the comparison of the amino acid sequences described above, CGTase excellent in the effect of increasing the trehalose content in the enzyme reaction solution, that is, common to the Paenibacillus microorganism-derived CGTase, and has the effect of increasing the trehalose content in the enzyme reaction solution. The following (a) to (d) are partial amino acid sequences not present in CGTase which are not so much, ie, CGTase derived from Geobacillus stearothermophilus, Bacillus mazellans and Thermoanaerobacter thermosulfrigenes, respectively. The amino acid sequence was observed.
_{(A) Gly-Ser-X} 1 -Ala-Ser-Asp;
(B) Lys-Thr-Ser-Ala-Val-Asn-Asn;
(C) Lys-Met-Pro-Ser-Phe-Ser-Lys;
(D) Val-Asn-Ser -Asn-X 2 -Tyr;
(However, X ₁ means Ala or Ser, and X ₂ means Ala or Thr.)

  From the above results, in the production of trehalose by the production method of the present invention, more preferable CGTase, that is, CGTase capable of making the trehalose content in the enzyme reaction solution more than 86.0% is the above (a) to ( It can be characterized as having a partial amino acid sequence of d).

  Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to these examples.

<Manufacture of powder containing trehalose dihydrate crystals>
Corn starch was suspended in water to a concentration of 30%, and calcium carbonate was added to the suspension to a final concentration of 0.1% to adjust the pH to 6.0. Thermostable α-amylase (trade name “Termamyl 60L”, sold by Novozymes Japan Co., Ltd.) was added thereto at 0.2% per starch mass and reacted at 98 to 100 ° C. for 15 minutes to gelatinize and liquefy the starch. The obtained liquefied starch solution was autoclaved at 125 ° C. for 15 minutes, then cooled to 57 ° C., and the recombinant α-glycosyl trehalose producing enzyme and the recombinant trehalose releasing enzyme prepared by the method of Experiment 1-1 were added thereto. The partially purified preparation is added to 5 units and 20 units, respectively, per gram of starch, and further 1,500 units of isoamylase (produced by Hayashibara Co., Ltd.) per gram of starch and 15 units of the method of Experiment 1-2. The CGTase derived from Paenibacillus illinoisensis NBRC15959 strain prepared in the above was added, and further reacted for about 50 hours. Next, this reaction solution was heated at 97 ° C. for 60 minutes to inactivate the enzyme, then adjusted to pH 4.5, and a glucoamylase agent (trade name “Glucoteam # 20000”, sold by Nagase ChemteX Corporation). ) Was added for 10 units per gram of starch and reacted for 24 hours to obtain a reaction solution having a trehalose purity, that is, a trehalose content of 86.4% in terms of anhydride. The reaction solution thus obtained is heated to deactivate the enzyme, decolorized and filtered with activated carbon by a conventional method, and the filtrate is desalted with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type). Concentrated under reduced pressure to obtain a concentrated liquid having a solid concentration of about 85%. Take this in an auxiliary crystal can and add 2% of reagent-grade trehalose dihydrate crystal-containing powder (trade name “Trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by Hayashibara Co., Ltd.) as a seed crystal. The temperature was 55 ° C, and the mixture was naturally cooled to 15 ° C over 24 hours with gentle stirring to crystallize trehalose dihydrate crystals. Crystals are recovered with a basket-type centrifuge, and the crystals are sprayed and washed with about 5% purified water based on the weight of the mass kit, then aged and dried at 50 ° C. for 2 hours, and air at 20 ° C. is blown for 10 minutes. Cool, grind, 99.1% trehalose, 0.6% D-glucose, 0.09% 4-O-α-glucosyl trehalose in terms of anhydride, and 6-O-α-glucosyl A powder containing trehalose dihydrate crystals containing 0.12% trehalose was obtained with a starch yield of about 40%.

According to the production method of this example, the trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 40%. The trehalose dihydrate crystal-containing powder produced by this production method has a crystallinity of 87.5% for the trehalose dihydrate crystal, an average crystallite size of 2,610 mm, and a reducing power of the whole powder of 0.7%. Met. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. When the particle size distribution of the product was measured, particles having a particle size of 53 μm or more and less than 425 μm were 75.3%, particles having a particle size of 53 μm or more and less than 300 μm were 66.1%, and particles having a particle size of 425 μm or more were 10.2. % Was included. This product can be used as a food material, a cosmetic material, a quasi-drug material, a pharmaceutical material, and the like in the same manner as a conventional food-grade trehalose dihydrate crystal-containing powder.

<Manufacture of powder containing trehalose dihydrate crystals>
The reaction time was set to 40 hours using 10 units and 40 units of recombinant α-glycosyl trehalose-producing enzyme and recombinant trehalose-releasing enzyme per gram of starch, respectively. Except for using CGTase derived from Illinois NBRC15379 strain, a trehalose production reaction and a glucoamylase treatment were carried out in the same manner as in Example 1, whereby trehalose purity, that is, a trehalose content in terms of anhydride was 86.2%. A liquid was obtained. The obtained reaction solution is heated to deactivate the enzyme, and decolorized and filtered with activated carbon by a conventional method. The filtrate is desalted with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type), and concentrated under reduced pressure. Thus, a concentrated solution having a solid concentration of about 85% was obtained. Take this in an auxiliary crystal can and add 1% of reagent-grade trehalose dihydrate crystal-containing powder (trade name “trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by Hayashibara Co., Ltd.) as a seed crystal. Pseudo control where the trehalose containing solution is cooled to 60 ° C. and then cooled with gentle agitation for 12 hours from 60 ° C. to 50 ° C., 6 hours from 50 ° C. to 40 ° C., and 6 hours from 40 ° C. to 15 ° C. By cooling to 15 ° C. in a total of 24 hours by a cooling method, dihydrated crystals of trehalose were crystallized. Crystals are recovered with a basket-type centrifuge, and the crystals are sprayed and washed with about 5% purified water based on the weight of the mass kit, then aged and dried at 50 ° C. for 2 hours, and air at 20 ° C. is blown for 20 minutes. Cool and grind, 99.4% trehalose, 0.08% D-glucose, 0.05% 4-O-α-glucosyl trehalose and 6-O-α-glucosyl in terms of anhydride A powder containing trehalose dihydrate crystals containing 0.07% trehalose was obtained with a starch yield of about 43%.

The crystallinity of the trehalose dihydrate-containing powder of this trehalose dihydrate crystal was 94.1%, the average crystallite size was 3,340 mm, and the reducing power of the whole powder was 0.18%. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. When the particle size distribution of this product was measured, 85.1% of particles having a particle size of 53 μm or more and less than 425 μm, 74.7% of particles having a particle size of 53 μm or more and less than 300 μm, and 5.8 of particles having a particle size of 425 μm or more were measured. % Was included. Using this product, a caking test was performed by the same method as in Experiment 2-2, Experiment 3-2, etc., and it was determined that “no caking” (−). Further, when the solubility in water was tested by the same method as in Experiment 5, it was determined that the solubility was “good”.

  According to the production method of this example, the trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 43%. In addition, the trehalose dihydrate crystal-containing powder produced by this production method is compared with the conventional food-grade trehalose dihydrate crystal-containing powder that is commercially available as a food material (trade name “Treha”, sold by Hayashibara Co., Ltd.). Even though there is no significant difference in trehalose purity, it is a powder that is significantly harder to set than conventional food-grade trehalose dihydrate crystal-containing powders, and is easy to store and handle. This product is similar to the conventional food grade trehalose dihydrate crystal-containing powder in that it is a trehalose dihydrate crystal-containing powder, and it is easy to store and handle, so it is a food material, cosmetic material, quasi-drug. It can be more suitably used as a raw material, a pharmaceutical material, and the like.

<Manufacture of powder containing trehalose dihydrate crystals>
A trehalose production reaction was carried out in the same manner as in Example 1 except that CGTase derived from Paenibacillus publi NBRC13638 strain prepared by the method of Experiment 1-2 was used as CGTase. Anhydrous reaction in the reaction solution after glucoamylase treatment was performed. The trehalose content in terms of product was 86.1%. The reaction solution thus obtained is heated to deactivate the enzyme, decolorized and filtered with activated carbon by a conventional method, and the filtrate is desalted with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type). Concentrated under reduced pressure to obtain a concentrated liquid having a solid concentration of about 85%. Take this in an auxiliary crystal can and add 1% of reagent-grade trehalose dihydrate crystal-containing powder (trade name “trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by Hayashibara Co., Ltd.) as a seed crystal. Trehalose dihydrated crystals were cooled for 24 hours by a two-stage pseudo-controlled cooling method in which the temperature was 60 ° C. and the mixture was gently stirred for 60 hours from 60 ° C. to 45 ° C. and cooled from 45 ° C. to 20 ° C. over 9 hours. Was crystallized. Crystals are recovered with a basket-type centrifuge, and the crystals are sprayed and washed with about 5% purified water based on the weight of the mass kit, then aged and dried at 50 ° C. for 2 hours, and air at 20 ° C. is blown for 10 minutes. Cool, grind, 99.3% trehalose, 0.3% D-glucose, 0.07% 4-O-α-glucosyl trehalose in terms of anhydride, and 6-O-α-glucosyl A powder containing trehalose dihydrate crystals containing 0.12% trehalose was obtained with a starch yield of about 43%.

This trehalose dihydrate crystal-containing powder had a crystallinity of 93.4% for the trehalose dihydrate crystal, an average crystallite size of 3,210 mm, and a reducing power of the whole powder of 0.4%. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. When the particle size distribution of this product was measured, 77.9% of particles having a particle size of 53 μm or more and less than 425 μm, 68.4% of particles having a particle size of 53 μm or more and less than 300 μm, and 8.8 of particles having a particle size of 425 μm or more were measured. % Was included. Using this product, a caking test was performed by the same method as in Experiment 2-2, Experiment 3-2, etc., and it was determined that “no caking” (−). Further, when the solubility in water was tested by the same method as in Experiment 5, it was determined that the solubility was “good”.

  According to the production method of this example, the trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 43%. In addition, the trehalose dihydrate crystal-containing powder produced by this production method is compared with the conventional food-grade trehalose dihydrate crystal-containing powder that is commercially available as a food material (trade name “Treha”, sold by Hayashibara Co., Ltd.). Even though there is no significant difference in trehalose purity, it is a powder that is significantly harder to set than conventional food-grade trehalose dihydrate crystal-containing powders, and is easy to store and handle. This product is similar to the conventional food grade trehalose dihydrate crystal-containing powder in that it is a trehalose dihydrate crystal-containing powder, and it is easy to store and handle, so it is a food material, cosmetic material, quasi-drug. It can be more suitably used as a raw material, a pharmaceutical material, and the like.

<Manufacture of powder containing trehalose dihydrate crystals>
The trehalose production reaction and glucoamylase treatment were carried out in the same manner as in Example 1 except that tapioca starch was used as the raw material starch, and CGTase derived from Paenibacillus amylolyticus NBRC15957 strain obtained by the method of Experiment 1-2 was used as CGTase. As a result, a reaction solution having a trehalose purity, that is, a trehalose content in terms of anhydride of 86.2% was obtained. The obtained reaction solution is heated to deactivate the enzyme, and decolorized and filtered with activated carbon by a conventional method. The filtrate is desalted with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type), and concentrated under reduced pressure. Thus, a concentrated liquid having a solid concentration of about 86% was obtained. Take this in an auxiliary crystal can and add 1% of reagent-grade trehalose dihydrate crystal-containing powder (trade name “trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by Hayashibara Co., Ltd.) as a seed crystal. The trehalose-containing solution is cooled to 60 ° C. for 8 hours, from 50 ° C. to 50 ° C. for 8 hours, from 50 ° C. to 35 ° C. for 8 hours, and from 35 ° C. to 15 ° C. for 8 hours. The dihydrate crystal of trehalose was crystallized by cooling to 15 ° C. in a total of 24 hours by the pseudo controlled cooling method. Crystals are recovered with a basket-type centrifuge, and the crystals are sprayed and washed with about 5% purified water based on the weight of the mass kit, then aged and dried at 50 ° C. for 2 hours, and air at 20 ° C. is blown for 20 minutes. Cool, grind, 99.2% trehalose, 0.07% D-glucose, 0.04% 4-O-α-glucosyl trehalose and 6-O-α-glucosyl in terms of anhydride A powder containing trehalose dihydrate crystals containing 0.06% trehalose was obtained at a starch yield of about 42%.

This trehalose dihydrate crystal-containing powder had a crystallinity of 93.1% for the trehalose dihydrate crystal, an average crystallite size of 3,160 mm, and a reducing power of the whole powder of 0.15%. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. The particle size distribution of this product was measured. As a result, 78.8% of particles having a particle size of 53 μm or more and less than 425 μm, 71.0% of particles having a particle size of 53 μm or more and less than 300 μm, and 8.4 of particles having a particle size of 425 μm or more were obtained. % Was included. Using this product, a caking test was performed by the same method as in Experiment 2-2, Experiment 3-2, etc., and it was determined that “no caking” (−). Further, when the solubility in water was tested by the same method as in Experiment 5, it was determined that the solubility was “good”.

  According to the production method of this example, the trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 42%. In addition, the trehalose dihydrate crystal-containing powder produced by this production method is compared with the conventional food-grade trehalose dihydrate crystal-containing powder that is commercially available as a food material (trade name “Treha”, sold by Hayashibara Co., Ltd.). Even though there is no significant difference in trehalose purity, it is a powder that is significantly harder to set than conventional food-grade trehalose dihydrate crystal-containing powders, and is easy to store and handle. This product is similar to the conventional food grade trehalose dihydrate crystal-containing powder in that it is a trehalose dihydrate crystal-containing powder, and it is easy to store and handle, so it is a food material, cosmetic material, quasi-drug. It can be more suitably used as a raw material, a pharmaceutical material, and the like.

<Preparation of Recombinant CGTase and Mutant CGTase and Production of Trehalose Dihydrate Crystal-Containing Powder Using them>
Instead of CGTase derived from Paenibacillus illinoisensis NBRC15379 strain used in Example 2, a recombinant (wild type) CGTase obtained by expressing Escherichia coli as a host for the CGTase gene derived from the same strain, and the CGTase gene. Two types of mutant CGTase were prepared by introducing site-specific mutation by the method, and one amino acid residue in the encoded amino acid sequence was replaced with another amino acid residue, and used for the production of powder containing trehalose dihydrate crystals. It was.

<Preparation of recombinant CGTase>
Using the CGTase gene (base sequence shown by SEQ ID NO: 7 in the Sequence Listing) derived from the same strain that the present inventors have cloned and retained from Paenibacillus illinoisensis NBRC15379 strain, mutation is performed without changing the encoded amino acid sequence. After introducing or deleting a restriction enzyme site or the like, it is recombined into an expression plasmid vector “pRSET-A” (manufactured by Invitrogen), for expression including a gene encoding a natural type (wild type) CGTase Recombinant DNA was made. The resulting recombinant DNA “pRSET-iPI” is used to transform Escherichia coli BL21 (DE3) (manufactured by Stratagene) by a conventional method, and a transformant “BL21-RSET-iPI” retaining the recombinant DNA is used. Acquired. Subsequently, the transformant was added to T medium containing 100 μl / ml of ampicillin Na salt (containing 12 g of bacto-tryptone, 24 g of bacto-east extract, 5 ml of glycerol, 17 mM monopotassium phosphate, 72 mM dipotassium phosphate per liter of medium). ) At 37 ° C. for 24 hours. The cells obtained by centrifuging the culture solution are crushed using an ultrasonic crusher (trade name “Ultra Sonic Homogenizer UH-600”, manufactured by MST Co., Ltd.), and the supernatant of the crushed solution obtained by centrifugation. When CGTase activity (starch degradation activity) was measured and converted per 1 ml of the culture solution, an enzyme activity of about 12.8 units / ml was obtained. After obtaining the crude enzyme solution of recombinant CGTase by subjecting the supernatant of the disrupted solution to ammonium sulfate salting out and dialysis according to a conventional method, anion exchange column chromatography using DEAE-Toyopearl 650S gel (manufactured by Tosoh Corporation) and The product was purified by hydrophobic column chromatography using butyl-Toyopearl 650M gel (manufactured by Tosoh Corporation) to obtain a partially purified preparation of recombinant CGTase.

<Preparation of mutant CGTase>
A single amino acid substitution mutant CGTase is obtained by introducing a site-specific mutation into the natural (wild-type) CGTase gene derived from the Paenibacillus illinoisensis NBRC15379 strain by a conventional method and expressing the obtained mutant CGTase gene in Escherichia coli. Two types were prepared. When introducing an amino acid substitution into the CGTase, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, which is the amino acid sequence of CGTase derived from Paenibacillus illinoisensis NBRC15379 strain, is changed from the 133th aspartic acid residue to the 138th aspartic acid residue. Histidine residue (Asp133-His138), 223rd glycine residue to 231st histidine residue (Gly223-His231), 255th glutamic acid residue to 258th leucine residue (Glu255-Leu258), 321st The 326th aspartic acid residue (Phe321 to Asp326) from the phenylalanine residue, that is, four conserved regions conserved in common to the enzymes classified into the α-amylase family, and the 259th 264th aspartic acid residue from lysine residue (Gly269 to Asp264), 331th lysine residue to 337th asparagine residue (Lys331 to Asn337), 375th lysine residue to 381st lysine residue ( Lys 375 to Lys 381) from the 567th valine residue to the 572nd tyrosine residue (Val567 to Tyr572), that is, the amino acids to the partial amino acid sequences (a) to (d) described above that are unique to Paenibacillus microorganism-derived CGTase Substitution mutations were avoided, and mutation sites were selected from other sites.

  Two types of mutant CGTase (G178R) in which the 178th glycine residue in SEQ ID NO: 1 in the sequence listing is substituted with an arginine residue and mutant CGTase (Y454H) in which the 454th tyrosine residue is substituted with a histidine residue Decided to prepare. Synthetic oligos having base sequences shown by SEQ ID NOs: 8 and 9 in the sequence listing, using the recombinant DNA “pRSET-iPI” carrying the natural (wild-type) CGTase gene derived from Paenibacillus illinoisensis NBRC15379 strain as a template Using the nucleotides as sense primer and antisense primer, respectively, a commercially available “QuickChange Site-Directed Mutagenesis Kit” (manufactured by Stratagene) introduces site-specific mutation into CGTase gene by conventional PCR method and DpnI method. As a result, a recombinant DNA “pRSET-iPI (G178R)” encoding a mutant CGTase (G178R) was obtained. Further, a recombinant DNA “encoding mutant CGTase (Y454H)” was prepared in the same manner as described above except that the synthetic oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 10 and 11 in the sequence listing were used as the sense primer and the antisense primer, respectively. pRSET-iPI (Y454H) "was obtained.

  Escherichia coli BL21 (DE3) (manufactured by Stratagene) was transformed by a conventional method using recombinant DNAs “pRSET-iPI (G178R)” and “pRSET-iPI (Y454H)” each having a mutant CGTase gene, Transformants “BL21-RSET-iPI (G178R)” and “BL21-RSET-iPI (Y454H)” retaining the respective recombinant DNAs were obtained. These transformants were cultured in the same manner as in the case of “BL21-RSET-iPI”, crushed and then partially purified to obtain partially purified preparations of the respective mutant CGTases. Incidentally, CGTase activity (starch degrading activity) was measured for each cell disruption supernatant and converted to enzyme activity per 1 ml of the culture solution. As a result, “BL21-RSET-iPI (G178R)” was about 10.3 units / unit. ml, “BL21-RSET-iPI (Y454H)”, it was about 13.7 units / ml.

<Manufacture of powder containing trehalose dihydrate crystals>
Recombinant (wild type) CGTase obtained above, G178R (CGTase having the amino acid sequence shown by SEQ ID NO: 14 in the sequence listing) as a mutant CGTase, or Y454H (amino acid shown by SEQ ID NO: 15 in the sequence listing) A trehalose dihydrate crystal-containing powder was produced in the same manner as in Example 2 except that CGTase having a sequence) was used. Trehalose content in reaction solution obtained using each CGTase, sugar composition in obtained trehalose dihydrate crystal-containing powder, starch yield, crystallinity of trehalose dihydrate crystal, average crystallite diameter, powder The total reducing power and particle size distribution were measured, and the powder was subjected to a caking test by the same method as in Experiment 2-2 and Experiment 3-2 and a solubility test in water by the same method as in Experiment 5. The results are summarized in Table 6.

  As shown in Table 6, even when recombinant CGTase or 1 amino acid substitution mutant CGTase was used, the trehalose content in the enzyme reaction solution was 86.0% or more equivalent to that of natural CGTase, According to the same production method, approximately the same trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 42% to 43%. In addition, the trehalose dihydrate crystal-containing powder produced by this production method is commercially available as a food material or the like, similar to the trehalose dihydrate crystal-containing powder produced using natural CGTase in Examples 1 to 4. Despite the fact that there is no significant difference in trehalose purity compared to the food-grade trehalose dihydrate crystal-containing powder (trade name “Treha”, sold by Hayashibara Co., Ltd.), the conventional food-grade trehalose dihydrate crystal-containing powder Compared with, it is a powder that is hardly hardened, and is easy to store and handle.

<Manufacture of powder containing trehalose dihydrate crystals>
In the crystallization process of trehalose dihydrate crystal, using a general-purpose programmed constant temperature circulator for a crystallization system, a temperature-controlled heat medium is passed through a jack of an auxiliary crystal can to change the temperature from 60 ° C. to 20 ° C. 2] by applying a controlled cooling method in which cooling is performed over a period of 24 hours with a 20-stage cooling profile approximated to 2), except that trehalose dihydrate crystals are crystallized by the same method as in Example 2. A hydrous crystal-containing powder is produced, and 99.5% of trehalose, 0.05% of D-glucose, 0.03% of 4-O-α-glucosyl trehalose in terms of anhydride, and 6-O-α- A powder containing trehalose dihydrate crystals containing 0.05% glucosyl trehalose was obtained at a starch yield of about 44%.

The trehalose dihydrated crystal-containing powder of this trehalose dihydrated crystal had a crystallinity of 95.6%, an average crystallite size of 3,480 mm, and a reducing power of the whole powder of 0.14%. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. When the particle size distribution of this product was measured, 82.1% of particles having a particle size of 53 μm or more and less than 425 μm, 77.4% of particles having a particle size of 53 μm or more and less than 300 μm, and 8.1 of particles having a particle size of 425 μm or more were measured. % Was included. Using this product, a caking test was performed by the same method as in Experiment 2-2, Experiment 3-2, etc., and it was determined that “no caking” (−). Further, when the solubility in water was tested by the same method as in Experiment 5, it was determined that the solubility was “good”.

  According to the production method of this example, the trehalose dihydrate crystal-containing powder can be produced with a high starch yield of about 44%. In addition, the trehalose dihydrate crystal-containing powder produced by this production method is compared with the conventional food-grade trehalose dihydrate crystal-containing powder that is commercially available as a food material (trade name “Treha”, sold by Hayashibara Co., Ltd.). Even though there is no significant difference in trehalose purity, it is a powder that is significantly harder to set than conventional food-grade trehalose dihydrate crystal-containing powders, and is easy to store and handle. This product is similar to the conventional food grade trehalose dihydrate crystal-containing powder in that it is a trehalose dihydrate crystal-containing powder, and it is easy to store and handle, so it is a food material, cosmetic material, quasi-drug. It can be more suitably used as a raw material, a pharmaceutical material, and the like.

Comparative example

<Manufacture of powder containing trehalose dihydrate crystals>
As CGTase, a trehalose production reaction and a glucoamylase treatment were carried out in the same manner as in Example 1 except that a CGTase enzyme agent derived from Giobacillus stearothermophilus Tc-91 strain (produced by Hayashibara Co., Ltd.) was used. The trehalose content in terms of anhydride in the reaction solution after the glucoamylase treatment was 82.2%. This reaction solution was decolorized and filtered with activated carbon by a conventional method, and the filtrate was desalted with a cation exchange resin (H ⁺ type) and an anion exchange resin (OH ⁻ type), concentrated under reduced pressure, and a solid concentration of about 84%. It was set as the concentrate. Take this in an auxiliary crystal can and add 1% of reagent-grade trehalose dihydrate crystal-containing powder (trade name “trehalose 999”, code number: TH224, trehalose purity 99.9% or more, sold by Hayashibara Co., Ltd.) as a seed crystal. The temperature was 55 ° C. Next, this trehalose-containing solution was naturally cooled from 55 ° C. to 15 ° C. over 20 hours with gentle stirring, thereby crystallizing dihydrate-containing trehalose crystals. Crystals are recovered with a basket-type centrifuge, and the crystals are sprayed and washed with about 5% purified water based on the weight of the mass kit, then aged and dried at 50 ° C. for 2 hours, and air at 20 ° C. is blown for 20 minutes. Cool and grind, 98.3% trehalose, 0.9% D-glucose, 0.08% 4-O-α-glucosyl trehalose in terms of anhydride, and 6-O-α-glucosyl Although a trehalose dihydrate crystal-containing powder containing 0.12% trehalose was obtained, the starch yield was about 31%.

This trehalose dihydrate crystal-containing powder had a crystallinity of 84.8% for the trehalose dihydrate crystal, an average crystallite diameter of 2,460 mm, and a reducing power of the whole powder of 1.1%. Incidentally, the measurement of the crystallinity was conducted using Hermance method was performed using the analytical values H ₁₀₀ and H ₀ obtained in experiments 4-2. When the particle size distribution of this product was measured, 76.7% of particles having a particle size of 53 μm or more and less than 425 μm, 68.8% of particles having a particle size of 53 μm or more and less than 300 μm, and 11.5% of particles having a particle size of 425 μm or more were measured. % Was included. Using this product, a caking test was performed in the same manner as in Experiment 2-2, Experiment 3-2, and the like, and it was determined that “somewhat solidified” (±). Moreover, when the solubility in water was tested by the same method as in Experiment 5, it was determined that the solubility was “good”.

<Preparation of α-glycosyl trehalose synthase mutant>
A site-specific mutation was introduced into the natural (wild-type) α-glycosyltrehalose synthase gene cloned by the present inventors from the Sulfolobus acidocardarius ATCC33909 strain, and the obtained mutant α -Four kinds of 1 amino acid substitution α-glycosyl trehalose synthase mutants were prepared by expressing the glycosyl trehalose synthase gene in E. coli.

  By introducing a site-specific mutation into the α-glycosyl trehalose-producing enzyme gene by a conventional PCR method or DpnI method using a commercially available “QuickChange Site-Directed Mutagenesis Kit” (Stratagene), in the sequence listing The 192nd arginine residue in SEQ ID NO: 1 is an alanine residue (R192A), the 275th aspartic acid residue is an alanine residue (D275A), the 400th tyrosine residue is a serine residue (Y400S), Alternatively, four mutant genes encoding α-glycosyl trehalose synthase mutants in which the 402th arginine residue was substituted with a serine residue (R402S) were prepared.

  Recombinant DNAs “pRSET-iST (R192A)”, “pRSET-iST (D275A)”, “pRSET-iST (Y400S)” and “pRSET-iST (R402S)” each holding this mutant gene are used. Transformants “BL21-RSET-iST (R192A)” and “BL21-RSET-iST (D275A)” that transform E. coli BL21 (DE3) (Stratagene) by the method and retain the respective recombinant DNA , “BL21-RSET-iST (Y400S)” and “BL21-RSET-iST (R402S)” were obtained. These transformants were each T medium containing 100 μl / ml of ampicillin Na salt (containing 12 g of bacto-tryptone, 24 g of bacto-east extract, 5 ml of glycerol, 17 mM monopotassium phosphate, 72 mM dipotassium phosphate per liter of medium) And aerobic culture at 37 ° C. for 24 hours. The cells obtained by centrifuging the culture solution are crushed using an ultrasonic crusher (trade name “Ultra Sonic Homogenizer UH-600”, manufactured by MST Co., Ltd.), and the supernatant of the crushed solution obtained by centrifugation. The α-glycosyl trehalose producing enzyme activity was measured. Next, the supernatant of the disrupted solution was precipitated with ammonium sulfate and dialyzed according to a conventional method to obtain a crude enzyme solution of an α-glycosyl trehalose-producing enzyme mutant, and then a DEAE-Toyopearl 650S gel (manufactured by Tosoh Corporation) was used. The product was purified by ion exchange column chromatography and hydrophobic column chromatography using butyl-Toyopearl 650M gel (manufactured by Tosoh Corporation) to obtain a purified preparation of the α-glycosyl trehalose-producing enzyme mutant.

  When the enzyme activity and substrate specificity of the obtained four kinds of α-glycosyl trehalose-producing enzyme mutants were examined, the enzyme activity was slightly reduced to 40 to 80% of that of the natural type (wild type) enzyme. There was no significant change in specificity. These α-glycosyl trehalose-producing enzyme mutants can be used in the same manner as natural (wild-type) enzymes in the method for producing trehalose of the present invention.

  As described above, according to the method for producing trehalose dihydrate crystal-containing powder of the present invention, starch is used as a raw material, and trehalose that is high in purity and difficult to consolidate as in conventional food grade trehalose dihydrate crystal-containing powder. A dihydrate crystal-containing powder can be produced with a high starch yield. In particular, when the controlled cooling method or the pseudo controlled cooling method is applied in the crystallization process of the trehalose dihydrate crystal, a higher-purity trehalose dihydrate crystal-containing powder can be produced at a higher starch yield. . As described above, the production method of the present invention makes it possible to produce trehalose dihydrate crystal-containing powder more efficiently on an industrial scale using starch, which is a limited resource although it is abundant, as a raw material. And its industrial utility is exceptional. In addition, the trehalose dihydrate crystal-containing powder produced by the production method of the present invention to which the controlled cooling method or the pseudo controlled cooling method is applied is a powder that hardly significantly solidifies compared with the conventional food-grade trehalose dihydrate crystal-containing powder. It has excellent industrial applicability that it can be used in various applications as a powdery food material, cosmetic material, quasi-drug material, or pharmaceutical material that is easier to handle. . The industrial utility of the present invention that exhibits such remarkable effects is extremely great.

In FIG.
a: Diffraction angle (2θ) 13.7 ° (mirror index (hkl): 101) used for calculation of crystallite diameter b: Diffraction angle (2θ) 17.5 ° (mirror) used for calculation of crystallite diameter Diffraction peak (index: 220) c: diffraction angle (2θ) used for calculation of crystallite diameter 21.1 ° (mirror index: 221) diffraction peak d: diffraction angle (2θ) used for calculation of crystallite diameter Diffraction peak of 9 ° (Miller index: 231) e: Diffraction angle (2θ) used for calculation of crystallite diameter 25.9 ° (Miller index: 150) Diffraction peak In FIG.
a: Control cooling curve b: Linear cooling c: Natural cooling curve

Claims (4)

Α-glycosyl trehalose producing enzyme and trehalose releasing enzyme are allowed to act on liquefied starch together with starch debranching enzyme and cyclomaltodextrin / glucanotransferase, and then glucoamylase is allowed to act to obtain an α, α-trehalose-containing sugar solution. A step of crystallizing α, α-trehalose dihydrate crystals from the sugar solution, and collecting the crystallized α, α-trehalose dihydrate crystals by centrifugation, aging and drying the crystals. A method for producing α, α-trehalose dihydrate crystal-containing powder, in the step of obtaining an α, α-trehalose-containing sugar solution, α-glycosyl trehalose producing enzyme and trehalose-free enzyme derived from a microorganism belonging to the genus Sulfolobus, and Paenibacillus genus Microbe-derived cyclomaltodextrin glucanotransferase Without a fractionation step by column chromatography by Being, alpha of the sugar solution, the alpha-trehalose content and 86.0% greater on a dry solid basis, and the alpha, alpha-trehalose two hydrous crystalline Characterized in that the step of crystallizing is carried out by controlled cooling method or pseudo controlled cooling method, containing 98.0% by mass or more of α, α-trehalose in terms of anhydride , based on the powder X-ray diffraction profile A method for producing an α, α-trehalose dihydrate crystal-containing powder, wherein the calculated crystallinity of α, α-trehalose dihydrate crystal is 90.0% or more .   The method for producing an α, α-trehalose bihydrate crystal-containing powder according to claim 1, wherein the Sulfolobus microorganism is Sulfolobus acidcardarius or Sulfolobus solfataricus.   The method for producing an α, α-trehalose dihydrate crystal-containing powder according to claim 1 or 2, wherein the α-glycosyl trehalose-producing enzyme and trehalose-releasing enzyme derived from a microorganism belonging to the genus Sulfolobus is a recombinant enzyme or a mutant enzyme.   4. The method for producing an α, α-trehalose bihydrate crystal-containing powder according to claim 1, wherein the Paenibacillus microorganism-derived cyclomaltodextrin glucanotransferase is a recombinant enzyme or a mutant enzyme.

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