CN1756769A - Cereal composition containing probiotic isomalto-oligosaccharides and methods for its manufacture and use - Google Patents

Abstract

Methods for producing isomalto-oligosaccharide containing substrate, tuber and grain compositions are described. The method comprises the following steps: contacting a substrate, tuber or grain containing ungelatinized starch with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglycosidic enzyme, wherein said steps and (b) occur at a temperature less than or equal to the starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having enzymatically produced isomalto-oligosaccharides, wherein said oligosaccharides are derived from said grain. The maltogenic enzyme may be exogenous or endogenous to the grain. The contacting steps may be sequential or simultaneous. The invention also describes cereal flour, oral rehydration solutions, beer additives, food, feed, beverage additives, containing the cereal compositions prepared as described.

Description

Cereal composition containing probiotic isomalto-oligosaccharides and methods for its manufacture and use

technical field

The present invention describes cereal compositions containing isomalto-oligosaccharides and methods for their manufacture. The method involves deriving isomaltooligosaccharides from starch contained in cereals.

Background technique

Isomaltooligosaccharides ("IMOs") are mixed linkage oligosaccharides containing a mixture of 1,4α and/or 1,6α glycosidic linkages. They are also known as randomly linked oligosaccharides ("ALOs"). Isomaltoligosaccharides contain a large number of branched oligosaccharides such as isomaltose, 4-alpha-glucosylmaltose, isomaltotriose, isomaltotetraose, iso-4-alpha-glucosylmaltose and more highly branched oligosaccharides .

There is a market demand for products containing IMOs. IMO products are sold in powder or liquid form, depending on the intended use. A potential use is in the food sector. Examples of IMO products are: condiments (mayonnaise, vinegar, soup bases, etc.), confectionery (candies, chewing gum, chocolate, ice cream, sherbet, syrups), processed fruits and vegetables (jams, marmalades, fruit sauces, pickles), meat or fish products (ham, sausages, etc.), baked goods (bread, cakes, cookies, puff pastry), pre-cooked foods (salads, cooked beans, etc.), canned and Bottled food, convenience food (instant coffee, instant cake base, etc.), and beverages, including alcoholic beverages (alcoholic beverages, seju, fruit wine, sake, beer [International Publication No. WO 02/20712 A1], etc.) also includes non-alcoholic beverages (coffee, fruit juices, honey drinks, aerated or carbonated drinks, lemonade, cola). Isomaltooligosaccharides can be further used as ingredients in animal feed and pet feed. Non-food application areas are cosmetics and pharmaceuticals (cigarettes, lipsticks, toothpaste, internal medicines, etc.).

Isomaltooligosaccharides belong to a class of oligosaccharides classified as functional health food oligosaccharides ("FHFO"). Representative IMOs include fructo-oligosaccharides, galacto-oligosaccharides, xylose-oligosaccharides and gentio-oligosaccharides. IMOs are classified as "prebiotics" when taken orally on a regular daily basis, which is associated with an improvement in the overall health status of humans and animals. Prebiotics are defined as non-digestible substances (eg, dietary fibers) that exert some biological effect on humans by selectively stimulating the growth or biological activity of beneficial microorganisms present in the gut or introduced into the gut during therapy. (Przemyslaw Jan Tomasik and Piotr Tomasik. 2003 American Association of Cereal Chemists, Inc. 80(2): 113-117). The "prebiotic" effect of oligosaccharides is to increase the number of bifidobacteria and lactobacilli ("prebiotics") in the large intestine and to reduce the concentration of putrifactive bacteria. Bifidobacteria have been associated with several health-promoting properties, such as inhibition of pathogen growth, either through acid formation or through antimicrobial activity. They are also associated with various effects of this class, such as modulation of the immune system (antineoplastic properties), reduction of triglyceride and cholesterol levels, production of vitamins (B complex), reduction of blood ammonia levels, prevention of translocation, normalization after antimicrobial therapy Intestinal flora, produce digestive enzymes, reduce side effects associated with antibiotics (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(3) (1992), 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.

Isomalt oligosaccharides are synthesized by enzymatically catalyzed transglucosylation reaction using D-glucosyltransferase (E.C.2.4.1.24, transglucosidase, α-glucosidase). This enzyme catalyzes hydrolysis and transfer reactions when incubated with α-D-gluco-oligosaccharides. The transfer to 6-OH (hydroxyl group 6 of the glucose molecule) occurs most frequently, isomaltose from D-glucose, or 4-alpha-glucosylmaltose from maltose. The enzyme can also catalyze the transfer to 2-OH or 3-OH of D-glucose to form kojibiose (2-glucose-α-glucoside) or nigerose, or return to 4-OH to form maltose. As a result of the transglucosidase reaction, maltooligosaccharides are converted to isomaltoligosaccharides, forming a class of oligosaccharides that contain a higher proportion of glucose moieties attached to the primary hydroxyl group at the non-reducing end of the glucose molecule Groups are linked, for example, by α-D-1,6 glycosidic linkages. The transglucosidase from Aspergillus niger acts only on oligosaccharides with a low degree of 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). The degree of polymerization refers to the number of glucose units. For example, a diglucosyl molecule such as maltose has a DP of 2. These sugars have received increasing attention as food additives because they help prevent dental caries (Oshima, et. al 1988. The caries inhibitory effects of gos-sugar in vitro and rates experiments. Microbial Immunol. 32, 1093- 1105), and can be used as a growth factor (prebiotic) of Bifidobacteria to improve human gut microbes (Komoto, et.al 1988; Effect of Isomalto-oligosaccharides on human fecal flora, Bifidobacteria Micro flora 7, 61-69) .

Isomaltooligosaccharides can be obtained in different ways. For example, treatment of high dry solids concentration, ie, 60-80% glucose syrup, with glucoamylase resulted in the formation of isomaltooligosaccharides with a DP of mainly 2. The presence of high solids levels forces the reaction to reverse from its normal direction in favor of hydrolysis.

Cereals, including wheat, barley, etc., are excellent in the economical production of many value-added functional food ingredients such as wheat flour, starch, starch hydrolysates (glucose, fructose, high maltose syrup, etc.) and wheat gluten (wheat gluten) raw materials. In the production of antibiotics, drugs, vaccines, biochemicals such as ethanol (both beverages and fuels), amino acids, organic acids, etc., and more recently in the production of functional health food oligosaccharides called isomaltooligosaccharides , syrups containing high levels of maltose are also used as carbon sources in many microbial fermentations. In traditional methods of producing starch hydrolysates, such as maltose syrup, the insoluble granular starch is separated from other cellular components of wheat prior to hydrolysis, usually by starch liquefaction and maltogenic alpha-amylases. Maltose is a disaccharide composed of two glucosyl residues linked by an α1-4D-glycosidic bond, and it is the smallest substance in the maltooligosaccharide family. It is mass-produced as syrups, powders and crystals of various purity levels. Various maltose syrups have received significant attention for use in the brewing, baking, canning of soft drinks, confectionary and other commercial applications in the food and beverage industry. In Japan, ultra-pure maltose is used as an intravenous nutrient. Catalytic reduction of maltose forms maltitol, which is considered a low-calorie sweetener. Recently, high maltose syrup has become an important raw material for the industrial production of isomalto-oligosaccharides (J.K.Shitty and O.J.Lantero, 1999 "Transglucosylation of Malto-oligosaccharides." Paper presented at 50th Starch Convention, Detmold, Germany).

In traditional methods of producing starch hydrolysates such as high maltose syrup, a thermostable liquefying alpha-amylase from Bacillus licheniformis or Bacillus stearothermophilus [EC 3.2.1.2 , α(1,4)-glucan glucan hydrolase] will separate the insoluble starch. Purified starch (refined) is hydrolyzed by suspending insoluble granular starch (30%-50% dissolved solids content [dsb]) in water and heating to a temperature between 85°C and 120°C to dissolve the starch and make it readily by enzymatic hydrolysis. The liquefied starch is further treated to produce a starch hydrolyzate with a different carbohydrate composition using a maltose-forming enzyme, such as fungal alpha-amylase (sold under the trade name CLARASE L by Genencor International, Palo Alto, CA) for products containing less than 55% maltose syrup, beta amylase (sold by Genencor International, Palo Alto, CA under the trade name OPTIMAL BBA) was used to produce a syrup containing 55% to 62% maltose content and less than 1% glucose. For higher levels of maltose syrup, >62%, the combined addition of debranching enzyme (sold by Genencor International, Palo Alto, CA under the trade name OPTIMAX L-1000) and beta-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 method has been reported for the conversion of granular starch (refined) to a soluble hydrolyzate by incubation with bacterial alpha-amylase at temperatures below the starch gelatinization temperature (Leach et.al 1978; U.S. Patent No. 4,113,509), followed by hydrolysis by β-amylase to produce high maltose syrup (Leach et.al 1975; US Patent No. 3,922,196). However, the syrup produced by this method contains only 55% maltose and very high levels of maltotriose. European Patent Application 0905256 (Christophersen, et.al 2000) and U.S. Patent 5,141,859 (Nimmi, et.al 1992), describe the production of high maltose syrup using liquefied starch (after gelatinization, hydrolyzed with thermostable alpha amylase) . This process is cumbersome, expensive and requires the separation of starch from other cellular components, the cost of added maltose-forming enzymes, high temperature treatment and long reaction times. European patent application 0350737 A2 (Shinke, et.al 1989) discloses a method for producing maltose syrup by hydrolyzing the α-amylase from corn, wheat, potato and sweet potato at 60°C using alpha-amylase from Bacillus stearothermophilus. Granular (purified) starch without the traditional liquefaction step (gelation followed by liquefaction at high temperature). However, this hydrolyzed starch results in maltose concentrations ranging from 50% to 55%. The syrup also contained very high levels of maltotriose (30-36%). Regardless of the source of the starch, this method results in a ratio of maltose to maltotriose of less than 2.0. Due to the difficulty of metabolizing maltotriose, maltose syrup containing high levels of maltotriose is not a preferred substrate for carbon supply in many microbial fermentations including yeast ethanol fermentation (J.K. Shetty and O.J. Lantero, 1999 "Transglucosylation of Malto-oligosaccharides . ", the paper was released on the 50th starch conference in Detmold, Germany). US Patent 6,361,809 describes a process for the production of maltose and limited dextrins by treating purified granulated waxy cornstarch with the hydrolytic enzyme, maltogenase alpha-amylase from Bacillus stearothermophilus classified as EC 3.2.1.133, followed by the application of supernatant Separation of maltose by filtration. Evaporation of dilute permeate containing maltose is expensive due to high energy costs and high risk of microbial contamination.

Traditionally, grains such as wheat, malt, sorghum (Sorghum spp.), millet (Pampillium), and especially whole grains, have been used as sources of macro- and micro-elements, proteins, fibers, and vitamins. Carrier, used in nutritional products. Grains Most grains seem to be too easily digested to be effective as prebiotics or even as nutraceuticals. It has been suggested that genetically modified, less digestible cereals, suitable as prebiotics, be designed to modulate the gut microbiota (Gibson, G.R, and Roberfroid, M.B. 1995, Dietary modulation of the human colonic microflora: Introducing the concept of prebiotics. J. Nutr. 125, 1401-1412).

There has been interest in methods of producing cereal compositions containing isomaltooligosaccharides, in the hope that such methods would not require separation of starch from other cereal components and/or decontamination of the substrate prior to transglucosidation action. Isomaltooligosaccharides can be enzymatically derivatized from source substrates such as grains or tubers by subjecting starch to high temperatures under steam pressure. Low pH methods for minimizing microbial contamination have also been of interest. The present invention addresses these concerns.

Summary of the invention

The present invention describes a method for producing an isomaltooligosaccharide cereal composition comprising: (a) contacting a cereal containing ungelatinized starch with a maltogenic enzyme and a starch liquefying enzyme , to generate maltose; (b) contacting said maltose with a transglucosidic enzyme (transglucosidic enzyme), wherein the temperature at which said step (a) and said step (b) occurs is lower than or equal to the starch gelatinization temperature; and (c) Obtaining a cereal composition with enzymatically produced isomaltooligosaccharides, wherein said oligosaccharides originate from said cereals.

Optionally, in one embodiment, steps (a) and (b) occur simultaneously. In another embodiment, the method further comprises the step of drying said cereal composition. In another embodiment, the grain is selected from wheat, rye, barley, malt and rice. In another embodiment, the grain is selected from sorghum, millet and rice. In another embodiment, the maltogenic enzyme is beta amylase. In another embodiment, said malting enzyme is endogenous to said grain. In another embodiment, said malting enzyme is exogenous to said grain. In another embodiment, the starch liquefying enzyme is an alpha-amylase derived from Bacillus. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidase is transglucosidase. In another embodiment, the transglucosidase is derived from Aspergillus. In another embodiment, the transglucosidase is derived from Aspergillus niger. Other embodiments of the invention include cereal compositions produced according to the methods described above. Another embodiment of the present invention includes a food additive comprising the above described cereal composition.

The present invention also describes a method for producing isomalto-oligosaccharide-fortified cereal flour at or below the gelatinization temperature, wherein the ungelatinized cereal with endogenous maltogenic enzymes is contacted with a solubilizing enzyme selected from Bacillus enzyme) to produce maltose syrup. Exposure of maltose syrup to transglucosidase yields a matrix (tuber or grain) composition comprising isomalt-saccharides derived therefrom.

The present invention also describes a process for the production of isomalto-oligosaccharide-fortified cereal flour at or below the gelatinization temperature, wherein ungelatinized cereals (wheat, barley, etc.) Ungelatinized grains (eg, sorghum, millet, or rice) having endogenous maltogenic enzymes are mixed, and the grain mixture is contacted with a lytic enzyme selected from Bacillus to produce a maltose syrup. Contacting this maltose syrup with transglucosidase produces a matrix (tuber or grain) composition comprising isomalt-saccharides derived therefrom.

The invention also describes a method for making a wheat grain composition comprising: (a) contacting ungelatinized wheat grain containing endogenous maltogenic beta amylase with a starch liquefying alpha amylase from Bacillus , to produce maltose; (b) contacting said maltose with a transglucosidase, wherein said steps (a) and (b) occur at a temperature lower than the gelatinization temperature of wheat; A wheat grain composition of isomaltooligosaccharides, wherein said oligosaccharides are derived from said ungelatinized grains.

Optionally, in another embodiment, the method applies the method described above to manufacture a cereal composition for use in the manufacture of a food additive. Another embodiment includes the cereal composition made thereby. Another embodiment includes a cereal flour comprising the cereal composition as described above. Another embodiment includes isomaltooligosaccharides made according to the method described above. Another embodiment includes an oral rehydration solution comprising the above-described isomaltooligosaccharides. Another embodiment includes a cereal composition comprising ungelatinized cereal and at least one isomaltooligosaccharide, wherein said isomaltooligosaccharide is enzymatically derived from said ungelatinized cereal. In another embodiment, the cereal composition contains greater than 1% by weight of at least one isomaltooligosaccharide.

Brief description of the drawings

Figure 1 is a flowchart describing the production of isomalto-oligosaccharide fortified flour.

Figure 2 is another flowchart describing the production of isomaltooligosaccharide enriched wheat flour.

Detailed description of the invention

definition

The term "grain" refers to plants which are classified as cereals or monocotyledonous plants belonging to the order Gramineae, especially monocotyledonous plants belonging to the family Poaceae. Examples of plants belonging to this class are those selected from the genera Triticum (wheat), Hordeum (barley); Triticum (rye); Zea mays (corn (corn) or maize (maize)); Avena (oats) , Buckwheat (buckwheat); Sorghum (Sorghum or milo), Panicum or Setaria (millet or barnyardgrass); or Oryza (rice).

For example, in one embodiment, the term "wheat" refers to a plant that is or was classified as a strain of Triticum aestivum.

For example, in one embodiment, the term "barely" refers to a plant that is or was classified as a strain of Hordeum vulgare.

For example, in one embodiment, the term "rye" refers to a plant that is or was classified as a strain of Secale cereal.

For example, in one embodiment, the term "corn" refers to a plant that is or was classified as a strain of Zea mays.

For example, in one embodiment, the term "oats" refers to plants that are or were classified as Avena sativa strains.

For example, in one embodiment, the term "buckwheat" refers to a plant that is or was classified as a buckwheat (Fagopyrum esculentum) strain.

For example, in one embodiment, the term "sorghum" refers to a plant that is or was classified as a Sorghum bicolor strain.

For example, in one embodiment, the term "millet" refers to plants that are or were classified as Panicum miliaceum or Setaria italic strains.

For example, in one embodiment, the term "rice" refers to a plant that is or was classified as an Orvza sativa strain.

The term "tuber" refers to a starch storage organ (eg, potato, sweet potato, yam, cassava, etc.), which is formed by the distal expansion of an underground stem or root.

For example, in one embodiment, the term "potato" refers to a plant that is or was classified as a Solanum tuberosum strain.

For example, in one embodiment, the term "sweet potato" refers to a plant that is or was classified as a line of ipom [oe]a batatas.

For example, in one embodiment, the term "yam" refers to a plant that is or was classified as a strain of Dioscorea sativa, D. villosa, C. batatas.

The term substrate (also sometimes referred to herein as "substrate") refers to substances that can be converted enzymatically to maltose and thus to IMOs. The term "substrate" includes, for example, cereals and tubers. Further, the term substrate includes all forms of grains (hulled or not) or tubers, such as whole grains, cracked grains, meal and flour, and any plant part.

The term "starch" refers to any substance consisting of complex polysaccharide carbohydrates of plants, consisting of amylose and amylopectin of the formula (C6H10O5)x, where X can be any number.

The term "granular strach" refers to uncooked (raw) starch, which has not been gelatinized. The term "gelatinization" refers to the dissolution of starch molecules to form a viscous suspension.

The term ""substrate", "grain" or "tuber" containing ungelatinized starch" means an ungelatinized substrate, grain or tuber which has not been subjected to a temperature above the gelatinization temperature of starch, said starch The gelatinization temperature causes gelatinization or liquefaction of the starch contained in the substrate.

The term "maltose" refers to a disaccharide having two glucosyl residues linked by an α1,4D-glycosidic bond.

The term "isomaltose" refers to a disaccharide having two glucosyl residues linked by an α1,6D-glycosidic bond.

The term "isomalto-oligosaccharide (IMO)" refers to a sugar having at least two glucosyl residues linked by an α1,6 glycosidic bond at the non-reducing end. Furthermore, the term may refer to irregularly linked oligosaccharides, sugars having both α1,6 and α1,4 glycosidic linkages. Representative isomaltoligosaccharides include isomaltose, 4-α-glucosylmaltose (panose), and isomalto-triose.

The term "isomalto-oligosaccharide" cereal composition refers to a cereal composition characterized by a level of isomalto-sugars of at least 1% (w/w %) of the total sugar content, as determined by HPLC determined by chromatographic methods.

The term "maltogenic enzyme" refers to an enzyme that converts starch into maltose. Representative malting enzymes include alpha and beta amylases of fungal, bacterial and plant origin.

The term "amylases" refers to enzymes that catalyze the hydrolysis of starch.

The term "α-amylases" refers to enzymes of (E.C.) 3.2.1.1 class and/or 3.2.1.133 class which catalyze the hydrolysis of α-1,4-glycosidic bonds. These enzymes are also described as those that exohydrolyze or endohydrolyze 1,4-alpha-D-glycosidic linkages in polysaccharides containing 1,4-alpha-linked D-glucose units. Another term used to describe these enzymes is glycogenase. Representative enzymes include alpha-1,4-glucan 4-glucanohydrolase glucanohydrolase.

The term "beta-amylases" refers to enzymes of the class (E.C.) 3.2.1.2, which catalyze the hydrolysis of alpha-1,4-glucosidic bonds, releasing maltose units. These enzymes have also been described as those which hydrolyze 1,4-α-D-glycosidic linkages in polysaccharides so as to remove consecutive maltose units from the non-reducing ends of the chains.

The term "transglucosidic" enzyme refers to an enzyme that catalyzes both hydrolysis and transfer reactions when incubated with αD-gluco-oligosaccharides. Representative enzymes include transglucosidases and/or those of the (E.G.) 2.4.1.24 class, eg, D-glucosyltransferases. These enzymes are also known as 1,4-α-glucan-α-glucosyltransferases and oligoglucan branched glycosyltransferases.

The term debranching enzyme refers to an enzyme that catalyzes the hydrolysis of alpha-1,6-glycosidic bonds. In this regard, enzymes of the class E.C.3.2.1.41 are useful. A representative enzyme of this class is pullanase, also known as α-dextrin endo-1,6-α-glucosidase, limit dextrinase, debranching enzyme, pullulan 1,6 - Glucanohydrolase. Other representative enzymes of class (E.C.) 3.2.1.41, for example, pullulanases (pullulanases), [α-(1-6)-glucan 6-glucanohydrolase, also known as α-( 1,6)-glucosidase]).

The term "starch gelatinizing temperatures" refers to temperatures high enough to effectively liquefy or gelatinize granular starch. Heating the starch in water causes the starch granules to swell. At sufficient solids concentrations, the swollen particles take up most of the space and a viscous mass called paste results. The dissolution of starch molecules is called gelatinization. Gelation is accompanied by a loss of birefringence. The term starch gelatinization 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. Representative starch liquefying enzymes include alpha amylases of the class (E.C.) 3.2.1.1.

The term "endogenous" refers to an enzyme present in the grain or tuber without resorting to adding the malting enzyme to the grain.

The term "exogenous" refers to enzymes that are not present in the grain. Representative exogenous enzymes include, for example, enzymes that are not present in wild-type substrates such as rice, millet, and the like.

The term "total sugar content" refers to the total amount of sugar present in a starch, cereal or tuber composition.

The term "IMO Number (IMO No.)" is calculated as the sum of isomaltose, 4-α-glucosylmaltose, isomaltotriose and branched sugars with a DP greater than 3. The IMO number provides an indication of the amount of IMO compound present in the compound or solution.

The term "ratio of branched sugars (RBS)" refers to the ratio of the level of maltose (DP2) present in the cereal to the level of maltotriose (DP3) present in the resulting cereal composition.

The term "Degrees of Diastatic Power" (DP°) unit refers to the amount of enzyme contained in a 5% solution of an enzyme preparation sample of 0.10 ml at 20°C (68°F) and 100 ml of substrate After 1 hour of incubation, sufficient reducing sugars are formed to reduce 5 ml of Fehling's solution.

The term "DE" or "dextrose equivalent" is an industry standard for determining the concentration of total reducing sugars that, when calculated, are converted to D-glucose, calculated as Dry weight calculation (on a dry weight basis). The DE of unhydrolyzed granular starch is essentially 0, and that of D-glucose is 100.

The term "total sugar content" refers to the total content of sugar present in the starch composition.

The terms "dry solid basis" and "dsb" refer to the total amount (expressed in %) of compounds in a slurry, such as flour, calculated on a dry weight basis.

The term "Brix" refers to the well-known hydrometer scale used to determine the sugar content of a solution at a specific temperature. Thus, the term "Brix" refers to the measure of dissolved sugar in a solution. The Brix scale value measures the grams of sucrose present per 100 grams of aqueous sugar solution (total dissolved solids content). For example, a measurement of 1.00 Brix means that the sugar in solution is approximately 10 mg/ml. The measurement of Brix is usually done by using a hydrometer or a refractometer.

The term "degree of polymerization (DP)" refers to the number (n) of anhydroglucopyranose units in a particular carbohydrate. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4 ⁺ means a polymer with a degree of polymerization greater than 3.

The term "enzymatically produced" means that the substrate is enzymatically catalyzed into IMO, which is different from the chemical synthesis or organic chemical synthesis of 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 yeast. Vegetative growth of filamentous fungi is elongated by means of mycelium, and carbon catabolism is obligately aerobic. In the present invention, the filamentous fungal parental cells may be, but are not limited to, cells of the following genus: Trichoderma, for example, Trichoderma reesei (formerly classified as Trichoderma longibrachiae (T. longibrachiatum), currently known as Hypocrea jecorina), Trichodermaviride, Trichoderma koningii, Trichoderma harzianum; Penicillium sp.; Humicola Humicola sp., including Humicola insolens and Humicola grisea; Chrysosporium sp., including C. lucknowense; Gliocladium sp.; Aspergillus sp., including Aspergillus oryzae (A.oryzae), Aspergillus nidulans (A.nidulans), Aspergillus niger (A.niger) and Aspergillus awamori (A.awamori); Fusarium sp., Neurospora sp., meat Hypocrea sp. and Emericella sp. See also Innis et al., (1985) Sci. 228:21-26.

The term "Aspergillus" or "Aspergillus" refers to any mold formerly or currently classified as Aspergillus.

The term "bacteria" refers to Bacillus, including but not limited to B. subtilis, B. amyloliquefaciesn, B. lentus, B. Carlsberg, lichen Bacillus (B. licheniformis) and Bacillus stearothermophilus (B. stearothermophilus).

The term "plant origin" refers to an enzyme derived, extracted, isolated, expressed from a plant source such as barley malt, soybean, wheat or barley.

The term "contacting" refers to placing the individual enzyme(s) in close enough proximity to their respective substrates so that the enzyme(s) can convert the substrates to the desired end product. product. Those skilled in the art will recognize that contacting can be effected by mixing the enzyme, or a solution of the plurality of enzymes, with the respective substrates.

The term "incubating" means mixing a matrix containing a substrate with a respective enzyme, for example, liquefaction enzyme or maltogenic or transglucosidase enzyme, under specific conditions for a defined period of time .

The term "enzymatic conversion" refers to the modification of a rice substrate to produce soluble hydrolyzed granular rice starch, preferably 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 in a liquid or solid medium under suitable conditions. In one embodiment, culturing refers to the fermentative bioconversion of a granular starch substrate into glucose syrup or other desired end product (typically in a vessel or reactor).

For example, in one embodiment, the term alpha-amylase unit is defined as the amount of alpha-amylase that hydrolyzes 1 micromole of starch substrate in 1 minute under standard assay conditions of pH 5.2 and 40°C.

For example, in one embodiment, the term beta-amylase unit is defined as the amount of beta-amylase that hydrolyzes 1 micromole of starch substrate in 1 minute 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 that converts 1 micromole of maltose substrate in 1 minute under standard assay conditions of pH 4.8 and 37°C.

In another embodiment, the term transglucosidase unit is defined as a transglucosidase that generates 1 micromole of 4-alpha-glucosylmaltose per minute under standard analytical conditions of pH 4.8 and 37°C quantity.

For example, in one embodiment, the term one Liquefon unit (LU) is a measure of the digestion time required for a solution of iodine to produce a color change, which represents the amount of starch substrate paste under standard analytical conditions of pH 5.6 and 25°C. Definitive period of refinement.

"ATCC" means the American Type Culture Collection, located in Manassas, VA 20108 (ATCC, www/atcc.org).

"NRRL" means the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (formerly known as the USDA Northern Regional Research Laboratory) , Peoria, ILL.

"NCBI" means the National Center for Biotechnology Information, Natl Library Med. (www.ncbi.nlm.nih.gov/).

"A", "an" and "the" include plural reference unless the context clearly dictates otherwise.

The present invention describes a method for the manufacture of an isomaltogenic oligosaccharide substrate, cereal or tuber composition comprising: (a) contacting a substrate containing ungelatinized starch, e.g. Cereals or tubers to produce maltose; (b) contacting said maltose with a transglucosidase enzyme, wherein said step (a) and said step (b) occur at a temperature lower than or equal to the starch gelatinization temperature; and ( c) Obtaining a substrate, cereal or tuber composition with enzymatically produced isomaltooligosaccharides, wherein said oligosaccharides originate from said substrate, cereal or tuber. Figure 1 depicts one embodiment of the invention.

The invention also describes a method of making an isomaltooligosaccharide-rich matrix, cereal or tuber composition, powder, oral rehydration solution, and/or food additive at a temperature equal to or lower than the gelatinization temperature, which will have Or a substrate containing ungelatinized starch and containing an endogenous maltogenic enzyme is contacted with a lytic enzyme selected from Bacillus to produce a maltose syrup. The maltose syrup is then contacted with a transglucosidase at a temperature equal to or lower than the gelatinization temperature or liquefaction temperature to produce a cereal composition having isomaltooligosaccharides. In one embodiment, the cereal composition is characterized by more than 60% maltose and a branched sugar ratio of greater than 2.0 to 1.0 in the sugar composition. Conversion from substrate to IMO can occur enzymatically.

The invention also describes a method for producing an isomaltogenic oligosaccharide substrate, grain or tuber composition, the method comprising: (a) contacting a starch-containing substrate, cereal or tuber with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting maltose with a transglucosidic enzyme, wherein said step (a) and said step (b) occur at a temperature lower than or equal to the starch gelatinization temperature; and (c) obtaining isomalt with enzymatic generation A substrate, cereal or tuber composition of oligosaccharides, wherein the oligosaccharides are derived from the substrate, cereal or tubers. The invention may optionally further describe an additional step of separating the insoluble constituents from the soluble constituents. The invention further describes the 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 (sorghum milo), millet (sorghum), and rice. In another embodiment, the maltogenic enzyme is beta amylase. In another embodiment, the malting enzyme is endogenous to the grain. In another embodiment, the starch liquefying enzyme is an alpha amylase derived from Bacillus. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidic enzyme is transglucosidase. In another embodiment, the transglucosidase is derived from Aspergillus. In another embodiment, the Aspergillus is Aspergillus niger. The invention also describes cereal compositions, food supplements, oral rehydration solutions and/or powders produced according to the methods described above.

In another embodiment, the invention describes a method for the manufacture of a wheat grain composition comprising: (a) combining ungelatinized wheat grain with endogenous maltogenic beta-amylase with Bacillus-derived Starch liquefaction α-amylase is contacted to generate maltose; (b) contacting maltose with transglucosidase, wherein the temperature at which said step (a) and said step (b) occurs is lower than or equal to the starch gelatinization temperature; and (c ) obtaining a wheat cereal composition having enzymatically generated isomaltooligosaccharides, wherein said oligosaccharides are derived from said ungelatinized cereals. Figure 2 depicts an embodiment of the invention.

In another embodiment, the methods described above may be used to manufacture food additives, bakery products, oral rehydration solutions and/or powders. In another embodiment, the maltogenic enzyme is beta amylase. In another embodiment, the malting enzyme is endogenous to the grain. In another embodiment, the starch liquefying enzyme is an alpha amylase derived from a bacterial source. In another embodiment, the bacterial source is Bacillus. In another embodiment, the starch liquefying enzyme is derived from Bacillus licheniformis or Bacillus stearothermophilus. In another embodiment, the transglucosidic enzyme is transglucosidase. In another embodiment, the transglucosidase is derived from a fungal source. In another embodiment, the fungal source is Aspergillus. In another embodiment, the Aspergillus is Aspergillus niger. The invention also describes cereal compositions, food supplements, oral rehydration solutions and/or powders produced according to the methods described above. The cereal composition may contain greater than 1% by weight of at least one isomaltooligosaccharide. The at least one isomaltooligosaccharide may be selected from isomaltose, 4-α-glucosylmaltose, and isomaltotriose. In a further embodiment of the invention the endogenous maltogenic enzyme is selected from beta amylase or alpha amylase. In yet a further embodiment of the invention, the lytic enzyme is a liquefied alpha-amylase derived from Bacillus. In yet a further embodiment of the invention, the liquefying amylase is derived from Bacillus licheniformis or Bacillus stearothermophilus.

Matrix (or substrate) (substrates)

The invention includes a starch-containing substrate, such as a starch-containing grain or tuber, contacted with a maltogenic enzyme and a starch liquefying enzyme to produce maltose. The term substrate refers to a substance that can be converted enzymatically to maltose and thus to MIO. A representative substrate may be at least one substrate selected from cereals and tubers. The maltose may be in the form of a maltose-enriched syrup or slurry.

Starch exists in two forms, amylose, a linear chain polysaccharide, and amylopectin, a branched chain polysaccharide. Amylose contains long unbranched chains in which all D-glucose units are composed of α-1,4-linkages (“α-1,4-linkages” or “1,4-α-D-glucosyl linkages” )connect. Amylopectin is highly branched, the glycosidic linkages of the backbone are α-1,4 linkages, but the glycosidic linkages of the branch points are α-1,6 linkages. The main constituents of starch can be enzymatically hydrolyzed in two different ways. Amylose can be hydrolyzed by α-amylases (E.C.3.2.1.1), e.g., α-(1-4)-glucan 4-glucanohydrolase (α-(1-4)-glucan 4-glucanohydrolase) . Alpha amylases hydrolyze α-(1,4) linkages to produce a mixture of glucose, maltose, maltotriose and higher sugars. Amylose can also be hydrolyzed by β-amylase (E.C.3.2.1.2) [α(1,4)-glucan maltohydrolase, 1,4-α-D-glucan maltohydrolase]. This enzyme cuts off consecutive maltose units from the non-reducing end to quantitatively produce maltose. Alpha and beta amylases also hydrolyze amylopectin. Neither α-amylase nor β-amylase can hydrolyze the α(1-6) bond at the branch position of amylopectin. The end product of the exhaustive action of β-amylase on amylopectin is a large, highly branched core structure or β-limited dextrin. Debranching enzymes (E.C.3.2.1.41, e.g., pullulanases, [α-(1-6)-glucan 6-glucanohydrolase, also known as α-(1,6)-glucosidase]) can Hydrolyzes α-(1-6) bonds at branch positions. Therefore, the combined action of β-amylase and α1,6 glucosidase can completely degrade amylopectin into maltose and glucose, resulting in maltose content as high as 60%, 65%, 79%, 75%, 80% or higher.

For the purposes of the present invention, the starch-containing substrate may be cereals or tubers or mixtures thereof. A grain may be any cereal crop or seed that contains starch. The substrate can be ground, ground or otherwise reduced in size to increase the surface area of the substrate in contact with the respective enzyme. For example, the substrate can be wet milled or dry milled, as desired. In one embodiment of the invention the starch is granular starch. Grains contemplated for use in the present invention include any grain currently used in baking, pasta, or other applications. Contemplated representative grains include, but are not limited to, at least one selected from the group consisting of wheat (Triticum genus, including but not limited to, T. monococcum, T. turgidum, spelt ( T. spelta) and/or common wheat (T. aestivum)), barley (e.g., Hordeum vulgare and the varieties described in Table 1 of U.S. Patent 6,492,576), rye (Secale sp.), including But not limited to rye (S. cereal)), corn (Zea mays, including but not limited to Zea mays), buckwheat (Buckwheat, including but not limited to buckwheat (F. esculentum)), malt (for example, Malted barley), sorghum (Sorghum genus, including but not limited to Sorghum bicolor or otherwise known as milo sorghum, millet (Prunus barnyardii) (Porticum and Setaria, including but not limited to P. milaceum ); Setaria, including but not limited to chestnut (S. italica)) and rice (Oryza, including but not limited to Oryza sativa). It is contemplated that both wild-type plants and transgenic plants with beneficial properties can be used as Starch-containing substrates, said beneficial properties such as increased levels of endogenous enzymes or the presence of exogenous enzymes.

Germinated cereals, e.g., malt, are used as one of the key ingredients in many food and healthy drink formulations due to their high nutritional value, e.g., malt-containing foods (Table A). Germination results in the synthesis and activation of endogenous malting and proteolytic enzymes. Therefore, sprouted cereals are good sources of grains containing endogenous malting enzymes. Malt powder and malt extract are also used as a source of digestive enzymes in brewing and baking applications. Germination of barley, however, renders the grain components too digestible to be effective as prebiotics or even as nutraceuticals, since they tend to be fully digested before reaching the lower gastrointestinal tract. Unfortunately, the beneficial effects of prebiotic compounds are most recognized in the lower GI tract. Thus, converting the highly digestible malt-sugars to the less digestible isomalt-sugars enables the modified malt to act as a prebiotic, allowing the malt to reach the lower GI tract and provide additional functionality and health benefit. For example, Table A provides suitable commercially available food products containing malt extract.

Form A

Commercial Foods Containing Malt Extract brand name manufacturer Place HORLICKS Glaxosmithkline Punjab, India MALTOVA Glaxosmithkline Punjab, India VIVA Glaxosmithkline Punjab, India BOURNAVITA Cadbury Mumbai, India BOOST Jagjit Industries Punjab, India MILO Nestle New Delhi, India

Therefore, the use of malt as a starch-containing substrate converts some of the granular starch contained in this substrate into other beneficial forms of oligosaccharides, eg IMO.

Alternatively, the starchy substrate may be tubers. Contemplated tubers include potato (Solanum sp., including but not limited to S. tuberosum), sweet potato (Ipomoea sp. including but not limited to Ipomoea batatas), cassava [tapioca, cassava] (Manihot sp., including but not limited to cassava (Manihot esculenta, Manihot aipi, and Manihot utilissima)) and/or taro root (Colocasia sp., including but not limited to C. esculenta or C. macrorhiza).

The starch-containing matrix may be an aqueous slurry characterized by a dissolved solids (DS) concentration of 10% to 50%. In another embodiment, the starch-containing matrix is characterized as having a DS concentration of 2% to 90%. In another embodiment, the starch-containing matrix is characterized as having a DS concentration of 5% to 70%. In another embodiment, the starch-containing matrix is characterized as having a DS concentration of 10% to 60%. In another embodiment, the starch-containing matrix is characterized as having a DS concentration of 20% to 40%. In another embodiment, the starch-containing matrix is characterized as having a DS concentration of 25% to 35%.

In another embodiment of the invention, the pH of the starch-containing substrate is between 1.00 and 9.00. In another embodiment of the invention, the pH of the starch-containing substrate is between 2.00 and 8.00. In another embodiment of the invention, the pH of the starch-containing substrate is between 3.00 and 7.50. In another embodiment of the invention, the pH of the starch-containing substrate is between 4.00 and 6.50. In another embodiment of the invention, the pH of the starch-containing substrate is between 4.25 and 5.75.

enzyme

The present invention involves contacting a starch-containing substrate with a maltogenic enzyme and a starch liquefying enzyme to produce maltose. "Malting" means that enzymes can enzymatically convert starch to maltose. Representative malting enzymes include alpha amylases and beta amylases. As previously described, amylose can be hydrolyzed by alpha-amylases (E.C.3.2.1.1), eg, alpha-(1-4)-glucan 4-glucanohydrolase. Alpha amylases hydrolyze α-(1,4)-linkages to produce a mixture of glucose, maltose, maltotriose and higher sugars. Amylose can also be hydrolyzed by β-amylases (E.C.3.2.1.2) [α(1,4)-glucan maltohydrolase, 1,4-α-D-glucan maltohydrolase]. This enzyme cuts off continuous maltose from the non-reducing end to produce a certain amount of maltose. Alpha and beta amylases also hydrolyze amylopectin.

alpha amylase

In some embodiments encompassed by the invention, the alpha amylase is a fungal enzyme or a microbial enzyme having an E.C. number, E.C.3.2.1.1-3, especially E.C.3.2.1.1. In some embodiments, the alpha amylase is a thermotolerant fungal alpha amylase. Suitable alpha-amylases may be naturally occurring as well as recombinant and mutated alpha-amylases. In some embodiments, the alpha amylase is derived from Bacillus. Preferred Bacillus include Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis and Bacillus stearothermophilus. In a particularly preferred embodiment, the alpha amylase is derived from Aspergillus. Preferred Aspergillus species include Aspergillus niger and Aspergillus oryzae. Also refer to NCIB 11837 for strains.

Commercially available alpha-amylases useful in the methods of the invention include CLARASE L ([Aspergillus oryzae] Genencor International Inc.) and NOVAMYL ([Bacillus stearothermophilus] Novozyme Biotech.).

Those skilled in the art understand that the amount of alpha-amylase used in the method of the present invention depends on the enzymatic activity of the alpha-amylase. Generally, alpha amylase is added to 1 metric ton (MT) of starch-containing substrate in an amount of about 0.01 to 5.0 kg. In some embodiments, alpha amylase is added in an amount of 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, also about 0.5 to 1.5 kg per MT. In further embodiments, other amounts are used. For example, typically, an amount of between about 0.01 to 1.5 kg of CLARASE L (Genencor International Inc.) is added to 1 MT of starch. In other embodiments, the enzyme is added in an amount of between about 0.05 and 1.0 kg; between about 0.1 and 0.6 kg; between about 0.2 and 0.6 kg and between about 0.4 and 0.6 kg per MT of starch CLARASEL.

β-amylase

In some embodiments encompassed by the invention, the maltogenic enzyme is beta amylase. Although alpha amylases are maltogenic in the sense that contacting a starch-containing substrate with alpha amylases provides maltose, the use of beta amylases is useful because contact with granular starch provides more More maltose than other sugars such as glucose. In some embodiments, the beta amylase is a plant or microbial enzyme having an E.C. number, E.C. 3.2.1.2 (eg, those beta amylases described in US Patents 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 mutated beta-amylases. The term "bacterial" refers to enzymes derived from bacilli, such as B. subtilis, B. licheniformis, B. stearothermophilus, B. coagulans, B. amyloliquefaciens and/or B. lentus. Particularly preferred beta amylases are derived from the Bacillus strains Bacillus stearothermophilus, Bacillus amyloliquefaciens and Bacillus licheniformis. See also NCIB 11608 strain. The term "plant origin" means that the enzyme is derived, extracted, isolated, expressed in a plant source, eg barley malt, soybean, wheat or barley.

Commercially available beta amylases contemplated for use in the methods of the invention include OPTIMAL BBA, Spezyme DBA, and OPTIMAL ME (Genencor International Inc.). Other commercially available wheat beta amylases are also useful in the methods of the invention.

In some embodiments, the starch-containing substrate, eg, wheat, rye, barley, malt, contains endogenous maltogenic enzymes at levels sufficient to generate sufficient maltose for conversion to isomaltooligosaccharides. The term "endogenous" refers to enzymes present in the grain or tubers without the addition of maltogenic enzymes to the grain or genetic modification of the grain to provide maltogenic enzymes.

In embodiments where the starch-containing substrate contains no or low levels of endogenous maltogenic enzymes, for example, the substrate is rice, millet, sorghum and/or corn, any exogenous maltogenic enzymes, etc. Quantitative addition is also contemplated by the inventors. Exogenous maltogenic enzymes may be added, for example, by genetically manipulating host cells to express sufficient levels of maltogenic enzymes, and/or by providing maltogenic enzyme concentrates or maltogenic enzyme materials from another source. The term exogenous maltogenic enzymes refers to maltogenic enzymes that are not present in the grain. In this embodiment, the maltogenic enzyme is contacted with the substrate in sufficient amount to produce maltose.

In one embodiment, the amount of exogenous maltogenic enzyme contacted with the starch-containing substrate is from 0.050 to 5.000 degree of saccharification power ("DP°") units per gram of maltogenic enzyme. In another embodiment of the present invention, 0.100 to 2.000 DP° units/gram of maltogenic enzyme is contacted with the starch-containing cereal. In yet another embodiment, 0.100 to 3.000 DP° units/gram of maltogenic enzyme is contacted with the starch-containing grain.

In another embodiment, the amount of exogenous maltogenic enzyme contacted to the starch-containing substrate is expressed in kilograms of maltogenic enzyme per metric ton of substrate. In one embodiment, the amount of exogenous maltogenic enzyme contacted to the substrate is about 0.05 kg of maltogenic enzyme per metric ton of dry solid basis ("kg/mt dsb"). In another embodiment, the amount of exogenous maltogenic enzyme is about 0.1 kg of maltogenic enzyme per metric ton of dry solids content ("kg/mt dsb"). In other embodiments, 0.2, 0.4, 0.6, 0.8 and/or 1.0 kg/mt dsb provides sufficient maltogenic enzymes, e.g., beta-amylase.

In another embodiment, the amount of exogenous maltogenic enzyme contacted to the starch-containing substrate is expressed in kilograms of maltogenic enzyme per metric ton of substrate. In one embodiment, the amount of exogenous maltogenic enzyme contacted to the substrate is about 0.05 kg maltogenic enzyme per metric ton of 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 of dissolved starch fraction ("kg/mt dsb"). In other embodiments, 0.2, 0.4, 0.6, 0.8 and/or 1.0 kg/mt dissolved starch fraction provides sufficient maltogenic enzymes, eg, beta-amylase.

In another embodiment, the amount of maltogenic enzyme to be contacted with the grain is expressed in maltogenic enzyme units. Assays for determining malting activity include those described in the Examples and those describing beta-amylase activity. The term DP° unit refers to the amount of enzyme contained in 0.10 ml of a 5% sample enzyme preparation solution that, when incubated with 100 ml of substrate at 20°C (68F°) for 1 hour, will produce sufficient reducing sugars to reduce Fehling's solution 5ml.

In another embodiment, grains with endogenous malting enzymes (barley, wheat, etc.) may be blended with those grains requiring exogenous malting enzymes. The inventors contemplate a 30:70, 60:40, 50:50, 60:40, 70:30 mixture of cereals with endogenous malting enzymes and cereals utilizing exogenous malting enzymes, condition is the presence of maltogenic enzymes (either endogenous or exogenous) in sufficient quantities in the mixture. The use of endogenous sources of malting enzymes reduces the amount of exogenous enzymes added to or in contact with the grain mix.

Starch liquefying enzymes

A starch liquefying enzyme is contacted with the starch to reduce the viscosity of the liquefied or dissolved starch. In one embodiment of the invention, the starch liquefying enzyme is an enzyme selected from E.C.3.2.1.1, eg, alpha amylase. Representative alpha amylases can be derived, isolated or extracted from bacterial sources. In one embodiment, the bacterial source is Bacillus. In another embodiment, Bacillus-derived alpha-amylases include those derived from at least one bacterial source selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus coagulans, Bacillus amylobacter and Bacillus lentus. Those enzymes in Bacillus licheniformis and Bacillus stearothermophilus are particularly useful. The inventors contemplate other amylases such as, but not limited to, those of EC 3.2.1.133 (US Patent No. 6,361,809). Other amylases contemplated by the inventors include enzymes characterized by increased oxidation or thermostability, including mutant or genetically modified or variant amylases, including those in US Pat. 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 Bacillus stearothermophilus strain ATCC 39709. Such enzymes include those available under the tradenames "SPEZYME AA" or "SPEZYME FRED", "SPEZYME LT300" and "SPEZYME LT75" from Genencor International (Palo Also, California, USA). Other such enzymes include the alpha-amylase from Bacillus stearothermophilus sold under the trade names GZYME G997, GC007, and the alpha-amylase from Bacillus licheniformis sold under the trade name GC262SP, also available from Genencor International.

Maltogenic enzymes and starch liquefying enzymes are contacted with starchy grains to produce maltose. As will be appreciated by those skilled in the art, the amount 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, the starch liquefying enzyme is contacted with the starch-containing grain in an amount of 0.01 to 25 Liquefon units/gm. In another embodiment, 1 to 10 Liquefon units/gm of starch liquefying enzyme is contacted with the starch-containing grain. One Liquefon unit (LU) is a measure of the digestion time required to produce a color change in an iodine solution, which indicates a specific period of dextrinization of a starch substrate under specific 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, 0.6, 0.8, or 1.0 kg of starch liquefying enzyme is added per metric ton of grain (kg/mt dissolved starch component). In one embodiment, 0.1 kg of starch liquefying enzyme is added per metric ton of dissolved starch fraction of the grain (kg/mt dissolved starch fraction). In other embodiments, 0.2, 0.4, 0.6, 0.8, or 1.0 kg of starch liquefying enzyme is added per metric ton of grain (kg/mt dissolved starch component). Assays for determining starch liquefaction activity include those described in the Examples herein. Representative assays for the determination of alpha amylase activity are also described in US Patent Nos. 5,763,385; 5,824,532; 5,958,739 and/or 6,008,026, which are incorporated herein by reference.

Transglucosidic enzyme

Contacting maltose with a transglucosidic enzyme results in a cereal composition having enzymatically produced isomaltooligosaccharides derived from starchy cereals. When incubated with α-D-gluco-oligosaccharides, the transglucosidic enzyme catalyzes the hydrolysis and transfer reactions to produce isomaltose, 4-α-glucosylmaltose, kojibiose or nigerose. The increased amount of DP2 disaccharides suggested the presence of these sugars and thus conversion mediated by transglucosidases. The transglucosidase (E.C. 2.4.1.24) may be transglucosidase. Representative transglucosidases include TRANSGLUCOSIDASE L-1000 (Genencor International, Inc.) and TRANSGLUCOSIDE L of AmanoEnzymes, Inc. (Nagoya, Japan). In one embodiment, the transglucosidic enzyme is derived from a filamentous fungal source, eg, Aspergillus. The transglucosidic enzyme derived from Aspergillus can be derived from Aspergillus niger. In one embodiment, the Aspergillus niger strain is ATCC14916.

In one embodiment, a sufficient amount of the transglucosidase is contacted with a substrate, such as starchy grain, to produce maltose. As understood by those skilled in the art, the amount of transglucosidic enzyme used in the method 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 starch-containing grain. In another embodiment of the invention, 0.05 TGU to 10.00 TGU/gm of transglucosidase is contacted with the starchy grain. In yet another embodiment of the invention, 0.10 TGU to 5.00 TGU/gm of transglucosidase is contacted with the starch-containing grain. The term TGU refers to the enzyme activity required to produce 1 micromole of 4-alpha-glucosylmaltose per minute under the conditions of the assay.

In one embodiment, 0.05 to 6.00 kg of transglucosidase is added per metric ton of grain (kg/mtdsb). In another embodiment, 0.10 to 5.00 kg of transglucosidase is added per metric ton of grain (kg/mtdsb). In another embodiment, 0.25 to 3.00 kg of transglucosidase is added per metric ton of grain (kg/mtdsb). In another embodiment, 0.50 to 1.50 kg of transglucosidase is added per metric ton of grain (kg/mtdsb). Other assays for determining transglucosidase activity include those described in the Examples and those described in Shetty, J., et al (U.S. Patent 4,575,487 (1986), entitled "Method for determination of transglucosidase"), which incorporated herein by reference.

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 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 the action of transglucosidases, malto-oligosaccharides are converted to isomalt-oligosaccharides, forming a new class of polysaccharides containing a higher proportion of Glucosyl residues. Isomalt-oligosaccharides produced by this method include isomaltose, 4-α-glucosylmaltose, isomalt-triose, isomalt-tetraose, isomalt-pentaose, isomalt-hexose and isomalt-heptose sugar. These sugars have received increasing attention as food additives because they help prevent dental caries (Oshima, et.al 1988, Thecaries inhibitory effects of gos-sugar in vitro and rat experiments. Microbial Immunol. 32.1093-1105)) and improve Human intestinal flora, 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 determine IMO production, experimental and/or other analytical methods may be used to determine the amount of IMO produced. One method of determining the level of IMO produced includes high performance liquid chromatography (HPLC). For example, analysis of the mixture can provide hints on the levels of various sugars produced by the method. A useful assessment is the degree of polymerization (DP) of the mixture. The term degree of polymerization is a measure of the relative amount of the number of glucose residues in the molecule. For example, glucose (one glucosyl unit, lowest level of aggregation) is generally considered DP1. Isomalto-oligosaccharides are generally referred to as DP2 (two glucose units). In one embodiment, the cereal 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 An isomalto-oligosaccharide. In one embodiment, said at least one isomalt-oligosaccharide is selected from isomaltose, 4-alpha-glucosylmaltose and/or isomalto-triose. In one embodiment, the amount of isomalto-oligosaccharides produced in the cereal composition is between 1% and 99% of the cereal composition. In one embodiment, the amount of isomalto-oligosaccharides produced in the cereal composition is between 1% and 90% of the cereal composition. In one embodiment, the amount of isomalto-oligosaccharides produced in the cereal composition is between 1% and 80% of the cereal composition. In one embodiment, the amount of isomalto-oligosaccharides produced in the cereal composition is between 1% and 70% of the cereal composition. In one embodiment of the invention, the total sugar present in the cereal composition following the above procedure comprises a maltose level of greater than 50%, greater than 60%, greater than 70% or greater than 80% of the total sugar content. Maltose levels 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 the maltose (DP2) present in the cereal to the level of maltotriose (DP3) present in the resulting cereal composition. Higher PBS values suggest the presence of higher amounts of maltose and thus a more complete conversion of starch to maltose, as opposed to the presence of other less desirable end products such as maltotriose. In one embodiment, the RBS ratio is greater than 2.0. In one embodiment, the RBS ratio is greater than 3.0, greater than 4.0. Representative ranges include RBS ratios of 2.0 to 50.00, 2.0 to 30.00, and/or 2.0 to 10.00. Various RBS ratios are described in the Examples. It has been indicated that liquefied starches are hydrolyzed by commercial beta-amylases (barley or wheat), usually yielding maltose contents between 55% and 65%. In order to obtain a maltose content greater than 50% using liquefied starch, a very low starting DE with the addition of debranching enzymes and/or liquefied starch was previously required. Optionally, the addition of debranching enzymes can be used to increase maltose production. The term debranching enzyme refers to an enzyme that catalyzes the hydrolysis of alpha-1,6-bonds. In this regard, enzymes of the class E.C.3.2.1.41 are useful. A representative enzyme of this class is pullanase, also known as alpha-dextrin endo-1,6-alpha glucosidase, limit dextrinase, debranching enzyme, pullulan 1,6-glucanohydrolase.

Contacting the starchy grain with a maltogenic enzyme produces maltose and contacting the maltose with a transglucosidic enzyme occurs at a temperature below the gelatinization temperature of the starch of the grain used. The substrate is contacted or incubated with the respective enzyme for a period of at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours and/or at least 36 hours. A period of at least one mentioned 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 time period during which maltose or substrate is converted to IMO. The transglycosidic enzyme may be contacted or added separately or simultaneously with the substrate such as starch-containing grain, the maltogenic enzyme such as alpha-amylase or beta-amylase, and/or the liquefying enzyme such as alpha-amylase. In one embodiment, the transglycosidic enzyme and the liquefying enzyme are added simultaneously. Thus in one embodiment step (a) and step (b) are carried out simultaneously. In another embodiment, step (a) and step (b) are performed sequentially or separately. In another embodiment, step (a) is performed earlier than step (b). The term gelatinization temperature refers to the temperature at which the starch contained in the grain changes phase or forms a gel. Although the exact temperature varies from grain to grain, temperatures sufficient to gelatinize the starch include temperatures 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. Representative temperatures above the indicated gelatinization temperature include 45°C to 120°C, 50°C to 110°C, 50° to 100°C. In one embodiment, eg, wheat, the gelatinization temperature is such that the grain is maintained at a temperature below, eg, selected from below 50°C to 70°C, in another embodiment, below 55°C to 65°C. °C, in another embodiment, is less than 60 °C. For example, the gelatinization temperatures of corn, potato, wheat, cassava, waxy corn, sorghum, rice, Saigon rice, arrowroot, arrowroot amylomaize, and/or sweet potato have been described as shown in Table 1 (Beynum , G.M.A and Roels, J.A., Starch Conversion Technology (Marcel Dekker, Inc., NewYork, NewYork (1985), pp.15-45):

Table 1 starch Kolfler gelatinization temperature range (℃) Brabenberg gelatinization temperature (℃) corn 62-67-72 75-80 potato 58-63-68 60-65 wheat 58-61-64 80-85 cassava 59-64-69 65-70 waxy corn 63-68-72 65-70 sorghum 68-74-78 75-80 rice 68-74-78 70-75 Saigon Rice 60-66-72 65-70 Arrowroot 62-66-70 starch corn 67-80-92 90-95 sweet potato 58-65-72 65-70

In another embodiment of the invention, after the incubation period, the slurry may be subjected to rapid heating for a period sufficient to stop further enzymatic activity, but not to gelatinize or liquefy the slurry. For example, the slurry may be heated to a temperature of 80°C, 85°C, 90°C, 95°C, or 100°C for a duration of 5-60 minutes, 10.0 to 40.0 minutes, or 30.0 minutes.

Another embodiment of the present invention further comprises the step of separating the slurry into insolubles and solubles. The separation step can be any chromatographic method known in the art, such as, but not limited to, HPLC, size exclusion and/or charge chromatography. Filtration can be used to separate soluble from insoluble. The insolubles or the whole slurry can be subjected to the drying step described later in this application. In another embodiment, the solubles obtained from the separation step can be concentrated by evaporation, eg, rotary evaporation, pan drying, and the like. The evaporated concentrate can be charcoaled (filtered through charcoal particles) and/or further chromatographed to provide an isolated IMO liquid concentrate. The isolated IMO concentrate may have an IMO concentration of greater than 75%, greater than 80%, greater than 90%, greater than 95%, greater than 97%, and/or greater than 99%.

Another embodiment of the present invention is the application or incorporation of this syrup (enzymatically derived from isomalt-oligosaccharides with a matrix of ungelatinized starch) in oral rehydration solutions. The amount of isomalto-oligosaccharide may be the amount or formulation described in US Pat. Nos. 4,981,687; 5,096,894 and/or 5,733,579.

Another embodiment of the present invention is to dry the aforementioned isomalto-oligosaccharide base, cereal or tuber composition to produce a powder containing the cereal composition. Typically, the drying step is accelerated by heating. By applying suitable drying methods such as but not limited to spray dryers, tray dryers, tumble dryers, drum dryers or box dryers, the cereal composition can be dried to the desired moistness. level. Other drying methodologies can be applied, such as spray drying, reduced pressure evaporative drying.

Powders or other dry powders are obtained therefrom by drying cereal compositions, slurries, separated insolubles and/or separated solubles. The obtained powder or powder can be incorporated into compositions requiring isomalto-oligosaccharides, for example, food products (breakfast cereals, dry bread), food additives and bakery products. The term food additive refers to isomalto-oligosaccharides dispersed on materials, used as ingredients in the manufacture of other foods, and/or applied as topical ingredients added to foods.

In another embodiment, dry powders can be incorporated into food supplements. The dry powder may be incorporated into a dietary supplement in any acceptable addition or form. Dietary supplements may be formulated in matrices in a form suitable for oral administration, such as, but not limited to, powders, crystals, granules, microparticles (including particles on the micron scale, such as microspheres and microcapsules), granules (including Micron-scale particles), beads, microbeads, pellets, pellets, microtablets, compressed tablets or ground tablets (tablet triturates), molded tablets or tablet triturates (tablet triturates), can also be formulated in In capsules, which are hard or soft, and contain the composition in the form of powder, granules, beads, solution or suspension. Dietary supplements may also be formulated as solutions or suspensions in aqueous liquids for oral administration, as liquids for incorporation into capsules, or in any other convenient form suitable for administration or for rectal administration, such as Suppository, enema, or other convenient form. The isomalto-oligosaccharide composition may also be provided as a controlled release system.

Dietary supplement formulations may also include any type of acceptable excipients, additives or carriers. 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, sugar powder, bentonite, microcrystalline cellulose or hydroxypropyl methylcellulose, may be added to the isomalto-oligosaccharide composition to increase the volume of the composition. Also, binders such as, but not limited to, starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, gum arabic, sodium alginate, Irish moss (carrageen) extract, panwar gum, ghatti ) glue, isapgol husks glue, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, magnesium aluminum silicate (modified) (veegum) and starch arabinogalactan, polyethylene glycol, methylcellulose Glycerin, glyceryl monostearate, and waxes can be added to the formulation to increase its adhesive properties.

Additionally, lubricants such as, but not limited to, glyceryl monostearate, talc, magnesium stearate 15, calcium stearate, stearic acid, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, chloride Sodium chloride, leucine, carbowax, sodium lauryl sulfate, and magnesium lauryl sulfate can be added to the formulation. Also, glidants such as, but not limited to, colloidal silica, manganese silicate, or mica may be added to improve the flow properties of the powder formulation. Finally, disintegrants such as, but not limited to, starches, clays, celluloses, alginic acid, resins, cross-linked polymers (e.g., croscarmellose, crospovidone, and sodium starch glycolate), silicon Magnesium aluminum acid (modified), methylcellulose, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp, carboxymethyl Cellulose, or sodium lauryl sulfate with starch, may also be added to assist in the breakdown of the formulation in the stomach or intestines.

Another embodiment of the present invention is the application of the novel substrates, tubers or cereal compositions described herein in the production of flour for use in various bakery products. The term bakery product relates to both fermented and unfermented products. The term "fermented" refers to a baked product to which yeast has been applied during the baking process. Whereas the term "unleavened" refers to a baked product in which no yeast has been applied during the baking process. Representative articles include bread, biscuits, cakes, pies, pastries, naan, bagels, pasta, crackers, rolls, donuts, pita bread, and pastries. Representative matzos include matzos, chapati, breakfast cereals and tortillas. Another embodiment of the present invention is the application of the new cereal composition to pasta, such as noodles (short macaroni, solid vermicelli, lasagna, corn noodles, etc.). Another embodiment of the present invention is a substrate, tuber or cereal composition prepared according to the above method. Another embodiment of the present invention is flour comprising a substrate, tuber or cereal composition prepared according to the above method. Another embodiment of the present invention is an oral rehydration solution comprising the above-mentioned isomalto-oligosaccharides. According to the method described above, powders comprising substrates, tubers or cereal compositions can be produced. Another embodiment of the present invention is the substrate, tuber or cereal composition prepared according to the above method.

Another embodiment of the present invention is a substrate, tuber or cereal composition prepared according to the above method. Another embodiment of the present invention is the application of the novel cereal composition in a fermented/beer wort or substrate. For example, the new grain composition can be used in beer fermentation as described, as described in International Publication No. WO02/20712 A1, which is hereby incorporated by reference. The new cereal composition can also incorporate beer additives.

The isomalto-oligosaccharide-containing matrix can also be subjected to an additional step to recover maltose by extraction and separation of the resulting maltose, eg maltose syrup. The syrup may be extracted and/or separated from the cereal composition by methods familiar in the art, such as in US Pat. Nos. 3,922,196 and 4,113,509, which are incorporated herein by reference.

Another way to increase the sweetness or the isomalto-oligosaccharide content is to treat the resulting isomalto-oligosaccharide syrup (in soluble or immobilized form) with hydrolytic enzymes, preferably or even exclusively Hydrolyzes malto-oligosaccharides with little or no affinity for isomalto-oligosaccharides. Examples of such enzymes are glucoamylases from Aspergillus niger or other sources such as Aspergillus or Rhizopus, which preferably hydrolyze 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).

Enzymes such as α-D-glucopyranosidase from Bacillus stearothermophilus can also be used. This enzyme cannot hydrolyze isomalto-oligosaccharides and only degrades malto-oligosaccharides present in isomalto-oligosaccharide-rich syrups (Suzuki Y., Shinji M., Nobuyuki E., Biochimica et Biophysica Acta, 787 (1984) , 281-289). Other alpha-D-glucosidases known as maltases may also be used. For example, maltase from yeast only hydrolyzes maltose and to a lesser extent maltotriose (Kelly C.T., Fogarty W.M., Process Biochemistry, May/June (1983), 6-12). After hydrolysis of malto-oligosaccharides to glucose, the syrup can be enriched in isomalto-oligosaccharides by chromatographic techniques or by nanofiltration or ultrafiltration.

The following examples are intended to illustrate the main embodiments of the invention.

Example

The following specific examples further illustrate the compositions and methods of the present invention. It should be understood that these examples are for illustrative purposes only and that they can be applied to other suitable substances rich in starch and containing endogenous maltogenic enzymes, for example, wheat, rice, barley, malt, potatoes, sweet potatoes and the like.

Enzyme activity assay

Transglycosidase activity was determined by the method of Shetty, J., et al, 1986 (US Pat. No. 4,575,487). Beta-amylase activity was determined by hydrolyzing the starch substrate at pH 4.6 and 20°C for 30 minutes. The reducing sugar groups produced by hydrolysis are determined in a titration procedure using basic ferricyanide. Amylase activity is expressed as the degree of DP, one unit of which refers to the amount of enzyme contained in 0.1 ml of a 5% solution of the sample enzyme preparation, when it is incubated with 100 ml of substrate at 20 ° C for 1 hour, it will generate enough to reduce 5 ml Reducing sugars of Fehlings' solution.

The α-amylase activity was determined based on the end-point assay kit provided by Megazyme (Aust.) Pty.Ltd. A vial of substrate (p-nitrophenylmaltoheptapaside, BPNPG7) was dissolved in 10 ml sterile water, followed by an assay buffer (50 mM maleic acid buffer, pH 6.7, 5 mM calcium chloride, 0.002% A 1:4 dilution was performed in Tween 20). Analysis was performed by adding 10 μl of amylase to 790 μl of substrate in a transparent vessel at 25°C. The rate of hydrolysis was measured as the rate of change in absorbance at 410 nm after a delay of 75 seconds. The analysis is linear up to a rate of 0.2 absorbance units/minute.

Using bovine serum albumin standard, based on the method of Bradford, Anal. Biochem., Vol.72, p.248 (1976), the protein concentration of α-amylase was determined with standard Bio-RadAssay (Bio-Rad Laboratories).

Substrate (matrix)

Wheat flour, used as a substrate (substrate) in all examples, was purchased from retail stores. Other tuber or cereal substrates, eg rice and barley used as substrates can be purchased from commercial sources (Huai An Liujun Food processing company, Jiangshu province, China).

Oligosaccharide analysis

It was maintained at 60°C and equipped with a Refractive Index (RI) detector (ERC-7515A , RI Detector from The Anspec Company, Inc.), to determine the composition of oligosaccharide reaction products. Diluted sulfuric acid (0.01 N) was used as mobile phase at a flow rate of 0.6 ml per minute. 20 microliters of the 4.0% solution was injected onto the column. The column separates sugars according to their molecular weight. For example, DP1 means monosaccharides, such as glucose; DP2 means disaccharides, such as maltose; DP3 means trisaccharides, such as maltotriose; DP4 ⁺ means the degree of polymerization (DP) is 4 or more oligosaccharides. The term higher sugar ("Hr. Sugar") refers to sugars with a DP greater than 3.

For iso-saccharides or branched sugars, pass through HPLC (Agilent 1010, PaloAlto, California, USA) equipped with an HPLC column (Shodex Rspak Oligosaccharide Column #DC-613), which is maintained at 50°C And equipped with a Refractive Index (RI) detector (ERC-7515A, RI Detector, from The Anspec Company, Inc.), the reaction product was determined. 70 (acetonitrile): 25 (methanol): 5 water was used as mobile phase and the flow rate was 2.5 ml per minute. Inject 20 microliters of the 4.0% solution onto the column. The column separates sugars according to their molecular weight. Standard sugars, glucose, maltose, maltotriose, isomaltose, 4-α-glucosylmaltose and isomalt-triose (Sigma Chemicals, St. Louis, Missouri, USA) were used to calibrate the column.

Example 1

Compare the α-amylase from Bacillus licheniformis (a kind of α-amylase sold by Genencor International, Palo Alto, CA, trade name is GC262SP) and the α-amylase from Bacillus stearothermophilus (by Genencor International, Palo Alto, CA). An alpha-amylase sold by CA under the trade name GC007) utilizes wheat flour for the production of maltose. 150 grams of wheat flour from a commercial retail source was suspended in 450 ml of deionized water. The suspension was stirred at room temperature for 15 minutes to mix well (pH 5.5). The pH was adjusted with 6.0N sulfuric acid (H ₂ SO ₄ ). The resulting suspension was kept in a water bath, maintained at 60°C, and stirred for uniform mixing prior to enzyme addition. Under continuous stirring at 60°C, approximately 6000 LU/g of amylase from Bacillus stearothermophilus (0.6 kg of GC007 [from Genencor International. Inc.]/metric ton (Mt.) starch dsb) and amylase from Bacillus licheniformis were added, respectively. Amylase 15,100 LU/g (0.6 kg of GC262SP [from Genencor International. Inc.]/Mt. starch dsb) and incubated. Samples were collected at various pre-determined time intervals and analyzed for total sugar components by high pressure liquid chromatography (HPLC). At predetermined time intervals, 2 ml samples were withdrawn from each container using plastic pipettes and transferred to centrifuge tubes. Samples were centrifuged at 8000 rpm for 3 minutes. The supernatant was collected from the centrifuge tube and a few drops were added to the sample well of a Lecia AR200 (Leica Microsystems, Inc., Buffalo, NY, USA) digital handheld refractometer and recorded (Table 2). The Brix (measured as dissolved sugar) of the solutions was determined (Table 2).

Table 2

Comparison of maltose production by liquefied alpha-amylases during incubation of wheat flour, pH 5.5, 60°C Enzyme & Dosage SUGAR & BRIX Incubation time, 1 hour, 60℃ sugar percentage After heating at 80°C for 30 minutes 2 hours 4 hours 6 hours Gelatinization No alpha amylase added DP1 3.86 3.70 3.74 DP2 54.87 56.24 56.78 DP3 2.83 3.44 3.96 high sugar 38.44 36.62 35.52 BRIX 11.00 12.70 14.10 Bacillus stearothermophilus [GC007] 0.6kg/MT starch.dsb DP1 2.67 2.89 3.23 3.17 DP2 65.10 67.44 69.08 65.83 DP3 10.62 12.18 13.07 13.42 high sugar 21.61 17.49 14.62 17.58 BRIX 18.50 19.40 20.00 22.70 Bacillus licheniformis [GC262SP] 0.6kg/MT starch.dsb DP1 2.86 3.59 3.96 4.03 DP2 62.43 64.36 65.88 58.14 DP3 13.19 14.82 15.93 16.69 high sugar 21.52 17.23 14.23 21.14 BRIX 17.40 18.40 19.10 22.60

The results in Table 2 show that incubation of wheat flour with alpha-amylase from Bacillus stearothermophilus produced a higher maltose content than when incubated with alpha-amylase from Bacillus licheniformis. Incubation of wheat flour with α-amylase resulted in a significant increase in dissolved solids due to hydrolysis of granular starch compared to incubation of wheat flour without the addition of α-amylase. It is interesting to note here that the reaction product of the alpha-amylase from Bacillus stearothermophilus leads to a higher ratio of maltose to glucose and maltose to maltotriose than the alpha-amylase from Bacillus licheniformis. These results suggest that the alpha-amylase from B. licheniformis is a particularly useful enzyme in producing syrups with a high maltose content.

Example 2

Effect of concentration of Bacillus licheniformis alpha-amylase (alpha-amylase sold by Genencor international, Palo Alto, CA under the trade name GC 007) on maltose production during incubation with wheat flour. The experimental conditions were the same as those described in Example 1. In addition, Bacillus stearothermophilus (6,000 units/gram) was added at 0.1 Kg, 0.2 Kg and 0.6 Kg/MT starch dsb. The results are summarized in Table 3.

table 3

Effect of α-amylase [GC007] concentration on maltose production during incubation of wheat flour, pH 5.5, 60°C GC007 concentration DP&BRIX Incubation time, 1 hour, sugar percentage at 60°C 2 hours 4 hours 6 hours 24 hours 0.1kg/MT starch.dsb DP1 1.87 2.10 2.27 3.70 DP2 65.56 64.25 65.63 70.22 DP3 5.92 7.15 8.03 11.68 high sugar 29.65 26.50 24.07 14.40 BRIX 17.80 19.20 19.70 21.80 0.2kg/MT starch.dsb DP1 1.71 2.13 2.37 3.90 DP2 62.47 64.34 65.44 69.30 DP3 6.75 7.78 8.58 12.27 high sugar 29.07 25.75 23.61 14.53 BRIX 18.00 19.20 19.80 21.80 0.6kg/MT starch.dsb DP1 2.67 2.89 3.23 3.17 DP2 65.10 67.44 69.08 65.83 DP3 10.62 12.18 13.03 13.42 high sugar 21.61 17.49 14.62 17.58 BRIX 18.50 19.40 20.00 22.70

No significant effect of different levels of alpha amylase on maltose content or on dissolved solids was observed during wheat flour incubation. Therefore, for further optimization studies, 0.1 Kg GC007/MT starch, dsb was applied.

Example 3

150 g 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 with 6.0N H ₂ SO ₄ . Shake the slurry well to mix evenly, and adjust the pH until it stabilizes at the specified pH value. GC007 was added to each experimental group in an amount of 0.1 Kgs/MT starch dsb and incubated at 60°C. Samples were taken at different predetermined time intervals for determination of sugar composition and brix, as described in Example 1. (Table 4).

Table 4

Effect of pH on Maltose Yield During Incubation of Wheat Flour and GC007 pH DP&BRIX Incubation time, 1 hour, 60℃ sugar percentage 2 hours 4 hours 6 hours 24 hours 5.0 DP1 2.59 3.22 3.77 5.17 DP2 63.87 66.89 67.41 65.77 DP3 10.90 13.23 14.25 16.87 high sugar 22.64 16.66 14.57 12.19 BRIX 17.70 18.80 19.20 20.90 4.5 DP1 2.29 2.80 3.31 4.37 DP2 63.86 67.44 68.03 65.97 DP3 10.93 13.30 14.44 17.56 high sugar 22.92 16.46 14.22 19.10 BRIX 17.60 18.60 19.10 20.70 4.0 DP1 1.71 1.79 2.11 3.17 DP2 58.19 60.23 61.82 62.95 DP3 11.16 13.47 14.87 18.67 high sugar 28.93 24.51 21.74 15.21 BRIX 17.60 18.60 19.00 19.50

When the pH value of the wheat flour was lowered from 5.5 during incubation, the maltose content increased and reached a maximum at pH 4.5, about 68%, followed by a decrease at pH 4.0. During starch hydrolysis using plant beta amylase, it was an unexpected result that a maltose content greater than 60% was produced without the addition of a debranching enzyme. Hydrolysis of liquefied starch by commercial beta-amylases (barley or wheat) typically yields maltose contents between 55% and 60%. In order to obtain maltose content greater than 60% using liquefied starch, debranching enzymes need to be added or a very low starting DE of the liquefied starch is required. It is important to note here that the method described in the present invention allows maltose manufacturers to operate at pH 4.5 and 60°C, reducing the high risk of microbial contamination with current methods.

Example 4

Suspend 150 grams of wheat flour in 450 ml of deionized water, and adjust the pH of the slurry to pH 4.5. Shake the slurry well to mix _well and adjust the pH with 6.0N _H2SO4 until the pH is stable. The resulting suspension was kept in a water bath maintained at 60°C and shaken for homogeneous mixing prior to enzyme addition. Add starch liquefaction enzyme, for example, Bacillus stearothermophilus alpha amylase, which is sold under the trade name "GC 007" (Genencor International, Inc.), in an amount of 0.1 Kg/MT, dsb. Then, debranching enzyme pullulanase sold under the trade name OPTIMAX L-1000 (Genencor International, Inc.) was added in an amount of 0.25Kg, 0.5Kg and 1.0Kg/MT dsb, and incubated at 60°C . Samples were taken at different predetermined time intervals (2, 4, 6 and 24 hours) to determine sugar composition and brix, as described in Example 1. Record the results (Table 5).

table 5

During the incubation of wheat flour and GC007 (0.1Kg/MT, dsb), the effect of debranching enzyme (OPTIMAX L-1000) on wheat

Effect of malt sugar production, pH 4.5, 60°C OPTIMAX Concentration DP&BRIX Incubation time, 1 hour, 60℃ sugar percentage 2 hours 4 hours 6 hours 24 hours 0.0 DP1 1.21 1.38 1.47 1.99 DP2 62.77 64.69 66.24 67.16 DP3 6.29 7.32 8.64 10.97 high sugar 29.73 26.61 23.65 19.90 BRIX 17.5 18.6 19.4 20.40 0.25Kg/MTdbs DP1 1.28 1.33 1.90 1.94 DP2 66.61 69.97 72.01 73.64 DP3 8.18 10.03 10.68 13.58 high sugar 23.93 18.67 15.41 10.86 BRIX 17.70 18.60 19.40 20.80 0.50Kg/MTdbs DP1 1.41 1.41 1.64 2.01 DP2 70.50 73.35 74.46 74.32 DP3 9.62 11.26 11.94 14.31 high sugar 18.47 13.98 11.96 9.36 BRIX 17.70 18.70 19.80 20.80 1.00Kg/MT dbs DP1 1.47 1.56 1.58 2.02 DP2 72.90 75.54 76.08 75.21 DP3 10.05 11.35 11.95 13.53 high sugar 15.48 11.55 10.39 9.24 BRIX 17.60 18.80 19.70 21.70

Maltogenic enzymes such as beta amylases or starch liquefying alpha amylases such as GC007 are unable to hydrolyze the alpha 1-6 glycosidic bond, which is the branch point in branched amyloid starch substrates. Therefore, in common practice, during the incubation of the starch substrate and the beta-amylase, the debranching enzyme, pullulanase (OPTIMAX L-1000, from Genencor International Inc), is added for greater than 65% maltose production. The effect of OPTIMAX L-1000 concentration during the incubation of wheat flour and GC007 was investigated and the results are shown in Table 5. Addition of OPTIMAXL-1000 resulted in significantly higher maltose (DP2) levels (>75%) compared to the control.

Example 5

Industrially, it is common to apply high maltose syrups produced by enzymatic processes using high temperature (>90°C) enzyme liquefaction of starch substrates, followed by treatment with glucosyltransferases to produce isomalto-oligosaccharide syrups practice. This example illustrates the conversion of granular starch in wheat flour to isomalto-oligosaccharides 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. Then, shake for 15 min to mix well, then, adjust the pH to _4.5 with 6.0 N _H2SO4 . The resulting suspension was kept in a water bath maintained at 60°C and shaken to mix well before adding the enzyme. Add a starch liquefying enzyme, such as Bacillus stearothermophilus alpha amylase ([GC007 provided by Genencor International] (0.1 Kgs/MT dsb) and a debranching enzyme such as pullulanase (OPTIMAX L-1000 provided by Genencor International) (0.5 kgs/MT dsb). Then, the suspension was divided into two equal parts. For one of the two parts, Aspergillus niger transglucosidase (Genencor International) sold under the trade name "TRANSGLUCOSIDASE L-500" was added, The amount was 1.0 Kg/MT dsb and was kept in a water bath at 60°C (sample 1).Another aliquot was first incubated at 60°C for four hours before adding Aspergillus niger sold under the trade name "TRANSGLUCOSIDASE L-500" Transglucosidase (Genencor International), the amount is 1.0Kg/MT dsb, and keeps in water bath, maintains at 60 ℃ (sample 2).The results shown in table 6 suggest that the conversion of substrate to IMO occurs, which is accompanied by or It was not accompanied by pre-incubation of the substrate (wheat flour) prior to the addition of transglucosidase.

Table 6

  During the incubation of wheat flour with GC007 and OPTIMAXL-1000, the formation of isomalto-oligosaccharides,

pH4.5, 60℃, using TRANSGLUCOSIDASE L-500 sample Reaction time glucose maltose Isomaltose Maltotriose 4-α-Glucosyl Maltose Isomaltotriose branched sugar G3+ IMO number 1 17 14.74 30.27 2.73 6.82 23.05 7.13 14.65 47.5610 2 17 13.92 33.17 2.32 7.70 23.69 4.88 14.32 45.21 1 48 16.90 20.04 7.98 10.29 22.35 3.95 17.95 52.23 2 48 14.96 23.59 6.85 12.17 16.65 5.57 19.66 48.73 1 72 16.78 21.21 5.78 8.51 22.83 4.17 20.24 53.02 2 72 15.24 25.44 4.99 8.88 18.48 7.52 19.03 50.0115

IMO number: Calculated as the sum of isomaltose, 4-alpha-glucosylmaltose, isomaltotriose and branched sugars greater than DP3.

Incubation of modified wheat flour with a high content of maltose and a transglucosidase produces isomalto-oligosaccharides of the same composition as produced by traditional methods. The method is simple, economical and can be easily scaled up for commercial production.

Example 6

It is common knowledge that grains such as wheat, barley and rye contain high levels of beta-amylase. Incubation of these grains at 55°C-60°C, pH 5.5, typically forms a syrup containing greater than 50% maltose. 28% slurries of wheat, barley and rye flour, respectively, were prepared by adding 280 gm of each powder to 720 gm deionized water. Bacillus stearothermophilus alpha amylase (eg, Bacillus stearothermophilus alpha amylase sold under the trade name "GC007" by Genencor International) was added to each preparation in an amount of 0.2 kg/MT powder. Then, the pH was adjusted to 5.5 with 6.0 N _H2SO4 and incubated at 60 °C _for 4.5 h. Then, the pH of the incubated sample was adjusted to 4.5 with _6.0 NH SO ₄ , with 1.25 kg of transglucosidase (for example, transglucosidase sold under the trade name "TRANSGLUCOSIDASE L-500" by Genencor International)/MT powder Add the amount of transglucosidase. Afterwards, the slurry was incubated in a 60°C water bath for 48 hours. Samples were then centrifuged and analyzed for IMO composition (Table 7), as described in Example 1.

Table 7

After incubation with GC007 and "TRANSGLUCOSIDASE L-500", the yield of wheat, barley and rye

Soluble sugar components Cereals/cereals deal with time (hours) Soluble sugar component % IMO number .10 glucose maltose Isomaltose maltose 4-α-Glucosyl Maltose Isomaltotriose branched sugar G3+ wheat TgL-500 ^* 48 21.92 19.37 9.97 3.67 26.52 3.12 14.85 54.46 barley TgL-500 ^* 48 25.43 6.68 16.50 7.72 12.82 0.30 30.54 60.1615 rye TgL-500 ^* 48 22.18 10.39 11.15 3.21 22.93 0.01 30.14 64.2220

Tg L-500 ^* refers to "TRANSGLUCOSIDASE L-500"

Example 7

In one experiment, 140 grams of malt (Cargill Malt/Schreier-Malting Company, Wisconsin, USA) was mixed with 360 grams of distilled water. The slurry was shaken at room temperature for 15.0 minutes to mix well, then the pH was adjusted to 4.5 with diluted acetic acid. After the pH stabilized, the slurry was kept in a water bath maintained at 60°C. Incubation was continued for 30 minutes with constant shaking and a 2 ml sample was withdrawn for Brix and HPLC analysis (0, time). Add TRANSGLUCOSIDASE L-500 at 1.5kg/MT of malt and incubate at 60°C. During the incubation period at different predetermined time intervals, eg, 2, 4, 6, 12 and 24 hours, samples were taken to determine the Brix and IMO components (Table 8), as described in Example 1.

Table 8

Soluble carbohydrate composition of malt after incubation with TRANSGLUCOSIDASE L-500 Cereals/cereals time (hours) Brix Soluble sugar component% IMO number glucose maltose Isomaltose Maltotriose 4-α-Glucosyl Maltose Isomaltotriose Branch G3+ malt 0 15.70 16.42 33.87 - 14.04 - - 35.66 - malt 2 16.50 19.59 21.91 0.75 7.70 9.38 2.87 31.40 44.40 malt 4 16.70 21.82 19.70 0.90 6.16 12.35 1.42 30.94 45.62 malt 6 16.90 24.43 18.29 1.00 4.63 11.50 2.51 30.73 45.84 malt 12 17.00 28.37 15.47 3.93 4.32 8.36 2.68 29.10 43.98 malt twenty four 17.00 33.24 14.94 5.96 2.34 5.50 3.34 26.15 40.95

The results in Table 8 show that commercially available malt extracts can be used as suitable substrates for the production of malt extracts containing isomalto-oligosaccharides. The reaction time and the IMO sugar composition of the malt extract can be adjusted by controlling the dosage of the enzyme. The addition of maltogenic enzymes can increase the IMO content of the resulting composition.

Example 8: Sorghum, millet and rice (exogenous malting enzyme)

In another experiment, 280 grams of sorghum, millet and rice powders were respectively mixed with 720 grams of deionized water. The pH of the suspension was adjusted to 5.5 and Bacillus stearothermophilus alpha-amylase sold under the trade name "GC007" (Genencor International) was added in an amount of 0.5 kg/mt powder. After uniform mixing, the suspension was kept in a water bath maintained at 75°C. During the 6 hour incubation, the reaction mixture was continuously stirred. Then, the temperature was lowered to 60°C and beta-amylase (sold under the trade name OPTIMALBBA by Genencor International) was added in an amount of 1.0 kg/mt of powder. The incubation was continued for another 10-15 hours (sample was taken for Brix and HPLC). After the indicated time, the pH was lowered to _4.5 with 6.0 N _H2SO4 and Aspergillus niger transglucosidase (sold under the trade name "TRANSGLUCOSIDASE L-500" by Genencor International) was added in an amount of 1.0 kg/mt powder. At 24 hours and 48 hours, samples were taken for analysis (Table 9).

Table 9

Soluble carbohydrate fractions of sorghum, millet and rice after "TRANSGLUCOSIDASE L-500" treatment Cereals/cereals deal with time (hours) Brix Soluble sugar component % glucose maltose Isomaltose Maltotriose 4-α-Glucosyl Maltose Isomaltotriose branched sugar G3+ IMO number sorghum TgL-500 ^* twenty four 20.20 18.70 23.50 0.60 5.00 15.90 0.00 35.70 52.20 48 21.40 19.17 22.45 0.66 4.56 16.34 0.29 36.01 53.03 Millet TgL-500 ^* twenty four 23.50 38.00 16.40 5.00 1.70 8.30 2.50 28.0 43.90 48 23.9 40.20 17.13 5.36 0.14 6.08 5.49 24.26 41.19 rice TgL-500 ^* twenty four 25.50 15.90 22.90 0.60 6.80 18.90 1.70 33.20 54.40 48 26.00 17.83 20.30 0.54 4.58 22.13 0.38 34.21 51.25

As shown in Figure 9, IMO values of 41 to 54% (52.20, 53.03, 43.90, 41.19, 54.40 and 51.25) were obtained.

Example 9: Mixed Cereal/Cereal Compositions

The data for wheat in Example 5 and for barley and rye in Example 6 show a considerable amount of endogenous maltogenic enzyme activity which results in the formation of a very high maltose syrup. On the other hand, cereals known not to contain endogenous maltogenic enzymes, eg, sorghum, millet, and rice, require the addition of exogenous maltogenic enzymes in order to produce substrates suitable for transglucosidase treatment. In this experiment, we investigated the supplementation of sorghum and rice with maltogenic cereals such as wheat or barley to convert starch into a substrate with high maltose levels. In a typical experiment, a 15% suspension of sorghum and rice was prepared by suspending 140 g of powder in 720 g of deionized water. The pH was adjusted to 5.5 with _6.0 N _H2SO4 , and Bacillus stearothermophilus alpha amylase, sold under the trade name "GC007" (Genencor International), was added in an amount of 0.5/MT powder. Afterwards, the resulting suspension was maintained at 75°C in a water bath. Stirring of the suspension was continued for 6 hours. Then, the temperature was lowered to 60°C. By adding barley flour, the solids content of the powder, eg pre-treated rice flour, is increased from 15% to 30%. Similarly, wheat was added to pretreated sorghum to a final concentration of 30%. Then, the incubation was continued for another 10-12 hours at 60°C. The pH was lowered to 4.5 and TRANSGLUCOSIDASE L-500 was added at 1.0 kg/mt of powder. Incubation was continued at 60°C for 24 hours and 48 hours. Samples were taken for HPLC analysis and brix; results are shown in Table 10.

Table 10

Millet and barley; soluble carbohydrate fraction of rice and wheat Cereals/cereals time (hours) Brix IMO number glucose maltose Isomaltose 4-α-Glucosyl Maltose Maltotriose Isomaltotriose branched sugar G3+ Wheat and Sorghum (50:50) twenty four 16.1 48.70 29.50 18.60 2.90 1.00 13.00 3.40 29.50 48 16.5 45.27 34.68 17.09 5.03 0.51 6.20 6.42 27.63 Rice and barley (50:50) twenty four 21.00 51.00 19.50 18.40 1.00 4.20 20.20 1.10 34.7 48 21.4 56.20 21.60 18.21 1.3 2.92 17.96 2.08 37.86

As shown in Table 10 above, a mixture of millet and barley and rice and wheat resulted in an IMO of 45% to 56% in the resulting suspension after the incubation period described above.

It should be understood that the examples and implementations described here are for the purpose of illustration only, and various modifications or changes to them are suggested to those skilled in the art, and they are included in the spirit and spirit of the present application. within the scope and scope of the claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all subject matter.

Claims (17)

1. be used to make the method for isomalto Oligosaccharide grain compositions, described method comprises:

(a) the ungelatinized cereal that will contain starch contact with the starch liquefacation enzyme with producing maltase, generation maltose;

(b) described maltose is contacted with the enzyme that changes glucosides, wherein said step (a) and step (b) are in the temperature generation that is lower than or equals starch gelatinization temperature; With

(c) acquisition has the grain compositions of the isomalto Oligosaccharide of enzymatic generation, and wherein said oligosaccharides derives from described cereal.

2. method according to claim 1, wherein said step (a) and (b) generation simultaneously.

3. method according to claim 1 further comprises the step of dry described grain compositions.

4. method according to claim 1, wherein said cereal is selected from wheat, rye, barley and Fructus Hordei Germinatus.

5. method according to claim 1, wherein said cereal is selected from broomcorn millet, jowar and rice.

6. method according to claim 1, wherein said product maltase is a βDian Fenmei.

7. method according to claim 1, wherein said product maltase is endogenic for described cereal.

8. method according to claim 1, wherein said starch liquefacation enzyme are the αDian Fenmeis from genus bacillus.

9. method according to claim 8, wherein said starch liquefacation enzyme is from Bacillus licheniformis (Bacilluslicheniformis) or bacstearothermophilus (Bacillus stearothermophilus).

10. method according to claim 1, the enzyme of wherein said commentaries on classics glucosides is a transglucosidase.

11. method according to claim 10, wherein said transglucosidase derives from aspergillus tubigensis.

12. method according to claim 11, wherein said aspergillus tubigensis are aspergillus niger (Aspergillus niger).

13. according to the grain compositions that claim 1 produced.

14. food additive, it contains grain compositions according to claim 13.

15. flour, it contains grain compositions according to claim 13.

16. isomalto Oligosaccharide according to claim 1 manufacturing.

17. oral rehydrated solution, it contains the isomalto Oligosaccharide of with good grounds claim 16.

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