Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-02-28
2002-08-20
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S072000, C435S099000, C435S100000, C536S123130, C127S038000, C127S040000
Reexamination Certificate
active
06436678
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention is in the field of oligosaccharides, and in particular, the invention pertains to the preparation of a high-purity maltose product.
BACKGROUND OF THE INVENTION
Maltose, an alpha 1-4 linked disaccharide, is a commercially important chemical with numerous industrial and consumer uses, for instance, as a sweetener or in the preparation of maltitol, a different sweetener in confectioneries intended for the low calorie or diabetic market. Maltose is also commercially important in the brewing industry, wherein it is provided as a fermentable component of brewing worts. In these applications, it is desirable to provide maltose in a substantially pure form, i.e., to the substantial exclusion of other carbohydrates. For instance, it is desirable in brewing applications to provide maltose to the substantial exclusion of dextrose. Although dextrose is fermentable, dextrose begins to inhibit yeast growth when present in the brewing wort, for reasons believed to relate to metabolic disturbances of the yeast used in the fermenting process. Similarly, in the preparation of maltitol, it is commercially desirable to provide maltose in a substantially pure form, so that sorbitol and higher molecular weight hydrogenated sugars such as maltotritol are not formed upon hydrogenation of the maltose. For these reasons, the production of maltose in a highly pure form is highly commercially desirable. It is particularly desirable to avoid substantial amounts of glucose, maltotriose, or other lower molecular weight sugars, because of the difficulty in separating maltose from such other carbohydrates.
Generally, maltose is prepared via the enzymatic hydrolysis of starch, whereby certain enzymes, known as beta amylases, convert starch into maltose. Natural starches are composed of two molecules, amylose, a linear saccharide, and amylopectin, a branched starch molecule which for a given starch ordinarily is of higher molecular weight than amylose and in which approximately 4% of the glucoside bonds are alpha 1-6 bonds. With the exception of so-called waxy (corn) or glutinous (rice) specialty starches, most starches found in nature are composed of between 20% and 30% amylose. In the case of amylose, beta-amylase enzymes catalyze hydrolysis from the non-reducing end of the molecule, thus releasing maltose units sequentially until the reducing end is reached. Presumably, if the amylose molecule contains an even number of glucose units, the sole product will be maltose; however, if the amylose molecule contains an odd number of glucose units, then, in addition to maltose, one glucose molecule is released per amylose molecule from the reducing end. In addition to glucose, maltotriose is often seen as an end product due to the relatively slow conversion of maltotriose to glucose and maltose. Moreover, if the amylose were present as a large number of short molecules, such as would be the case if the amylose had been partially hydrolyzed with an acid or alpha-amylase enzyme, then relatively larger amounts of glucose and maltotriose would result upon treatment of the short-chain amylose with beta-amylase. With respect to amylopectin, beta-amylase enzymes release maltose by sequential hydrolysis, but when the sequential hydrolysis reaches an alpha 1-6 branch point, the hydrolysis stops because the enzyme is unable to hydrolyze the branch point. As a result, the highest yield of maltose obtainable from amylopectin is about 50%. The remaining 50% of the original amylopectin exists as large, essentially water-soluble molecules.
For the foregoing reasons, the purity of the maltose product obtained via enzymatic hydrolysis of a starch is related to the number of amylose molecules per given weight of starch, and is limited by the extent of branching in the amylopectin component of the starch. Because of the production of by-products such as glucose and maltotriose, it is difficult to obtain a high-purity maltose from starch without subsequent purification steps. Conventionally, chromatographic processes have been employed to separate maltose from the glucose and other by-products of the beta-amylase hydrolysis product. Chromatographic separations are expensive and difficult to perform, however, thus resulting in an increased cost for a high-purity maltose product.
In recognition of this problem, the prior art has provided a number of attempts to improve the yield of maltose from starch. For instance, yields of maltose from amylopectin can be significantly increased by cleaving (hydrolyzing) the amylopectin structure between branch points with acid or with an alpha-amylase enzyme. This can be done either before treating the starch with a beta-amylase enzyme, or concurrently therewith. By cleaving between branch points, additional non-reducing ends are produced, thus providing more sites for attack by the beta-amylase enzyme. Using this technique, yields of maltose can be increased, but in addition to maltose, small branched molecular products undesirably are produced. Another strategy for increasing yields of maltose for amylopectin is the use of enzymes that hydrolyze the alpha 1-6 branch points. Enzymes such as pullulanse and isoamylase are known; such enzymes are generally known as alpha 1-6 glucosidases.
Such techniques are somewhat effective in improving the yield of maltose. For example, if normal starch (containing about 25% amylose and 75% amylopectin) is liquefied using conventional alpha-amylase or acid liquefaction techniques, and then treated with barley beta-amylase, a yield of about 55% maltose can be expected, the yield depending somewhat on the extent of prior hydrolysis with alpha-amylase. Barley malt-derived beta-amylase enzymes, which contain an alpha-amylase component, will provide a maltose yield of closer to 60%. The use of starch de-branching enzymes will provide even higher yields, ranging up to 75% maltose. Other attempts have focused on the development of new enzymes for use in the production of maltose from starch. One such enzyme (Maltogenase, from Novozymes A/S) is used in combination with an alpha-amylase enzyme, a beta-amylase enzyme, and a pullulanse enzyme to obtain maltose from starch. The maximum yield of maltose using these four enzymes appears to be about 80%.
The use of multiple enzymes on a commercial scale is expensive, both in connection with the purchase of such enzymes and the removal of the enzymes from the maltose product thus prepared. In addition, although the yield of maltose is high relative to other known processes, still the product obtained via the use of such enzymes contains substantial amounts of dextrose and of higher order oligosaccharides (typically having a degree of polymerization (DP) ranging from 3 to about 10). The presence of such other saccharides is undesirable for many purposes, in particular in brewing applications and in maltitol production, and thus expensive chromatographic processes are still required before a high-purity maltose product is obtained. In addition, although it may be feasible to develop new enzymes in an effort to further increase the yield of maltose, such development of new enzymes is extraordinarily expensive, and it is by no means certain that such new enzymes will be more effective than known techniques in preparing maltose products.
For the foregoing reasons, the prior processes for preparation of maltose discussed hereinabove suffer from a number of drawbacks. The present invention seeks to address these drawbacks by providing a process for the production of maltose from starch.
THE INVENTION
It has now been found that the treatment of starch with an enzyme that consists essentially of a beta-amylase enzyme, and which is to the substantial exclusion or complete exclusion of alpha-amylase enzymes and de-branching enzymes, will yield a product mixture that includes maltose and that is substantially free of glucose and of other malto-oligosaccharides having a DP from 3-10. The maltose may be readily separated from the product mixture thus formed via ultrafiltration to yield a high-puri
Antrim Richard L.
Lee Clark P.
Grain Processing Corporation
Leydig , Voit & Mayer, Ltd.
Prats Francisco
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