Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-29
2002-11-12
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S123100, C536S123120, C435S074000, C435S101000, C435S105000, C435S124000, C435S127000
Reexamination Certificate
active
06479657
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for producing crystal 1-kestose, specifically a process for producing large crystal grains of crystal 1-kestose by vacuum crystallization or cold crystallization at a high yield. The present invention also relates to an enzyme for efficiently producing 1-kestose, and a process for producing crystal 1-kestose using said enzyme.
2. Description of the Related Art
The molecular structure of 1-kestose, a type of trisaccharide, is the same as that of sucrose except that the fructose half of 1-kestose is coupled with another fructose at position 1 via a &bgr;-2,1 bond. Fructooligosaccharides are characterized by little cariosity, indigestibility to biological digestive enzymes, and the specific facilitation of the growth of Bifidobacterium in the intestines, as demonstrated by some of the present inventors of the present invention (Japanese Patent Publication No. 53834/1984). It is believed that 1-kestose, which is a component of fructooligosaccharides, also has the same characteristics.
Commercially available crystalline oligosaccharides have been limited to raffinose, which has small crystal grain sizes of about 0.1 to 0.3 mm lengthwise. The currently most well-known crystalline sugar, sucrose, is produced mostly as granulated sugar, which is composed primarily of large crystals of about 0.25 to 1 mm, and soft sugar, which is a mixture of large and medium crystals of about 0.1 to 0.5 mm. In oligosaccharides, large grains are also favored for high utility.
Although both 1-kestose and sucrose have columnar crystals, the crystals of 1-kestose are more rectangular than the almost cubic crystals of sucrose. Therefore, 1-kestose should have larger crystals than sucrose in order to show physical properties similar to those of granulated sugar.
In a previously disclosed method for producing crystal 1-kestose, aqueous solution of 1-kestose of Brix 85 or higher and a purity of 70% or higher is heated to a temperature of 80° C. or higher; a suspension of microcrystals is added to the solution; the temperature is lowered to 65 to 75° C. to deposit crystals; and, while the temperature is maintained at 60 to 80° C., the deposited crystal 1-kestose is recovered (Japanese Patent Publication No. 70075/1994).
This method has a disadvantage in that the yield of crystals is low at about 30 to 40% because the concentration and purity of the crystallizing solution drop as crystals deposit. Further, this method unavoidably involves the deposition of microcrystals with increasing the viscosity of the solution. Thus, the solution should be heated during the recovery of crystals. Further, the grain sizes of the obtained crystals are not uniform and generally small.
On the other hand, 1-kestose may be produced from sucrose, making use of the activity of fructose transferase. In one of such known methods, sucrose solution adjusted to a concentration of 50% or higher is allowed to react at pH 4.0 to 7.0 in the presence of an enzyme having fructose transferase activity which is derived from an Asperaillus or Fusarium microorganism (Japanese Patent Publication No. 62184/1988). In another known method, an enzyme having fructose transferase activity derived from an AsTeraillus microorganism is allowed to react at pH 4.0 to 7.0, a temperature of 25 to 65° C., and a sucrose concentration of 20 to 70% (Japanese Patent Laid-Open Publication No. 268190/1986).
However, these methods are aimed primarily to obtain a mixture of a trisaccharide (1-kestose), a tetrasaccharide (nystose), and a pentasaccharide (fructosyl nystose), i.e., fructooligosaccharides, from sucrose. The rates of conversion from sucrose to 1-kestose in this method are only 36 to 41% at maximum (refer to Table 3 in Japanese Patent Publication No. 62184/1988, and Examples in Japanese Patent Laid-Open Publication No. 268190/1986). Also, in these methods, 11 to 23% of nystose is produced along with 1-kestose, leaving 11 to 23% of non-reacted sucrose, while 13 to 32% of monosaccharides such as fructose or glucose are produced as byproducts (ibid.).
It should be noted that the presence of monosaccharides, sucrose, and nystose is disadvantageous for the following reason: In the two-component simulated-moving bed chromatographic separation, which is used generally for producing isomerized sugar, etc., a solution containing two or more sugars of different molecular weights is divided into two fractions according to the difference in their molecular weights. If this method is used to obtain 1-kestose of high purity from a solution containing 1-kestose, the solution should be either (1) divided into two fractions, i.e., a fraction containing sugars with lower molecular weights than 1-kestose, and one containing those with molecular weights equal to or higher than that of 1-kestose, including 1-kestose; or (2) divided into two fractions, i.e., a fraction containing sugars with higher molecular weights than 1-kestose, and one containing those with molecular weights equal to or lower than that of 1-kestose, including 1-kestose. In the former case, for example, as the fraction contains sugars of higher molecular weights than that of 1-kestose, the purity of 1-kestose in the fraction is inevitably low depending on the content of such sugars. When the solution contains high contents of sugars whose molecular weights are higher than that of 1-kestose, e.g., nystose, with the two-component simulated-moving bed chromatographic separation, two or more steps of operations are required, such as a second chromatographic separation of the resultant fraction, in order to obtain 1-kestose solution at a high purity (e.g., 80% or higher), which is necessary for crystallization. In the latter case, the process has the same disadvantage when the contents of monosaccharides and sucrose are high. Therefore, a need exists for a method using enzymatic reactions in which the production of monosaccharides, sucrose, nystose, etc., is reduced, that is, a method in which 1-kestose can be selectively produced, and the conditions and a novel enzyme for this method.
Among Penicillium microorganisms, it has been suggested that
Penicillium frequentans
has an enzyme having fructose transferase activity (Japanese Patent Laid-Open Publication No. 293494/1992). However, this report only states that the microorganism produces fructose transferase, does not mention the rate of conversion to 1-kestose.
A method for selectively producing 1-kestose using a Scopulariopsis microorganism that produces 1-kestose in the presence of sucrose while consuming glucose has been reported (Japanese Patent Publication No. 47197/1993, Japanese Patent Publication No. 41600/1992). After the microorganism is incubated in a medium containing sucrose to produce 1-kestose, 1-kestose is recovered from the culture. According to the publication, the rate of conversion from sucrose to 1-kestose is as high as 60%. However, since the method uses the whole fungus body, the total sugar concentration in the culture, i.e., the sucrose concentration at the beginning of incubation, should be as low as about 15%. In addition, high contents of proteins and other impurities should be removed during purification.
Thus, methods for producing 1-kestose more selectively, particularly a novel enzyme and novel conditions for reaction, are solicited.
SUMMARY OF THE INVENTION
Inventors have now found that the viscosity of the fraction can be made low enough to enable the separation of crystals as solids from the solution at room temperature without heating the solution, by a controlled concentration procedure and temperature control based on what is called vacuum crystallization or cold crystallization. It has also be found that larger crystals of 1-kestose are obtained at a high yield. The inventors have also found certain microorganisms produce enzymes which efficiently produce 1-kestose. The present invention is based on these findings.
Thus, the object of the present invention is to provide a process for producing crystal 1-kestose wherein crystal
Adachi Takashi
Hirayama Masao
Kawakami Takashi
Matsumoto Hitoshi
Nakada Yuko
Meiji Seika Kaisha Ltd.
Owens Howard
Wilson James O.
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