Title of Invention	A METHOD FOR MAKING AN ISOMALTO-OLIGOSACCHARIDE GRAIN COMPOSITION
Abstract	Methods for the production of substrate, tuber, and grain compositions containing isomalto-oligosaccharides are described. The methods comprise sequentially or concurrently (a) contacting a substrate, tuber or grain containing ungelatinized starch with an exogenous or endogenous maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto- oligosaccharide, wherein the oligosaccharide is derived from the grain. Figures 1 and 2.

Title of Invention

A METHOD FOR MAKING AN ISOMALTO-OLIGOSACCHARIDE GRAIN COMPOSITION

Abstract

Methods for the production of substrate, tuber, and grain compositions containing isomalto-oligosaccharides are described. The methods comprise sequentially or concurrently (a) contacting a substrate, tuber or grain containing ungelatinized starch with an exogenous or endogenous maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto- oligosaccharide, wherein the oligosaccharide is derived from the grain. Figures 1 and 2.

Full Text	METHOD FOR MAKING AN ISOMALTO-OLIGOSACCHARIDE GRAIN COMPOSITION TECHNICAL FIELD The present invention describes grain compositions containing isomalto- oligosaccharides and methods for making the same. The method includes the derivation of the isomalto-oligosaccharides from the starch contained within the grain. BACKGROUND OF THE INVENTION Isomalto-oligosaccharides ("IMOs") are mixed linkage oligosaccharides, having mixtures of 1,4 alpha and/or 1,6 alpha glucosidic linkages. They are also known as anomalously linked oligosaccharides ("ALOs"). Isomalto-oligosaccharides contain a substantial amount of branched oligo-saccharides such as isomaltose, panose, isomaltotriose, isomaltotetraose, isopanose and hiaher branched oligo- saccharides. There is a market demand for products containing IMO's. I MO products are sold in powder or liquid form, depending on the intended application. The potential applications are situated in the food area. Examples of IMO products are: seasonings (mayonnaise, vinegar, soup base etc.), confectionery (candy, chewing gum, chocolate, ice cream, sherbet, syrup), processed foods of fruits and vegetables (jam, marmalade, fruit sauces, pickles), meat or fish foods (ham, sausage, etc.), bakery products (bread, cake, cookie, pastry), precooked foods (salad, boiled beans, etc.), canned and bottled foods, convenience foods (instant coffee, instant cake base, etc.), and beverages, both alcoholic (liquor, seju, wine, sake, beer [International Publication No. WO 02/20712 A1], etc.) and non- alcoholic (coffee, juice, nectar, aerated or carbonated drinks, lemonade, cola). Isomalto-oligosaccharide can further be applied as ingredients in animal feed and pet foods. Non-food application areas are cosmetics and medicine (cigarette, lipstick, toothpaste, internal medicine, etc.). Isomalto-oligosaccharides belong to a group of oligosaccharides classified as functional-health food oligosaccharides ("FHFO"). Exemplary IMO's include fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides and gentio-oligosaccharides. IMO's have been linked to the increase of the general well being of humans and animals when taken orally on a regular dally basis and are classified as "prebiotics". Prebiotics are defined as non-digestible substances (e.g., dietary fiber) that exert some biological effect on humans by selective stimulation of growth or bioactivity of beneficial microorganisms either present or therapeutically introduced to the intestine. (Przemyslaw Jan Tomasik and Plotr Tomasik. 2003 American Association of Cereal Chemists, Inc. 80(2): 113-117). The "prebiotic" action of the oligosaccharides is to increase the numbers of bifidobacteria and lactobacilli ("prebiotic") in the large intestine and to reduce the concentration of putrifactive bacteria. Bifidobacteria are associated with some health promoting properties like the inhibition of the growth of pathogens, either by acid formation or by anti-microbial activity. They are also associated with such diverse effects as the modulation of the immune system (anti-tumor properties), the reduction of the levels of triglycerides and cholesterol, the production of vitamins (B group), the reduction of blood ammonia concentrations, the prevention of translocation, the restoration of the normal gut flora after anti-microbial therapy, the production of digestive enzymes, the reduction of antibiotic associated side effects (Kohmoto T., Fukui F., Takaku H., Machida Y., et al., Bifidobacteria Microflora, 7(2)(1988),61-69; Kohmoto K., Tsuji K., Kaneko T., Shiota M., et al., Biosc. Biotech. Biochem., 56(6)(1992),937-940; Kaneko T., Kohmoto T., Kikuchi H., Fukui F., et al., Nippon Nogeikagaku Kaishi, 66(8)(1992),1211-1220, Park J-H, Jin-Young Y., Ok-Ho S., Hyun-Kyung S., et al., Kor. J. Appl. Microbiol. Biotechnol., 20(3X1992). 237- 242). Modler, H.W., 1992, "Compounds which enhance the growth of Prebiotic Bacteria", presented at the International Roundtable on Animal Feed Biotechnology, Ottawa, Ontario, Canada. The isomalto-oligosaccharides are synthesized by an enzyme catalyzed transglucosylation reaction using a D-glucosyltransferase (E.C. 2.4.1.24, transglucosidase, alpha-glucosidase). This enzyme catalyzes both hydrolytic and transfer reactions on incubation with alpha-D-gluco-oligosaccharides. The transfer occurs most frequently to 6-OH (hydroxyl group 6 of the glucose molecule), producing isomaltose from D-glucose, or panose from maltose. The enzyme can also transfer to the 2-OH or 3-OH of D-glucose to form kojibiose or nigerose, or back to 4-OH to reform maltose. As a result of transglucosidase reactions, the malto-oligosaccharides are converted into isomalto- oligosaccharides resulting in a class of oligosaccharides containing a higher proportion of glucose moieties linked to a primary hydroxy! group of a glucose molecule from the non-reducing end, e.g., by alpha-D-1,6 glucosidic linkages. The transglucosidase from A. niqer acts only on oligosaccharides with a low degree polymerization (DP) (McCleary B. V., Gibson T. S., Carbohydrate Research 185(1989)147-162; Benson C. P., Kelly C.T., Fogarty W. M., J. Chem. Tech. Biotechnol., 32(1982)790-798; Pazur J. H., Tominaga Y., DeBrosse C. W., Jackman L. M., Carbohydrate Research, 61(1978) 279-290). Degree of polymerization refers to the number of dextrose units. For example, a di- glucosyl molecule, for example maltose, has a DP of 2. These sugars are receiving increased attention as food additives because they help prevent dental caries (Oshima, et.al 1988. The caries inhibitory effects of gos-sugar in vitro and rat experiments. Microbial Immunol. 32,1093-1105) and improve human intestinal microflora acting as a growth factor (prebiotic) for bifidobacteria (Komoto.et.al 1988; Effect of Isomalto-oligosaccharides on human fecal flora Bifidobacteria Micro flora 7,61-69). Isomalto-oligosaccharides can be obtained in different ways. For example glucose syrups at high dry solids concentration i.e. 60-80% are treated with glucoamylase resulting in the formation of isomalto-oligosaccharides mainly DP2. The high solids levels are present to force the reaction to reverse from the normal direction in favor of hydrolysis. Grains, including wheat, barley, etc., are excellent raw materials in the commercial production of many value added functional food ingredients such as wheat flour, starch, starch hydrolysates (glucose, fructose, high maltose syrup, etc.) and wheat gluten. Syrup containing a high level of maltose is also used in many microbial fermentations as a carbon source in the production of antibiotics, pharmaceuticals, vaccines, biochemical, such as alcohol (both potable and fuel), amino acids, organic acids, etc and recently in the production of functional health -food oligosaccarides called isomalto-oligosaccharides. In a conventional process for the production of starch hydrolysate, such as maltose syrups, the insoluble granular starch is generally separated from other cellular components of wheat prior to the hydrolysis by starch liquefying and maltogenic alpha amylases enzymes. Maltose is a disaccharide consisting of two glucosyl residues linked by a 1-4 D-glucosidic linkage and is the smallest in the family of malto-oligosaccharides. It is produced on a large scale as syrup, powder and crystals in several grades of purity. Various maltose syrups are drawing considerable interest for commercial applications in brewing, baking, soft drink canning, confectionary and other food and beverage industries. Ultra pure maltose is used as an intravenous nutrient in Japan. Catalytic reduction of maltose results in maltitol, which is considered to be a low calorie sweetener. Recently, high maltose syrup has become a key raw material for industrial production of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999 Transglucosylation of Malto-oligosaccharides." Paper presented at 50th Starch Convention, Detmold, Germany). In a conventional process for the production of starch hydrolysate such as high maltose syrup, the insoluble starch is separated prior to the hydrolysis by thermostable liquefying alpha amylases [EC 3.2.1.2,alpha (1,4)-glucan glucanohydrolase] derived either from Bacillus licheniformis or Bacillus stearothermophilus. Hydrolysis of the purified starch (refined) is carried out by suspending insoluble granular starch in water (30-35 % dissolved solid basis [dsb]) and heated to a temperature of between 85° C and 120 ° C to solubilize the starch and making it susceptible for enzymatic hydrolysis. The liquefied starch is further processed to manufacture starch hydrolysate with different carbohydrate composition using specific maltose producing enzymes such as fungal alpha amylase (sold under the tradename CLARASE L from Genencor International, Palo Alto, CA) for syrup containing less than 55% maltose, p Amylase (sold under the tradename OPTIMALT BBA from Genencor International, Palo Alto, CA) for syrup containing maltose content between 55% and 62% and less than1% glucose. For higher levels of maltose syrup, >62%, addition of debranching enzyme (sold under the tradename OPTIMAX L-1000 from Genencor International, Palo Alto, CA) in conjunction p Amylase is useful. (Faigh, J.; Duan, G.; Strohm, B. and Shetty, J. (2002) "Production of Maltose, High Maltose & Very High Maltose Syrups," Technical Bulletin, Genencor International Inc.). A process for converting granular starch (refined) into soluble hydrolysate by incubating with bacterial alpha amylase at a temperature below the starch gelatinization temperature (Leach et.al 1978; US Pat. No. 4,113,509) and subsequent hydrolysis by beta amylase to produce high maltose syrup have been reported (Leach et.al 1975;US Pat. No. 3,922,196), However the syrup produced by such process resulted in only 55 % maltose of the total sugar content, with a very high level of maltotriose. The process for producing high maltose syrup using liquefied starch (gelatinized followed by hydrolysis using thermostable alpha amylase) is described in European Patent Application #0905256 (Christophersen, et.al 2000) and U.S. Pat. No. 5,141,859 (Nimmi, et.al 1992). The process is cumbersome, expensive and it requires the separation of starch from other cellular components, high cost of the additional maltose producing enzymes, high temperature treatment and longer reaction time. European Patent Application #0350737 A2 (Shinke, et.al 1989) disclosed a process for producing maltose syrup by hydrolyzing a granular (purified) starch from corn, wheat, potato and sweet potato at 60° C without the conventional liquefaction step (gelatinization followed by liquefaction at high temperature) using an alpha amylase from Bacillus stearothermophilus. However, the hydrolyzed starch resulted in a maltose concentration ranging from 50% to 55%. The syrup also contained very high level of maltotriose (30-36%). The process resulted in a ratio of maitose to maltotriose less than 2.0 irrespective of the source of the starch. Maltose syrup containing a high level of maltotriose is not a preferred substrate as carbon feed in many microbial fermentations including the alcohol fermentation by yeast because of the difficulties in metabolizing maltotriose. Maltose is a preferred donor of glucosyl residue in the transglucosylation reaction catalyzed by glucosyltransferases in the production of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999 "Transglucosylation of Malto-oligosaccharides." Paper presented at 50th Starch Convention, Detmold, Germany). U.S. Pat. No. 6,361,809 described a method for producing maltose and a limit dextrin by treating the purified granular waxy maize starch with a hydrolase, maltogenase alpha amylase classified as EC 3.2.1.133 from Bacillus stearothermophilus followed by separating the maltose using ultra filtration process. Evaporation of the dilute permeate containing the maltose is expensive because of high energy cost and also faces a very high risk of microbial contamination. Traditionally grains such as wheat, malt, sorghum (milo), millet (ragi), particularly whole grains are used in nutrition as carriers of macro- and micro- elements, proteins, fiber and vitamins. The majority of cereal grains appeared to be too readily digested to play an effective role as prebiotics or even as nutraceuticals. It has been suggested that designing genetically modified, less digestible cereals suitable as prebiotics to manipulate gut microflora (Gibson, G.R, and Roberfroid, M. B. 1995, Dietary modulation of the human colonic micrflora: Introducing the concept of prebiotics. J. Nutr. 125, 1401-1412). There is a continuing interest in methods for producing grain compositions with isomalto-oligosaccharides enzymatically derived from the source substrate, e.g., grain or tuber, without having to separate the starch from other grain components and/or subject the starch of the substrate to high temperatures of jet cooking prior to transglucosidation action. There is also a continuing interest in low DH processes for minimizing the risk of microbial contamination. The present invention addresses these interests. SUMMARY OF THE INVENTION The present invention describes a method for making an isomalto- oligosaccharide grain composition said method comprising:(a) contacting a ungelatinized starch containing grain with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a grain composition having an enzymatically produced isomalto oligosaccharide, wherein said oligosaccharide is derived from said grain. Optionally, in one embodiment, the steps (a) and (b) occur concurrently. In another embodiment, the method further includes a step of drying said grain composition. In another embodiment the grain is selected from the group consisting of wheat, rye, barley, malt and rice. In another embodiment the grain is selected from the group consisting of sorghum, millet and rice. In another embodiment, the maltogenic enzyme is a beta amylase. In another embodiment, the maltogenic enzyme is endogenous to said grain. In another embodiment, the maltogenic enzyme is exogenous to said grain. In another embodiment, the starch liquefying enzyme is an alpha amylase derived from a Bacillus. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidic enzyme is a transglucosidase. In another embodiment, the transglucosidase is derived from Aspergillus. In another embodiment, the transglucosidase derived from Asoergillus niger. Another embodiment of the present invention includes a grain composition produced according to above described method. Another embodiment of the present invention includes a food additive comprising said grain composition described above. The present invention also describes a method for making an isomalto- oligosaccharides enriched flours at temperatures at or below the gelatiniziation temperature wherein an ungelatinized grain having an endogenous maltogenic enzyme are contacted with a solubilizing enzyme selected from Bacillus to produce a maltose syrup. The maltose syrup is contacted with a transglucosidase to produce a substrate (tuber or grain) composition including isomalto-saccharides derived therefrom. The present invention also describes a method for making an isomalto- oligosaccharides enriched flours at temperatures at or below the gelatiniziation temperature wherein an ungelatinized grain having an endogenous maltogenic enzyme (wheat, barley, etc.) are mixed with ungelatinized grain not having endogenous maltogenic enzymes (e.g., sorghum, miller or rice), the grain mixture being contacted with a solubilizing enzyme selected from Bacillus to produce a maltose syrup. The maltose syrup is contacted with a transglucosidase to produce a substrate (tuber or grain) composition including isomalto-saccharides derived therefrom. The present invention also describes a method for making a wheat grain composition said method comprising: (a) contacting an ungelatinized wheat grain having an endogenous maltogenic beta-amylase and a starch liquefying alpha amylase from Bacillus to produce maltose; (b) contacting said maltose with a transglucosidase, wherein said steps (a) and step (b) occur at a temperature less than wheat gelatinizing temperature; and (c) obtaining a wheal grain composition having an enzymatically produced isomalto-oligosaccharide, wherein said oligosaccharide is derived from said ungelatinized grain. Optionally, in another embodiment the method uses the above method for making a grain composition for making a food additive. Another embodiment includes a grain composition made accordingly. Another embodiment includes a flour comprising the grain composition described above. Another embodiment includes an isomalto-saccharide made according to the method described above. Another embodiment includes an oral rehydration solution comprising the isomalto- oligosaccharide above. Another embodiment includes a grain composition comprising an ungelatinized grain and at least one isomalto- oligosaccharide, wherein said isomalto-oligosaccharide is enzymatically derived from said ungelatinized grain. In another embodiment the grain composition contains greater than 1 % by weight of at least one isomalto-oligosaccharide. ACCOMPANYING DESCRIPTION OF THE/FIGURES FIG. 1 is a flowchart describing the production of isomalto-oligosaccharide enriched flour. FIG. 2 is another flowchart describing the production of isomalto-oligosaccharide enriched wheat flour. DETAILED DESCRIPTION OF THE INVENTION Definitions The term "grain" refers to a plant which is classified as a cereal or as a monocotyledonous plant belonging to the Poales order, in particular the family Poaceae. Examples of plants belonging thereof are plants selected from the genuses Triticum (wheat), Hordeum (barley); Secale (rye); Zea (com or maize); Avena (oats), Faqoprvum (buckwheat); Sorghum (sorghum or milo), Panicum or Setaria (millet or ragi); or Oryza (rice). For example, in one embodiment, the term "wheat" refers to a plant which is classified or once was classified as a strain of Triticum aestivum. For example, in one embodiment, the term "barley" refers to a plant which is classified or once was classified as a strain of Hordeum vulqare. For example, in one embodiment, the term "rye" refers to a plant which is classified or once was classified as a strain of Secale cereale. For example, in one embodiment, the term "corn" refers to a plant which is classified or once was classified as a strain of Zea mays. For example, in one embodiment, the term "oats" refers to a plant which is classified or once was classified as a strain of Avena sativa. For example, in one embodiment, the term "buckwheat" refers to a plant which is classified or once was classified as a strain of Fagoprvum esculentum. For example, in one embodiment, the term "sorghum" refers to a plant which is classified or once was classified as a strain of Sorghum bicolor. For example, in one embodiment, the term "millet" refers to a plant which is classified or once was classified as a strain of Panicum miliaceum or Setaria italica. For example, in one embodiment, the term "rice" refers to a plant which is classified or once was classified as a strain of Orvza sativa. The term tuber refers to a starchy storage organ (for example a potato, sweet potato, yam, manioc, etc) formed by swelling of an underground stem or the distal end of a root. For example, in one embodiment, the term "potato" refers to a plant which is classified or once was classified as a strain of Solanum tuberosum. For example, in one embodiment, the term "sweet potato" refers to a plant which is classified or once was classified as a strain of Ipomroela balatas. For example, in one embodiment, the term "yam" refers to a plant which is classified or once was classified as a strain of Dioscorea sativa, D. villosa, C. batatas. The term substrate refers to materials that can be enzymatically converted to maltose and thus IMO's. The term "substrate" includes, for example, grains and tubers. Furthermore, the term substrate includes all forms of the grain (polished or unpolished) or tuber, such as whole grains, broken grains, grits and flour and any plant part. The term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C6H10O5)x, wherein X can be any number. The term "granular starch" refers to uncooked (raw) starch, which has not been subject to gelatinization. The term "gelatinization" refers to solubilization of a starch molecule to form a viscous suspension. The phrases "substrate", "grain" or "tuber" containing ungelatinized starch" refer to an ungelatinized substrate, grain or tuber that is not subjected to temperatures greater than the starch gelatinization temperatures which result in effecting a gelatinization or liquefaction of the starch contained within the substrate. The term "maltose" refers to a disaccharide having two glucosyl residues linked by an alpha 1-4 D-glucosidic linkage The term "isomaltose" refers to a disaccharide having two glucosyl residues linked by an alpha 1,6 D-glucosidic linkage. The term "isomalto-oligosaccharide" (IMO) refers to sugars having at least two glucosyl residues linked by alpha 1,6 glucosidic linkages at the non-reducing end. In addition term term refers to anomalously linked oligosaccharides, saccharides having both alpha 1,6 and alpha 1,4 glucosidic linkages. Exemplary isomalto-oligosaccharides include isomaltose, panose, and isomalto-triose The term "isomalto-oligosaccharide" grain composition refers to grain compositions characterized by isomalto-sugars level of at least 1% (w/w %) of the total sugar content as determined by high performance liquid chromatographic methods. The term "maltogenic enzyme" refers to an enzyme that converts starch to maltose. Exemplary maltogenic enzymes include fungal, bacterial and plant derived alpha amylases and beta-amylases. The term "amylases" refers to enzymes that catalyze the hydrolysis of starches. The term "alpha-amylase" refers to enzymes of the class (E.C.) 3.2.1.1 and/or 3.2.1.133 that catalyze the hydrolysis of alpha-1,4-glucosidic linkages. These enzymes have also been described as those effecting the exo or endohydrolysis of 1,4-alpha-D-glucosidic linkages in polysaccharides containing 1,4-alpha-linked D-glucose units. Another term used to describe these enzymes is glycogenase. Exemplary enzymes include alpha-1,4-glucan 4-giucanohydrase glucanohydrolase. The term "beta-amylase" refers to enzymes of the class (E.C.) 3.2.1.2 that catalyze the hydrolysis of alpha-1,4 glucosidic linkages releasing maltose units. These enzymes have also been described as those effecting the hydrolysis of 1,4-alpha-D-glucosidic linkages in polysaccharides so as to remove successive maltose units from the non-reducing end of chains. The term "transglucosidic" enzyme refers to an enzyme that catalyzes both hydrolytic and transfer reactions in incubation with alpha D-gluco- oligosaccharides. Exemplary enzymes include transglucosidases and/or those of the class (E.C.) 2.4.1.24, e.g., D-glucosyltransferase. These enzymes have also been refered to as 1,4-alpha-glucan 6-alpha-glucosyltransferase and oligoglucan-branching glycosyltransferase. The term debranching enzyme refers to enzymes that catalyze the hydrolysis of alpha-1,6-linkages. An enzyme of the class E.C.3.2.1.41 is useful in this regard. An exemplary enzyme of this class is a pullanase, also known as alpha-dextrin endo-1,6- alpha glucosidase, limit dextrinase, debranching enzyme, amylopectin 1,6-glucanohydrolase. Additional exemplary enzymes of the class (E.C.) 3.2.1.41, e.g., pullulanases, [alpha-(1-6)-glucan 6-giucanohydrolase, also called alpha-(1,6)-glucosidase]). The term "starch gelatinizing temperatures" refers to a temperature sufficiently high to effect liquefying or gelatinization of granular starch. Heating a starch in water causes the starch granules to swell. At sufficient solids concentration, the swollen granules occupy most of the space and a viscous mass, called a paste, results. Solubilization of starch molecule is called gelatinization. Gelatinization is accompanied by a loss of birefringence. The term starch gelatinizing temperature refers to the temperature at which gelatinization occurs. The term "starch liquefying enzyme" refers to an enzyme that effects the fluidization of granular starch. Exemplary starch liquefying enzymes include alpha amylases of the class (E.C.) 3.2.1.1. I The term "endogenous" refers to the enzyme being present in the grain or tuber \ without having to resort to adding the maltogenic enzyme to the grain. The term "exogenous" enzyme refers to an enzyme that is not present within the grain. Exemplary exogenous enzymes include, for example, maltogenic enzymes not present in the wild-type substrate, e.g., rice, millet, etc. The term "total sugar content" refers to the total amount of sugar present in a starch, grain or tuber composition. The term "IMO No." is calculated as the sum of isomaltose, panaose, isomaltotriose and branched sugars greater than DP3. The IMO Number provides an indication of the amount of IMO compounds present in the compound or solution. The term "ratio of branched sugars" ("RBS") refers to the ratio of maltose (DP2) present in the grain as compared to the level of maltotriose (DP3) present in the resultant grain composition. The term "Degrees of Diastatic Power" (DPo) unit refers to the amount of enzyme contained in 0.10 ml of a 5% solution of the sample enzyme preparation that will produce sufficient reducing sugars to reduce 5 ml of Fehling's solution when the sample is incubated with 100 ml of substrate for 1 hour at 20° C (68° F). The term "DE" or "dextrose equivalent" is an industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE that is essentially 0 and D- glucose has a DE of 100. The term "total sugar content" refers to the total sugar content present in a starch composition. The terms "dry solid basis" and "dsb" refer to the total amount of compound, e.g., flour, of a slurry (in %) on a dry weight basis. The terms "dry solid content", "dry solid granular starch", "dry solid starch" and "(dss)" refer to the total starch of a slurry (in %) on a dry weight basis. The term "Brix" refers to a well known hydrometer scale for measuring the sugar content of a solution at a given temperature. Thus the term "Brix" refers to a measure of the solubilized sugars in solution. The Brix scale measures the number of grams of sucrose present per 100 grams of aqueous sugar solution (the total solubilized solid content). For example, a measurement of 1.00 Brix refers to about 10 mg/ml of sugar in solution. Brix measurements are frequently made by use of a hydrometer or refractometer. The term "degree of polymerization (DP)" refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DP1 are the monosaccharides, such as glucose and fructose. Examples of DP2 are the disaccharides, such as maltose and sucrose. A "DP4+" denotes polymers with a degree of polymerization of greater than 3. The term "enzymatically produced" refers to enzymatic catalysis of the substrate to the IMO as opposed to chemical or organic chemical synthesis of the IMO. The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORY MYCOLOGY, New York: Wiley). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. In the present invention, the filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, e.g., Trichoderma reesei (previously classified as T.longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicola insolens and Humicola grisea; Chrysosporium sp., including C. lucknowense; Gliocladium sp.; Aspergillus sp., including A. oryzae, A. nidulans, A. niger, and A. awamori; Fusarium sp., Neurospora sp., Hypocrea sp., and Emericella sp. Reference is also made to Innis et al., (1985) Sci. 228:21 -26. The term "Aspergillus" or "Aspergillus sp." refers to any fungal strain, which had previously been classified as Aspergillus or is currently classified as Aspergillus. The term "bacterial" refers to Bacillus species of, but not limited to B. subtilis. B. amvloliouefaciesn. B. lentus. B. Carlsberg. B. licheniformis. and B. stearothermophilus The term "plant origin" refers to the enzyme being derived, extracted, isolated, expressed from a plant source, for example from barley malt, soybean, wheat or barley. The term "contacting" refers to the piacing of the respective enzyme[s] in sufficiently close proximity to the respective substrate to enable the enzyme[s] to convert the substrate to the desired end-product. Those skilled in the art will recognize that mixing solutions of the enzyme or enzymes with the respective substrates can effect contacting. The term "incubating" refers to mixing a substrate containing substrate with the respective enzymes, e.g., liquefying or maltogenic or transglucosidase under given conditions for a defined period of time. The term "enzymatic conversion" refers to the modification of a rice substrate to yield soluble hydrolyzed granular rice starch and preferably to yield glucose. The term "slurry" refers to an aqueous mixture containing insoluble granular starch. Sometimes the terms "slurry" and "suspension" are used interchangeably herein. The term "culturing" refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium. In one embodiment, culturing refers to fermentative bioconversion of a granular starch substrate to glucose syrup or other desired end products (typically in a vessel or reactor). For example, in one embodiment, the term alpha amylase enzyme unit is defined as the amount of alpha amylase which hydrolyzes 1 micromole of starch substrate in 1 min under standard assay conditions of pH 5.2 and 40°C. For example, in one embodiment, the term beta amylase enzyme unit is defined as the amount of beta amylase which hydrolyzes 1 micromole of starch substrate in 1 min under standard assay conditions of pH 4.6 and 20°C. For example, in one embodiment, the term transglucosidase unit is defined as the amount of transglucosidase which converts 1 micromole of maltose substrate in 1 min under standard assay conditions of pH 4.8 and 37°C. In another embodiment, the term transglucosidase unit is defined as the amount of transglucosidase which produces 1 micromole of panose per minute under standard assay conditions of pH 4.8 and 37° C. For example, in one embodiment, the term one Liquefon Unit (LU) is the measure of digestion time required to produce a color change with iodine solution, indicating a definite stage of dextrinization of starch substrate under standard assay conditions of pH 5.6 and 25 ° C. "ATCC" refers to American Type Culture Collection located at Manassas, VA 20108 (ATCC, www/atcc.org). "NRRL" refers to the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (and previously known as USDA Northern Regional Research Laboratory), Peoria, ILL. "NCBI" refers to the National Center for Biotechnology Information, Natl Library Med. (www.ncbi.nlm.nih.gov/). "A", "an" and "the" include plural references unless the context clearly dictates otherwise. The present invention describes a method for making an isomalto- oligosaccharide substrate, grain or tuber composition said method comprising:(a) contacting a ungelatinized starch containing substrate, e.g., a grain or a tuber, with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto-oligosaccharide, wherein said oligosaccharide is derived from said substrate, grain or tuber. An embodiment of the present invention is depicted in Fig.1. The present invention also describes a method for making an isomalto- oligosaccharide-enriched substrate, grain or tuber compositions, flours, oral rehydrating solutions, and/or food additives, at temperatures at or below the gelatiniziation temperature wherein a substrate having or containing an ungelatinized starch and having endogenous maltogenic enzyme are contacted with a solubilizing enzyme selected from Bacillus to produce a maltose syrup. The maltose syrup is then contacted with a transgiucosidase at a temperature at or less than gelatinization or liquefaction temperatures to produce a grain composition having isomalto-oligosaccharides. In one embodiment, the grain composition is characterized by a sugar composition of greater than 60% maltose and a ratio of branched sugars of greater than 2.0 to 1.0. The conversion of the substrate to the IMO can be enzymatically produced. The present invention also describes a method for making an isomalto- oligosaccharide substrate, grain or tuber composition, the method comprising: (a) contacting a substrate, grain or tuber containing a starch with a maltogenic enzyme and a starch liquefying enzyme to produce a maltose; (b) contacting the maltose with a transglucosidic enzyme, wherein the steps (a) and step (b) occur at a temperature less than or at starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto-oligosaccharide, wherein the oligosaccharide is derived from the substrate, grain or tuber. The invention optionally further describes an additional step of separating soluble constituents from insoluble constituents. The invention further describes an additional step of drying the substrate, grain or tuber composition. In one embodiment the grain is selected from the group consisting of wheat, rye, barley, malt, buckwheat, sorghum (milo), millet (ragi) and rice. In another embodiment, the maltogenic enzyme is a beta amylase. In another embodiment, the maltogenic enzyme is endogenous to the grain. In another embodiment, the starch liquefying enzyme is an alpha amylase derived from a Bacillus. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidic enzyme is a transgiucosidase. In another embodiment. The transgiucosidase is derived from Aspergillus. In another embodiment, the Aspergillus is Aspergillus niger. The invention also describes a grain composition, a food additive, oral rehydration solution, food product and/or a flour produced according to above described method. In another embodiment, the invention describes a method for making a wheat grain composition said method comprising: (a) contacting an ungelatinized wheat grain having an endogenous maltogenic beta-amylase and a starch liquefying alpha amylase from Bacillus to produce maltose; (b) contacting said maltose with a transglucosidase, wherein said steps (a) and step (b) occur at a temperature less than wheat gelatinizing temperature; and (c) obtaining a wheat grain composition having an enzymatically produced isomalto-oligosaccharide, wherein said oligosaccharide is derived from said ungelatinized grain. An embodiment of the present invention is depicted in Fig. 2. In another embodiment, the above described method can be used to make a food additive, a bakery product, oral rehydration solution and/or a flour. In another embodiment, the maltogenic enzyme is a beta amylase. In another embodiment, the maltogenic enzyme is endogenous to the grain. In another embodiment, the starch liquefying enzyme is an alpha amylase derived from a bacterial source. In one embodiment the bacterial source is a Bacillus sp.. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidic enzyme is a transglucosidase. In another embodiment. The transglucosidase is derived from a fungal source. In one embodiment the fungal source is an Aspergillus sp. In another embodiment, the Aspergillus is Aspergillus niger. The invention also describes a grain composition, a food additive, oral rehydration solution and/or a flour produced according to above described method. The grain composition could contain greater than 1% by weight of at least one isomalto-oligosaccharide. The at least one isomalto- oligosaccharide can be selected from the group consisting of isomaltose, panose, isomalto-triose. In a further embodiment of the invention, the endogenous maltogenic enzyme is selected from beta amylase or alpha amylase. In a still further embodiment of the invention, the solubilizing enzyme is a liquefying alpha amylase derived from a Bacillus. In a still further embodiment of the invention the liquefying amylase is derived from Bacillus licheniformis or Bacillus stearothermophilus. Substrates The present invention includes a substrate containing a starch, for example a grain or a tuber containing a starch that is contacted with a maltogenic enzyme and a starch liquefying enzyme to produce maltose. The term substrate refers to materials that can be enzymatically converted to maltose and thus IMO's. Exemplary substrates can be at least one substrate selected from the group consisting of grains and tubers. The maltose can be in the form of a maltose rich syrup or slurry. Starch occurs in two forms, amylose, a linear chain polysaccharide, and amylopectin, a branched chain polysaccharide. Amylose contains long unbranched chains in which all the D-glucose units are linked by alpha-1,4- linkages ("a-1,4 linkages" or "1,4-a-D-glucosyl linkages"). Amylopectin is highly branched, the backbone glucosidic linkage being a-1,4, but the branch points being a-1,6 linkages. The major components of starch can be enzymatically hydrolyzed in two different ways. Amylose can be hydrolyzed by a-amylases (E.C. 3.2.1.1), e.g., a-(1-4)-glucan 4-glucanohydrolase. Alpha amylases hydrolyze the a-(1,4)-linkages to yield a mixture of glucose, maltose, maltotriose and higher sugars. Amylose can also be hydrolyzed by a beta-amlylase (E.C. r 3.2.1.2) [alpha(1,4)-glucan maltohydrolase, 1,4-a-D-glucan maltohydrolase ]. This enzyme cleaves away successive maltose units beginning from the non- reducing end to yield maltose quantitatively. The alpha and beta amylases also hydrolyze amylopectin. Neither the alpha and beta amylases can hydrolyze the alpha (1-6) linkages at the branch points of amylopectin. The end product of exhaustive beta-amylase action on amylopectin is a large, highly branched core or beta limit dextrin. A debranching enzyme (E.C. 3.2.1.41 .e.g., pullulanases, [a-(1-6)-glucan 6-glucanohydrolase, also called a-(1,6)-glucosidase]) can hydrolyze the a-(1-6) linkages at the branch points. Thus the combined action of p-amylase and the a 1,6-glucosidase can therefore completely degrade amylopectin to maltose and glucose, resulting in a maltose content as high as 60%, 65%, 79%, 75%, 80% or higher of the total sugar content. For the purposes of this invention, the substrate containing starch can be a grain or a tuber or mixtures thereof. The grain can be any cereal or seed containing starch. The substrate can be milled, ground or otherwise reduced in size to increase the surface area of the substrate for contacting with the respective enzymes. For example, the substrate can be wet or dry milled as desired. In one embodiment of the present invention, the starch is granular starch. Grains contemplated for use within the present invention includes any grain currently used in baking, pasta or other uses. Exemplary grains contemplated by the inventors include, but are not limited to at least one selected from the group consisting of wheat (Triticum sp. Including, but not limited to T. monococcum. T. turgidum. T. spelta. and/or T. aestivum). barley (e.g., Hordeum vulaare. and the varieties described in U.S.Pat. No. 6,492,576, Table 1), rye (Secale sp.. including but not limited to S. cereale). corn (Zea sp.. including, but not limited to Zea mays), buckwheat (Faqoprvum sp., including, but not limited to F esculentum), malt (for example, germinated barley), sorghum (Sorghum sp.. including, but not limited to Sorghum bicolor') or otherwise known as milo, millet (ragi) (Panicum sp.and Setaria sp.. including, but not limited to P. milaceum: and Setaria sp., including, but not limited to Sitalica) and rice (Orvza sp.. including, but not limited to Orvza sativa). It is contemplated by the inventors that wild-type and transgenic plants having beneficial attributes, such as increased enzyme levels of endogenous enzymes or the presence of exogenous enzymes are also useful as starch containing substrates. Germinated cereals, for example, malt, are used as one of the key ingredients in many food and health drink formulations because of their high nutritive value, e.g., malt containing food products (TABLE A). Germination results in the synthesis and activation of endogenous maltogenic and proteolytic enzymes. Thus germinated cereals are a good source of grains containing endogenous maltogenic enzymes. Malt flour and malt extract are also used as a source of digestive enzymes in brewing and baking applications. However, germination of the barley renders the cereal grain components too readily digestible to play an effective role as a prebiotic or even as a nutraceuticals, since they tend to be digested in their entirety before arrival in the lower gastrointestinal tract. Unfortunately, the beneficial effects of prebiotic compounds are best realized in the lower gastrointestinal tract. Therefore, converting the highly digestible malto- sugars into less digestible isomalto sugars allows for the use of the modified malt to play a role as a prebiotic, allowing the malt to arrive in the lower gastrointestinal tract and provide additional functional and health benefits. For Thus the use of malt as a starch containing substrate converts some of the granular starch contained within the substrate to an additional beneficial form of the oligosaccharide, e.g., an IMO. In addition, the substrate containing starch can be a tuber. Tubers contemplated by the inventors include potato (Solanum sp.. including, but not limited to S tuberosum), sweet potato (Ipomoea sp.. including, but not limited to Ipomoea batatas), manioc [tapioca, cassava] (Manihot sp., including, but not limited to Manihot esculenta, Manihot aipi and Manihot utilissima) and/or taro root (Colocasia sp., including, not limited to C. esculenta or C. macrorhiza). The amount of substrate containing starch can be an aqueous slurry characterized by having a concentration of 10 to 50% dissolved solids (DS). In another embodiment, the substrate containing starch is characterized by having a concentration of 2 -90% DS. In another embodiment, the substrate containing starch is characterized by having a concentration of 5 -70% DS. In another embodiment, the substrate containing starch is characterized by having a concentration of 10-60% DS. In another embodiment, the substrate containing starch is characterized by having a concentration of 20-40% DS. In another embodiment, the substrate containing starch is characterized by having a concentration of 25-35% DS. In another embodiment of the invention, the pH of the substrate containing starch is between 1.00 to 9.00. In another embodiment of the invention, the pH of the substrate containing starch is between 2.00 to 8.00. In another embodiment of the invention, the pH of the substrate containing starch is between 3.00 to 7.50. In another embodiment of the invention, the pH of the substrate containing starch is between 4.00 to 6.50. In another embodiment of the invention, the pH of the substrate containing starch is between 4.25 to 5.75. Enzymes The present invention includes contacting the substrate containing starch with a maltogenic and a starch liquefying enzyme to produce maltose. By maltogenic is meant that the enzyme is able to enzymatically convert starch to maltose. Exemplary maltogenic enzymes include alpha amylases and beta amylases. As described before, amylose can be hydrolyzed by D-amylases (E.C. 3.2.1.1), e.g., D-(1-4)-glucan 4-glucanohydrolase. Alpha amylases hydrolyzethe D-(1,4)- linkages to yield a mixture of glucose, maltose, maltotriose and higher sugars. Amylose can also be hydrolyzed by a beta-amlylase (E.C. 3.2.1.2) [alpha(1,4)- glucan maltohydrolase, 1,4-D-D-glucan maltohydrolase ]. This enzyme cleaves away successive maltose units beginning from the non-reducing end to yield maltose quantitatively. The alpha and beta amylases also hydrolyze amylopectin. Alpha Amylases - In some of the embodiments encompassed by the invention, the alpha amylase is a funal or microbial enzyme having an E.C. number, E.C. 3.2.1.1-3 and in particular E.C. 3.2.1.1. In some embodiments, the alpha amylase is a thermostable fungal alpha amylase. Suitable alpha amylases may be naturally occurring as well as recombinant and mutant alpha amylases. In some embodiments, the alpha amylase is derived from a Bacillus species. Preferred Bacillus species include Bacillus amyloliquefaciens, B. lentus, B. licheniformis, and B. stearothermophilus. In particularly preferred embodiments, the alpha amylase is derived from a Aspergillus species. Preferred Aspergillus species include Aspergillus niqer and Aspergillus orvzae. Also reference is made to strains having NCIB 11837. Commercially available alpha amylases contemplated for use in the methods of the invention include; CLARASE L (fAspergilus orvzael Genencor International Inc.) and NOVAMYL ([B stearothermophilus] Novozyme Biotech.). As understood by those in the art, the quantity of alpha amylase used in the methods of the present invention will depend on the enzymatic activity of the alpha amylase. In general, an amount of about 0.01 to 5.0 kg of the alpha amylase is added to a metric ton (MT) of the substrate containing starch. Although in some embodiments the alpha amylase is added in an amount about 0.05 to 4.0 kg per MT. In other embodiments, the alpha amylase is added in an amount of about 0.1 to 2.5 kg per MT and also about 0.5 to 1.5 kg per MT. In further embodiments, other quantities are utilized. For example, generally an amount of between about 0.01 to 1.5 kg of CLARASE L (Genencor International Inc.) is added to a MT of starch. In other embodiments, the enzyme is added in an amount between about 0.05 to 1.0 kg; between about 0.1 to 0.6 kg; between about 0.2 to 0.6 kg and between about 0.4 to 0.6 kg of CLARASE L per MT of starch. Beta Amylase In some of the embodiments encompassed by the invention, the maltogenic enzyme is a beta amylase. While alpha amylases are maltogenic in the sense that contacting alpha amylases with a substrate containing starch would provide maltose, the use of beta amylases are useful in that their contact with granular starch would provide a greater amount of maltose to the exclusion of other saccharides, e.g., glucose. In some embodiments, the beta amylase is a plant or microbial enzyme having ah E.C. number, E.C. 3.2.1.2 (for example those beta amylases described in US 4,970,158 and 4,647,538). In some embodiments, the beta amylase is a thermostable bacterial beta amylase. Suitable beta amylases may be naturally occurring as well as recombinant and mutant beta amylases. The term bacterial refers to the enzyme being derived from Bacillus sp.. e.g., B. subtilis. B. licheniformis. B. stearothermophilus. B^ coaqulans. B. amyloliquefaciens. and/or B. lentus. Particularly preferred beta amylases are derived from Bacillus strains B. stearothermophilus, B. amyloliquefaciens and B. licheniformis. Also reference is made to strains having NCIB 11608. The term plant origin refers to the enzyme being derived, extracted, isolated, expressed from a plant source, for example from barley malt, soybean, wheat or barley. Commercially available beta amylases contemplated for use in the methods of the invention include; OPTIMALT BBA, Spezyme DBA, and OPTIMALT ME (Genencor International Inc.). Other commercially available wheat beta amylases are also useful in the methods of the invention. In some embodiments, the substrate containing starch, e.g., wheat, rye, barley, malt, comprises an endogenous maltogenic enzyme at sufficient levels to produce sufficient maltose for conversion to isomalto oligosaccharides.. The term "endogenous" refers to the enzyme being present in the grain or tuber without having to resort to adding the maltogenic enzyme to the grain, or the grain being genetically engineered to provide maltogenic enzymes. In embodiments where the substrate containing starch does not contain an endogenous maltogenic enzyme or has low endogenous levels of maltogenic enzymes, e.g., rice, millet, sorghum, and/or corn, the addition of an equivalent amount of any exogenous maltogenic enzyme is also contemplated by the inventors. The exogenous maltogenic enzyme can be added, for example by genetically manipulating the host cell to express sufficient levels of maltogenic enzyme, and/or providing a maltogenic enzyme concentrate or material from another source. The term exogenous maltogenic enzyme refers to a maltogenic enzyme that is not present within the grain. In this embodiment, a sufficient amount of maltogenic enzyme is contacted with the substrate to produce maltose. In one embodiment, the amount of exogenous maltogenic enzyme contacted with the substrate containing starch is between 0.050 to 5.000 Degrees of Diastatic Power ("DP0") units /gm of maltogenic enzyme. In another embodiment of the invention, 0.100 to 2.000 DP0 units/gm of maltogenic enzyme is contacted with the grain containing a starch. In still another embodiment of the invention, 0.100 to 3.000 DP0 units/gm of maltogenic enzyme is contacted with the grain containing a starch. In another embodiment, the amount of exogenous maltogenic enzyme contacted with the substrate containing starch is expressed in kilograms of maltogenic enzyme per metric ton of substrate. In one embodiment, the amount of exogenous maltogenic enzyme contacted with the substrate is about 0.05 kg of maltogenic enzyme per metric ton dry solids basis ("kg/mt dsb"). In another embodiment, the amount of exogenous maltogenic enzyme is about 0.1 kg of maltogenic enzyme per metric ton dry solids basis ("kg/mt dsb"). In other embodiments 0.2, 0.4, 0.6, 0.8. and/or 1.0 kg/mt dsb provide sufficient amounts of maltogenic enzyme, e.g., (3-amylase. In another embodiment, the amount of exogenous maltogenic enzyme contacted with the substrate containing starch is expressed in kilograms of maltogenic enzyme per metric ton of substrate. In one embodiment, the amount of exogenous maltogenic enzyme contacted with the substrate is about 0.05 kg of maltogenic enzyme per metric ton dissolved starch basis ("kg/mt dsb"). In another embodiment, the amount of exogenous maltogenic enzyme is about 0.1 kg of maltogenic enzyme per metric ton dissolved starch basis ("kg/mt dsb"). In other embodiments 0.2, 0.4, 0.6, 0.8. and/or 1.0 kg/mt dissolved starch basis provide sufficient amounts of maltogenic enzyme, e.g., p-amylase. In another embodiment, the amount of maltogenic enzyme to be contacted with the grain is in terms of maltogenic enzyme units. Assays useful to determine the maltogenic activity include those described in the examples and those describing \|3-amylase activity. The term DP° unit refers to the amount of enzyme contained in 0.10 ml of a 5% solution of the sample enzyme preparation that will produce sufficient reducing sugars to reduce 5 ml of Fehling's solution when the sample is incubated with 100 ml of substrate for 1 hour at 20° C (68° F). In another embodiment, a grain having endogenous maltogenic enzymes (barley, wheat, etc.) can be mixed with those grains needing exogenous maltogenic enzymes. Mixtures of 30:70, 60:40, 50:50, 60:40, 70:30 grains having an endogenous maltogenic enzyme: grains utilizing exogenous maltogenic enzyme sources are contemplated by the inventors, so long as sufficient amounts of maltogenic enzymes are present in the mixture (endogenous or exogenous sources). Use of endogenous sources of maltogenic enzymes can reduce the amount of exogenous enzymes added or contacted with the grain mixture. Starch liquefying enzymes A starch liquefying enzyme is contacted with the starch to reduce the viscosity of the liquefied or solubilized starch. In one embodiment of the invention the starch liquefying enzyme is an enzyme selected from the E.G. 3.2.1.1, e.g., alpha amylases. Exemplary alpha-amylases can be derived, isolated or extracted from a bacterial source. In one embodiment, the bacterial source is a Bacillus. In another embodiment, the alpha-amylases derived from Bacillus include those derived from at least one bacterial source selected from B. subtilis. B. licheniformis, B. stearothermophilus. B. coaqulans. B. amvloliquefaciens. and B.. lentus. Those of Bacillus licheniformis and Bacillus stearothermophilus are especially useful. Other amylases are contemplated by the inventors, for example, but not limited to those of EC 3.2.1.133 (U.S.Pat. No. 6,361,809). Other amylases contemplated by the inventors include those characterized by increased oxidative or thermostability, including those mutants or genetically modified or variant amylases described in U.S.Patent Nos. 5,763,385; 5,824,532; 5,958,739; and/or 6,008,026. Useful alpha amylases are those derived from B. licheniformis strains NCIB 8059, ATCC 6598, ATCC 6634, ATCC 8480, ATCC 9945A, ATCC 11945. Useful alpha amylases are those derived from B. stearothermophilus strains ATCC 39709. Such enzymes are identified by the trade names "SPEZYME AA" or "SPEZYME FRED", "SPEZYME LT300", and "SPEZYME LT75", available from Genencor International (Palo Also, California, USA). Other such enzymes include alpha amylases from Bacillus stearothermophilus sold under the tradename GZYME G997, GC007 and from Bacillus licheniformis sold under the tradename GC262 SP, also available from Genencor International. Contacting the grain containing starch with the maltogenic enzyme and the starch liquefying enzyme produces maltose. As understood by those in the art, the quantity of starch liquefying enzyme used in the methods of the present invention will depend on the enzymatic activity of the starch liquefying enzyme. In one embodiment, 0.01 to 25 Liquefon Units/gm of starch liquefying enzyme is contacted with the grain containing starch. In another embodiment, 1 to 10 Liquefon Units/gm of starch liquefying enzyme is contacted with the grain containing a starch. One Liquefon Unit (LU) is the measure of digestion time required to produce a color change with iodine solution, indicating a definite stage of dextrinization of starch substrate under specified conditions. In one embodiment, 0.1 kg of starch liquefying enzyme is added per metric ton of grain dissolved solid basis (kg/mt dsb). In other embodiments, 0.2, 0.4, 0.4, 0.8, or 1.0 kg of starch liquefying enzyme is added per metric ton of grain (kg/mt dissolved starch basis). In one embodiment, 0.1 kg of starch liquefying enzyme is added per metric ton of grain dissolved starch basis (kg/mt dissolved starch basis). In other embodiments, 0.2, 0.4, 0.4, 0.8, or 1.0 kg of starch liquefying enzyme is added per metric ton of grain (kg/mt dissolved starch basis). Assays useful to determine the starch liquefying activity include those described in the examples herein. Exemplary assays for the determination of a-amylase activity are also described in U.S. Pat. Nos. 5,763,385; 5,824,532; 5,958,739; and/or 6,008,026 which are incorporated by reference herein. Transglucosidic enzyme Contacting the maltose with a transglucosidic enzyme obtains a grain composition having an enzymatically produced isomalto-oligosaccharide, derived from the grain containing starch. The transglucosidic enzyme catalyzes hydrolytic and transfer reactions on incubation with alpha-D-gluco- oligosaccharides to produce isomaltose, panose, kojibiose or nigerose. The presence of these sugars and thus conversion by the transglucosidic enzyme is indicated in an increased amount of DP2 disaccharides. The transglucosidic enzyme (E.C. 2.4.1.24) can be transglucosidase. Exemplary transglucosidase enzymes are identified as TRANSGLUCOSIDASE L-1000 (Genencor International, Inc.) and TRANSGLUCOSIDE L by Amano Enzymes, Inc., (Nagoya, Japan). In one embodiment the transglucosidic enzyme is derived from a filamentous fungal source, e.g., Aspergillus SP. The transglucosidic enzyme that is derived from Aspergillus can be derived from Aspergillus niger. In one embodiment, the Aspergillus niger strain is ATCC14916. In this embodiment, a sufficient amount of the transglucosidic enzyme is contacted with the substrate, e.g. the grain containing a starch to produce maltose. As understood by those in the art, the quantity of transglucosidic enzyme used in the methods of the present invention will depend on the enzymatic activity of the alpha amylase. In one embodiment, 0.01 to 25.00 transglucosidase units ("TGU")/gm of transglucosidase is contacted with the grain containing a starch. In another embodiment of the invention, 0.05 TGU to 10.00 TGU/gm of transglucosidase is contacted with the grain containing a starch. In still another embodiment of the invention, 0.10 to 5.00 TGU /gm of grain is contacted with the grain containing a starch. The term TGU refers to the activity of the enzyme required to produce one micromole of panose per minute under the conditions of the assay. In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt dsb). In another embodiment, 0.10 to 5.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt dsb). In another embodiment, 0.25 to 3.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt dsb). In another embodiment, 0.50 to 1.50 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt dsb). Additional assays useful to determine the transglucosidic activity include those described in the Examples and in Shetty, J., et al (U.S. Pat. No. 4,575,487 (1986) entitled, "Method for determination of transglucosidase"), which are incorporated by reference herein. In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is added per metric ton of dissolved starch (kg/mt starch dsb). In another embodiment, 0.10 to 5.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt starch dsb). In another embodiment, 0.25 to 3.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt starch dsb). In another embodiment, 0.50 to 1.50 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt starch dsb). As a result of transglucosidase reactions, the malto-oligosaccharides are converted to isomalto-oligosaccharides resulting in a new class of polysaccharides containing higher proportion of glucosyl residues linked to a primary hydroxyl group of a glucose molecule from the non-reducing end . Isomalto-oligosaccharides produced by this method include isomaltose, panose, isomalto-triose, isomalto-tetrose, isomalto-pentose, isomalto-hexose and isomalto-heptose. These sugars are receiving increased attention as food additives because they help prevent dental caries (Oshima, et.al 1988, The caries inhibitory effects of gos-sugar in vitro and rat experiments. Microbial Immunol. 32.1093-1105 )) and improve human intestinal microflora acting as a growth factor (prebiotic) for bifidobacteria (Komoto.et.al 1988; Effect of Isomalto- oligosaccharides on human fecal flora .Bifidobacteria Micro flora 7,61-69). To ascertain the production of the IMO's, assays and/or other analytical methods can be used to determine the amount of IMO produced. One method for determining the levels of IMO produced includes high performance liquid chromatography (HPLC). For example, analysis of the mixture can provide an indication of the levels of the various sugars produced by the process. A useful rating is the degree of polymerization (DP) of the mixture. The term degree of polymerization is a measure of ther relative amounts of the number of glucose residues in the molecule. For example, glucose (one glucosyl unit, the lowest level of polymerization) is usually found as DP1. Isomalto-oligosaccharides are usually found in DP2 (two glucosyl units). In one embodiment, the grain composition contains greater than at least 1%, at least 5%, at least 25%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% by weight of at least one isomalto-oligosaccharide. In one embodiment the at least one isomalto-oligosaccharide is selected from the group of isomaltose, panose and/or isomalto-triose. In one embodiment, the amount of isomalto- oligosaccharides produced in the grain composition is between 1% and 99% of the grain composition. In one embodiment, the amount of isomalto- oligosaccharides produced in the grain composition is between 1 % and 90% of the grain composition. In one embodiment, the amount of isomalto- oligosaccharides produced in the grain composition is between 1 % and 80% of the grain composition. In one embodiment, the amount of isomalto- oligosaccharides produced in the grain composition is between 1% and 70% of the grain composition. In one embodiment of the present invention, the total sugar present in the grain composition after the above described procedure includes a level of maltose in the total sugar content of greater than 50%, greater than 60%, greater than 70%, or greater than 80%. Levels of maltose greater than 50% include ranges from 50% to 85%, from 55% to 80%, and/or from 60% to 75%. The term RBS ratio refers to the ratio of maltose (DP2) present in the grain as compared to the level of maltotriose (DPS) present in the resultant grain composition. A higher RBS value indicates a higher amount of maltose present and thus a more complete conversion of the starch to maltose as opposed to the presence of other less desireable end-products, e.g., maltotriose. In one embodiment, the RBS ratio is greater than 2.0. In one embodiments, the RBS ratio is greater than 3.0, greater than 4.0. Exemplary ranges include an RBS ratio of 2.0 to 50.0, 2.0 to 30.0 and/or 2.0 to 10.0. Various RBS ratios are described in the examples. It is noted that the hydrolysis of liquefied starch by commercial beta-amylases (barley or wheat) generally produces a maltose content between 55% and 65%. For maltose content greater than 50% using liquefied starch, the addition of debranching enzyme and/or a very low starting DE of the liquefied starch was previously required. Optionally, the addition of a debranching enzyme can be used to increase the production of maltose. The term debranching enzyme refers to enzymes that catalyze the hydrolysis of a-1,6-linkages. An enzyme of the class E.C.3.2.1.41 is useful in this regard. An exemplary enzyme of this class is a pullanase, also known as a-dextrin endo-1,6- a glucosidase, limit dextrinase, debranching enzyme, amylopectin 1,6-glucanohydrolase. Contacting the grain containing starch with a maltogenic enzyme to produce the maltose and the contacting of the maltose with a transglucosidic enzyme occurs at a temperature less than the gelatinization temperature of the starch of the grain used. The enzymes are contacted or incubated with the respective enzymes for an incubation time of at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours and/or at least 36 hours. The period of at least a noted time refers to a period of 12-80 hours, at least 18-60 hours and/or at least 24-48 hours. The term incubation time refers to the period of time for the conversion of maltose or the substrate to IMO's. The transglucosidic enzyme can be contacted or added separately or concurrently with the substrate, e.g. grain containing starch, the maltogenic enzyme, e.g., the alpha amylase or beta amylase, and/or the liquefying enzyme, e.g., the alpha amylase. In one embodiment, the transglucosidic enzyme is added concurrently with the liquefying enzyme. Thus in one embodiment, steps (a) and (b) are performed concurrently. In another embodiment, the steps (a) and (b) are performed sequentially or separately. In another embodiment, the step (a) is performed before step (b). The term gelatinization temperature refers to the temperature at which the starch contained within the grain changes phases or gelatinizes. While the specific temperature varies from grain to grain, temperatures sufficient to effect the gelatinization of starch include those greater than 45° C, greater than 50° C, greater than 60° C, greater than 70° C, greater than 80° C, and/or greater than 90° C. Exemplary temperatures greater than the indicated gelatinization temperatures include 45° C to 120° C, 50° C to 110° C, 50° to 100° C. In one embodiment, e.g. wheat, the gelatinization temperature is a temperature the grain is kept below, e.g. a temperature selected from below 50° C to 70° C, in another embodiment below 55° C to 65° C, and in another embodiment, below 60° C. For example, gelatinization temperatures have been described for corn, potato, wheat, tapioca, waxy maize, sorghum, rice sago, arrowroot amylomaize and/or sweet potato as shown in Table 1 (Beynum, G.M.A In another embodiment of the present invention, the slurry, after the incubation time, can be subjected to a flash heat period sufficient to halt further enzymatic activity, but not gelatinize or liquefy the slurry. For example, the slurry can be heated to a temperature of 80°, 85°, 90° 95° or 100° C for a period of 5-60 minutes, 10.0 to 40.0 minutes or 30.0 minutes. Another embodiment of the present invention further includes the step of separating the slurry into insolubles and solubles. The separating step can be by any chromatographic method known in the arts, for example, but not limited to HPLC, size exclusion and/or charge chromatography. Filtering can be used to separate the insolubles from the solubles. The insolubles or entire slurry can be subjected to the drying steps described later in this application. In another embodiment, the solubles resulting from the separating step can be concentrated by evaporation, for example by roto-evaporation, tray drying, etc. The evaporated concentrate can be subjected to carbon treatment (filtered through carbon granules) and /or further chromatographic treatment to provide an isolated IMO liquid concentrate. The isolated IMO concentrate can have an IMO concentration of greater than 75%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, and/or greater than 99%. Another embodiment of the present invention is the use or incorporation of such syrup (the isomalto-oligosaccharides enzymatically derived from the substrate having ungelatinized starch) in oral rehydration solutions. The amount of the isomalto-saccharides can be in the amounts or formulations as described as U.S.Pat. Nos. 4,981,687; 5,096,894; and/or 5,733,579. Another embodiment of the present invention is the drying of the aforementioned isomalto-oligosaccharide substrate, grain or tuber composition to produce a powder including the grain composition. Typically, this drying step is accelerated by heating. The grain composition can be dried to a desired moisture level by using a suitable drying method, for example, but not limited to a spray dryer, tray dryer, tumble dryer, drum dryer or cabinet dryer. Other drying methodologies can be used, for example spray drying, evaporative drying under reduced pressure. By drying the grain composition, slurry, separated insolubles, and/or separated solubles, a flour or other dried powder is obtained therefrom. The resulting powder or flour can be incorporated into compositions in which the presence of isomalto-oligosaccharides is desired, for example in food stuffs (breakfast cereals, pastas), food additives and baked goods. The term food additive refers to the use of the isomalto-oligosaccharides as a sprinkle on material, as an ingredient for use in the manufacture of other foods, and/or a topical ingredient added to the food. In another embodiment, the dried powder can be incorporated into food supplements. The incorporation of the dried powder into a food supplement can be provided in any acceptable supplement or form. The dietary supplements can be formulated for oral administration in a matrix as, for example but not limited to, drug powders, crystals, granules, small particles (which include particles sized on the order of micrometers, such as microspheres and microcapsules), particles (which include particles sized on the order of millimeters), beads, microbeads, pellets, pills, microtablets, compressed tablets or tablettriturates, molded tablets or tablet triturates, and in capsules, which are either hard or soft and contain the composition as a powder, particle, bead, solution or suspension. The dietary supplement can also be formulated for oral administration as a solution or suspension in an aqueous liquid, as a liquid incorporated into a gel capsule or as any other convenient form for administration or for rectal administration, as a suppository, enema or other convenient form. The isomalto- oligosaccharide composition can also be provided as a controlled release system. The dietary supplement formulation can also include any type of acceptable exicipients, additives or vehicles. For example, but not by way of limitation, diluents or fillers, such as dextrates, dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose, inositol, powdered sugar, bentonitc, microcrystalline cellulose, or hydroxypropyl methylcellulose may be added to isomalto-oligosacccharide composition to increase the bulk of the composition. Also, binders, such as, but not limited to, starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone,Veegurn and starch arabogalactan, polyethylene glycol, ethylcellulose, glycerylmonostearate and waxes, may be added to the formulation to increase its cohesive qualities. Additionally, lubricants, such as, but not limited to, glyceryl monostereate, talc, magnesium15 stearate, calcium stearate, stearic acid, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, carbowax, sodium laurylsulfate, and magnesium lauryl sulfate may be added to the formulation. Also, glidants, such as but not limited to, colloidal silicon dioxide, magnesium silicate or talc may be added to improve the flow characteristics of a powdered formulation. Finally, disintegrants, for example, but not limited to, starches, clays, celluloses, algins, gums, crosslinked polymers (e.g.,croscarmelose, crospovidone, and sodium starch glycolate), Veegum, methylcellulose, agar,benton:ite, cellulose and wood products, natural sponge, cation-exchange resins, alginicacid, guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfate with starchmay also be added to facilitate disintegration of the formulation in the stomach or intestine. Another embodiment of the present invention is the use of the novel substrate, tuber or grain composition described herein in the production of flour for use in various baked goods. The term baked goods refers to leavened and unleavened goods. The term leavened refers to baked goods using yeast in the baking process. Whereas the term unleavened means baked goods not using yeast in the baking process. Exemplary goods include bread, cookies, cakes, pies, biscuits, naan, bagels, pasta, crackers, rolls, donuts, pitas and pastries. Exemplary unlevened goods include matzoh, chapathi, breakfast cereals and torillas. Another embodiment of the present invention is the use of the novel grain compositions in pasta, for example, noodles (penne, spagetti, lasagna, udon, etc.). Another embodiment of the present invention is a substrate, tuber or grain composition made according to the above described method. Another embodiment of the present invention is a flour comprising the substrate, tuber or grain composition made according to the above described method. Another embodiment of the present invention is an oral rehydration solution comprising the isomalto-oligosaccharide described above. Flour comprising the substrate, tuber or grain composition can be made according to the above described method. Another embodiment of the present invention is a substrate, tuber or grain composition made according to the above described method. Another embodiment of the present invention is a substrate, tuber or grain composition made according to the above described method. Another embodiment of the present invention is the use of the novel grain compositions in fermentive/beer worts or substrates. For example, the novel grain composition can be used as described in beer fermentation as described in International Publication No WO 02/20712 A1, which is incorporated by reference herein. The novel grain compositions can also be incorporated in beer adjuncts. The isomalto-oligosaccharide containing substrate can also be subjected to an additional step of recovering the maltose by extraction and isolation of the generated maltose, for example as a maltose syrup. The syrup can be extracted and/or isolated from the grain composition by methods familiar in the art, for example in U.S.Pat. Nos. 3,922,196 and 4,113,509, which are incorporated by reference herein. Another route to enhance the sweetness or the isomalto-oligosaccharide content, is to treat the produced isomalto-oligosaccharide syrup with a hydrolase (in soluble or immobilised form) which hydrolyses preferentially or even exclusively malto-oligosaccharides, and has only a small or even no affinity for isomalto-oligosaccharides. Examples of such an enzyme is glucoamylase from A. niqer or other sources like Aspergillus sp. or Rhizopus sp. which preferentially hydrolyses malto-oligosaccharides ( Manjunath P., Shenoy B. C, Raghavendra Rao M. R., Journal of Applied Biochemistry, 5(1983),235-260; Meagher M. M., et al., Biotechnology and Bioengineering, 34(1989), 681-693; Pazur J. H., Kleppe K., The Journal of Biological Chemistry, 237(4)(1962),1002-1006; Hiromi K., Nitta Y., et al.,Biochimica et Biophysica Acta, 302(1973),362-37). Also an enzyme like the alpha-D-glucopyranosidase from Bacillus stearothermophilus can be applied. This enzyme is not capable of hydrolysing isomalto-oligosaccharides and will only degrade the malto-oligosaccharides present in the isomalto-oligosaccharide rich syrup (Suzuki Y., Shinji M., Nobuyuki E., Biochimica et Biophysica Acta, 787(1984),281 -289). Also other alpha-D-glucosidases which are called maltases can be used. The maltase from yeast for example will only hydrolyse maltose and to a lesser extent maltotriose (Kelly C. T., Fogarty W. M., Process Biochemistry, May/June(1983),6-12). After the hydrolysis of the malto-oligosaccharides to glucose, the syrup can be enriched in isomalto-oligosaccharides by a chromatographic technique or by nano- or ultra-filtration. The following examples serve to illustrate the main embodiments of this invention. EXAMPLES The following specific examples further illustrate the compositions and the methods of the invention. It is to be understood that these examples are for illustrative purposes only and can be applied to any other suitable materials rich in starch and containing endogenous maltose producing enzyme, for example, wheat, rice, barley, malt, potato, sweet potato, etc. Enzyme Activity Determination The transglucosidase activity is measured by the method of Shetty, J., et al, 1986 (U.S. Pat. No. 4,575,487). The beta amylase activity was measured by a 30-minute hydrolysis of a starch substrate at pH 4.6 and 20° C. The reducing sugar groups produced on hydrolysis are measured in titrimetirc procedure using alkaline ferricyanide. One unit of diastase activity, expressed as degrees DP refers to the amount of enzyme, contained in 0.1 ml of 5% solution of the sample enzyme preparation, that will produce sufficient reducing sugars to reduce 5 mL of Fehlings' solution when the sample is incubated with 100 mL of the substrate for 1 hour at 20 C. The alpha amylase activity was developed based on an end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd. A vial of substrate (p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10ml of sterile water followed by a 1:4 dilution in assay buffer (50mM maleate buffer, pH 6.7, 5mM calcium chloride, 0.002% Tween20). Assays were performed by adding 10µl of amylase to 790µl of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the rate of change of absorbance at 41 Onm, after a delay of 75 seconds. The assay was linear up to rates of 0.2 absorption units/min. a-Amylase protein concentration was measured using the standard Bio-Rad Assay (Bio-Rad Laboratories) based on the method of Bradford, Anal. Biochem.. Vol. 72, p. 248 (1976) using bovine serum albumin standards. Substrates: The wheat flour used as substrates in all examples, was purchased from retail commercial stores . Other tuber or grain substrates, e.g., rice and barley used as substrates may be purchased from commercial sources (Huai An Liujun Food processing company, Jiangshu province, China). Oligosaccharide Analysis The composition of the reaction products of oligosaccharides was measured by HPLC (Agilent 1010, Palo Alto, California, USA) equipped with a HPLC column (Rezex 8 u8% H, Monosaccharides), maintained at 60°C fitted with a refractive index (Rl) detector (ERC-7515A, Rl Detector from The Anspec Company, Inc.). Dilute sulfuric acid (0.01 N) was used as the mobile phase at a flow rate of 0.6 ml per minute. Twenty microliter of 4.0% solution was injected on to the column. The column separates based on the molecular weight of the saccharides. For example a designation of DP1 is a monosacchride, such as glucose; a designation of DP2 is a disaccharide, such as maltose; a designation of DP3 is a trisaccharide, such as maltotriose and the designation DP4+ is an oligosaccharide having a degree of polymerization (DP) of 4 or greater. The term Higher sugar ("Hr. Sugar") refers to sugars having DP greater than 3. For iso-saccharides or branched sugars.the reaction products were measured by HPLC (Agilent 1010, Palo Alto, California, USA) equipped with a HPLC column (Shodex Rspak Oligosaccharide Column #DC-613), maintained at 50°C fitted with a refractive index (Rl) detector (ERC-7515A, Rl Detector from The Anspec Company, Inc.). 70 (Acetonitrile):25 (methanol):5 Water was used as the mobile phase at a flow rate of 2.5 ml per minute. Twenty microliter of 4.0% solution was injected on to the column. The column separates based on the molecular weight of the saccharides. The standard sugars, glucose, maltose, maltotriose, isomaltose, panose and isomalto-triose (Sigma Chemicals, St. Louis, Missouri, USA) were used to calibrate the column. EXAMPLE 1 Maltose production from wheat flour by alpha amylase from Bacillus licheniformis (an alpha amylase sold under the tradename GC262SP by Genencor International, Palo Alto, CA) and Bacillus stearothermophilus (an alpha amylase sold under the tradename GC007 by Genencor International, Palo Alto, CA) were compared. One hundred fifty grams of wheat flour from commercial retail sources was suspended in 450 ml of deionized water. The suspension was stirred for 15 minutes at room temperature for uniform mixing (pH 5.5). The pH was adjusted with 6.0 N sulphuric acid (H2SO4). The resultant suspension was kept in a water bath maintained at 60° C and stirred for uniform mixing before the enzymes were added.. About 6,000 LU/g of amylase from Bacillus stearothermophilus (0.6 kg of GC007 [from Genencor International. Inc.]/Metric ton (Mt) starch dsb) and 15,100 LU/g of amylase from Bacillus licheniformis (0.6 kg GC262 SP from [Genencor International. Inc.]/Mt. starch dsb) were added separately and incubated with constant stirring at 60° C . Samples were withdrawn at different predetermined intervals of time and analyzed for total sugar composition using high-pressure liquid chromatography (HPLC). Two ml of sample was taken from each container at a predetermined time interval using a plastic pipette and transferred to a centrifuge tube. The sample was centrifuged at 8000 prm for 3 minutes. The supernatent was withdrawn from the centrifuge tube and a few drops were placed into a sample well of a Lecia AR200 (Leica Microsystems, Inc., Buffalo, NY, USA) digital hand held refractometer and recorded (Table 2). The Brix (as a measure of the dissolved sugars) of the solution was determined (Table 2). Table 2 The results in the table 2 showed that wheat flour incubated with alpha amylase from Bacillus stearothermophilus gave a higher maltose content compared to the maltose content from the incubation with alpha amylase from Bacillus licheniformis. Incubation of wheat flour with alpha amylases resulted in a significant increase in the dissolved solids due to the hydrolysis of the granular starch compared to incubation of wheat flour without alpha amylase addition. It is interesting to note here that the reaction product of alpha amylase from Bacillus stearothermophilus resulted in a higher ratio of maltose to glucose and maltose to maltotriose compared to alpha amylase from Bacillus licheniformis. These results indicate that the alpha amylase from Bacillus stearothermophilus is an especially useful enzyme for producing very high maltose syrup. EXAMPLE 2 Effect of Bacillus stearothermophilus alpha amylase (alpha amylase sold under the tradename GC 007by Genencor International, Palo Alto, CA) concentration on the maltose production during incubation with wheat flour. The experimental conditions were identical as explained in Example 1. In addition, Bacillus stearothermophilus (6,000 Units/g) was added at 0.1 Kg, 0.2 Kg and 0.6 Kg /MT of starch dsb. The results are summarized in table 3. Table 3 Effect of Alpha Amylase [GC007] Concentration on Maltose Yield during incubation of wheat flour, pH 5.5, 60° C EXAMPLE 3 One hundred fifty grams of wheat flour was suspended in 450 ml of deionized water and the pH was adjusted to pH 5.00, 4.50 and 4.00 using 6.0 N H2S04. The slurry was stirred well for uniform mixing and the pH was adjusted until the specified pH was stabilized. GC007 was added at 0.1 Kgs/MT, starch dsb to each of the trial and incubated at 60° C. The samples were withdrawn at different predetermined intervals of time and the composition of the sugar and brix were measured as described in Example 1. (Table 4). The maltose content increased with decreasing pH of the incubation of the wheat flour from pH 5.5 and reached maximum of about 68% at pH 4.5 followed by a decrease at pH 4.0. This is an unexpected result showing the production of maltose content greater than 60 % without the addition of a debranching enzyme during the hydrolysis of starch with plant beta amylases. The hydrolysis of liquefied starch by commercial Beta amylases (barley or wheat) generally produces maltose content between 55% and 60 %. For maltose content greater than 60 % using liquefied starch, the addition of debranching enzyme and or a very low starting DE of the liquefied starch are required. It is also important to note here that the process described in this invention allows maltose manufacturers to process at pH 4.5 and 60° C that reduces the high risk of microbial contamination of the current process. EXAMPLE 4 One hundred fifty grams of wheat flour was suspended in 450 ml of deionized water and the pH of the slurry was adjusted to pH 4.5. The slurry was stirred well for uniform mixing and the pH was adjusted with 6.0 N H2SO4 until the pH was stabilized. The resultant suspension was kept in a water bath maintained at 60° C and stirred for uniform mixing before the enzymes were added. A starch liquefying enzyme, e.g., a Bacillus stearothermophilus alpha amylase sold under the tradename "GC 007" (Genencor International, Inc.) was added at 0.1 Kg/MT, dsb. A debranching enzyme, a pullulanase sold under the tradename OPTIMAX L-1000 (Genencor International, Inc.) was then added at 0.25 Kg, 0.5 Kg and 1.0 Kg/M T dsb and incubated at 60° C. The samples were withdrawn at predetermined different intervals of time (2, 4, 6 and 24 hours) and the composition of the sugar and brix were measured as described in Example 1. The results were recorded (Table 5). Maltogenic enzymes (such as beta amylases) or starch liquefying alpha amylases (such as GC007) can not hydrolyze the alpha 1-6 glucosidic linkages, the branch point in the amylopectin of the starch substrate. So it is a common practice to add debranching enzyme, pullulanase (OPTIMAX L-1000 from Genencor International Inc) for producing maltose greater than 65 % during the incubation of starch substrate with beta amylase. The effect of OPTIMAX L-1000 concentration during the incubation of wheat flour with GC007 was studied and the results were shown in Table 5. OPTIMAX L-1000 addition resulted in a significantly higher level (>75 %) of maltose (DP2) compared to the control. EXAMPLE 5 It is generally the common practice in the industry to use high maltose syrup produced by an enzymatic process using a high temperature (>90° C) enzyme liquefied starch substrate followed by treating with enzyme glucosyltransferase for producing isomalto-oligosaccharides syrups. This example illustrates the process of converting the granular starch in the wheat flour into isomalto-oligosaccahrides in a single step. In this example, 275 grams of wheat flour was placed in a flask and 688 ml of deionized water was added. It was then stirred for 15 minutes for uniform mixing and the pH was then adjusted to pH 4.5 using 6.0 N H2SO4. The resultant suspension was kept in a water bath maintained at 60 C and stirred for uniform mixing before the enzymes were added. A starch liquefying enzyme, e.g., Bacillus stearothermophilus alpha amylase ([GC007 supplied by Genencor International] (0.1 Kgs /MT dsb) and a debranching enzyme, e.g., a pullulanase (OPTIMAX L-1000 supplied by Genencor International) (0.5 kgs/MT dsb) were added. The suspension was then divided into two equal parts. To one of the parts, an Aspergillus niqer transglucosidase sold under the tradename TRANSGLUCOSIDASE L-500" (Genencor International) was added at 1.0 Kg/MT dsb and kept in a wafer bath maintained at 60° C (Sample1). The other part was incubated first for four hours at 60°, followed by the addition of Aspergillus niaer transglucosidase sold under the tradename "TRANSGLUCOSIDASE L-500" (Genencor International) at 1.0 Kg/MT dsb and maintenance in a water bath maintained at 60° C (Sample 2). The results shown in Table 6 indicate that conversion of the substrate to IMO's occurs with or without preincubation of the substrate (wheat flour) prior to the addition of the transglucosidase. Incubation of modified wheat flour containing high content of maltose with Translucosidase produced isomalto-oligosaccharides identical to the composition produced by the conventional process. The process is simple, economical and can be easily scaled to commercial production EXAMPLE 6 It is common knowledge that cereals like wheat, barley and rye contain high levels of beta-amylase. Incubation of these cereals at 55°-60° C, pH 5.5 generally results in syrups containing greater than 50% maltose. A 28% slurry of wheat, barley and rye flour, respectively, was each prepared by adding 280 grams of the respective flour to 720 gm of deionized water. To each of these preparations, a Bacillus stearothermophilus alpha amylase (e.g., a Bacillus stearothermophilus alpha amylase sold under the trademark "GC007" by Genencor International) was added at 0.2 kg/MT of the flour. The pH was then adjusted to pH 5.5 using 6.0 N H2SO4 and incubated at 60° C for 4.5 hours. The pH of the incubated samples was then adjusted to pH 4.5 using 6 N H2SO4 and 1.25 kg of transglulcosidase (e.g., a transglucosidase sold under the tradename TRANSGLUCOSIDASE L-500 by Genencor International) / MT of the flour was added. The slurries were then incubated at 60° C water bath for 48 hours. The samples were then centrifuged and analyzed for I MO composition (Table 7) as set forth in Example 1. EXAMPLE 7 In an experiment, 140 grams of malt (Cargill Malt/Schreier-Malting Company, Wisconsin, USA) was mixed with 360 grams of distilled water. The slurry was stirred for 15.0 minutes at room temperature for uniform mixing and pH was then adjusted to pH 4.5 using dilute acetic acid. After stabilization of the pH, the slurry was kept in a water bath maintained at 60° C. Incubation was continued for 30.0 minutes with constant stirring and a 2 ml sample was withdrawn for Brix and HPLC analysis (0, time). Transglucosidase L-500 was added at 1.5 kg/MT malt and incubated at 60° C. Samples were withdrawn at different predetermined intervals of time during incubation, e.g., 2, 4, 6, 12,and 24 hours, to ascertain Brix and IMO composition (Table 8) as described in Example 1. The results in Table 8 showed that the commercial malt extract can be used as a suitable substrate for producing malt extract containing isomalto- oligosaccharides. The reaction time and the composition of IMO sugars of the malt extract could be adjusted by controlling the enzyme dosage. The addition of maltogenic enzymes can increase IMO content of the resulting composition. EXAMPLE 8: Sorghum, Millet and Rice (Exogenous Maltogenic enzymes) In another experiment, 280 grams of sorghum, millet and rice flour were each taken separately and mixed separately with 720 grams of deionized water. The pH of the suspension was adjusted to pH 5.5 and a Bacillus stearotherphilus alpha amylase sold under the trademark "GC007" (Genencor International) was added at 0.5 kg/mt of the flour. After uniform mixing, the suspension was kept in a water bath maintained at 75° C. The reaction mixture was continuously stirred during incubation for 6 hours. The temperature was then reduced to 60° C and a beta-amylase (sold under the tradename OPTIMALT BBA by Genencor International) was added at 1.0 kg/mt of the flour. The incubation was continued for additional 10-15 hours (a sample was taken for Brix and HPLC). After the specified time, the pH was reduced to pH 4.5 by 6N H2SO4 and an Aspergillus niger transglucosidase (sold under the tradename "TRANSGLUCOSIDASE L-500" by Genencor International) was added at 1.0 kg/mt flour. Samples were taken at 24 and 48 hrs. for analysis (Table 9). As shown in Table 9, IMO Numbers of 41 to 54 % (52.20, 53.03, 43.90, 41.19, 54.40, and 51.25) were achieved. EXAMPLE 9: Mixed Grain/Cereals Composition The data in Example 5 for wheat and Example 6 for barley and rye showed considerable amount of endogeneous maltogenic enzyme activity resulting in a syrup containing very high maltose. On the other hand, grains known to not contain endogenous maltogenic enzymes, for example sorghum, millet and rice, required the addition of exogenous maltogenic enzyme for producing the substrate suitable for transglucosidase treatment. In this experiment we studied the supplementation of maltogenic enzyme containing cereals like wheat or barley with sorghum and rice for converting the starch to substrates containing high maltose levels. In a typical experiment, a 15% suspension of sorghum and rice was prepared by suspending 140 grams of the flour in 720 grams of deionized water. The pH was adjusted to pH 5.5 using 6 N H2SO4 and Bacillus sterarothermophilus alpha amylase sold under the trademark "GC007" (Genencor International) was added at 0.5 kg/mt of the flour. The resultant suspension was then left in a water bath maintained at 75° C. The suspension was stirred continuously for 6 hours. The temperature was then reduced to 60° C. Solid content of flour, e.g., pre-treated rice flour, was increased from 15% to 30% by the addition of barley flour. Similarly, wheat was added to pre-treated sorghum to a final concentration to reach 30%. The incubation was then continued for an additional 10-12 hrs. at 60° C. The pH was reduced to 4.5 and TRANSGLUCOSIDASE L-500 was added at 1.0 kg/mt flour. The incubation at 60° C was continued for 24 hours and 48 hours. The samples were taken for HPLC analysis and brix; the results are shown in Table 10. As shown in Table 10 above, the mixtures of millet and barley; and rice and wheat resulted in 45 to 56 % IMO in the resultant suspension after the above described incubation periods. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. WE CLAIM : 1. A method for making an isomalto-oligosaccharide grain composition said method comprising: (a) contacting an ungelatinized starch in grain (insoluble starch) such as herein described with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature, such as herein described; and (c) obtaining a grain composition having an enzymatically produced isomalto oligosaccharide, wherein said oligosaccharide is present in said grain. 2. The method as claimed in claim 1, wherein said steps (a) and (b) occur concurrently. 3. The method as claimed in claim 1, optionally comprising the step of drying said grain composition. 4. The method as claimed in claim 1, wherein said grain is selected from the group consisting of wheat, rye, barley, and malt. 5. The method as claimed in claim 1, wherein said grain is selected from the group consisting of millet, sorghum and rice. 6. The method as claimed in claim 1, wherein said maltogenic enzyme is a beta amylase. 7. The method as claimed in claim 1, wherein said maltogenic enzyme is endogenous to said grain. 8. The method as claimed in claim 1, wherein said starch liquefying enzyme is an alpha amylase obtained from a Bacillus. 9. The method as claimed in claim 8, wherein said starch liquefying enzyme is obtained from Bacillus licheniformis or Bacillus stearothermophilus. 10. The method as claimed in claim 1, wherein said transglucosidic enzyme is a transglucosidase. 11. The method as claimed in claim 10, wherein said transglucosidase is obtained from Aspergillus. 12. The method as claimed in claim 11, wherein said Aspergillus is Aspergillus niger. 13. A grain composition produced as claimed in claim 1. 14. A food additive comprising said grain composition as claimed in claim 13. 15. A flour comprising said grain composition as claimed in claim 13. 16. An isomalto oligosaccharide made as claimed in claim 1. 17. An oral rehydration solution comprising the isomalto oligosaccharide as claimed in claim 16. Methods for the production of substrate, tuber, and grain compositions containing isomalto-oligosaccharides are described. The methods comprise sequentially or concurrently (a) contacting a substrate, tuber or grain containing ungelatinized starch with an exogenous or endogenous maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto- oligosaccharide, wherein the oligosaccharide is derived from the grain. Figures 1 and 2.

Full Text

METHOD FOR MAKING AN ISOMALTO-OLIGOSACCHARIDE GRAIN COMPOSITION
TECHNICAL FIELD
The present invention describes grain compositions containing isomalto-
oligosaccharides and methods for making the same. The method includes the
derivation of the isomalto-oligosaccharides from the starch contained within the
grain.
BACKGROUND OF THE INVENTION
Isomalto-oligosaccharides ("IMOs") are mixed linkage oligosaccharides, having
mixtures of 1,4 alpha and/or 1,6 alpha glucosidic linkages. They are also known
as anomalously linked oligosaccharides ("ALOs"). Isomalto-oligosaccharides
contain a substantial amount of branched oligo-saccharides such as isomaltose,
panose, isomaltotriose, isomaltotetraose, isopanose and hiaher branched oligo-
saccharides.
There is a market demand for products containing IMO's. I MO products are sold
in powder or liquid form, depending on the intended application. The potential
applications are situated in the food area. Examples of IMO products are:
seasonings (mayonnaise, vinegar, soup base etc.), confectionery (candy,
chewing gum, chocolate, ice cream, sherbet, syrup), processed foods of fruits
and vegetables (jam, marmalade, fruit sauces, pickles), meat or fish foods (ham,
sausage, etc.), bakery products (bread, cake, cookie, pastry), precooked foods
(salad, boiled beans, etc.), canned and bottled foods, convenience foods (instant
coffee, instant cake base, etc.), and beverages, both alcoholic (liquor, seju, wine,
sake, beer [International Publication No. WO 02/20712 A1], etc.) and non-
alcoholic (coffee, juice, nectar, aerated or carbonated drinks, lemonade, cola).
Isomalto-oligosaccharide can further be applied as ingredients in animal feed

and pet foods. Non-food application areas are cosmetics and medicine
(cigarette, lipstick, toothpaste, internal medicine, etc.).
Isomalto-oligosaccharides belong to a group of oligosaccharides classified as
functional-health food oligosaccharides ("FHFO"). Exemplary IMO's include
fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides and
gentio-oligosaccharides. IMO's have been linked to the increase of the general
well being of humans and animals when taken orally on a regular dally basis and
are classified as "prebiotics". Prebiotics are defined as non-digestible
substances (e.g., dietary fiber) that exert some biological effect on humans by
selective stimulation of growth or bioactivity of beneficial microorganisms either
present or therapeutically introduced to the intestine. (Przemyslaw Jan Tomasik
and Plotr Tomasik. 2003 American Association of Cereal Chemists, Inc. 80(2):
113-117). The "prebiotic" action of the oligosaccharides is to increase the
numbers of bifidobacteria and lactobacilli ("prebiotic") in the large intestine and to
reduce the concentration of putrifactive bacteria. Bifidobacteria are associated
with some health promoting properties like the inhibition of the growth of
pathogens, either by acid formation or by anti-microbial activity. They are also
associated with such diverse effects as the modulation of the immune system
(anti-tumor properties), the reduction of the levels of triglycerides and
cholesterol, the production of vitamins (B group), the reduction of blood
ammonia concentrations, the prevention of translocation, the restoration of the
normal gut flora after anti-microbial therapy, the production of digestive
enzymes, the reduction of antibiotic associated side effects (Kohmoto T., Fukui
F., Takaku H., Machida Y., et al., Bifidobacteria Microflora, 7(2)(1988),61-69;
Kohmoto K., Tsuji K., Kaneko T., Shiota M., et al., Biosc. Biotech. Biochem.,
56(6)(1992),937-940; Kaneko T., Kohmoto T., Kikuchi H., Fukui F., et al., Nippon
Nogeikagaku Kaishi, 66(8)(1992),1211-1220, Park J-H, Jin-Young Y., Ok-Ho S.,
Hyun-Kyung S., et al., Kor. J. Appl. Microbiol. Biotechnol., 20(3X1992). 237-
242). Modler, H.W., 1992, "Compounds which enhance the growth of Prebiotic
Bacteria", presented at the International Roundtable on Animal Feed
Biotechnology, Ottawa, Ontario, Canada.

The isomalto-oligosaccharides are synthesized by an enzyme catalyzed
transglucosylation reaction using a D-glucosyltransferase (E.C. 2.4.1.24,
transglucosidase, alpha-glucosidase). This enzyme catalyzes both hydrolytic
and transfer reactions on incubation with alpha-D-gluco-oligosaccharides. The
transfer occurs most frequently to 6-OH (hydroxyl group 6 of the glucose
molecule), producing isomaltose from D-glucose, or panose from maltose. The
enzyme can also transfer to the 2-OH or 3-OH of D-glucose to form kojibiose or
nigerose, or back to 4-OH to reform maltose. As a result of transglucosidase
reactions, the malto-oligosaccharides are converted into isomalto-
oligosaccharides resulting in a class of oligosaccharides containing a higher
proportion of glucose moieties linked to a primary hydroxy! group of a glucose
molecule from the non-reducing end, e.g., by alpha-D-1,6 glucosidic linkages.
The transglucosidase from A. niqer acts only on oligosaccharides with a low
degree polymerization (DP) (McCleary B. V., Gibson T. S., Carbohydrate
Research 185(1989)147-162; Benson C. P., Kelly C.T., Fogarty W. M., J. Chem.
Tech. Biotechnol., 32(1982)790-798; Pazur J. H., Tominaga Y., DeBrosse C. W.,
Jackman L. M., Carbohydrate Research, 61(1978) 279-290). Degree of
polymerization refers to the number of dextrose units. For example, a di-
glucosyl molecule, for example maltose, has a DP of 2. These sugars are
receiving increased attention as food additives because they help prevent dental
caries (Oshima, et.al 1988. The caries inhibitory effects of gos-sugar in vitro and
rat experiments. Microbial Immunol. 32,1093-1105) and improve human
intestinal microflora acting as a growth factor (prebiotic) for bifidobacteria
(Komoto.et.al 1988; Effect of Isomalto-oligosaccharides on human fecal flora
Bifidobacteria Micro flora 7,61-69).
Isomalto-oligosaccharides can be obtained in different ways. For example
glucose syrups at high dry solids concentration i.e. 60-80% are treated with
glucoamylase resulting in the formation of isomalto-oligosaccharides mainly
DP2. The high solids levels are present to force the reaction to reverse from the
normal direction in favor of hydrolysis.

Grains, including wheat, barley, etc., are excellent raw materials in the
commercial production of many value added functional food ingredients such as
wheat flour, starch, starch hydrolysates (glucose, fructose, high maltose syrup,
etc.) and wheat gluten. Syrup containing a high level of maltose is also used in
many microbial fermentations as a carbon source in the production of antibiotics,
pharmaceuticals, vaccines, biochemical, such as alcohol (both potable and fuel),
amino acids, organic acids, etc and recently in the production of functional health
-food oligosaccarides called isomalto-oligosaccharides. In a conventional
process for the production of starch hydrolysate, such as maltose syrups, the
insoluble granular starch is generally separated from other cellular components
of wheat prior to the hydrolysis by starch liquefying and maltogenic alpha
amylases enzymes. Maltose is a disaccharide consisting of two glucosyl
residues linked by a 1-4 D-glucosidic linkage and is the smallest in the family of
malto-oligosaccharides. It is produced on a large scale as syrup, powder and
crystals in several grades of purity. Various maltose syrups are drawing
considerable interest for commercial applications in brewing, baking, soft drink
canning, confectionary and other food and beverage industries. Ultra pure
maltose is used as an intravenous nutrient in Japan. Catalytic reduction of
maltose results in maltitol, which is considered to be a low calorie sweetener.
Recently, high maltose syrup has become a key raw material for industrial
production of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999
Transglucosylation of Malto-oligosaccharides." Paper presented at 50th Starch
Convention, Detmold, Germany).
In a conventional process for the production of starch hydrolysate such as high
maltose syrup, the insoluble starch is separated prior to the hydrolysis by
thermostable liquefying alpha amylases [EC 3.2.1.2,alpha (1,4)-glucan
glucanohydrolase] derived either from Bacillus licheniformis or Bacillus
stearothermophilus. Hydrolysis of the purified starch (refined) is carried out by
suspending insoluble granular starch in water (30-35 % dissolved solid basis
[dsb]) and heated to a temperature of between 85° C and 120 ° C to solubilize
the starch and making it susceptible for enzymatic hydrolysis. The liquefied
starch is further processed to manufacture starch hydrolysate with different

carbohydrate composition using specific maltose producing enzymes such as
fungal alpha amylase (sold under the tradename CLARASE L from Genencor
International, Palo Alto, CA) for syrup containing less than 55% maltose, p
Amylase (sold under the tradename OPTIMALT BBA from Genencor
International, Palo Alto, CA) for syrup containing maltose content between 55%
and 62% and less than1% glucose. For higher levels of maltose syrup, >62%,
addition of debranching enzyme (sold under the tradename OPTIMAX L-1000
from Genencor International, Palo Alto, CA) in conjunction p Amylase is useful.
(Faigh, J.; Duan, G.; Strohm, B. and Shetty, J. (2002) "Production of Maltose,
High Maltose & Very High Maltose Syrups," Technical Bulletin, Genencor
International Inc.).
A process for converting granular starch (refined) into soluble hydrolysate by
incubating with bacterial alpha amylase at a temperature below the starch
gelatinization temperature (Leach et.al 1978; US Pat. No. 4,113,509) and
subsequent hydrolysis by beta amylase to produce high maltose syrup have
been reported (Leach et.al 1975;US Pat. No. 3,922,196), However the syrup
produced by such process resulted in only 55 % maltose of the total sugar
content, with a very high level of maltotriose. The process for producing high
maltose syrup using liquefied starch (gelatinized followed by hydrolysis using
thermostable alpha amylase) is described in European Patent Application
#0905256 (Christophersen, et.al 2000) and U.S. Pat. No. 5,141,859 (Nimmi,
et.al 1992). The process is cumbersome, expensive and it requires the
separation of starch from other cellular components, high cost of the additional
maltose producing enzymes, high temperature treatment and longer reaction
time. European Patent Application #0350737 A2 (Shinke, et.al 1989) disclosed
a process for producing maltose syrup by hydrolyzing a granular (purified) starch
from corn, wheat, potato and sweet potato at 60° C without the conventional
liquefaction step (gelatinization followed by liquefaction at high temperature)
using an alpha amylase from Bacillus stearothermophilus. However, the
hydrolyzed starch resulted in a maltose concentration ranging from 50% to 55%.
The syrup also contained very high level of maltotriose (30-36%). The process
resulted in a ratio of maitose to maltotriose less than 2.0 irrespective of the

source of the starch. Maltose syrup containing a high level of maltotriose is not
a preferred substrate as carbon feed in many microbial fermentations including
the alcohol fermentation by yeast because of the difficulties in metabolizing
maltotriose. Maltose is a preferred donor of glucosyl residue in the
transglucosylation reaction catalyzed by glucosyltransferases in the production
of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999
"Transglucosylation of Malto-oligosaccharides." Paper presented at 50th Starch
Convention, Detmold, Germany). U.S. Pat. No. 6,361,809 described a method
for producing maltose and a limit dextrin by treating the purified granular waxy
maize starch with a hydrolase, maltogenase alpha amylase classified as EC
3.2.1.133 from Bacillus stearothermophilus followed by separating the maltose
using ultra filtration process. Evaporation of the dilute permeate containing the
maltose is expensive because of high energy cost and also faces a very high
risk of microbial contamination.
Traditionally grains such as wheat, malt, sorghum (milo), millet (ragi), particularly
whole grains are used in nutrition as carriers of macro- and micro- elements,
proteins, fiber and vitamins. The majority of cereal grains appeared to be too
readily digested to play an effective role as prebiotics or even as nutraceuticals.
It has been suggested that designing genetically modified, less digestible cereals
suitable as prebiotics to manipulate gut microflora (Gibson, G.R, and Roberfroid,
M. B. 1995, Dietary modulation of the human colonic micrflora: Introducing the
concept of prebiotics. J. Nutr. 125, 1401-1412).
There is a continuing interest in methods for producing grain compositions with
isomalto-oligosaccharides enzymatically derived from the source substrate, e.g.,
grain or tuber, without having to separate the starch from other grain
components and/or subject the starch of the substrate to high temperatures of jet
cooking prior to transglucosidation action. There is also a continuing interest in
low DH processes for minimizing the risk of microbial contamination. The
present invention addresses these interests.

SUMMARY OF THE INVENTION
The present invention describes a method for making an isomalto-
oligosaccharide grain composition said method comprising:(a) contacting a
ungelatinized starch containing grain with a maltogenic enzyme and a starch
liquefying enzyme to produce maltose; (b) contacting said maltose with a
transglucosidic enzyme, wherein said steps (a) and step (b) occur at a
temperature less than or at a starch gelatinization temperature; and (c) obtaining
a grain composition having an enzymatically produced isomalto
oligosaccharide, wherein said oligosaccharide is derived from said grain.
Optionally, in one embodiment, the steps (a) and (b) occur concurrently. In
another embodiment, the method further includes a step of drying said grain
composition. In another embodiment the grain is selected from the group
consisting of wheat, rye, barley, malt and rice. In another embodiment the grain
is selected from the group consisting of sorghum, millet and rice. In another
embodiment, the maltogenic enzyme is a beta amylase. In another
embodiment, the maltogenic enzyme is endogenous to said grain. In another
embodiment, the maltogenic enzyme is exogenous to said grain. In another
embodiment, the starch liquefying enzyme is an alpha amylase derived from a
Bacillus. In another embodiment, the starch liquefying enzyme is derived from
Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment,
the transglucosidic enzyme is a transglucosidase. In another embodiment, the
transglucosidase is derived from Aspergillus. In another embodiment, the
transglucosidase derived from Asoergillus niger. Another embodiment of the
present invention includes a grain composition produced according to above
described method. Another embodiment of the present invention includes a food
additive comprising said grain composition described above.
The present invention also describes a method for making an isomalto-
oligosaccharides enriched flours at temperatures at or below the gelatiniziation
temperature wherein an ungelatinized grain having an endogenous maltogenic

enzyme are contacted with a solubilizing enzyme selected from Bacillus to
produce a maltose syrup. The maltose syrup is contacted with a
transglucosidase to produce a substrate (tuber or grain) composition including
isomalto-saccharides derived therefrom.
The present invention also describes a method for making an isomalto-
oligosaccharides enriched flours at temperatures at or below the gelatiniziation
temperature wherein an ungelatinized grain having an endogenous maltogenic
enzyme (wheat, barley, etc.) are mixed with ungelatinized grain not having
endogenous maltogenic enzymes (e.g., sorghum, miller or rice), the grain
mixture being contacted with a solubilizing enzyme selected from Bacillus to
produce a maltose syrup. The maltose syrup is contacted with a
transglucosidase to produce a substrate (tuber or grain) composition including
isomalto-saccharides derived therefrom.
The present invention also describes a method for making a wheat grain
composition said method comprising: (a) contacting an ungelatinized wheat
grain having an endogenous maltogenic beta-amylase and a starch liquefying
alpha amylase from Bacillus to produce maltose; (b) contacting said maltose
with a transglucosidase, wherein said steps (a) and step (b) occur at a
temperature less than wheat gelatinizing temperature; and (c) obtaining a wheal
grain composition having an enzymatically produced isomalto-oligosaccharide,
wherein said oligosaccharide is derived from said ungelatinized grain.
Optionally, in another embodiment the method uses the above method for
making a grain composition for making a food additive. Another embodiment
includes a grain composition made accordingly. Another embodiment includes a
flour comprising the grain composition described above. Another embodiment
includes an isomalto-saccharide made according to the method described
above. Another embodiment includes an oral rehydration solution comprising
the isomalto- oligosaccharide above. Another embodiment includes a grain
composition comprising an ungelatinized grain and at least one isomalto-
oligosaccharide, wherein said isomalto-oligosaccharide is enzymatically derived

from said ungelatinized grain. In another embodiment the grain composition
contains greater than 1 % by weight of at least one isomalto-oligosaccharide.
ACCOMPANYING
DESCRIPTION OF THE/FIGURES
FIG. 1 is a flowchart describing the production of isomalto-oligosaccharide
enriched flour.
FIG. 2 is another flowchart describing the production of isomalto-oligosaccharide
enriched wheat flour.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "grain" refers to a plant which is classified as a cereal or as a
monocotyledonous plant belonging to the Poales order, in particular the family
Poaceae. Examples of plants belonging thereof are plants selected from the
genuses Triticum (wheat), Hordeum (barley); Secale (rye); Zea (com or maize);
Avena (oats), Faqoprvum (buckwheat); Sorghum (sorghum or milo), Panicum or
Setaria (millet or ragi); or Oryza (rice).
For example, in one embodiment, the term "wheat" refers to a plant which is
classified or once was classified as a strain of Triticum aestivum.
For example, in one embodiment, the term "barley" refers to a plant which is
classified or once was classified as a strain of Hordeum vulqare.
For example, in one embodiment, the term "rye" refers to a plant which is
classified or once was classified as a strain of Secale cereale.
For example, in one embodiment, the term "corn" refers to a plant which is
classified or once was classified as a strain of Zea mays.

For example, in one embodiment, the term "oats" refers to a plant which is
classified or once was classified as a strain of Avena sativa.
For example, in one embodiment, the term "buckwheat" refers to a plant which
is classified or once was classified as a strain of Fagoprvum esculentum.
For example, in one embodiment, the term "sorghum" refers to a plant which is
classified or once was classified as a strain of Sorghum bicolor.
For example, in one embodiment, the term "millet" refers to a plant which is
classified or once was classified as a strain of Panicum miliaceum or Setaria
italica.
For example, in one embodiment, the term "rice" refers to a plant which is
classified or once was classified as a strain of Orvza sativa.
The term tuber refers to a starchy storage organ (for example a potato, sweet
potato, yam, manioc, etc) formed by swelling of an underground stem or the
distal end of a root.
For example, in one embodiment, the term "potato" refers to a plant which is
classified or once was classified as a strain of Solanum tuberosum.
For example, in one embodiment, the term "sweet potato" refers to a plant which
is classified or once was classified as a strain of Ipomroela balatas.
For example, in one embodiment, the term "yam" refers to a plant which is
classified or once was classified as a strain of Dioscorea sativa, D. villosa, C.
batatas.
The term substrate refers to materials that can be enzymatically converted to
maltose and thus IMO's. The term "substrate" includes, for example, grains and
tubers. Furthermore, the term substrate includes all forms of the grain (polished

or unpolished) or tuber, such as whole grains, broken grains, grits and flour and
any plant part.
The term "starch" refers to any material comprised of the complex
polysaccharide carbohydrates of plants, comprised of amylose and amylopectin
with the formula (C6H10O5)x, wherein X can be any number.
The term "granular starch" refers to uncooked (raw) starch, which has not been
subject to gelatinization. The term "gelatinization" refers to solubilization of a
starch molecule to form a viscous suspension.
The phrases "substrate", "grain" or "tuber" containing ungelatinized starch" refer
to an ungelatinized substrate, grain or tuber that is not subjected to temperatures
greater than the starch gelatinization temperatures which result in effecting a
gelatinization or liquefaction of the starch contained within the substrate.
The term "maltose" refers to a disaccharide having two glucosyl residues linked
by an alpha 1-4 D-glucosidic linkage
The term "isomaltose" refers to a disaccharide having two glucosyl residues
linked by an alpha 1,6 D-glucosidic linkage.
The term "isomalto-oligosaccharide" (IMO) refers to sugars having at least two
glucosyl residues linked by alpha 1,6 glucosidic linkages at the non-reducing
end. In addition term term refers to anomalously linked oligosaccharides,
saccharides having both alpha 1,6 and alpha 1,4 glucosidic linkages. Exemplary
isomalto-oligosaccharides include isomaltose, panose, and isomalto-triose
The term "isomalto-oligosaccharide" grain composition refers to grain
compositions characterized by isomalto-sugars level of at least 1% (w/w %) of
the total sugar content as determined by high performance liquid
chromatographic methods.

The term "maltogenic enzyme" refers to an enzyme that converts starch to
maltose. Exemplary maltogenic enzymes include fungal, bacterial and plant
derived alpha amylases and beta-amylases.
The term "amylases" refers to enzymes that catalyze the hydrolysis of starches.
The term "alpha-amylase" refers to enzymes of the class (E.C.) 3.2.1.1 and/or
3.2.1.133 that catalyze the hydrolysis of alpha-1,4-glucosidic linkages. These
enzymes have also been described as those effecting the exo or endohydrolysis
of 1,4-alpha-D-glucosidic linkages in polysaccharides containing 1,4-alpha-linked
D-glucose units. Another term used to describe these enzymes is glycogenase.
Exemplary enzymes include alpha-1,4-glucan 4-giucanohydrase
glucanohydrolase.
The term "beta-amylase" refers to enzymes of the class (E.C.) 3.2.1.2 that
catalyze the hydrolysis of alpha-1,4 glucosidic linkages releasing maltose units.
These enzymes have also been described as those effecting the hydrolysis of
1,4-alpha-D-glucosidic linkages in polysaccharides so as to remove successive
maltose units from the non-reducing end of chains.
The term "transglucosidic" enzyme refers to an enzyme that catalyzes both
hydrolytic and transfer reactions in incubation with alpha D-gluco-
oligosaccharides. Exemplary enzymes include transglucosidases and/or those
of the class (E.C.) 2.4.1.24, e.g., D-glucosyltransferase. These enzymes have
also been refered to as 1,4-alpha-glucan 6-alpha-glucosyltransferase and
oligoglucan-branching glycosyltransferase.
The term debranching enzyme refers to enzymes that catalyze the hydrolysis of
alpha-1,6-linkages. An enzyme of the class E.C.3.2.1.41 is useful in this regard.
An exemplary enzyme of this class is a pullanase, also known as alpha-dextrin
endo-1,6- alpha glucosidase, limit dextrinase, debranching enzyme, amylopectin
1,6-glucanohydrolase. Additional exemplary enzymes of the class (E.C.)

3.2.1.41, e.g., pullulanases, [alpha-(1-6)-glucan 6-giucanohydrolase, also called
alpha-(1,6)-glucosidase]).
The term "starch gelatinizing temperatures" refers to a temperature sufficiently
high to effect liquefying or gelatinization of granular starch. Heating a starch in
water causes the starch granules to swell. At sufficient solids concentration, the
swollen granules occupy most of the space and a viscous mass, called a paste,
results. Solubilization of starch molecule is called gelatinization. Gelatinization
is accompanied by a loss of birefringence. The term starch gelatinizing
temperature refers to the temperature at which gelatinization occurs.
The term "starch liquefying enzyme" refers to an enzyme that effects the
fluidization of granular starch. Exemplary starch liquefying enzymes include
alpha amylases of the class (E.C.) 3.2.1.1.
I The term "endogenous" refers to the enzyme being present in the grain or tuber
\ without having to resort to adding the maltogenic enzyme to the grain.
The term "exogenous" enzyme refers to an enzyme that is not present within the
grain. Exemplary exogenous enzymes include, for example, maltogenic
enzymes not present in the wild-type substrate, e.g., rice, millet, etc.
The term "total sugar content" refers to the total amount of sugar present in a
starch, grain or tuber composition.
The term "IMO No." is calculated as the sum of isomaltose, panaose,
isomaltotriose and branched sugars greater than DP3. The IMO Number
provides an indication of the amount of IMO compounds present in the
compound or solution.
The term "ratio of branched sugars" ("RBS") refers to the ratio of maltose (DP2)
present in the grain as compared to the level of maltotriose (DP3) present in the
resultant grain composition.

The term "Degrees of Diastatic Power" (DPo) unit refers to the amount of
enzyme contained in 0.10 ml of a 5% solution of the sample enzyme preparation
that will produce sufficient reducing sugars to reduce 5 ml of Fehling's solution
when the sample is incubated with 100 ml of substrate for 1 hour at 20° C (68°
F).
The term "DE" or "dextrose equivalent" is an industry standard for measuring the
concentration of total reducing sugars, calculated as D-glucose on a dry weight
basis. Unhydrolyzed granular starch has a DE that is essentially 0 and D-
glucose has a DE of 100.
The term "total sugar content" refers to the total sugar content present in a
starch composition.
The terms "dry solid basis" and "dsb" refer to the total amount of compound,
e.g., flour, of a slurry (in %) on a dry weight basis.
The terms "dry solid content", "dry solid granular starch", "dry solid starch" and
"(dss)" refer to the total starch of a slurry (in %) on a dry weight basis.
The term "Brix" refers to a well known hydrometer scale for measuring the sugar
content of a solution at a given temperature. Thus the term "Brix" refers to a
measure of the solubilized sugars in solution. The Brix scale measures the
number of grams of sucrose present per 100 grams of aqueous sugar solution
(the total solubilized solid content). For example, a measurement of 1.00 Brix
refers to about 10 mg/ml of sugar in solution. Brix measurements are frequently
made by use of a hydrometer or refractometer.
The term "degree of polymerization (DP)" refers to the number (n) of
anhydroglucopyranose units in a given saccharide. Examples of DP1 are the
monosaccharides, such as glucose and fructose. Examples of DP2 are the
disaccharides, such as maltose and sucrose. A "DP4+" denotes polymers with a
degree of polymerization of greater than 3.

The term "enzymatically produced" refers to enzymatic catalysis of the substrate
to the IMO as opposed to chemical or organic chemical synthesis of the IMO.
The term "filamentous fungi" refers to all filamentous forms of the subdivision
Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORY MYCOLOGY,
New York: Wiley). These fungi are characterized by a vegetative mycelium with
a cell wall composed of chitin, cellulose, and other complex polysaccharides.
The filamentous fungi of the present invention are morphologically,
physiologically, and genetically distinct from yeasts. Vegetative growth by
filamentous fungi is by hyphal elongation and carbon catabolism is obligatory
aerobic. In the present invention, the filamentous fungal parent cell may be a
cell of a species of, but not limited to, Trichoderma, e.g., Trichoderma reesei
(previously classified as T.longibrachiatum and currently also known as
Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma
harzianum; Penicillium sp.; Humicola sp., including Humicola insolens and
Humicola grisea; Chrysosporium sp., including C. lucknowense; Gliocladium sp.;
Aspergillus sp., including A. oryzae, A. nidulans, A. niger, and A. awamori;
Fusarium sp., Neurospora sp., Hypocrea sp., and Emericella sp. Reference is
also made to Innis et al., (1985)
Sci. 228:21 -26.
The term "Aspergillus" or "Aspergillus sp." refers to any fungal strain, which had
previously been classified as Aspergillus or is currently classified as Aspergillus.
The term "bacterial" refers to Bacillus species of, but not limited to B. subtilis. B.
amvloliouefaciesn. B. lentus. B. Carlsberg. B. licheniformis. and B.
stearothermophilus
The term "plant origin" refers to the enzyme being derived, extracted, isolated,
expressed from a plant source, for example from barley malt, soybean, wheat or
barley.
The term "contacting" refers to the piacing of the respective enzyme[s] in
sufficiently close proximity to the respective substrate to enable the enzyme[s] to

convert the substrate to the desired end-product. Those skilled in the art will
recognize that mixing solutions of the enzyme or enzymes with the respective
substrates can effect contacting.
The term "incubating" refers to mixing a substrate containing substrate with the
respective enzymes, e.g., liquefying or maltogenic or transglucosidase under
given conditions for a defined period of time.
The term "enzymatic conversion" refers to the modification of a rice substrate to
yield soluble hydrolyzed granular rice starch and preferably to yield glucose.
The term "slurry" refers to an aqueous mixture containing insoluble granular
starch. Sometimes the terms "slurry" and "suspension" are used interchangeably
herein.
The term "culturing" refers to growing a population of microbial cells under
suitable conditions in a liquid or solid medium. In one embodiment, culturing
refers to fermentative bioconversion of a granular starch substrate to glucose
syrup or other desired end products (typically in a vessel or reactor).
For example, in one embodiment, the term alpha amylase enzyme unit is
defined as the amount of alpha amylase which hydrolyzes 1 micromole of starch
substrate in 1 min under standard assay conditions of pH 5.2 and 40°C.
For example, in one embodiment, the term beta amylase enzyme unit is defined
as the amount of beta amylase which hydrolyzes 1 micromole of starch
substrate in 1 min under standard assay conditions of pH 4.6 and 20°C.
For example, in one embodiment, the term transglucosidase unit is defined as
the amount of transglucosidase which converts 1 micromole of maltose
substrate in 1 min under standard assay conditions of pH 4.8 and 37°C.
In another embodiment, the term transglucosidase unit is defined as the amount
of transglucosidase which produces 1 micromole of panose per minute under
standard assay conditions of pH 4.8 and 37° C.
For example, in one embodiment, the term one Liquefon Unit (LU) is the
measure of digestion time required to produce a color change with iodine

solution, indicating a definite stage of dextrinization of starch substrate under
standard assay conditions of pH 5.6 and 25 ° C.
"ATCC" refers to American Type Culture Collection located at Manassas, VA
20108 (ATCC, www/atcc.org).
"NRRL" refers to the Agricultural Research Service Culture Collection, National
Center for Agricultural Utilization Research (and previously known as USDA
Northern Regional Research Laboratory), Peoria, ILL.
"NCBI" refers to the National Center for Biotechnology Information, Natl Library
Med. (www.ncbi.nlm.nih.gov/).
"A", "an" and "the" include plural references unless the context clearly dictates
otherwise.
The present invention describes a method for making an isomalto-
oligosaccharide substrate, grain or tuber composition said method
comprising:(a) contacting a ungelatinized starch containing substrate, e.g., a
grain or a tuber, with a maltogenic enzyme and a starch liquefying enzyme to
produce maltose; (b) contacting said maltose with a transglucosidic enzyme,
wherein said steps (a) and step (b) occur at a temperature less than or at a
starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber
composition having an enzymatically produced isomalto-oligosaccharide,
wherein said oligosaccharide is derived from said substrate, grain or tuber. An
embodiment of the present invention is depicted in Fig.1.
The present invention also describes a method for making an isomalto-
oligosaccharide-enriched substrate, grain or tuber compositions, flours, oral
rehydrating solutions, and/or food additives, at temperatures at or below the
gelatiniziation temperature wherein a substrate having or containing an
ungelatinized starch and having endogenous maltogenic enzyme are contacted
with a solubilizing enzyme selected from Bacillus to produce a maltose syrup.

The maltose syrup is then contacted with a transgiucosidase at a temperature at
or less than gelatinization or liquefaction temperatures to produce a grain
composition having isomalto-oligosaccharides. In one embodiment, the grain
composition is characterized by a sugar composition of greater than 60%
maltose and a ratio of branched sugars of greater than 2.0 to 1.0. The
conversion of the substrate to the IMO can be enzymatically produced.
The present invention also describes a method for making an isomalto-
oligosaccharide substrate, grain or tuber composition, the method comprising:
(a) contacting a substrate, grain or tuber containing a starch with a maltogenic
enzyme and a starch liquefying enzyme to produce a maltose; (b) contacting the
maltose with a transglucosidic enzyme, wherein the steps (a) and step (b) occur
at a temperature less than or at starch gelatinization temperature; and (c)
obtaining a substrate, grain or tuber composition having an enzymatically
produced isomalto-oligosaccharide, wherein the oligosaccharide is derived from
the substrate, grain or tuber. The invention optionally further describes an
additional step of separating soluble constituents from insoluble constituents.
The invention further describes an additional step of drying the substrate, grain
or tuber composition. In one embodiment the grain is selected from the group
consisting of wheat, rye, barley, malt, buckwheat, sorghum (milo), millet (ragi)
and rice. In another embodiment, the maltogenic enzyme is a beta amylase. In
another embodiment, the maltogenic enzyme is endogenous to the grain. In
another embodiment, the starch liquefying enzyme is an alpha amylase derived
from a Bacillus. In another embodiment, the starch liquefying enzyme is derived
from Bacillus licheniformis or Bacillus stearothermophilus. In another
embodiment, the transglucosidic enzyme is a transgiucosidase. In another
embodiment. The transgiucosidase is derived from Aspergillus. In another
embodiment, the Aspergillus is Aspergillus niger. The invention also describes a
grain composition, a food additive, oral rehydration solution, food product and/or
a flour produced according to above described method.
In another embodiment, the invention describes a method for making a wheat
grain composition said method comprising: (a) contacting an ungelatinized

wheat grain having an endogenous maltogenic beta-amylase and a starch
liquefying alpha amylase from Bacillus to produce maltose; (b) contacting said
maltose with a transglucosidase, wherein said steps (a) and step (b) occur at a
temperature less than wheat gelatinizing temperature; and (c) obtaining a wheat
grain composition having an enzymatically produced isomalto-oligosaccharide,
wherein said oligosaccharide is derived from said ungelatinized grain. An
embodiment of the present invention is depicted in Fig. 2.
In another embodiment, the above described method can be used to make a
food additive, a bakery product, oral rehydration solution and/or a flour. In
another embodiment, the maltogenic enzyme is a beta amylase. In another
embodiment, the maltogenic enzyme is endogenous to the grain. In another
embodiment, the starch liquefying enzyme is an alpha amylase derived from a
bacterial source. In one embodiment the bacterial source is a Bacillus sp.. In
another embodiment, the starch liquefying enzyme is derived from Bacillus
licheniformis or Bacillus stearothermophilus. In another embodiment, the
transglucosidic enzyme is a transglucosidase. In another embodiment. The
transglucosidase is derived from a fungal source. In one embodiment the fungal
source is an Aspergillus sp. In another embodiment, the Aspergillus is
Aspergillus niger. The invention also describes a grain composition, a food
additive, oral rehydration solution and/or a flour produced according to above
described method. The grain composition could contain greater than 1% by
weight of at least one isomalto-oligosaccharide. The at least one isomalto-
oligosaccharide can be selected from the group consisting of isomaltose,
panose, isomalto-triose. In a further embodiment of the invention, the
endogenous maltogenic enzyme is selected from beta amylase or alpha
amylase. In a still further embodiment of the invention, the solubilizing enzyme
is a liquefying alpha amylase derived from a Bacillus. In a still further
embodiment of the invention the liquefying amylase is derived from Bacillus
licheniformis or Bacillus stearothermophilus.
Substrates
The present invention includes a substrate containing a starch, for example a
grain or a tuber containing a starch that is contacted with a maltogenic enzyme

and a starch liquefying enzyme to produce maltose. The term substrate refers to
materials that can be enzymatically converted to maltose and thus IMO's.
Exemplary substrates can be at least one substrate selected from the group
consisting of grains and tubers. The maltose can be in the form of a maltose
rich syrup or slurry.
Starch occurs in two forms, amylose, a linear chain polysaccharide, and
amylopectin, a branched chain polysaccharide. Amylose contains long
unbranched chains in which all the D-glucose units are linked by alpha-1,4-
linkages ("a-1,4 linkages" or "1,4-a-D-glucosyl linkages"). Amylopectin is highly
branched, the backbone glucosidic linkage being a-1,4, but the branch points
being a-1,6 linkages. The major components of starch can be enzymatically
hydrolyzed in two different ways. Amylose can be hydrolyzed by a-amylases
(E.C. 3.2.1.1), e.g., a-(1-4)-glucan 4-glucanohydrolase. Alpha amylases
hydrolyze the a-(1,4)-linkages to yield a mixture of glucose, maltose, maltotriose
and higher sugars. Amylose can also be hydrolyzed by a beta-amlylase (E.C.
r
3.2.1.2) [alpha(1,4)-glucan maltohydrolase, 1,4-a-D-glucan maltohydrolase ].
This enzyme cleaves away successive maltose units beginning from the non-
reducing end to yield maltose quantitatively. The alpha and beta amylases also
hydrolyze amylopectin. Neither the alpha and beta amylases can hydrolyze the
alpha (1-6) linkages at the branch points of amylopectin. The end product of
exhaustive beta-amylase action on amylopectin is a large, highly branched core
or beta limit dextrin. A debranching enzyme (E.C. 3.2.1.41 .e.g., pullulanases,
[a-(1-6)-glucan 6-glucanohydrolase, also called a-(1,6)-glucosidase]) can
hydrolyze the a-(1-6) linkages at the branch points. Thus the combined action
of p-amylase and the a 1,6-glucosidase can therefore completely degrade
amylopectin to maltose and glucose, resulting in a maltose content as high as
60%, 65%, 79%, 75%, 80% or higher of the total sugar content.
For the purposes of this invention, the substrate containing starch can be a grain
or a tuber or mixtures thereof. The grain can be any cereal or seed containing
starch. The substrate can be milled, ground or otherwise reduced in size to
increase the surface area of the substrate for contacting with the respective
enzymes. For example, the substrate can be wet or dry milled as desired. In

one embodiment of the present invention, the starch is granular starch. Grains
contemplated for use within the present invention includes any grain currently
used in baking, pasta or other uses. Exemplary grains contemplated by the
inventors include, but are not limited to at least one selected from the group
consisting of wheat (Triticum sp. Including, but not limited to T. monococcum. T.
turgidum. T. spelta. and/or T. aestivum). barley (e.g., Hordeum vulaare. and the
varieties described in U.S.Pat. No. 6,492,576, Table 1), rye (Secale sp..
including but not limited to S. cereale). corn (Zea sp.. including, but not limited to
Zea mays), buckwheat (Faqoprvum sp., including, but not limited to F
esculentum), malt (for example, germinated barley), sorghum (Sorghum sp..
including, but not limited to Sorghum bicolor') or otherwise known as milo, millet
(ragi) (Panicum sp.and Setaria sp.. including, but not limited to P. milaceum: and
Setaria sp., including, but not limited to Sitalica) and rice (Orvza sp.. including,
but not limited to Orvza sativa). It is contemplated by the inventors that wild-type
and transgenic plants having beneficial attributes, such as increased enzyme
levels of endogenous enzymes or the presence of exogenous enzymes are also
useful as starch containing substrates.
Germinated cereals, for example, malt, are used as one of the key ingredients in
many food and health drink formulations because of their high nutritive value,
e.g., malt containing food products (TABLE A). Germination results in the
synthesis and activation of endogenous maltogenic and proteolytic enzymes.
Thus germinated cereals are a good source of grains containing endogenous
maltogenic enzymes. Malt flour and malt extract are also used as a source of
digestive enzymes in brewing and baking applications. However, germination of
the barley renders the cereal grain components too readily digestible to play an
effective role as a prebiotic or even as a nutraceuticals, since they tend to be
digested in their entirety before arrival in the lower gastrointestinal tract.
Unfortunately, the beneficial effects of prebiotic compounds are best realized in
the lower gastrointestinal tract. Therefore, converting the highly digestible malto-
sugars into less digestible isomalto sugars allows for the use of the modified
malt to play a role as a prebiotic, allowing the malt to arrive in the lower
gastrointestinal tract and provide additional functional and health benefits. For

Thus the use of malt as a starch containing substrate converts some of the
granular starch contained within the substrate to an additional beneficial form of
the oligosaccharide, e.g., an IMO.
In addition, the substrate containing starch can be a tuber. Tubers contemplated
by the inventors include potato (Solanum sp.. including, but not limited to S
tuberosum), sweet potato (Ipomoea sp.. including, but not limited to Ipomoea
batatas), manioc [tapioca, cassava] (Manihot sp., including, but not limited to
Manihot esculenta, Manihot aipi and Manihot utilissima) and/or taro root
(Colocasia sp., including, not limited to C. esculenta or C. macrorhiza).
The amount of substrate containing starch can be an aqueous slurry
characterized by having a concentration of 10 to 50% dissolved solids (DS). In
another embodiment, the substrate containing starch is characterized by having
a concentration of 2 -90% DS. In another embodiment, the substrate containing
starch is characterized by having a concentration of 5 -70% DS. In another

embodiment, the substrate containing starch is characterized by having a
concentration of 10-60% DS. In another embodiment, the substrate containing
starch is characterized by having a concentration of 20-40% DS. In another
embodiment, the substrate containing starch is characterized by having a
concentration of 25-35% DS.
In another embodiment of the invention, the pH of the substrate containing
starch is between 1.00 to 9.00. In another embodiment of the invention, the pH
of the substrate containing starch is between 2.00 to 8.00. In another
embodiment of the invention, the pH of the substrate containing starch is
between 3.00 to 7.50. In another embodiment of the invention, the pH of the
substrate containing starch is between 4.00 to 6.50. In another embodiment of
the invention, the pH of the substrate containing starch is between 4.25 to 5.75.
Enzymes
The present invention includes contacting the substrate containing starch with a
maltogenic and a starch liquefying enzyme to produce maltose. By maltogenic
is meant that the enzyme is able to enzymatically convert starch to maltose.
Exemplary maltogenic enzymes include alpha amylases and beta amylases. As
described before, amylose can be hydrolyzed by D-amylases (E.C. 3.2.1.1),
e.g., D-(1-4)-glucan 4-glucanohydrolase. Alpha amylases hydrolyzethe D-(1,4)-
linkages to yield a mixture of glucose, maltose, maltotriose and higher sugars.
Amylose can also be hydrolyzed by a beta-amlylase (E.C. 3.2.1.2) [alpha(1,4)-
glucan maltohydrolase, 1,4-D-D-glucan maltohydrolase ]. This enzyme cleaves
away successive maltose units beginning from the non-reducing end to yield
maltose quantitatively. The alpha and beta amylases also hydrolyze
amylopectin.
Alpha Amylases -
In some of the embodiments encompassed by the invention, the alpha amylase
is a funal or microbial enzyme having an E.C. number, E.C. 3.2.1.1-3 and in
particular E.C. 3.2.1.1. In some embodiments, the alpha amylase is a
thermostable fungal alpha amylase. Suitable alpha amylases may be naturally
occurring as well as recombinant and mutant alpha amylases. In some

embodiments, the alpha amylase is derived from a Bacillus species. Preferred
Bacillus species include Bacillus amyloliquefaciens, B. lentus, B. licheniformis,
and B. stearothermophilus. In particularly preferred embodiments, the alpha
amylase is derived from a Aspergillus species. Preferred Aspergillus species
include Aspergillus niqer and Aspergillus orvzae. Also reference is made to
strains having NCIB 11837.
Commercially available alpha amylases contemplated for use in the methods of
the invention include; CLARASE L (fAspergilus orvzael Genencor International
Inc.) and NOVAMYL ([B stearothermophilus] Novozyme Biotech.).
As understood by those in the art, the quantity of alpha amylase used in the
methods of the present invention will depend on the enzymatic activity of the
alpha amylase. In general, an amount of about 0.01 to 5.0 kg of the alpha
amylase is added to a metric ton (MT) of the substrate containing starch.
Although in some embodiments the alpha amylase is added in an amount about
0.05 to 4.0 kg per MT. In other embodiments, the alpha amylase is added in an
amount of about 0.1 to 2.5 kg per MT and also about 0.5 to 1.5 kg per MT. In
further embodiments, other quantities are utilized. For example, generally an
amount of between about 0.01 to 1.5 kg of CLARASE L (Genencor International
Inc.) is added to a MT of starch. In other embodiments, the enzyme is added in
an amount between about 0.05 to 1.0 kg; between about 0.1 to 0.6 kg; between
about 0.2 to 0.6 kg and between about 0.4 to 0.6 kg of CLARASE L per MT of
starch.
Beta Amylase
In some of the embodiments encompassed by the invention, the maltogenic
enzyme is a beta amylase. While alpha amylases are maltogenic in the sense
that contacting alpha amylases with a substrate containing starch would provide
maltose, the use of beta amylases are useful in that their contact with granular
starch would provide a greater amount of maltose to the exclusion of other
saccharides, e.g., glucose. In some embodiments, the beta amylase is a plant
or microbial enzyme having ah E.C. number, E.C. 3.2.1.2 (for example those
beta amylases described in US 4,970,158 and 4,647,538). In some

embodiments, the beta amylase is a thermostable bacterial beta amylase.
Suitable beta amylases may be naturally occurring as well as recombinant and
mutant beta amylases. The term bacterial refers to the enzyme being derived
from Bacillus sp.. e.g., B. subtilis. B. licheniformis. B. stearothermophilus. B^
coaqulans. B. amyloliquefaciens. and/or B. lentus. Particularly preferred beta
amylases are derived from Bacillus strains B. stearothermophilus, B.
amyloliquefaciens and B. licheniformis. Also reference is made to strains having
NCIB 11608. The term plant origin refers to the enzyme being derived,
extracted, isolated, expressed from a plant source, for example from barley malt,
soybean, wheat or barley.
Commercially available beta amylases contemplated for use in the methods of
the invention include; OPTIMALT BBA, Spezyme DBA, and OPTIMALT ME
(Genencor International Inc.). Other commercially available wheat beta
amylases are also useful in the methods of the invention.
In some embodiments, the substrate containing starch, e.g., wheat, rye, barley,
malt, comprises an endogenous maltogenic enzyme at sufficient levels to
produce sufficient maltose for conversion to isomalto oligosaccharides.. The
term "endogenous" refers to the enzyme being present in the grain or tuber
without having to resort to adding the maltogenic enzyme to the grain, or the
grain being genetically engineered to provide maltogenic enzymes.
In embodiments where the substrate containing starch does not contain an
endogenous maltogenic enzyme or has low endogenous levels of maltogenic
enzymes, e.g., rice, millet, sorghum, and/or corn, the addition of an equivalent
amount of any exogenous maltogenic enzyme is also contemplated by the
inventors. The exogenous maltogenic enzyme can be added, for example by
genetically manipulating the host cell to express sufficient levels of maltogenic
enzyme, and/or providing a maltogenic enzyme concentrate or material from
another source. The term exogenous maltogenic enzyme refers to a maltogenic
enzyme that is not present within the grain. In this embodiment, a sufficient

amount of maltogenic enzyme is contacted with the substrate to produce
maltose.
In one embodiment, the amount of exogenous maltogenic enzyme contacted
with the substrate containing starch is between 0.050 to 5.000 Degrees of
Diastatic Power ("DP0") units /gm of maltogenic enzyme. In another
embodiment of the invention, 0.100 to 2.000 DP0 units/gm of maltogenic enzyme
is contacted with the grain containing a starch. In still another embodiment of
the invention, 0.100 to 3.000 DP0 units/gm of maltogenic enzyme is contacted
with the grain containing a starch.
In another embodiment, the amount of exogenous maltogenic enzyme contacted
with the substrate containing starch is expressed in kilograms of maltogenic
enzyme per metric ton of substrate. In one embodiment, the amount of
exogenous maltogenic enzyme contacted with the substrate is about 0.05 kg of
maltogenic enzyme per metric ton dry solids basis ("kg/mt dsb"). In another
embodiment, the amount of exogenous maltogenic enzyme is about 0.1 kg of
maltogenic enzyme per metric ton dry solids basis ("kg/mt dsb"). In other
embodiments 0.2, 0.4, 0.6, 0.8. and/or 1.0 kg/mt dsb provide sufficient amounts
of maltogenic enzyme, e.g., (3-amylase.
In another embodiment, the amount of exogenous maltogenic enzyme contacted
with the substrate containing starch is expressed in kilograms of maltogenic
enzyme per metric ton of substrate. In one embodiment, the amount of
exogenous maltogenic enzyme contacted with the substrate is about 0.05 kg of
maltogenic enzyme per metric ton dissolved starch basis ("kg/mt dsb"). In
another embodiment, the amount of exogenous maltogenic enzyme is about 0.1
kg of maltogenic enzyme per metric ton dissolved starch basis ("kg/mt dsb"). In
other embodiments 0.2, 0.4, 0.6, 0.8. and/or 1.0 kg/mt dissolved starch basis
provide sufficient amounts of maltogenic enzyme, e.g., p-amylase.
In another embodiment, the amount of maltogenic enzyme to be contacted with
the grain is in terms of maltogenic enzyme units. Assays useful to determine
the maltogenic activity include those described in the examples and those
describing |3-amylase activity. The term DP° unit refers to the amount of

enzyme contained in 0.10 ml of a 5% solution of the sample enzyme preparation
that will produce sufficient reducing sugars to reduce 5 ml of Fehling's solution
when the sample is incubated with 100 ml of substrate for 1 hour at 20° C (68°
F).
In another embodiment, a grain having endogenous maltogenic enzymes
(barley, wheat, etc.) can be mixed with those grains needing exogenous
maltogenic enzymes. Mixtures of 30:70, 60:40, 50:50, 60:40, 70:30 grains
having an endogenous maltogenic enzyme: grains utilizing exogenous
maltogenic enzyme sources are contemplated by the inventors, so long as
sufficient amounts of maltogenic enzymes are present in the mixture
(endogenous or exogenous sources). Use of endogenous sources of
maltogenic enzymes can reduce the amount of exogenous enzymes added or
contacted with the grain mixture.
Starch liquefying enzymes
A starch liquefying enzyme is contacted with the starch to reduce the viscosity of
the liquefied or solubilized starch. In one embodiment of the invention the starch
liquefying enzyme is an enzyme selected from the E.G. 3.2.1.1, e.g., alpha
amylases. Exemplary alpha-amylases can be derived, isolated or extracted from
a bacterial source. In one embodiment, the bacterial source is a Bacillus. In
another embodiment, the alpha-amylases derived from Bacillus include those
derived from at least one bacterial source selected from B. subtilis. B.
licheniformis, B. stearothermophilus. B. coaqulans. B. amvloliquefaciens. and B..
lentus. Those of Bacillus licheniformis and Bacillus stearothermophilus are
especially useful. Other amylases are contemplated by the inventors, for
example, but not limited to those of EC 3.2.1.133 (U.S.Pat. No. 6,361,809).
Other amylases contemplated by the inventors include those characterized by
increased oxidative or thermostability, including those mutants or genetically
modified or variant amylases described in U.S.Patent Nos. 5,763,385;
5,824,532; 5,958,739; and/or 6,008,026. Useful alpha amylases are those
derived from B. licheniformis strains NCIB 8059, ATCC 6598, ATCC 6634,

ATCC 8480, ATCC 9945A, ATCC 11945. Useful alpha amylases are those
derived from B. stearothermophilus strains ATCC 39709. Such enzymes are
identified by the trade names "SPEZYME AA" or "SPEZYME FRED", "SPEZYME
LT300", and "SPEZYME LT75", available from Genencor International (Palo
Also, California, USA). Other such enzymes include alpha amylases from
Bacillus stearothermophilus sold under the tradename GZYME G997, GC007
and from Bacillus licheniformis sold under the tradename GC262 SP, also
available from Genencor International.
Contacting the grain containing starch with the maltogenic enzyme and the
starch liquefying enzyme produces maltose. As understood by those in the art,
the quantity of starch liquefying enzyme used in the methods of the present
invention will depend on the enzymatic activity of the starch liquefying enzyme.
In one embodiment, 0.01 to 25 Liquefon Units/gm of starch liquefying enzyme is
contacted with the grain containing starch. In another embodiment, 1 to 10
Liquefon Units/gm of starch liquefying enzyme is contacted with the grain
containing a starch. One Liquefon Unit (LU) is the measure of digestion time
required to produce a color change with iodine solution, indicating a definite
stage of dextrinization of starch substrate under specified conditions.
In one embodiment, 0.1 kg of starch liquefying enzyme is added per metric ton
of grain dissolved solid basis (kg/mt dsb). In other embodiments, 0.2, 0.4, 0.4,
0.8, or 1.0 kg of starch liquefying enzyme is added per metric ton of grain (kg/mt
dissolved starch basis). In one embodiment, 0.1 kg of starch liquefying enzyme
is added per metric ton of grain dissolved starch basis (kg/mt dissolved starch
basis). In other embodiments, 0.2, 0.4, 0.4, 0.8, or 1.0 kg of starch liquefying
enzyme is added per metric ton of grain (kg/mt dissolved starch basis). Assays
useful to determine the starch liquefying activity include those described in the
examples herein. Exemplary assays for the determination of a-amylase activity
are also described in U.S. Pat. Nos. 5,763,385; 5,824,532; 5,958,739; and/or
6,008,026 which are incorporated by reference herein.
Transglucosidic enzyme

Contacting the maltose with a transglucosidic enzyme obtains a grain
composition having an enzymatically produced isomalto-oligosaccharide,
derived from the grain containing starch. The transglucosidic enzyme catalyzes
hydrolytic and transfer reactions on incubation with alpha-D-gluco-
oligosaccharides to produce isomaltose, panose, kojibiose or nigerose. The
presence of these sugars and thus conversion by the transglucosidic enzyme is
indicated in an increased amount of DP2 disaccharides. The transglucosidic
enzyme (E.C. 2.4.1.24) can be transglucosidase. Exemplary transglucosidase
enzymes are identified as TRANSGLUCOSIDASE L-1000 (Genencor
International, Inc.) and TRANSGLUCOSIDE L by Amano Enzymes, Inc.,
(Nagoya, Japan). In one embodiment the transglucosidic enzyme is derived
from a filamentous fungal source, e.g., Aspergillus SP. The transglucosidic
enzyme that is derived from Aspergillus can be derived from Aspergillus niger.
In one embodiment, the Aspergillus niger strain is ATCC14916.
In this embodiment, a sufficient amount of the transglucosidic enzyme is
contacted with the substrate, e.g. the grain containing a starch to produce
maltose. As understood by those in the art, the quantity of transglucosidic
enzyme used in the methods of the present invention will depend on the
enzymatic activity of the alpha amylase. In one embodiment, 0.01 to 25.00
transglucosidase units ("TGU")/gm of transglucosidase is contacted with the
grain containing a starch. In another embodiment of the invention, 0.05 TGU to
10.00 TGU/gm of transglucosidase is contacted with the grain containing a
starch. In still another embodiment of the invention, 0.10 to 5.00 TGU /gm of
grain is contacted with the grain containing a starch. The term TGU refers to the
activity of the enzyme required to produce one micromole of panose per minute
under the conditions of the assay.
In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is added per
metric ton of grain (kg/mt dsb). In another embodiment, 0.10 to 5.00 kg of
transglucosidic enzyme is added per metric ton of grain (kg/mt dsb). In another
embodiment, 0.25 to 3.00 kg of transglucosidic enzyme is added per metric ton
of grain (kg/mt dsb). In another embodiment, 0.50 to 1.50 kg of transglucosidic
enzyme is added per metric ton of grain (kg/mt dsb). Additional assays useful to

determine the transglucosidic activity include those described in the Examples
and in Shetty, J., et al (U.S. Pat. No. 4,575,487 (1986) entitled, "Method for
determination of transglucosidase"), which are incorporated by reference herein.
In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is added per
metric ton of dissolved starch (kg/mt starch dsb). In another embodiment, 0.10
to 5.00 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt
starch dsb). In another embodiment, 0.25 to 3.00 kg of transglucosidic enzyme
is added per metric ton of grain (kg/mt starch dsb). In another embodiment, 0.50
to 1.50 kg of transglucosidic enzyme is added per metric ton of grain (kg/mt
starch dsb).
As a result of transglucosidase reactions, the malto-oligosaccharides are
converted to isomalto-oligosaccharides resulting in a new class of
polysaccharides containing higher proportion of glucosyl residues linked to a
primary hydroxyl group of a glucose molecule from the non-reducing end .
Isomalto-oligosaccharides produced by this method include isomaltose, panose,
isomalto-triose, isomalto-tetrose, isomalto-pentose, isomalto-hexose and
isomalto-heptose. These sugars are receiving increased attention as food
additives because they help prevent dental caries (Oshima, et.al 1988, The
caries inhibitory effects of gos-sugar in vitro and rat experiments. Microbial
Immunol. 32.1093-1105 )) and improve human intestinal microflora acting as a
growth factor (prebiotic) for bifidobacteria (Komoto.et.al 1988; Effect of Isomalto-
oligosaccharides on human fecal flora .Bifidobacteria Micro flora 7,61-69).
To ascertain the production of the IMO's, assays and/or other analytical methods
can be used to determine the amount of IMO produced. One method for
determining the levels of IMO produced includes high performance liquid
chromatography (HPLC). For example, analysis of the mixture can provide an
indication of the levels of the various sugars produced by the process. A useful
rating is the degree of polymerization (DP) of the mixture. The term degree of
polymerization is a measure of ther relative amounts of the number of glucose
residues in the molecule. For example, glucose (one glucosyl unit, the lowest
level of polymerization) is usually found as DP1. Isomalto-oligosaccharides are

usually found in DP2 (two glucosyl units). In one embodiment, the grain
composition contains greater than at least 1%, at least 5%, at least 25%, at least
40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% by
weight of at least one isomalto-oligosaccharide. In one embodiment the at least
one isomalto-oligosaccharide is selected from the group of isomaltose, panose
and/or isomalto-triose. In one embodiment, the amount of isomalto-
oligosaccharides produced in the grain composition is between 1% and 99% of
the grain composition. In one embodiment, the amount of isomalto-
oligosaccharides produced in the grain composition is between 1 % and 90% of
the grain composition. In one embodiment, the amount of isomalto-
oligosaccharides produced in the grain composition is between 1 % and 80% of
the grain composition. In one embodiment, the amount of isomalto-
oligosaccharides produced in the grain composition is between 1% and 70% of
the grain composition. In one embodiment of the present invention, the total
sugar present in the grain composition after the above described procedure
includes a level of maltose in the total sugar content of greater than 50%,
greater than 60%, greater than 70%, or greater than 80%. Levels of maltose
greater than 50% include ranges from 50% to 85%, from 55% to 80%, and/or
from 60% to 75%. The term RBS ratio refers to the ratio of maltose (DP2)
present in the grain as compared to the level of maltotriose (DPS) present in the
resultant grain composition. A higher RBS value indicates a higher amount of
maltose present and thus a more complete conversion of the starch to maltose
as opposed to the presence of other less desireable end-products, e.g.,
maltotriose. In one embodiment, the RBS ratio is greater than 2.0. In one
embodiments, the RBS ratio is greater than 3.0, greater than 4.0. Exemplary
ranges include an RBS ratio of 2.0 to 50.0, 2.0 to 30.0 and/or 2.0 to 10.0.
Various RBS ratios are described in the examples. It is noted that the hydrolysis
of liquefied starch by commercial beta-amylases (barley or wheat) generally
produces a maltose content between 55% and 65%. For maltose content
greater than 50% using liquefied starch, the addition of debranching enzyme
and/or a very low starting DE of the liquefied starch was previously required.
Optionally, the addition of a debranching enzyme can be used to increase the
production of maltose. The term debranching enzyme refers to enzymes that

catalyze the hydrolysis of a-1,6-linkages. An enzyme of the class E.C.3.2.1.41 is
useful in this regard. An exemplary enzyme of this class is a pullanase, also
known as a-dextrin endo-1,6- a glucosidase, limit dextrinase, debranching
enzyme, amylopectin 1,6-glucanohydrolase.
Contacting the grain containing starch with a maltogenic enzyme to produce the
maltose and the contacting of the maltose with a transglucosidic enzyme occurs
at a temperature less than the gelatinization temperature of the starch of the
grain used. The enzymes are contacted or incubated with the respective
enzymes for an incubation time of at least 12 hours, at least 18 hours, at least 24
hours, at least 30 hours and/or at least 36 hours. The period of at least a noted
time refers to a period of 12-80 hours, at least 18-60 hours and/or at least 24-48
hours. The term incubation time refers to the period of time for the conversion of
maltose or the substrate to IMO's. The transglucosidic enzyme can be
contacted or added separately or concurrently with the substrate, e.g. grain
containing starch, the maltogenic enzyme, e.g., the alpha amylase or beta
amylase, and/or the liquefying enzyme, e.g., the alpha amylase. In one
embodiment, the transglucosidic enzyme is added concurrently with the
liquefying enzyme. Thus in one embodiment, steps (a) and (b) are performed
concurrently. In another embodiment, the steps (a) and (b) are performed
sequentially or separately. In another embodiment, the step (a) is performed
before step (b). The term gelatinization temperature refers to the temperature at
which the starch contained within the grain changes phases or gelatinizes.
While the specific temperature varies from grain to grain, temperatures sufficient
to effect the gelatinization of starch include those greater than 45° C, greater
than 50° C, greater than 60° C, greater than 70° C, greater than 80° C, and/or
greater than 90° C. Exemplary temperatures greater than the indicated
gelatinization temperatures include 45° C to 120° C, 50° C to 110° C, 50° to 100°
C. In one embodiment, e.g. wheat, the gelatinization temperature is a
temperature the grain is kept below, e.g. a temperature selected from below 50°
C to 70° C, in another embodiment below 55° C to 65° C, and in another
embodiment, below 60° C. For example, gelatinization temperatures have been
described for corn, potato, wheat, tapioca, waxy maize, sorghum, rice sago,
arrowroot amylomaize and/or sweet potato as shown in Table 1 (Beynum, G.M.A

In another embodiment of the present invention, the slurry, after the incubation
time, can be subjected to a flash heat period sufficient to halt further enzymatic
activity, but not gelatinize or liquefy the slurry. For example, the slurry can be
heated to a temperature of 80°, 85°, 90° 95° or 100° C for a period of 5-60
minutes, 10.0 to 40.0 minutes or 30.0 minutes.
Another embodiment of the present invention further includes the step of
separating the slurry into insolubles and solubles. The separating step can be
by any chromatographic method known in the arts, for example, but not limited

to HPLC, size exclusion and/or charge chromatography. Filtering can be used to
separate the insolubles from the solubles. The insolubles or entire slurry can be
subjected to the drying steps described later in this application. In another
embodiment, the solubles resulting from the separating step can be
concentrated by evaporation, for example by roto-evaporation, tray drying, etc.
The evaporated concentrate can be subjected to carbon treatment (filtered
through carbon granules) and /or further chromatographic treatment to provide
an isolated IMO liquid concentrate. The isolated IMO concentrate can have an
IMO concentration of greater than 75%, greater than 85%, greater than 90%,
greater than 95%, greater than 97%, and/or greater than 99%.
Another embodiment of the present invention is the use or incorporation of such
syrup (the isomalto-oligosaccharides enzymatically derived from the substrate
having ungelatinized starch) in oral rehydration solutions. The amount of the
isomalto-saccharides can be in the amounts or formulations as described as
U.S.Pat. Nos. 4,981,687; 5,096,894; and/or 5,733,579.
Another embodiment of the present invention is the drying of the aforementioned
isomalto-oligosaccharide substrate, grain or tuber composition to produce a
powder including the grain composition. Typically, this drying step is accelerated
by heating. The grain composition can be dried to a desired moisture level by
using a suitable drying method, for example, but not limited to a spray dryer, tray
dryer, tumble dryer, drum dryer or cabinet dryer. Other drying methodologies can
be used, for example spray drying, evaporative drying under reduced pressure.
By drying the grain composition, slurry, separated insolubles, and/or separated
solubles, a flour or other dried powder is obtained therefrom. The resulting
powder or flour can be incorporated into compositions in which the presence of
isomalto-oligosaccharides is desired, for example in food stuffs (breakfast
cereals, pastas), food additives and baked goods. The term food additive refers
to the use of the isomalto-oligosaccharides as a sprinkle on material, as an
ingredient for use in the manufacture of other foods, and/or a topical ingredient
added to the food.

In another embodiment, the dried powder can be incorporated into food
supplements. The incorporation of the dried powder into a food supplement can
be provided in any acceptable supplement or form. The dietary supplements can
be formulated for oral administration in a matrix as, for example but not limited
to, drug powders, crystals, granules, small particles (which include particles
sized on the order of micrometers, such as microspheres and microcapsules),
particles (which include particles sized on the order of millimeters), beads,
microbeads, pellets, pills, microtablets, compressed tablets or tablettriturates,
molded tablets or tablet triturates, and in capsules, which are either hard or soft
and contain the composition as a powder, particle, bead, solution or suspension.
The dietary supplement can also be formulated for oral administration as a
solution or suspension in an aqueous liquid, as a liquid incorporated into a gel
capsule or as any other convenient form for administration or for rectal
administration, as a suppository, enema or other convenient form. The isomalto-
oligosaccharide composition can also be provided as a controlled release
system.
The dietary supplement formulation can also include any type of acceptable
exicipients, additives or vehicles. For example, but not by way of limitation,
diluents or fillers, such as dextrates, dicalcium phosphate, calcium sulfate,
lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose,
inositol, powdered sugar, bentonitc, microcrystalline cellulose, or hydroxypropyl
methylcellulose may be added to isomalto-oligosacccharide composition to
increase the bulk of the composition. Also, binders, such as, but not limited to,
starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, acacia gum,
sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of
isapgol husks, carboxymethylcellulose, methylcellulose,
polyvinylpyrrolidone,Veegurn and starch arabogalactan, polyethylene glycol,
ethylcellulose, glycerylmonostearate and waxes, may be added to the
formulation to increase its cohesive qualities.
Additionally, lubricants, such as, but not limited to, glyceryl monostereate, talc,
magnesium15 stearate, calcium stearate, stearic acid, hydrogenated vegetable

oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,
leucine, carbowax, sodium laurylsulfate, and magnesium lauryl sulfate may be
added to the formulation. Also, glidants, such as but not limited to, colloidal
silicon dioxide, magnesium silicate or talc may be added to improve the flow
characteristics of a powdered formulation. Finally, disintegrants, for example, but
not limited to, starches, clays, celluloses, algins, gums, crosslinked polymers
(e.g.,croscarmelose, crospovidone, and sodium starch glycolate), Veegum,
methylcellulose, agar,benton:ite, cellulose and wood products, natural sponge,
cation-exchange resins, alginicacid, guar gum, citrus pulp,
carboxymethylcellulose, or sodium lauryl sulfate with starchmay also be added
to facilitate disintegration of the formulation in the stomach or intestine.
Another embodiment of the present invention is the use of the novel substrate,
tuber or grain composition described herein in the production of flour for use in
various baked goods. The term baked goods refers to leavened and unleavened
goods. The term leavened refers to baked goods using yeast in the baking
process. Whereas the term unleavened means baked goods not using yeast in
the baking process. Exemplary goods include bread, cookies, cakes, pies,
biscuits, naan, bagels, pasta, crackers, rolls, donuts, pitas and pastries.
Exemplary unlevened goods include matzoh, chapathi, breakfast cereals and
torillas. Another embodiment of the present invention is the use of the novel
grain compositions in pasta, for example, noodles (penne, spagetti, lasagna,
udon, etc.). Another embodiment of the present invention is a substrate, tuber
or grain composition made according to the above described method. Another
embodiment of the present invention is a flour comprising the substrate, tuber or
grain composition made according to the above described method. Another
embodiment of the present invention is an oral rehydration solution comprising
the isomalto-oligosaccharide described above. Flour comprising the substrate,
tuber or grain composition can be made according to the above described
method. Another embodiment of the present invention is a substrate, tuber or
grain composition made according to the above described method.
Another embodiment of the present invention is a substrate, tuber or grain
composition made according to the above described method. Another

embodiment of the present invention is the use of the novel grain compositions
in fermentive/beer worts or substrates. For example, the novel grain
composition can be used as described in beer fermentation as described in
International Publication No WO 02/20712 A1, which is incorporated by
reference herein. The novel grain compositions can also be incorporated in beer
adjuncts.
The isomalto-oligosaccharide containing substrate can also be subjected to an
additional step of recovering the maltose by extraction and isolation of the
generated maltose, for example as a maltose syrup. The syrup can be extracted
and/or isolated from the grain composition by methods familiar in the art, for
example in U.S.Pat. Nos. 3,922,196 and 4,113,509, which are incorporated by
reference herein.
Another route to enhance the sweetness or the isomalto-oligosaccharide
content, is to treat the produced isomalto-oligosaccharide syrup with a hydrolase
(in soluble or immobilised form) which hydrolyses preferentially or even
exclusively malto-oligosaccharides, and has only a small or even no affinity for
isomalto-oligosaccharides. Examples of such an enzyme is glucoamylase from
A. niqer or other sources like Aspergillus sp. or Rhizopus sp. which preferentially
hydrolyses malto-oligosaccharides ( Manjunath P., Shenoy B. C, Raghavendra
Rao M. R., Journal of Applied Biochemistry, 5(1983),235-260; Meagher M. M., et
al., Biotechnology and Bioengineering, 34(1989), 681-693; Pazur J. H., Kleppe
K., The Journal of Biological Chemistry, 237(4)(1962),1002-1006; Hiromi K.,
Nitta Y., et al.,Biochimica et Biophysica Acta, 302(1973),362-37).
Also an enzyme like the alpha-D-glucopyranosidase from Bacillus
stearothermophilus can be applied. This enzyme is not capable of hydrolysing
isomalto-oligosaccharides and will only degrade the malto-oligosaccharides
present in the isomalto-oligosaccharide rich syrup (Suzuki Y., Shinji M.,
Nobuyuki E., Biochimica et Biophysica Acta, 787(1984),281 -289). Also other
alpha-D-glucosidases which are called maltases can be used. The maltase from
yeast for example will only hydrolyse maltose and to a lesser extent maltotriose
(Kelly C. T., Fogarty W. M., Process Biochemistry, May/June(1983),6-12).

After the hydrolysis of the malto-oligosaccharides to glucose, the syrup can be
enriched in isomalto-oligosaccharides by a chromatographic technique or by
nano- or ultra-filtration.
The following examples serve to illustrate the main embodiments of this
invention.
EXAMPLES
The following specific examples further illustrate the compositions and the
methods of the invention. It is to be understood that these examples are for
illustrative purposes only and can be applied to any other suitable materials rich
in starch and containing endogenous maltose producing enzyme, for example,
wheat, rice, barley, malt, potato, sweet potato, etc.
Enzyme Activity Determination
The transglucosidase activity is measured by the method of Shetty, J., et al,
1986 (U.S. Pat. No. 4,575,487).
The beta amylase activity was measured by a 30-minute hydrolysis of a starch
substrate at pH 4.6 and 20° C. The reducing sugar groups produced on
hydrolysis are measured in titrimetirc procedure using alkaline ferricyanide.
One unit of diastase activity, expressed as degrees DP refers to the amount of
enzyme, contained in 0.1 ml of 5% solution of the sample enzyme preparation,
that will produce sufficient reducing sugars to reduce 5 mL of Fehlings' solution
when the sample is incubated with 100 mL of the substrate for 1 hour at 20 C.
The alpha amylase activity was developed based on an end-point assay kit
supplied by Megazyme (Aust.) Pty. Ltd. A vial of substrate (p-nitrophenyl
maltoheptaoside, BPNPG7) was dissolved in 10ml of sterile water followed by a
1:4 dilution in assay buffer (50mM maleate buffer, pH 6.7, 5mM calcium chloride,
0.002% Tween20). Assays were performed by adding 10µl of amylase to 790µl
of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the
rate of change of absorbance at 41 Onm, after a delay of 75 seconds. The assay
was linear up to rates of 0.2 absorption units/min.

a-Amylase protein concentration was measured using the standard Bio-Rad
Assay (Bio-Rad Laboratories) based on the method of Bradford, Anal. Biochem..
Vol. 72, p. 248 (1976) using bovine serum albumin standards.
Substrates:
The wheat flour used as substrates in all examples, was purchased from retail
commercial stores . Other tuber or grain substrates, e.g., rice and barley used
as substrates may be purchased from commercial sources (Huai An Liujun Food
processing company, Jiangshu province, China).
Oligosaccharide Analysis
The composition of the reaction products of oligosaccharides was measured by
HPLC (Agilent 1010, Palo Alto, California, USA) equipped with a HPLC column
(Rezex 8 u8% H, Monosaccharides), maintained at 60°C fitted with a refractive
index (Rl) detector (ERC-7515A, Rl Detector from The Anspec Company, Inc.).
Dilute sulfuric acid (0.01 N) was used as the mobile phase at a flow rate of 0.6
ml per minute. Twenty microliter of 4.0% solution was injected on to the column.
The column separates based on the molecular weight of the saccharides. For
example a designation of DP1 is a monosacchride, such as glucose; a
designation of DP2 is a disaccharide, such as maltose; a designation of DP3 is a
trisaccharide, such as maltotriose and the designation DP4+ is an
oligosaccharide having a degree of polymerization (DP) of 4 or greater. The
term Higher sugar ("Hr. Sugar") refers to sugars having DP greater than 3.
For iso-saccharides or branched sugars.the reaction products were measured by
HPLC (Agilent 1010, Palo Alto, California, USA) equipped with a HPLC column
(Shodex Rspak Oligosaccharide Column #DC-613), maintained at 50°C fitted
with a refractive index (Rl) detector (ERC-7515A, Rl Detector from The Anspec
Company, Inc.). 70 (Acetonitrile):25 (methanol):5 Water was used as the mobile
phase at a flow rate of 2.5 ml per minute. Twenty microliter of 4.0% solution was
injected on to the column. The column separates based on the molecular weight

of the saccharides. The standard sugars, glucose, maltose, maltotriose,
isomaltose, panose and isomalto-triose (Sigma Chemicals, St. Louis, Missouri,
USA) were used to calibrate the column.
EXAMPLE 1
Maltose production from wheat flour by alpha amylase from Bacillus
licheniformis (an alpha amylase sold under the tradename GC262SP by
Genencor International, Palo Alto, CA) and Bacillus stearothermophilus (an
alpha amylase sold under the tradename GC007 by Genencor International,
Palo Alto, CA) were compared. One hundred fifty grams of wheat flour from
commercial retail sources was suspended in 450 ml of deionized water. The
suspension was stirred for 15 minutes at room temperature for uniform mixing
(pH 5.5). The pH was adjusted with 6.0 N sulphuric acid (H2SO4). The resultant
suspension was kept in a water bath maintained at 60° C and stirred for uniform
mixing before the enzymes were added.. About 6,000 LU/g of amylase from
Bacillus stearothermophilus (0.6 kg of GC007 [from Genencor International.
Inc.]/Metric ton (Mt) starch dsb) and 15,100 LU/g of amylase from Bacillus
licheniformis (0.6 kg GC262 SP from [Genencor International. Inc.]/Mt. starch
dsb) were added separately and incubated with constant stirring at 60° C .
Samples were withdrawn at different predetermined intervals of time and
analyzed for total sugar composition using high-pressure liquid chromatography
(HPLC). Two ml of sample was taken from each container at a predetermined
time interval using a plastic pipette and transferred to a centrifuge tube. The
sample was centrifuged at 8000 prm for 3 minutes. The supernatent was
withdrawn from the centrifuge tube and a few drops were placed into a sample
well of a Lecia AR200 (Leica Microsystems, Inc., Buffalo, NY, USA) digital hand
held refractometer and recorded (Table 2). The Brix (as a measure of the
dissolved sugars) of the solution was determined (Table 2).
Table 2

The results in the table 2 showed that wheat flour incubated with alpha amylase
from Bacillus stearothermophilus gave a higher maltose content compared to the
maltose content from the incubation with alpha amylase from Bacillus
licheniformis. Incubation of wheat flour with alpha amylases resulted in a
significant increase in the dissolved solids due to the hydrolysis of the granular
starch compared to incubation of wheat flour without alpha amylase addition. It
is interesting to note here that the reaction product of alpha amylase from
Bacillus stearothermophilus resulted in a higher ratio of maltose to glucose and
maltose to maltotriose compared to alpha amylase from Bacillus licheniformis.
These results indicate that the alpha amylase from Bacillus stearothermophilus
is an especially useful enzyme for producing very high maltose syrup.

EXAMPLE 2
Effect of Bacillus stearothermophilus alpha amylase (alpha amylase sold under
the tradename GC 007by Genencor International, Palo Alto, CA) concentration
on the maltose production during incubation with wheat flour. The experimental
conditions were identical as explained in Example 1. In addition, Bacillus
stearothermophilus (6,000 Units/g) was added at 0.1 Kg, 0.2 Kg and 0.6 Kg /MT
of starch dsb. The results are summarized in table 3.
Table 3
Effect of Alpha Amylase [GC007] Concentration on Maltose Yield during
incubation of wheat flour, pH 5.5, 60° C

EXAMPLE 3
One hundred fifty grams of wheat flour was suspended in 450 ml of deionized
water and the pH was adjusted to pH 5.00, 4.50 and 4.00 using 6.0 N H2S04.
The slurry was stirred well for uniform mixing and the pH was adjusted until the
specified pH was stabilized. GC007 was added at 0.1 Kgs/MT, starch dsb to
each of the trial and incubated at 60° C. The samples were withdrawn at
different predetermined intervals of time and the composition of the sugar and
brix were measured as described in Example 1. (Table 4).

The maltose content increased with decreasing pH of the incubation of the
wheat flour from pH 5.5 and reached maximum of about 68% at pH 4.5 followed
by a decrease at pH 4.0. This is an unexpected result showing the production of
maltose content greater than 60 % without the addition of a debranching enzyme
during the hydrolysis of starch with plant beta amylases. The hydrolysis of
liquefied starch by commercial Beta amylases (barley or wheat) generally

produces maltose content between 55% and 60 %. For maltose content greater
than 60 % using liquefied starch, the addition of debranching enzyme and or a
very low starting DE of the liquefied starch are required. It is also important to
note here that the process described in this invention allows maltose
manufacturers to process at pH 4.5 and 60° C that reduces the high risk of
microbial contamination of the current process.
EXAMPLE 4
One hundred fifty grams of wheat flour was suspended in 450 ml of deionized
water and the pH of the slurry was adjusted to pH 4.5. The slurry was stirred
well for uniform mixing and the pH was adjusted with 6.0 N H2SO4 until the pH
was stabilized. The resultant suspension was kept in a water bath maintained at
60° C and stirred for uniform mixing before the enzymes were added. A starch
liquefying enzyme, e.g., a Bacillus stearothermophilus alpha amylase sold under
the tradename "GC 007" (Genencor International, Inc.) was added at 0.1 Kg/MT,
dsb. A debranching enzyme, a pullulanase sold under the tradename OPTIMAX
L-1000 (Genencor International, Inc.) was then added at 0.25 Kg, 0.5 Kg and 1.0
Kg/M T dsb and incubated at 60° C. The samples were withdrawn at
predetermined different intervals of time (2, 4, 6 and 24 hours) and the
composition of the sugar and brix were measured as described in Example 1.
The results were recorded (Table 5).

Maltogenic enzymes (such as beta amylases) or starch liquefying alpha
amylases (such as GC007) can not hydrolyze the alpha 1-6 glucosidic linkages,
the branch point in the amylopectin of the starch substrate. So it is a common
practice to add debranching enzyme, pullulanase (OPTIMAX L-1000 from
Genencor International Inc) for producing maltose greater than 65 % during the
incubation of starch substrate with beta amylase. The effect of OPTIMAX L-1000
concentration during the incubation of wheat flour with GC007 was studied and
the results were shown in Table 5. OPTIMAX L-1000 addition resulted in a
significantly higher level (>75 %) of maltose (DP2) compared to the control.
EXAMPLE 5
It is generally the common practice in the industry to use high maltose
syrup produced by an enzymatic process using a high temperature (>90° C)
enzyme liquefied starch substrate followed by treating with enzyme
glucosyltransferase for producing isomalto-oligosaccharides syrups. This
example illustrates the process of converting the granular starch in the wheat
flour into isomalto-oligosaccahrides in a single step. In this example, 275 grams
of wheat flour was placed in a flask and 688 ml of deionized water was added. It
was then stirred for 15 minutes for uniform mixing and the pH was then adjusted
to pH 4.5 using 6.0 N H2SO4. The resultant suspension was kept in a water bath
maintained at 60 C and stirred for uniform mixing before the enzymes were

added. A starch liquefying enzyme, e.g., Bacillus stearothermophilus alpha
amylase ([GC007 supplied by Genencor International] (0.1 Kgs /MT dsb) and a
debranching enzyme, e.g., a pullulanase (OPTIMAX L-1000 supplied by
Genencor International) (0.5 kgs/MT dsb) were added. The suspension was then
divided into two equal parts. To one of the parts, an Aspergillus niqer
transglucosidase sold under the tradename TRANSGLUCOSIDASE L-500"
(Genencor International) was added at 1.0 Kg/MT dsb and kept in a wafer bath
maintained at 60° C (Sample1). The other part was incubated first for four hours
at 60°, followed by the addition of Aspergillus niaer transglucosidase sold under
the tradename "TRANSGLUCOSIDASE L-500" (Genencor International) at 1.0
Kg/MT dsb and maintenance in a water bath maintained at 60° C (Sample 2).
The results shown in Table 6 indicate that conversion of the substrate to IMO's
occurs with or without preincubation of the substrate (wheat flour) prior to the
addition of the transglucosidase.

Incubation of modified wheat flour containing high content of maltose with
Translucosidase produced isomalto-oligosaccharides identical to the
composition produced by the conventional process. The process is simple,
economical and can be easily scaled to commercial production
EXAMPLE 6
It is common knowledge that cereals like wheat, barley and rye contain
high levels of beta-amylase. Incubation of these cereals at 55°-60° C, pH 5.5
generally results in syrups containing greater than 50% maltose. A 28%
slurry of wheat, barley and rye flour, respectively, was each prepared by
adding 280 grams of the respective flour to 720 gm of deionized water. To
each of these preparations, a Bacillus stearothermophilus alpha amylase
(e.g., a Bacillus stearothermophilus alpha amylase sold under the trademark
"GC007" by Genencor International) was added at 0.2 kg/MT of the flour. The
pH was then adjusted to pH 5.5 using 6.0 N H2SO4 and incubated at 60° C for
4.5 hours. The pH of the incubated samples was then adjusted to pH 4.5
using 6 N H2SO4 and 1.25 kg of transglulcosidase (e.g., a transglucosidase
sold under the tradename TRANSGLUCOSIDASE L-500 by Genencor
International) / MT of the flour was added. The slurries were then incubated
at 60° C water bath for 48 hours. The samples were then centrifuged and
analyzed for I MO composition (Table 7) as set forth in Example 1.

EXAMPLE 7
In an experiment, 140 grams of malt (Cargill Malt/Schreier-Malting
Company, Wisconsin, USA) was mixed with 360 grams of distilled water. The
slurry was stirred for 15.0 minutes at room temperature for uniform mixing and
pH was then adjusted to pH 4.5 using dilute acetic acid. After stabilization of
the pH, the slurry was kept in a water bath maintained at 60° C. Incubation
was continued for 30.0 minutes with constant stirring and a 2 ml sample was
withdrawn for Brix and HPLC analysis (0, time). Transglucosidase L-500 was
added at 1.5 kg/MT malt and incubated at 60° C. Samples were withdrawn at
different predetermined intervals of time during incubation, e.g., 2, 4, 6,
12,and 24 hours, to ascertain Brix and IMO composition (Table 8) as
described in Example 1.

The results in Table 8 showed that the commercial malt extract can be used
as a suitable substrate for producing malt extract containing isomalto-
oligosaccharides. The reaction time and the composition of IMO sugars of the
malt extract could be adjusted by controlling the enzyme dosage. The
addition of maltogenic enzymes can increase IMO content of the resulting
composition.
EXAMPLE 8: Sorghum, Millet and Rice (Exogenous Maltogenic enzymes)
In another experiment, 280 grams of sorghum, millet and rice flour
were each taken separately and mixed separately with 720 grams of
deionized water. The pH of the suspension was adjusted to pH 5.5 and a
Bacillus stearotherphilus alpha amylase sold under the trademark "GC007"
(Genencor International) was added at 0.5 kg/mt of the flour. After uniform
mixing, the suspension was kept in a water bath maintained at 75° C. The
reaction mixture was continuously stirred during incubation for 6 hours. The
temperature was then reduced to 60° C and a beta-amylase (sold under the
tradename OPTIMALT BBA by Genencor International) was added at 1.0
kg/mt of the flour. The incubation was continued for additional 10-15 hours (a
sample was taken for Brix and HPLC). After the specified time, the pH was
reduced to pH 4.5 by 6N H2SO4 and an Aspergillus niger transglucosidase
(sold under the tradename "TRANSGLUCOSIDASE L-500" by Genencor
International) was added at 1.0 kg/mt flour. Samples were taken at 24 and
48 hrs. for analysis (Table 9).

As shown in Table 9, IMO Numbers of 41 to 54 % (52.20, 53.03, 43.90, 41.19,
54.40, and 51.25) were achieved.
EXAMPLE 9: Mixed Grain/Cereals Composition
The data in Example 5 for wheat and Example 6 for barley and rye showed
considerable amount of endogeneous maltogenic enzyme activity resulting in a
syrup containing very high maltose. On the other hand, grains known to not
contain endogenous maltogenic enzymes, for example sorghum, millet and rice,
required the addition of exogenous maltogenic enzyme for producing the
substrate suitable for transglucosidase treatment. In this experiment we studied
the supplementation of maltogenic enzyme containing cereals like wheat or
barley with sorghum and rice for converting the starch to substrates containing
high maltose levels. In a typical experiment, a 15% suspension of sorghum and
rice was prepared by suspending 140 grams of the flour in 720 grams of
deionized water. The pH was adjusted to pH 5.5 using 6 N H2SO4 and Bacillus
sterarothermophilus alpha amylase sold under the trademark "GC007"
(Genencor International) was added at 0.5 kg/mt of the flour. The resultant
suspension was then left in a water bath maintained at 75° C. The suspension
was stirred continuously for 6 hours. The temperature was then reduced to 60°
C. Solid content of flour, e.g., pre-treated rice flour, was increased from 15% to
30% by the addition of barley flour. Similarly, wheat was added to pre-treated
sorghum to a final concentration to reach 30%. The incubation was then
continued for an additional 10-12 hrs. at 60° C. The pH was reduced to 4.5 and
TRANSGLUCOSIDASE L-500 was added at 1.0 kg/mt flour. The incubation at
60° C was continued for 24 hours and 48 hours. The samples were taken for
HPLC analysis and brix; the results are shown in Table 10.

As shown in Table 10 above, the mixtures of millet and barley; and rice and
wheat resulted in 45 to 56 % IMO in the resultant suspension after the above
described incubation periods.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or changes in
light thereof will be suggested to persons skilled in the art and are to be
included within the spirit and purview of this application and scope of the
appended claims. All publications, patents, and patent applications cited herein
are hereby incorporated by reference in their entirety for all purposes.

WE CLAIM :
1. A method for making an isomalto-oligosaccharide grain composition said
method comprising:
(a) contacting an ungelatinized starch in grain (insoluble starch) such as
herein described with a maltogenic enzyme and a starch liquefying enzyme to
produce maltose;
(b) contacting said maltose with a transglucosidic enzyme, wherein said
steps (a) and step (b) occur at a temperature less than or at a starch gelatinization
temperature, such as herein described; and
(c) obtaining a grain composition having an enzymatically produced
isomalto oligosaccharide, wherein said oligosaccharide is present in said grain.

2. The method as claimed in claim 1, wherein said steps (a) and (b) occur
concurrently.
3. The method as claimed in claim 1, optionally comprising the step of drying
said grain composition.
4. The method as claimed in claim 1, wherein said grain is selected from the
group consisting of wheat, rye, barley, and malt.
5. The method as claimed in claim 1, wherein said grain is selected from the
group consisting of millet, sorghum and rice.
6. The method as claimed in claim 1, wherein said maltogenic enzyme is a
beta amylase.

7. The method as claimed in claim 1, wherein said maltogenic enzyme is
endogenous to said grain.
8. The method as claimed in claim 1, wherein said starch liquefying enzyme is
an alpha amylase obtained from a Bacillus.
9. The method as claimed in claim 8, wherein said starch liquefying enzyme is
obtained from Bacillus licheniformis or Bacillus stearothermophilus.
10. The method as claimed in claim 1, wherein said transglucosidic enzyme is a
transglucosidase.
11. The method as claimed in claim 10, wherein said transglucosidase is
obtained from Aspergillus.
12. The method as claimed in claim 11, wherein said Aspergillus is Aspergillus
niger.
13. A grain composition produced as claimed in claim 1.
14. A food additive comprising said grain composition as claimed in claim 13.
15. A flour comprising said grain composition as claimed in claim 13.
16. An isomalto oligosaccharide made as claimed in claim 1.
17. An oral rehydration solution comprising the isomalto oligosaccharide as
claimed in claim 16.

Methods for the production of substrate, tuber, and grain compositions
containing isomalto-oligosaccharides are described. The methods comprise
sequentially or concurrently (a) contacting a substrate, tuber or grain containing
ungelatinized starch with an exogenous or endogenous maltogenic enzyme and a
starch liquefying enzyme to produce maltose; (b) contacting said maltose with a
transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature
less than or at a starch gelatinization temperature; and (c) obtaining a substrate,
grain or tuber composition having an enzymatically produced isomalto-
oligosaccharide, wherein the oligosaccharide is derived from the grain.
Figures 1 and 2.

Documents:

1584-KOLNP-2005-FORM-27.pdf

1584-kolnp-2005-granted-abstract.pdf

1584-kolnp-2005-granted-assignment.pdf

1584-kolnp-2005-granted-claims.pdf

1584-kolnp-2005-granted-correspondence.pdf

1584-kolnp-2005-granted-description (complete).pdf

1584-kolnp-2005-granted-drawings.pdf

1584-kolnp-2005-granted-examination report.pdf

1584-kolnp-2005-granted-form 1.pdf

1584-kolnp-2005-granted-form 13.pdf

1584-kolnp-2005-granted-form 18.pdf

1584-kolnp-2005-granted-form 3.pdf

1584-kolnp-2005-granted-form 5.pdf

1584-kolnp-2005-granted-gpa.pdf

1584-kolnp-2005-granted-reply to examination report.pdf

1584-kolnp-2005-granted-specification.pdf

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Patent Number

228110

Indian Patent Application Number

1584/KOLNP/2005

PG Journal Number

05/2009

Publication Date

30-Jan-2009

Grant Date

28-Jan-2009

Date of Filing

09-Aug-2005

Name of Patentee

GENENCOR INTERNATIONAL INC.

Applicant Address

925 PAGE MILL ROAD, PALO ALTO, CA

Inventors:

#	Inventor's Name	Inventor's Address
1	LI FENG	925 PAGE MILL ROAD, PALO ALTO, CA 94304
2	VADAKOOT JULIUS	925 PAGE MILL ROAD, PALO ALTO, CA 94304
3	DUAN GANG	925 PAGE MILL ROAD, PALO ALTO, CA 94304
4	SHETTY JAYARAMA K	925 PAGE MILL ROAD, PALO ALTO, CA 94304

PCT International Classification Number

C07H

PCT International Application Number

PCT/US2004/007781

PCT International Filing date

2004-03-10

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/477,199	2003-06-09	U.S.A.
2	60/453,668	2003-03-10	U.S.A.