WO2017205337A1 - Procédé de cuisson et son procédé - Google Patents

Procédé de cuisson et son procédé Download PDF

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Publication number
WO2017205337A1
WO2017205337A1 PCT/US2017/033942 US2017033942W WO2017205337A1 WO 2017205337 A1 WO2017205337 A1 WO 2017205337A1 US 2017033942 W US2017033942 W US 2017033942W WO 2017205337 A1 WO2017205337 A1 WO 2017205337A1
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WIPO (PCT)
Prior art keywords
enzyme
starch
cereal flour
enzymes
composition
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Ceased
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PCT/US2017/033942
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English (en)
Inventor
Jayarama K. Shetty
Troy Thomas BOUTTE
Ashley BEECH
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International N&H Denmark ApS
EIDP Inc
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DuPont Nutrition Biosciences ApS
EI Du Pont de Nemours and Co
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Publication of WO2017205337A1 publication Critical patent/WO2017205337A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A21D13/062—Products with modified nutritive value, e.g. with modified starch content with modified sugar content; Sugar-free products
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/12—Replacer
    • A23V2200/132—Sugar replacer
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/28—Oligosaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/50—Polysaccharides, gums
    • A23V2250/51—Polysaccharide
    • A23V2250/5118—Starch

Definitions

  • the present invention relates to a method of preparing a dough composition using one or more enzymes. It also relates to compositions (for example, dough compositions, baked products, bread, and the like) obtainable using the present method. The invention also relates to the use of a composition prepared using the method for improving the properties of dough and baked products prepared from the dough. The invention also relates to a method for preparing a composition containing isomaltooligosaccharides containing higher proportion of branched sugars.
  • sequence listing submitted via EFS in compliance with 37 C.F.R. ⁇ 1 .52(e), is incorporated herein by reference.
  • the sequence listing text file submitted via EFS contains the file "20170511_NB41081 WOPCT_SequenceListingST25.txt” created on May 10, 2015, which is 112 KB (115,446 bytes) in size.
  • conditioners are well known in the baking industry.
  • the addition of conditioners to bread dough has resulted in improved machinability of the dough, improved texture, increased loaf volume, improved flavour, and increased softness (anti-staling) of the bread.
  • Chemicals such as ascorbic acid, iodates, peroxides, and potassium bromate have been suggested but have met with consumer resistance or are not permitted by regulatory agencies.
  • grains such as wheat, malt, sorghum (milo), millet, and particularly whole grains are used in nutrition as carriers of macro- and microelements: starch, proteins, fibre, vitamins, and minerals.
  • the majority of cereal grains appeared to be too readily digested to play an effective role as a prebiotic or even as a nutraceutical.
  • IMO isomaltooligosaccharides
  • oligosaccharides exhibiting this prebiotic activity, especially in Asia.
  • These oligosaccharides typically include inulin, levan, fructooligosaccharides (FOS) , galactooligosaccharides (GOS) ,
  • mannooligosaccharides MOS
  • IMO isomaltooligosaccharides
  • oligosaccharides are relatively new functional food ingredients that have great potential to improve the quality of many foods.
  • Isomaltooligosacchandes are generally regarded as safe (GRAS) by the US FDA.
  • Addition of IMO in the baking process can result in replacing 25 to 50 % of the sucrose used in the process.
  • Use of IMO results in dough expanding rapidly in fermenting state and volume becomes larger and stiffening of bread becomes slower due to high hygroscopicity of branched sugars (Food Chemical News, February 15, 1990) .
  • Functional food oligosaccharides which are classified as "prebiotic” are receiving increased attention in food, beverage, and animal nutrition due to their health benefit by promoting the growth of beneficial bacteria, such as Bifidobacteria or Lactobacilli, in the colon (Hideo Tomomatsu, Food Technology, (1994) 61 -65).
  • beneficial bacteria such as Bifidobacteria or Lactobacilli
  • US Patent 7,993,689 describes a process for making isomaltooligosaccharide- enriched flours at a temperature at or below the starch gelatinization temperature.
  • the process comprises contacting an ungelatinized wheat having an endogenous maltogenic enzyme, i.e. beta amylase, with or without a starch solubilizing microbial enzyme to produce high maltose syrup.
  • the maltose syrup was then contacted with a 1 ,4-a-glucan 6-a- glucosyltransferase to produce a substrate composition containing isomaltooligosacchandes.
  • the process described does not involve the in situ production of IMO nor the application of a hydrolysate in bread production .
  • WO 00/27215 describes a process for preparing dough and or baked products with a glucose isomerase.
  • US 2003/0077369 describes a method to increase sweetness in baked goods using a combination of added IMO and high intensity sweeteners. However, IMO used in this method is not produced in situ.
  • US 4,377,602 describes a method in which whole grain products are subjected to enzymatic hydrolysis in order to extract all nutritionally important proteins. This patent describes an application for added fibre in food products, but does not address the replacement of sugar for sweetness and browning of breads. The wheat syrup produced by the process may subsequently be used in a baking composition. However, the proportion of wheat used in the initial enzymatic hydrolysis to total wheat used in the entire procedure exceeds that used in the first portion of wheat flour in the present invention.
  • US 4,859,474 describes a method for enzymatically producing fructose from moistened cereal grains using cellulase and glucose isomerase. This method requires heating under pressure and drying the final product prior to use in baked goods. This method describes the production of fermentable sugars by hydrolysis of starch components of flour and also the in situ production of IMO. In addition, it does not specifically disclose the subsequent addition of the fermentable sugar mixture to a further portion of cereal flour for use in a baking composition.
  • EP154135A describes a multi-step method for preparing bread dough by (a) heating and enzymatically treating a portion of wheat flour using an a-amylase, to produce a soluble, partially hydrolysed starch; (b) adding glucoamylase to the soluble, partially hydrolysed starch prepared in step (a); and (c) adding the composition prepared in step (b) to a mixture of wheat flour, water, and scrap dough.
  • step (a) generally requires very high temperature cooking (greater than 90°C) for an extended period of time (typically over 10 hours): this results in both liquefaction and gelatinization of the starch and the high temperature above the starch gelatinization also results in the complete inactivation of wheat endogenous starch hydrolyzing enzyme activity.
  • subjecting the composition to temperatures above 75°C results in the denaturation of wheat gluten proteins (Eliasson, A.C. and Hegg, P-O., Cereal Chem. (1980) 57, 436-437) which may affect the gluten:starch complex structure in baked products.
  • a method of preparing a dough composition comprising:
  • said first portion of cereal flour comprises 5 to 50% by weight of the total weight of said first and second portions of cereal flour; and said water used in step (a) comprises 50% to 100% by weight of the total weight of the water used in said method.
  • a method of preparing a dough composition comprising:
  • step (c) adding a second portion of cereal flour; wherein said first portion of cereal flour comprises 5 to 50% by weight of the total weight of said first and second portions of cereal flour; and said water used in step (a) comprises 50% to 100% by weight of the total weight of the water used in said method.
  • a method of preparing a baked product comprising: preparing a dough composition according to the present method; and baking the dough composition to prepare the baked product.
  • composition is provided that is obtained or obtainable according to the present method.
  • a baked product is provided that is obtained or obtainable according to the present method.
  • a baked product can be prepared without added sugars.
  • a use is provided for the present methods and/or compositions for improving the properties of a dough and/or a baked product prepared from the dough.
  • Such improved properties include no added sugar; improved elasticity;
  • a use related to in situ IMO as an anti-staling agent is also provided.
  • SEQ ID NO: 1 is the amino acid sequence of an 1 ,4-a-glucan 6-a-glucosyltransferase from
  • SEQ ID NO: 2 is the amino acid sequence of an alpha-amylase from Geobacillus stearothermophilus.
  • SEQ ID NO: 3 is the amino acid sequence of a glucoamylase from Trichoderma reesei.
  • SEQ ID NO: 4 is the amino acid sequence of a variant glucoamylase derived from SEQ ID NO: 3.
  • SEQ ID NO: 5 is the amino acid sequence of an 1 ,4-a-glucan 6-a-glucosyltransferase from Aspergillus niger.
  • SEQ ID NO: 6 is the amino acid sequence of an alpha-amylase derived from Geobacillus stearothermophilus.
  • SEQ ID NO: 7 is the amino acid sequence of a glucose isomerase from Streptomyces rubiginosus.
  • SEQ ID NO: 8 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GEN BAN K® BAA23616.1 .
  • SEQ ID NO: 9 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GEN BANK® BAD06006.1 .
  • SEQ ID NO: 10 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GEN BAN K® BAA08125.1 .
  • SEQ ID NO: 1 1 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GENBANK® XP_001271891 .1 .
  • SEQ ID NO: 12 is the amino acid sequence of a1 ,4-a-glucan 6-a-glucosyltransferase from G EN BAN K® XP_001266999.1 .
  • SEQ ID NO: 13 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GENBANK® XP_75181 1 .1 .
  • SEQ ID NO: 14 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GEN BANK® XP 659621 .1 .
  • SEQ ID NO: 15 is the amino acid sequence of a 1 ,4-a-glucan 6-a-glucosyltransferase from GENBANK® XP_001216899.1 .
  • SEQ ID NO: 16 is the amino acid sequence of a1 ,4-a-glucan 6-a-glucosyltransferase from GENBANK® XP_001258585.1 .
  • Figure 1 is an ion chromatographic composition of IMO in the baking adjunct of the present invention as produced in Example 3.
  • Figure 2 is a chromatogram showing the effect of incubation time on the conversion of wheat flour starch to glucose and fructose under baking conditions.
  • Figures 3A to 3C shows the baked bread produced according to the methods of Examples 1 -3 together with a control with added sucrose: Fig. 3A showing colour; Fig. 3B showing volume; and Fig. 3C showing open cells.
  • Figure 4 is a chromatogram showing the effect of exo-peptidase (SUMIZYME ® Pf-G) during incubation of wheat flour with a-amylase (SPEZYME ® RSL) and 1 ,4-a-glucan 6-a- glucosyltransferase (TR-TG) at pH 5.6, 55°C and 5 hours on the production of free amino acids.
  • SUMIZYME ® Pf-G exo-peptidase
  • SPEZYME ® RSL a-amylase
  • TR-TG 1 ,4-a-glucan 6-a- glucosyltransferase
  • Figure 5 shows a comparison of baked bread with and without added Example 1 with added sugar and Example 7 (without added sugar).
  • BIOLOGY, 2ND ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
  • the term 'saccharide' in its broadest sense is intended to cover all saccharides (sugars), including naturally occurring and synthetic and semi-synthetic saccharides.
  • the term encompasses monosaccharides (i.e. saccharides that cannot be hydrolyzed into simpler sugars), disaccharides (i.e. compounds having two monosaccharide units (moieties) joined together by a glycoside bond), oligosaccharides (i.e. compounds having 3 to 10 monosaccharide units joined together by glycoside bonds in a branched or unbranched chain or a ring (optionally having a saccharide side chain) and polysaccharides, i.e. compounds having over 10 monosaccharide units joined together by a glycoside bond in a branched or unbranched chain or a ring (optionally having a saccharide side chain).
  • the saccharide may be bonded to other molecules, such as biomolecules, for example peptides / proteins, lipids and nucleic acids. However, it is preferred for the purposes of the present invention that the saccharide is formed from monosaccharide units only.
  • the saccharide is a monosaccharide, i.e. a saccharide that cannot be hydrolyzed into a simpler sugar.
  • the monosaccharide may have the D- or L- configuration, and may be an aldose or ketose.
  • the monosaccharide is a hexose, examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose and ketohexoses such as fructose, tagatose, psicose and sorbose.
  • aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose
  • ketohexoses such as fructose, tagatose, psicose and sorbose.
  • the hexose is glucose or galactose.
  • the monosaccharide is a pentose, examples of which include aldopentoses such as ribose, arabinose, xylose and lyxose and ketopentoses such as ribulose and xylulose.
  • aldopentoses such as ribose, arabinose, xylose and lyxose
  • ketopentoses such as ribulose and xylulose.
  • the pentose is arabinose or xylose.
  • the saccharide is a higher saccharide, i.e. a saccharide comprising more than one monosaccharide moiety joined together by glycoside bonds and which are generally hydrolysable into their constituent monosaccharides.
  • higher saccharides examples include disaccharides (2 monosaccharide moieties), oligosaccharides (3 to 10 monosaccharide moieties) and polysaccharides (more than 10 monosaccharide moieties).
  • the monosaccharide moieties which form the higher saccharide may be the same or different, and may each independently have the D- or L-configuration, and may each independently be aldose or ketose moieties.
  • degree of polymerization refers to the number (n) of
  • DP1 monosaccharide units in a given saccharide.
  • DP2 monosaccharides, such as glucose and fructose.
  • DP2 are the disaccharides, such as maltose and sucrose.
  • a DP4 + (>DP4) denotes polymers with a degree of polymerization of greater than 4.
  • the monosaccharide units which form the higher saccharide may have the same or different numbers of carbon atoms.
  • the monosaccharide moieties of the higher saccharide are hexose moieties, examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose and ketohexoses such as fructose, tagatose, psicose and sorbose.
  • the hexose moieties of such a higher saccharide are glucose moieties.
  • the monosaccharide moieties of the higher saccharide are aldopentose moieties such as ribose, arabinose, xylose and lyxose and ketopentoses such as ribulose and xylulose.
  • the pentose moieties of such a higher saccharide are arabinose or xylose moieties.
  • the monosaccharide moieties which form the higher saccharide are joined together by glycosidic bonds.
  • the glycosidic bonds may be 1 ,4'- glycosidic bonds (which may be 1 ,4'-a- or 1 ,4'-p-glycoside bonds), 1 ,6'-glycosidic bonds (which may be 1 ,6'-a- or ⁇ , ⁇ '- ⁇ -glycoside bonds), 1 ,2'- glycosidic bonds (which may be 1 ,2'-a- or 1 ,2'-p-glycoside bonds), or 1 ,3'-glycosidic bonds (which may be 1 ,3'-a- or 1 ,3'-p-glycoside bonds), or any combination thereof.
  • the higher saccharide comprises 2 monosaccharide units (i.e. a disaccharide).
  • suitable disaccharides include lactose, maltose, cellobiose, sucrose, trehalose, isomaltulose, and trehalulose.
  • the higher saccharide comprises 3 to 10 monosaccharide units (i.e. an oligosaccharide).
  • the monosaccharide units may be in a chain, which may be branched or unbranched: such oligosaccharides are referred to in this specification as 'chain oligosaccharides'.
  • oligosaccharides examples include maltooligosaccharides (as defined below) such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose; as well as fructooligosaccharides (FOS) which consist of short chains of fructose molecules; mannanoligosaccharides, isomaltooligosaccharides,
  • maltooligosaccharides as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and cellohepta
  • galactooligosaccharides galactooligosaccharides, and xylooligosaccharides.
  • the higher saccharide is a polysaccharide, comprising at least 10 monosaccharide units joined together by glycoside bonds.
  • polysaccharides comprise at least: 40, 100, 200, 500, 1000, 5000, 10000, 50000, 100000 or more monosaccharide units.
  • the polysaccharide comprises from 10 to 500000
  • the polysaccharide comprises from 100 to 1000 monosaccharide units. In other embodiments, the polysaccharide comprises from 1000 to 10000 monosaccharide units. In other embodiments, the polysaccharide comprises from 10000 to 100000 monosaccharide units. In some embodiments, the polysaccharide comprises from 40 to 3000, preferably 200 to 2500, monosaccharide units.
  • polysaccharides include starch and derivatives thereof (as defined herein; such as cationic or anionic, oxidised or phosphated starch), amylose, amylopectin, glycogen, cellulose or a derivative thereof (such as carboxymethyl cellulose), alginic acid or a salt or derivative thereof, polydextrose, pectin, pullulan, carrageenan, locust bean gum and guar and derivatives thereof (such as cationic or anionic guar).
  • starch and derivatives thereof as defined herein; such as cationic or anionic, oxidised or phosphated starch
  • amylose amylopectin
  • glycogen such as carboxymethyl cellulose
  • cellulose or a derivative thereof such as carboxymethyl cellulose
  • alginic acid or a salt or derivative thereof such as polydextrose, pectin, pullulan, carrageenan, locust bean gum and guar and derivatives thereof (such as cationic or anionic
  • fermentable sugar refers to mono or di-saccharides capable of undergoing fermentation using a suitable fermentation microorganism, typically yeast.
  • suitable fermentation microorganism typically yeast.
  • fermentable sugars include glucose, fructose and maltose.
  • starch refers to a polysaccharide consisting of a large number of glucose units joined by glycosidic bonds, having the formula (C 6 HioOs)x, wherein x can be any number. Typically, x ranges from 10 to 1 ,000,000; preferably x is from 50 to 500,000; and even more preferably x ranges from 100 to 100,000.
  • Starch consists of two types of molecules: amylose and amylopectin.
  • Amylose typically consists of a-D-glucose units, bound to each other through a (1 ⁇ 4) glycosidic bonds).
  • the chain typically comprises 50 to 50,000, preferably 100 to 10,000, more preferably 300 to 3,000 glucose units.
  • Amylopectin typically consists of glucose units linked in a linear way with a (1 ⁇ 4) glycosidic bonds and have branching with a (1 ⁇ 6) bonds occurring every 24 to 30 glucose units.
  • the chain typically comprises 1 ,000 to 1 ,000,000, preferably 200 to 200,000 glucose units.
  • the starch contains 15 to 30% amylose and 70 to 85% amylopectin by weight of the total weight of the starch.
  • the starch contains 20 to 25% amylose and 75 to 80% amylopectin by weight of the total weight of the starch.
  • Starch may be derived from any suitable plant-based material including, but not limited to grains, grasses, tubers and roots; and more specifically wheat, barley, corn, rye, rice, sorghum, legumes, cassava, millet, potato, sweet potato, and tapioca.
  • the starch is derived from wheat.
  • granular starch refers to uncooked (raw) starch, which has not been subject to gelatinization.
  • starch gelatinization means solubilization of a starch molecule to form a viscous suspension.
  • gelatinization temperature refers to the lowest temperature at which gelatinization of a starch substrate begins. The exact temperature depends upon the specific starch substrate and further may depend on the particular variety of plant species from which the starch is obtained and the growth conditions. Typically, the gelatinization temperature of starch ranges from 40 to 70°C, preferably from 50 to 60°C, and more preferably 55 to 60°C.
  • DE or "dextrose equivalent” is an industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Un- hydrolyzed granular starch has a DE that is essentially 0 and D-glucose has a DE of 100.
  • starch substrate refers to granular starch or liquefied starch using refined starch, whole ground grains or fractionated grains.
  • liquefied starch refers to starch which has gone through solubilization process using starch liquefaction process.
  • total sugar content refers to the total sugar content present in a starch composition.
  • ds refers to dissolved solids in a solution.
  • starch-liquefying enzyme refers to an enzyme that affects the hydrolysis or breakdown of granular starch.
  • Exemplary starch liquefying enzymes include a-amylases (E.C. 3.2.1 .1).
  • hydrolysis of starch refers to the cleavage of glycosidic bonds between the glucose moieties of starch, with water molecule being added across the cleaved glycosidic bond.
  • maltooligosaccharide means a disaccharide or oligosaccharide consisting of short chains of glucose units (typically 2 to 10, preferably 2 to 5 glucose units) bound to each other through a (1 ⁇ 4) glycosidic bonds.
  • maltooligosaccharides include maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose.
  • maltooligosaccharide refers to the reaction products derived from the hydrolysis of starch substrate.
  • IMO isomaltooligosaccharide
  • Isomaltooligosaccharides consisting of short chains of glucose units (typically 3 to 10, preferably 3 to 5 glucose units) bound to each other through alpha a (1 ⁇ 6) glycosidic bonds from the non- reducing ends.
  • Isomaltooligosaccharides are glucose oligomers with a-D-(1 ,6)-linkages. Examples of isomaltooligosaccharides may include isomaltose, panose, isomaltotriose, isomaltotetraose, isomaltopentaose, nigerose, kojibiose, and higher branched
  • contacting is generally understood to refer to the placing of the respective enzymes in sufficiently close proximity to the respective substrate to enable the enzymes to convert the substrate to the end product.
  • contacting will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.
  • the term "improved property" is defined herein as any property of a dough and/or a product obtained from the dough, particularly a baked product.
  • the improved property is increased strength of the dough.
  • the improved property is increased elasticity of the dough.
  • the improved property is reduced stickiness of the dough.
  • the improved property is increased extensibility of the dough.
  • the improved property is improved machinability of the dough.
  • the improved property is increased volume of the baked product.
  • the improved property is improved crumb structure of the baked product.
  • the improved property is improved anti- staling of the baked product.
  • the improved property is improved softness of the baked product.
  • the improved property is improved flavour of the baked product.
  • in situ production refers to production of the desired product (for example, a fermentable sugar or isomaltooligosaccharide) during the specified process, thereby eliminating the requirement for external addition of this product.
  • the term “in situ production” means that the required product is produced in step (a) and/or step (b) of the method of either aspect of the present invention, i.e.
  • the product is prepared when the first portion of cereal flour, water and one or more enzymes are mixed and allowed to act so as to convert the starch into the desired product (such as a fermentable sugar or isomaltooligosaccharide), but before the addition of the second portion of cereal flour and the subsequent steps to produce the dough, and any subsequent step to produce a baked product prepared from the dough.
  • the term "in situ production” means that the required product is produced in a baking step subsequent to the steps of the method of the present invention.
  • the term “in situ production” means that the required product is produced partially in step (a) and/or step (b) of the method of either aspect of the present invention, and partially in a baking step subsequent to the steps of the method of the present invention.
  • baking adjunct in its broadest sense refers to
  • the term "baking adjunct" means that the required product is produced in step (a) and/or step (b) of the method of either aspect of the present invention, i.e. the intermediate composition prepared from the first portion of cereal flour, water and one or more enzymes, and the enzymes are allowed to act so as to convert the starch into the desired product (for example, a fermentable sugar or isomaltooligosaccharide).
  • the baking adjunct is then added to the second portion of cereal flour used in the subsequent steps to produce the dough, and any subsequent step to produce a baked product prepared from the dough.
  • the term "effective amount” is defined herein as an amount of in situ production of fermentable sugars or branched oligosaccharides that is sufficient for proving a measurable effect on at least one property of interest of the dough and /or baked product.
  • the present inventors have identified that by in situ formation of fermentable sugars and/or IMO from wheat flour starch, it is possible to eliminate the need for addition of sugars (especially sucrose) to dough.
  • a method of preparing a dough composition comprising: (a) preparing a composition by mixing a first portion of cereal flour, water and one or more enzymes capable of converting starch present in said first portion of cereal flour into a fermentable sugar, said one or more enzymes being introduced into said composition in a single step; (b) allowing said enzyme to convert said starch into said fermentable sugar; and (c) addition of a second portion of cereal flour; wherein said first portion of cereal flour comprises 5 to 50% by weight of the total weight of said first and second portions of cereal flour; and said water used in step (a) comprises 50% to 100% by weight of the total weight of the water used in said method.
  • the method according confers the particular advantage that fermentable sugars (such as maltose, glucose or fructose) can be produced in situ by the action of the enzyme on the starch present in the wheat flour. This therefore avoids the requirement to add sucrose or other sugars to the composition.
  • fermentable sugars such as maltose, glucose or fructose
  • the present method requires the introduction of the one or more starch hydrolysing enzymes (particularly although not exclusively amylase, such as an alpha or beta amylase, and a glucoamylase) into the initial composition comprising water and the first portion of cereal flour in a single step. This avoids the requirement to heat the mixture to temperatures above wheat starch
  • gelatinization temperature i.e. around 60°C
  • processing time thereby avoiding gelatinization of the starch, deactivating the wheat endogenous enzymes, and denaturing the wheat gluten. It also enables the processing time to be reduced from over 10 hours to less than 4 hours.
  • a method of preparing a dough composition comprising: (a) preparing a composition by mixing a first portion of cereal flour, water and an enzyme(s) capable of converting starch present in said first portion of cereal flour into an isomaltooligosaccharide; (b) allowing said enzyme to convert said starch into said isomaltooligosaccharide; and (c) adding a second portion of cereal flour; wherein said first portion of cereal flour comprises 5 to 50% by weight of the total weight of said first and second portions of cereal flour; and said water used in step (a) comprises 50% to 100% by weight of the total weight of the water used in said method.
  • the isomaltooligosacchandes can be produced in situ production of IMO by the hydrolytic action of an enzymes on the starch in wheat flour, typically to produce maltose or maltooligosaccharides; followed by action of an enzyme (typically a 1 ,4-a-glucan 6-a- glucosyltransferase) on the maltose or maltooligosaccharides to produce IMO.
  • the IMO produced in situ can be used as a sweetening and/or softening agent in bread, thereby allowing sucrose to be removed from the formulation.
  • a dough composition and baked products prepared from the composition comprising an effective amount of functional oligosaccharides (in particular, containing a higher level of a-1 ,6 branched oligosaccharides, for example isomaltooligosacchandes, than was possible in the prior art) can be produced in situ under baking processing conditions.
  • functional oligosaccharides in particular, containing a higher level of a-1 ,6 branched oligosaccharides, for example isomaltooligosacchandes, than was possible in the prior art
  • the present method allows the production of fructose as well as in situ IMO from a hydrolysate made from wheat flour, water, amylase, glucoamylase, and/or 1 ,4-a-glucan 6-a-glucosyltransferase.
  • the present methods involve a single step for hydrolyzing starch and, in one aspect, isomerisation of the hydrolysis products.
  • the hydrolysis and/or isomerised products are used as baking adjuncts in the subsequent steps.
  • the starch used in the present methods may be derived from any suitable plant product.
  • the starch is derived from a cereal crop, typical examples of which include wheat, barley, oats, rye and maize.
  • the starch is derived from wheat.
  • the starch is granular wheat starch.
  • the starch used as a starting material is insoluble in the aqueous suspension of cereal flour and water to which the enzyme is added.
  • the starch is insoluble granular wheat starch.
  • the present method is carried out in situ in a food composition. In one embodiment, the present method is carried out in situ in a baking composition. [0083] In one embodiment, the present method uses enzymes which solubilize and hydrolyze the starch (such as wheat granular starch) are endogenous to wheat. Typically, the enzymes comprise a-amylase, ⁇ -amylase or mixtures thereof.
  • Step (a) of both aspects of the present method involves addition of water and the required enzyme to a first portion of cereal flour.
  • the first portion of cereal flour comprises only a minor part of the total cereal flour used in the baking process as a whole.
  • the proportion of the first cereal flour introduced at step (a) is expressed by weight of the total weight of cereal flour, i.e. the total of the first portion, introduced in step (a) and the second portion, introduced in step (c).
  • the first portion of cereal flour introduced at step (a) comprises 5 to 50% by weight of the total weight of said first and second portions of cereal flour.
  • the first portion of cereal flour introduced at step (a) comprises 10 to 40% by weight of the total weight of the first and second portions of cereal flour.
  • the first portion of cereal flour introduced at step (a) comprises 20 to 30% by weight of the total weight of the first and second portions of cereal flour.
  • Step (a) of both aspects of the present method involves addition of water and the required enzyme to a first portion of cereal flour.
  • the water used in step (a) comprises the major part of the total water used in the overall method, i.e. the method of each aspect of the invention comprising steps (a) , (b) and (c).
  • the water used in step (a) comprises 50% to 100% by weight of the total weight of the water used in the overall method.
  • the water used in step (a) comprises 80% to 100% by weight of the total weight of the water used in the overall method. More preferably, the water used in step (a) comprises 90% to 100% by weight of the total weight of the water used in the overall method. Even more preferably, the water used in step (a) comprises 95% to 100% by weight of the total weight of the water used in the overall method.
  • the water used in step (a) comprises 100% by weight of the total weight of the water used in the overall method.
  • the total amount of water used in the present method is a conventional amount used in the production of dough compositions. However, the addition in the first step of the method of only a minor portion of the flour, but the major part of the water, wherein the enzymes are added in a single step, has not previously been disclosed in the art. [0088] Typically, the total amount of water used in the present method ranges from 20 to 100 grams of water per 100 grams of cereal flour. Preferably, the total amount of water used in the method of the invention ranges from 40 to 80 grams of water per 100 grams of cereal flour. More preferably, the total amount of water used in the method of the invention ranges from 50 to 70 grams of water per 100 grams of cereal flour.
  • the total amount of water used ranges from 20 to 100 grams of water per 100 grams of wheat of the total used for making dough.
  • the total amount of water used ranges from 40 to 80 grams of water per 100 grams of wheat. More preferably, the total amount of water used ranges from 50 to 70 grams of water per 100 grams of wheat.
  • the method comprises heating the composition.
  • the heating step is comprised in step (b) and this typically comprises heating the composition prepared in step (a). Heating typically increases the rate at which the enzyme(s) are of carrying out its hydrolytic activity and, where required, the conversion of
  • the method is carried out below or at wheat starch gelatinization temperature. Typically, the method is carried out at a temperature below 60°C, such as below 58°C. In one embodiment, the method is carried out at a temperature of 40°C to 60°C, such as 50 to 58°C.
  • the present method comprises heating the composition for 1 to 24 hours. In one embodiment, the present method, typically step (b) thereof, comprises heating the composition for 2 to 6 hours.
  • the present method enables the in situ production of fermentable sugars by incubation of the enzyme with a cereal flour, especially wheat flour, in water.
  • a cereal flour especially wheat flour
  • the desired sugar is typically produced by the action of glucoamylase (gamma amylase) on the
  • the fermentable sugar comprises fructose
  • the desired sugar is typically produced by the further action of glucose isomerase to convert glucose into fructose.
  • step c) the composition produced in steps a) and b), containing the fermentable sugars and/or IMO, is then mixed with the second portion of cereal flour and, if required, any remaining water to produce the dough composition.
  • the second portion of cereal flour introduced at step (c) comprises 50 to 95% by weight of the total weight of said first and second portions of cereal flour.
  • the second portion of cereal flour introduced at step (a) comprises 60 to 90% by weight of the total weight of the first and second portions of cereal flour. More preferably, the second portion of cereal flour introduced at step (c) comprises 70 to 80% by weight of the total weight of the first and second portions of cereal flour.
  • the method further comprises shaping the dough composition.
  • the method further comprises baking the dough composition, particularly the shaped dough composition to form a baked product. Baking is carried out by conventional baking processes well known to those skilled in the art. Examples of baked products are set out below.
  • step (c) of the method is carried out in situ in a food composition. In one embodiment, step (c) of the method is carried out in situ in a baking composition.
  • the starch is hydrolyzed below or at wheat starch gelatinization temperature. Typically, the starch is hydrolyzed at a temperature below 60°C, such as below 58°C. In one embodiment, the starch is hydrolyzed at a temperature of 40°C to 60°C, such as 50 to 58°C.
  • the method comprises the steps of:
  • any subsequent baking step is typically carried out as per conventional baking process.
  • the cereal flour is wheat flour comprising wheat granular starch and the method comprises the steps of:
  • any subsequent baking step is typically carried out as per conventional baking process.
  • the method comprises the steps of:
  • a1 adding enzymes capable of converting starch into isomaltooligosaccharides (e.g. an amylase and a 1 ,4-a-glucan 6-a-glucosyltransferase) in addition to any wheat endogenous starch hydrolyzing enzymes present in the mixture;
  • isomaltooligosaccharides e.g. an amylase and a 1 ,4-a-glucan 6-a-glucosyltransferase
  • any subsequent baking step is typically carried out as per conventional baking process.
  • the cereal flour is wheat flour comprising wheat starch and the method comprises the steps of:
  • any subsequent baking step is typically carried out as per conventional baking process.
  • the present methods involve the use of one or more enzymes to carry out the hydrolysis of the starch used as a starting material and, if necessary, the isomerisation of glucose to fructose or the isomerisation of the maltooligosaccharides to IMO.
  • the enzymes used to carry out these functions are all well known to those skilled in the art.
  • the enzyme or enzymes used in the process are endogenous to the cereal flour. In one embodiment, the enzyme or enzymes used in the process are added to the cereal flour. In one embodiment, the enzyme or enzymes used in the process comprise a mixture of an enzyme or enzymes endogenous to the cereal flour and an enzyme or enzymes added to the flour. In one embodiment, the enzyme or enzymes used in the process comprise a mixture of up to 50% (by weight of the total weight of enzymes used) of an enzyme or enzymes endogenous to the cereal flour and more than 50% (by weight of the total weight of enzymes used) of an enzyme or enzymes added to the flour.
  • the enzyme or enzymes used in the process comprise a mixture of up to 25% (by weight of the total weight of enzymes used) of an enzyme or enzymes endogenous to the cereal flour and more than 75% (by weight of the total weight of enzymes used) of an enzyme or enzymes added to the flour. In one embodiment, the enzyme or enzymes used in the process comprise a mixture of more than 50% (by weight of the total weight of enzymes used) of an enzyme or enzymes endogenous to the cereal flour and up to 50% (by weight of the total weight of enzymes used) of an enzyme or enzymes added to the flour.
  • the enzyme or enzymes used in the process comprise a mixture of more than 75% (by weight of the total weight of enzymes used) of an enzyme or enzymes endogenous to the cereal flour and up to 25% (by weight of the total weight of enzymes used) of an enzyme or enzymes added to the flour.
  • the one or more enzymes comprise an enzyme having granular starch hydrolysing activity (GSHE) (as defined below) or a mixture thereof.
  • GSHE granular starch hydrolysing activity
  • the GSHE is an amylase.
  • the one or more enzymes is selected from the group consisting of an alpha amylase (E.C. 3.2.1 .1), a beta amylase (E.C. 3.2.1 .2) and a gamma amylase (glucoamylase; E.C. 3.2.1 .3), or a mixture of any thereof.
  • an alpha amylase E.C. 3.2.1 .1
  • a beta amylase E.C. 3.2.1 .2
  • a gamma amylase glucoamylase
  • said one or more enzymes comprises a mixture of an alpha amylase (E.C. 3.2.1 .1) and a gamma amylase (glucoamylase; E.C. 3.2.1 .3).
  • said one or more enzymes additionally comprises a glucose isomerase (E.C:5.3.1 .5).
  • said one or more enzymes comprises a 1 ,4-a-glucan 6-a-glucosyltransferase (E.C. 2.4.1 .24).
  • said 1 ,4-a- glucan 6-a-glucosyltransferase enzyme catalyses the transfer of a glucose moiety from a malto-oligosaccharide to the 6-OH position of another saccharide.
  • the one or more enzymes comprise a further enzyme having granular starch hydrolysing activity (GSHE) (as defined below) or a mixture thereof.
  • GSHE granular starch hydrolysing activity
  • the GSHE is an amylase.
  • the further enzyme selected from the group consisting of an alpha amylase (E.C. 3.2.1 .1), a beta amylase (E.C. 3.2.1 .2) and a gamma amylase
  • the enzyme used is of bacterial origin. In one embodiment, the enzyme used is of fungal origin. In one embodiment, the enzyme used is of animal origin. In one embodiment, the enzyme used is of plant origin.
  • the enzyme used is a recombinant enzyme.
  • the quantity of enzyme used in the methods of the present invention will depend on the nature and activity of the enzyme, the nature and amount of the cereal flour, and the desired reaction.
  • the enzyme is used in an amount of about 1 g to 10 kg of the enzyme per metric ton of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e. excluding the weight of the water used in this step).
  • the enzyme is used in an amount about 10 g to 5.0 kg per metric ton of the first portion of the cereal flour.
  • the enzyme is used in an amount about of about 0.5 kg to 2.0 kg per metric ton of the first portion of the cereal flour.
  • the enzyme is used in an amount of about 0.1 kg to 1 .0 kg per metric ton of the first portion of the cereal flour.
  • the enzyme used in the present invention may have side activities in addition to their primary activity as defined below.
  • the primary activity of the enzyme comprises more than 50% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 75% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 90% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 95% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 97% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 98% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises more than 99% of the total activity of the enzyme.
  • the primary activity of the enzyme comprises 100% of the total activity of the enzyme.
  • GSHEs Granular Starch Hydrolyzing Activity
  • the enzyme used is an enzyme having granular starch hydrolyzing activity.
  • enzyme having granular starch hydrolyzing activity or "GSHE” or “GSH” means an enzyme capable of catalysing the hydrolysis of granular starch.
  • the enzyme having GSH activity is an amylase (as defined below) .
  • the enzyme having GSH activity is an enzyme having glucoamylase activity (as defined below). In one embodiment, the enzyme having GSH activity is an enzyme having alpha-amylase activity (as defined below). In one embodiment, the enzyme having GSH activity is an enzyme having both glucoamylase activity and alpha- amylase activity. In one embodiment, the enzyme having GSH activity is a mixture of enzymes, at least one enzyme having both glucoamylase activity and at lease one, different enzyme having alpha-amylase activity.
  • the enzyme having GSH activity is of fungal origin. In one embodiment, the enzyme having GSH activity is of bacterial origin. In one embodiment, the enzyme having GSH activity is of plant origin. These enzymes have been recovered from fungal, bacterial and plant cells such as Bacillus sp. , Penicillium sp. , Humicola sp. , Trichoderma sp. Aspergillus sp. Mucor sp. and Rhizopus sp.
  • a particular group of enzymes having GSH activity include enzymes having glucoamylase activity and/or alpha-amylase activity (Tosi ef al, Can. J. Microbiol. (1993) 39: 846 -855).
  • a Rhizopus oryzae GSHE has been described in Ashikari ef a/. , Agric. Biol. Chem., (1986) 50: 957-964 and US patent 4,863,864.
  • a Humicola grisea GSHE has been described in Allison ef a/. , Curr. Genet. (1992) 21 : 225-229; International patent application publication no. WO 05/052148, and European patent application publication no. EP 171218A.
  • a GSHE may have glucoamylase activity and is derived from a strain of Humicola grisea, particularly a strain of Humicola grisea var. thermoidea (see US patent 4,618,579) .
  • the Humicola enzyme having GSH activity will have at least 85%, 90% , 92%, 94% , 95%, 96%, 97%, 98% OR 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 of WO 2005/052148.
  • a GSHE may have glucoamylase activity and is derived from a strain of Aspergillus awamori, particularly a strain of A. awamori var. kawachi.
  • the A. awamori var. kawachi enzyme having GSH activity will have at least 85%, 90%, 92% , 94%, 95%, 96%, 97% , 98% and 99% sequence identity to the amino acid sequence of SEQ ID NO: 6 of WO 2005/052148.
  • a GSHE may have glucoamylase activity and is derived from a strain of Rhizopus, such as R. niveus or R. oryzae.
  • Rhizopus such as R. niveus or R. oryzae.
  • the enzyme derived from the Koji strain R. niveus is sold under the trade name "CU CONC” or the enzyme from Rhizopus sold under the trade name GLUZYME® (Novozymes A/S).
  • the GSHE having glucoamylase activity is SPIRIZYMETM Plus (Novozymes A/S), which also includes acid fungal amylase activity.
  • a GSHE may have alpha-amylase activity and is derived from a strain of Aspergillus such as a strain of A. awamori, A. niger, A. oryzae, or A. kawachi and particularly a strain of A. kawachi.
  • the A. kawachi enzyme having GSHE activity will have at least 85%, 90% , 92%, 94%, 95% , 96%, 97%, 98% and 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 of WO 2005/1 18800 and WO 2005/00331 1 .
  • the enzyme having amylase activity or GSH activity is a hybrid enzyme, for example one containing a catalytic domain of an alpha amylase such as a catalytic domain of an Aspergillus niger alpha amylase, an Aspergillus oryzae alpha amylase or an Aspergillus kawachi alpha amylase and a starch binding domain of a different fungal alpha amylase or glucoamylase, such as an Aspergillus kawachi or a Humicola grisea starch binding domain.
  • an alpha amylase such as a catalytic domain of an Aspergillus niger alpha amylase, an Aspergillus oryzae alpha amylase or an Aspergillus kawachi alpha amylase and a starch binding domain of a different fungal alpha amylase or glucoamylase, such as an Aspergillus kawachi or a Humicola grisea starch
  • the hybrid enzyme having GSH activity may include a catalytic domain of a glucoamylase, such as a catalytic domain of an Aspergillus sp. , a Talaromyces sp. , an Althea sp. , a Trichoderma sp. or a Rhizopus sp. and a starch binding domain of a different glucoamylase or an alpha amylase.
  • Some hybrid enzymes having GSH activity are disclosed in WO 2005/00331 1 , WO 2005/045018; Shibuya et a/. , Biosci. Biotech. Biochem. (1992) 56; 1674- 1675, and Cornett et al.
  • the quantity of the enzyme having GSH activity used in the methods of the present invention will depend on nature and the activity of the enzyme having GSH activity, the nature and amount of the cereal flour, and the desired reaction.
  • the enzyme having GSH activity is a glucoamylase
  • the enzyme is used in an amount of about 0.01 Glucoamylase Activity Units (GAU) to 10.0 GAU per gram of the first portion of the cereal flour used in step (a) of the processes of the invention (calculated on a dry solids basis, i.e. excluding the water used in this step).
  • the glucoamylase is used in an amount about 0.5 to 5.0 GAU per gram dry solids of the first portion of the cereal flour. In some embodiments, the glucoamylase is used in an amount of about 1 .0 to 2.0 GAU per gram dry solids of the first portion of the cereal flour.
  • the units of glucoamylase enzyme activity can be calculated according to the assay methods set out below.
  • the enzyme used is an amylase.
  • amylase refers generally to an enzyme that catalyzes the hydrolysis of starches. All amylases are glycoside hydrolases and act on a-1 ,4-glycosidic bonds.
  • the amylase is an amylase selected from the group consisting of an alpha amylase (E.C. 3.2.1 .1), a beta amylase (E.C. 3.2.1 .2) and a gamma amylase or glucoamylase (E.C. 3.2.1 .3).
  • the amylase is an alpha amylase (E.C. 3.2.1 .1).
  • the amylase is a beta amylase (E.C. 3.2.1 .2).
  • the amylase is a gamma amylase or glucoamylase (E.C. 3.2.1 .3).
  • the amylase is an alpha-amylase.
  • Alpha amylases are classified in E.C. 3.2.1 .1 .
  • the term "alpha-amylase” refers to enzymes that catalyze the hydrolysis of a-1 ,4-glucosidic linkages in polysaccharides, particularly starch. Alternative names for this enzyme include 1 ,4-a-D-glucan glucanohydrolase or glycogenase.
  • Alpha-amylases can act at random locations along the starch chain, in order to break down the glucose chain, typically yielding: maltotriose and maltose from amylose; or maltose, glucose and "limit dextrin" from amylopectin.
  • the alpha amylase is of microbial origin. In some embodiments, the alpha amylase is of microbial origin.
  • the alpha amylase is of bacterial origin. [0137] In some embodiments, the alpha amylase is a thermostable bacterial alpha amylase. Suitable alpha amylases may be naturally occurring as well as recombinant and mutant alpha amylases.
  • the alpha amylase is derived from a bacterium of the genus Bacillus.
  • Preferred Bacillus species include B. subtilis, B. stearothermophilus, B. lentus, B. licheniformis, B. coagulans, and B. amyloliquefaciens (disclosed in United States patent Nos. US 5,763,385; US 5,824,532; US 5,958,739; US 6,008,026; and
  • alpha amylases are derived from Bacillus species selected from the group consisting of B. stearothermophilus, B. amyloliquefaciens, and B. licheniformis.
  • the enzyme is derived from the strains having the accession number ATCC 39709; ATCC 1 1945; ATCC 6598; ATCC 6634; ATCC 8480; ATCC 9945A, and NCIB 8059.
  • alpha amylases contemplated for use in the methods of the invention include; SPEZYMETM AA; SPEZYMETM FRED; GZYMETM G997 (Genencor International Inc.) and TERMAMYLTM 120-L, LC, SC and SUPRA (Novozymes).
  • the quantity of alpha amylase used in the methods of the present invention will depend on nature and activity of the alpha amylase enzyme, the nature and amount of the cereal flour, and the desired reaction. In general, an amount of about 0.01 to 5.0 kg of the alpha amylase is used per metric ton of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e. excluding the weight of the water used in this step). In some embodiments, the alpha amylase is used in an amount about 0.5 to 2.0 kg per metric ton of the first portion of the cereal flour. In some embodiments, the alpha amylase is used in an amount of about 0.1 to 1 .0 kg per metric ton of the first portion of the cereal flour.
  • the alpha amylase enzyme is used in an amount of about 0.01 units of alpha amylase activity (AAU) to 10 AAU per g of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e. excluding the weight of the water used in this step). In some embodiments, the enzyme is used in an amount about 0.1 to 2 AAU per g dry solids of the first portion of the cereal flour. In some embodiments, the enzyme is used in an amount of about 0.4 to 0.5 AAU per g dry solids of the first portion of the cereal flour.
  • the units of alpha amylase enzyme activity can be calculated according to the assay methods set out below.
  • GZYMETM 997 or SPEZYMETM FRED DuPont Industrial Bioscience
  • an amount of between about 0.01 to 1 .0 kg of GZYMETM 997 or SPEZYMETM FRED is added per metric ton of starch.
  • the enzyme is added in an amount between about 0.05 to 1 .0 kg; between about 0.1 to 0.6 kg; between about 0.2 to 0.6 kg and between about 0.4 to 0.6 kg of GZYMETM 997 and SPEZYMETM FRED per metric ton of starch.
  • the alpha amylase is of fungal origin.
  • Alpha amylases (1 ,4-a- D-glucan glucanohydrolase, E.C. 3.2.1 .1) of fungal origin are known in the art.
  • the alpha amylase is derived from a fungus of the genus Aspergillus or Rhizopus.
  • the alpha amylase is derived from a fungus of the species Aspergillus niger, Aspergillus fumigatus, Aspergillus awachi, Aspergillus awamori, Aspergillus clavatus, Aspergillus oryzae or Rhizopus oryzae.
  • Fungal alpha amylase from Aspergillus oryzae has been extensively used in the
  • alpha-amylases examples include CLARASE® from DuPont Industrial Bioscience and
  • the enzyme is a maltogenic enzyme.
  • maltogenic enzymes refers to an enzyme or mixture of enzymes capable of producing maltose, maltotriose and/or maltotetraose by hydrolysis of starch substrate, preferably in an amount of greater than 20% by weight of maltose, maltotriose and maltotetraose (the amount by weight being measured as the total weight of maltose, maltotriose and maltotetraose produced relative to the total weight of all sugars produced), more preferably during their optimum conditions of pH and temperature.
  • the maltogenic enzyme is an amylase (as defined above).
  • the maltogenic enzyme is an alpha amylase (E.C. 3.2.1 .1). In one embodiment, the maltogenic enzyme is a beta amylase (E.C. 3.2.1 .2). In one embodiment, the maltogenic enzyme is a mixture of an alpha amylase and a beta amylase.
  • the maltogenic enzyme may be a high DP2 forming enzyme, such as a fungal alpha amylase or a plant and microbial beta amylase (e.g. derived from barley, soya bean or wheat). In one embodiment, the maltogenic enzyme may be a high DP 3 forming enzyme, examples of which are of bacterial origin (e.g. derived from
  • the maltogenic enzyme may be a high DP4 forming enzyme, examples of which are of bacterial origin and include those derived from Pseudomonas species (OPTIMALTTM 4G; DuPont Industrial Biosciences).
  • the quantity of maltogenic enzyme used in the present methods will depend on nature and activity of the maltogenic enzyme, the nature and amount of the cereal flour, and the desired reaction. In general, an amount of enzyme added to the first portion of the wheat flour resulting in greater than 20% by weight of maltose, maltotriose, and maltotetraose; the amount by weight being measured as the total weight of maltose, maltotriose, and maltotetraose produced relative to the total weight of all sugars produced.
  • the maltogenic enzyme is an alpha amylase
  • the maltogenic enzyme is added to the first portion of the wheat flour in an amount of about resulting in greater than 20% by weight of maltose, maltotriose and maltotetraose.
  • the units of alpha-amylase enzyme activity can be calculated according to the assay methods set out below.
  • the amylase is a glucoamylase (E.C. 3.2.1 .3).
  • Glucoamylases also known as gamma amylases, ⁇ -amylases, 1 ,4-a-glucosidases or 1 ,4-a-D-glucan glucohydrolases, are enzymes that remove successive glucose units from the non-reducing ends of starch.
  • the enzyme can hydrolyse both linear and branched glycosidic linkages of starch, both the amylose and amylopectin portions.
  • the glucoamylase is of plant origin. In some embodiments, the glucoamylase is of bacterial origin. In some embodiments, the glucoamylase is of fungal origin.
  • Preferred glucoamylases used in the present methods are derived from fungal strains.
  • the glucoamylase is derived from a fungus of the genus Aspergillus, Rhizopus, Humicola or Mucor.
  • the glucoamylase is derived from a fungus of the species Aspergillus niger, Aspergillus awamori, Talaromyces, species and its variants, Rhizopus niveus, Rhizopus oryzae, Mucor miehei, Humicola grisea, Aspergillus shirousami, and Humicola (Thermomyces) lanuginosa. Examples of such enzymes are disclosed in, Boel et al. (1984) EMBO J. 3: 1097-1 102; WO 92/00381 ;
  • Enzymes having glucoamylase activity are commercially available. Examples include that produced from Aspergillus niger (OPTIDEXTM L-400 and G-ZYMETM G990 4X from DuPont Industrial Biosciences) or that produced from Rhizopus species (CU.CONCTM from Shin Nihon Chemicals, Japan and GLUCZYMETM from Amano Pharmaceuticals, Japan).
  • the glucoamylase enzyme is a recombinant glucoamylase enzyme.
  • examples include a glucoamylase enzyme obtainable (or obtained) from a Trichoderma host, such as those described in WO 2005/052148.
  • the Trichoderma host expresses a heterologous polynucleotide which encodes a Humicola grisea glucoamylase enzyme, particularly a Humicola grisea var.
  • thermoidea glucoamylase enzyme expresses a recombinant granular starch hydrolysing enzyme (GSHE) wherein the heterologous polynucleotide encodes a GSHE having at least 50% sequence identity with the sequence of the Trichoderma glucoamylase having SEQ ID NO: 3 of WO 2005/052148.
  • GSHE granular starch hydrolysing enzyme
  • This enzyme and its variants are commercially available as DIAZYME® -TGA from DuPont Industrial Biosciences.
  • the quantity of glucoamylase used in the methods will depend on nature and activity of the glucoamylase enzyme, the nature and amount of the cereal flour, and the desired reaction. In general, an amount of about 0.025 to 25.0 kg of the glucoamylase is used per metric ton of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e. excluding the weight of the water used in this step). In some embodiments, the glucoamylase is used in an amount about 0.5 to 15.0 kg per metric ton of the first portion of the cereal flour. In some
  • the glucoamylase is used in an amount of about 5.0 to 12.5 kg per metric ton of the first portion of the cereal flour.
  • the glucoamylase enzyme is used in an amount of about 0.01
  • Glucoamylase Activity Units GAU to 10.0 GAU per g of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e.
  • the weight of the water used in this step excluding the weight of the water used in this step.
  • glucoamylase is used in an amount of about 0.5 to 5.0 GAU per gram dry solids of the first portion of the cereal flour. In some embodiments, the glucoamylase is used in an amount of about 1 .0 to 2.0 GAU per gram dry solids of the first portion of the cereal flour.
  • the units of glucoamylase enzyme activity can be calculated according to the assay methods set out below.
  • the enzyme comprises a 1 ,4-a-glucan 6-a- glucosyltransferase (transglucosidase, EC 2.4.1 .24).
  • a 1 ,4-a-glucan 6-a-glucosyltransferase (systematic name: 1 ,4-a-D-glucan: 1 ,4-a-D-glucan(D-glucose) 6-a-D-glucosyltransferase) catalyzes both hydrolytic and transfer reactions on incubation with a-D- glucooligosaccharides. Transfer of a glucose residue occurs to the primary hydroxyl group (HO-6) of a glucose unit, producing isomaltose from D-glucose and panose from maltose.
  • HO-6 primary hydroxyl group
  • maltooligosaccharides resulting from the partial hydrolysis of starch are converted to isomaltooligosaccharides which contain high proportions of glucosyl residues linked by an a- D-1 ,6 linkages from the non-reducing end.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a 1 ,4-a-glucan 6-a- glucosyltransferase having an action on maltose which produces equimolar concentration of panose and glucose.
  • Any suitable 1 ,4-a-glucan 6-a-glucosyltransferase enzyme finds use in the present methods. Specific examples of such products include those described in Pazur et al., Carbohydr. Res. (1986), 149: 137-147 and in Nakamura et al., J. Biotechnol. (1997) 53: 75-84).
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is of plant origin. In some embodiments, the 1 ,4-a-glucan 6-a-glucosyltransferase is of bacterial origin. In some embodiments, the 1 ,4-a-glucan 6-a-glucosyltransferase is of fungal origin.
  • Preferred 1 ,4-a-glucan 6-a-glucosyltransferases are derived from fungal strains.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is derived from a fungus of the genus Aspergillus, Trichoderma or Neosartorya.
  • the 1 ,4-a- glucan 6-a-glucosyltransferase is derived from a fungus of the species Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus clavatus, Aspergillus fumigatus, Aspergillus nidulans, Neosartorya fischeri or Trichoderma reesei.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase enzyme that finds use in the present method is commercially available.
  • Examples of commercially available 1 ,4-a-glucan 6-a-glucosyltransferase enzymes include, but are not limited to enzymes obtained from Megazyme, Wicklow, Ireland, those from DuPont Industrial Bioscience or those from Amano International, Japan.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase enzyme is an Aspergillus niger 1 ,4-a-glucan 6-a-glucosyltransferase produced in Trichoderma reesei cells.
  • Specific example of such enzymes include TRANSGLUCOSIDASE L-2000 (from DuPont Industrial Bioscience) and Transglucosidase L (SEQ ID NO: 1 ; from Amano International, Japan).
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including, but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : BAA23616.1 ; Aspergillus niger; SEQ ID NO: 8), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase including, but not limited to a fungal 1 ,4-
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : BAD06006.1 (Aspergillus awamori), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : BAA08125.1 ( Aspergillus oryzae), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : XPJD01271891 .1 (Aspergillus clavatus), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase including but not limited to a fungal 1 ,4-a-glu
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : XP_001266999.1 (Neosartorya fischeri), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase including but not limited to a fungal 1 ,4-a-glu
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : XP_75181 1 .1 (Aspergillus fumigatus), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase including but not limited to a fungal 1 ,4-a-glu
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : XP_659621 .1 (Aspergillus nidulans), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase including but not limited to a fungal 1 ,4-a-
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK® database as accession number : XP_001216899.1 (Aspergillus terreus) or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase is a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase (including but not limited to a fungal 1 ,4-a-glucan 6-a- glucosyltransferase having an amino acid sequence deposited in NCBI's GENBANK ® database as accession number : XP_001258585.1 (Neosartorya fischeri), or a variant thereof that has an amino acid sequence that is at least about 70 % identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least 95 % identical, or at least 98% identical to a wild type fungal 1 ,4-a-glucan 6-a-glucosyltransferase.
  • the quantity of 1 ,4-a-glucan 6-a- glucosyltransferase used in the methods of the present invention will depend on the nature and activity of the 1 ,4-a-glucan 6-a-glucosyltransferase enzyme, the nature and amount of the cereal flour, and the desired reaction. In general, an amount of about 0.20 to 5.0 kg of the 1 ,4-a-glucan 6-a-glucosyltransferase is used per metric ton of the first portion of the cereal flour used in step (a) of the process of the invention (calculated on a dry solids basis, i.e. excluding the weight of the water used in this step).
  • the 1 ,4-a- glucan 6-a-glucosyltransferase is used in an amount about 0.5 to 2.0 kg per metric ton of the first portion of the cereal flour. In some embodiments, the 1 ,4-a-glucan 6-a- glucosyltransferase is used in an amount of about 1 .0 to 1 .5kg per metric ton of the first portion of the cereal flour.
  • the 1 ,4-a-glucan 6-a-glucosyltransferase enzyme is used in an amount of about 0.5 to 10 units of 1 ,4-a-glucan 6-a-glucosyltransferase activity (TGU) per gram of the first portion of the cereal flour used in step (a) of the present invention (calculated on a dry solids basis, i.e. excluding the water used in this step).
  • TGU 1 ,4-a-glucan 6-a-glucosyltransferase activity
  • the 1 ,4-a- glucan 6-a-glucosyltransferase is used in an amount about 1.0 to 5.0 TGU per gram dry solids of the first portion of the cereal flour.
  • the 1 ,4-a-glucan 6-a- glucosyltransferase is used in an amount of about 2.0 to 3.0 TGU per dry solids of the first portion of the cereal flour.
  • the units of 1 ,4-a-glucan 6-a-glucosyltransferase enzyme activity can be calculated according to the assay methods set out below. Glucose Isomerases (Xylose isomerases)
  • the enzyme comprises a glucose isomerase (E.C:5.3.1 .5).
  • Glucose isomerases catalyze the isomerization of glucose to fructose and are found in a large number of different microorganisms. Generally glucose isomerases are also described as xylose isomerases.
  • the glucose isomerase (xylose isomerase) is of plant origin. In some embodiments, the glucose isomerase (xylose isomerase) is of bacterial origin. In some embodiments, the glucose isomerase (xylose isomerase) is of fungal origin.
  • Preferred xylose isomerases are derived from bacterial strains.
  • the xylose isomerase is derived from a bacterium of the genus Streptomyces, Actinoplanes, Bacillus, or Flavobacterium.
  • the xylose isomerase is derived from a fungus, such as a species belonging to the class Basidiomycetes.
  • the xylose isomerase is derived from a bacterium of the genus Streptomyces.
  • the glucose isomerase is an immobilized glucose isomerase.
  • the high cost of glucose isomerase and the problems associated with using liquid glucose isomerase for commercial application limited the use of liquid glucose isomerase on a large scale production of high fructose corn syrup. This resulted in the development of immobilized glucose isomerase.
  • Examples of glucose isomerases include GENSWEET®-IGI from DuPont Industrial Bioscience and Sweetzyme®-T from Novozymes. Liquid glucose isomerase product, GENSWEET®-SGI from DuPont Industrial Bioscience is now
  • the quantity of glucose isomerase used in the methods of the present invention will depend on the nature and activity of the glucose isomerase enzyme, the nature and amount of the cereal flour, and the desired reaction. In general, an amount of about 2.0 to 20.0 kg of the glucose isomerase is used per metric ton of the first portion of the cereal flour used in step (a) of the process of the invention
  • the glucose isomerase is used in an amount about 5.0 to 15.0 kg per metric ton of the first portion of the cereal flour. In some embodiments, the glucose isomerase is used in an amount of about 8.0 to 12.5 kg per metric ton of the first portion of the cereal flour.
  • the glucose isomerase enzyme is used in an amount of about 5 to 500 units of glucose isomerase activity (GIU) per g of the first portion of the cereal flour used in step (a) of the present invention (calculated on a dry solids basis, i.e. excluding the water used in this step) .
  • the glucose isomerase is used in an amount about 25 to 100 GIU per g dry solids of the first portion of the cereal flour.
  • the glucose isomerase is used in an amount of 50 to 75 GIU per g dry solids of the first portion of the wheat flour.
  • the units of glucose isomerase enzyme activity can be calculated according to the assay methods set out below.
  • the glucose isomerase enzyme is the liquid glucose isomerase product GENSWEET®-SG I
  • this enzyme is typically used in an amount of about 0.5 to 10.0 kg per metric ton of the first portion of the cereal flour used in step (a) of the process of the present invention (calculated on a dry solids basis, i.e. excluding the water used in this step).
  • 3 and 6 kg of GENSWEET®-SGI is used per ton dry solids of the first portion of the cereal flour.
  • about 2 to 8 kg of GENSWEET®-SG I is used per ton dry solids of the first portion of the cereal flour.
  • about 5.0 to 8.0 kg of GENSWEET®-SGI is used per ton dry solids of the first portion of the cereal flour.
  • composition of the reaction products of oligosaccharides was measured by high pressure liquid chromatographic method (Beckman System Gold 32 Karat Fullerton, California, USA) equipped with a HPLC column (Rezex 8 u8% H, Monosaccharides) , maintained at 50 °C fitted with a refractive index (Rl) detector (ERC-7515A, Rl Detector from The Anspec Company, Inc.).
  • the column separates based on the molecular weight of the saccharides.
  • a designation of DP1 is a monosaccharide, such as glucose or fructose
  • a designation of DP2 is a disaccharide, such as maltose
  • a designation of DP3 is a monosaccharide, such as glucose or fructose
  • DP4 + is an oligosaccharide having a degree of polymerization (DP) of 4 or greater.
  • Sample preparation IMO syrup was diluted to a suitable concentration and filtered through a 0.22 mm membrane before analysis.
  • Data acquisition and integration were performed using Chromeleon 6.8 workstation.
  • GAU Glucoamylase Activity Units
  • the PNPG assay is based on the activity of glucoamylase enzyme to catalyze the hydrolysis of p-nitrophenyl-a-D-glucopyranoside (PNPG) to glucose and p-nitrophenol.
  • PNPG p-nitrophenyl-a-D-glucopyranoside
  • One Glucoamylase Unit is the amount of enzyme that will liberate one gram of reducing sugars calculated as glucose from a soluble starch substrate per hour under the specified conditions of the assay.
  • AAU Alpha Amylase activity Units
  • AAU was determined by the rate of starch hydrolysis, as reflected in the rate of decrease of iodine-staining capacity measured spectrophotometrically.
  • One AAU of bacterial alpha-amylase activity is the amount of enzyme required to hydrolyze 10 mg of starch per min under standardized conditions (pH 6.0 and 60°C).
  • Alpha-amylase activity can also be determined as soluble starch unit (SSU) and is based on the degree of hydrolysis of soluble potato starch substrate (4% DS) by an aliquot of the enzyme sample at pH 4.5 and 50°C. The reducing sugar content is measured using the DNS method as described in Miller, G. L. Anal. Chem. (1959) 31 :426 - 428.
  • TGU 6-a-glucosyltransferase Activity Units
  • TGU transglucosidase activity unit
  • the activity of soluble glucose isomerase is determined from the rate of conversion of glucose to fructose under controlled conditions for thirty minutes (2.6 M glucose in 200 mM maleate, 20 mM MgS0 4 .7H 2 0, 1 mM CoCI 2 .6H 2 0, pH 6.85). Glucose isomerase activity is then calculated by measuring the amount of fructose converted from glucose via HPLC.
  • One glucose isomerase activity Unit (GIU) is defined as that activity which will produce one micromole of fructose per minute under the conditions of this assay.
  • the intermediate product is a composition in which at least part, in some embodiments all, of the starch present in said first portion of cereal flour has been converted into a fermentable sugar. This is described in the examples below as a "baking adjunct".
  • the fermentable sugar produced in situ is selected from glucose, fructose and maltose.
  • the intermediate product is a composition in which at least part or all of the starch present in said first portion of cereal flour has been converted into an isomaltooligosacchande. This is also described in the examples below as a "baking adjunct".
  • the isomaltooligosacchande produced in situ has a degree of
  • said isomaltooligosacchande has a degree of polymerisation of less than 4. More preferably, said isomaltooligosacchande has a degree of polymerisation of less than 3. Most preferably, said isomaltooligosacchande is selected from the group consisting of isomaltose, panose, and mixtures thereof.
  • the present invention further relates to the use of the composition obtained or obtainable according to the methods of the present invention.
  • the composition is useful as a food ingredient, particularly though not exclusively for incorporation into flour compositions for preparing dough and baked products, such as bread, prepared from dough.
  • the composition may be used as - or in the preparation of - a food product.
  • the term "food product” is used in a broad sense - and covers food for humans as well as food for animals (i.e. a feed).
  • the food is for human consumption.
  • the food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • the composition according to the invention is added directly in the production of the food product. In other alternative embodiments, the composition according to the invention may be used without further separation or harvesting directly in production of food products.
  • the composition may also be used as a food ingredient.
  • food ingredient includes a formulation which is or can be added to functional foods or foodstuffs, for example, as a nutritional supplement and/or fibre supplement.
  • the term food ingredient as used here also refers to formulations which can be used at low levels in a wide variety of products that require gelling, texturising, stabilising, suspending, film-forming and structuring, retention of juiciness and improved mouthfeel, without adding viscosity.
  • the food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • the composition may be - or may be added to - food
  • the composition may be - or may be added to - functional foods.
  • functional food means food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a further beneficial effect to consumer. Accordingly, functional foods are ordinary foods that have components or ingredients (such as those described herein) incorporated into them that impart to the food a specific functional - e.g. medical or physiological benefit - other than a purely nutritional effect. Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that they are foods marketed as having specific health effects.
  • nutraceutical means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a therapeutic (or other beneficial) effect to the consumer.
  • the invention further provides a food product containing the present composition(s).
  • the composition comprises a dough composition.
  • dough is defined herein as a mixture of flour and other ingredients firm enough to knead or roll. This may be fresh, frozen, pre-baked, or prebaked.
  • the dough composition is baked to form a baked product.
  • baked products is defined herein as any product prepared from dough, either of a soft or a crisp character.
  • Examples of the baked products, whether of a white, light or dark type, which may be advantageously produced by the present invention are bread (in particular white, wholemeal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pasta, pita bread, tortillas, tacos, cakes, pancakes, biscuits, cookies, pie crusts, steamed bread, and crisp bread, and the like.
  • Suitable baking conditions are well known to the person skilled in the art.
  • a baked product is also provided (obtained) obtainable by baking the dough composition of the invention.
  • said baked product comprises bread.
  • ds or DS dry solids content
  • MT metric ton
  • a baking adjunct containing very high soluble sugars was prepared by incubating 200 grams of wheat flour slurred with 1 ,200 g water followed by dosing with an alpha-amylase (SEQ ID NO: 2; SPEZYME® RSL from DuPont) at 0.5 AAU/g ds at pH 5.6 and 60°C for 6 hours. Samples were withdrawn at 6 hours for HPLC.
  • SEQ ID NO: 2 an alpha-amylase
  • Table 1 A - Total soluble sugars (mg/g wheat flour)
  • Baking adjunct containing very high glucose was prepared by incubating 200 grams of wheat flour slurred with 1 ,200 g water followed by dosed with alpha amylase (SEQ ID NO: 2; SPEZYME ® RSL) at 0.5 AAU/g ds and glucoamylase (SEQ ID NO: 4; "TrGA CS4" variant from wild type glucoamylase SEQ ID NO: 3 from Trichoderma reesei; "GC321 ”) at 2 GAU/ g ds at pH 5.6 and 60°C for 6 hours. Samples were withdrawn at 6 hours for HPLC.
  • Table 2A shows that 487.8 mg of glucose were produced per 1 gram of wheat flour, indicating that most of starch in wheat flour was solubilized and converted to glucose by alpha-amylase and glucoamylase.
  • Total soluble sugars concentration (mg/g wheat flour, Table 2A) and the percentage of the solution sugars (Table 2B) were shown below.
  • a baking adjunct containing very high isomaltooligosaccharide was prepared by incubating 200 grams of wheat flour slurred with 1 ,200 g water followed by dosing with alpha amylase (SEQ ID NO:2; SPEZYME ® RSL) at 2 or 0.5 AAU/g ds and 1 ,4-a-glucan 6-a- glucosyltransferase (SEQ ID NO:5; TRANSGLUCOSIDASE L-2000 ("TG L-2000”) from DuPont Industrial Bioscience) at 2.5 TGU/g ds at pH 5.6 and 60°C for 6 hours. Samples were withdrawn at 6 hours for HPLC.
  • alpha amylase SEQ ID NO:2; SPEZYME ® RSL
  • SEQ ID NO:5 TRANSGLUCOSIDASE L-2000
  • TG L-2000 TRANSGLUCOSIDASE L-2000
  • Tables 3A and 3B show that significantly elevated trisaccharide (88.8 mg) was produced while 553.5 mg of total soluble sugars produced per 1 gram of wheat flour, indicating that most of starch in wheat flour was solubilized and converted to IMO by alpha- amylase and 1 ,4-a-glucan 6-a-glucosyltransferase.
  • baking adjuncts from Examples 1 , 2 and 3 were used to make dough by adding remaining 800 grams of wheat flour to each and mixed with other baking ingredients listed in Table 5. Additional sugar was added to Example 1 prior to making dough for baking.
  • White pan breads were prepared using a no-time dough system; as follows:
  • Sample 1 is a control made using 8% sucrose.
  • PANODAN ® 205 (Dupont) is a diacetyl tartaric acid ester of mono- and diglycerides (DATEM) made from edible, refined vegetable fat;
  • HP 75 is a distilled monoglyceride made from edible, fully hydrogenated palm based oil; and POWERBAKE ® 4205 is an enzyme blend having xylanase, oxidase and lipase activities and is produced by fermentation with selected microbial strains.
  • the wheat slurry was made as described in Example 1 (200 gram of wheat flour + 1 ,200 grams of water) .
  • the pH of the slurry was adjusted to pH 7.0 using sodium bicarbonate, then alpha-amylase (SEQ ID NO: 6; SPEZYME® Xtra (0.5 AAU/g wheat flour)), Trichoderma glucoamylase, (SEQ ID NO: 3; GC321 ®)(2.00 GAU / g wheat flour) and glucose isomerase (SEQ ID NO: 7; GENSWEET®-SGI glucose isomerase (DuPont Industrial Bioscience)) (2.5 mL) were added, then the slurry was incubated at 60°C with constant mixing.
  • the baked bread using baking adjunct prepared with GEN S WE ET®- SG I produced bread with slight to moderate sweetness, slightly more elastic, medium brown in colour, slightly more open and a decrease in loaf volume (6.06 cm 3 /g vs 6.38 cm 3 /g) compared to conventional bread with added sugar.
  • SUMIZYME ® PF-G (Shin Nihon Chemicals, Japan) containing exopeptidase activity during in situ production of fermentable sugars (Example 7) resulted in the hydrolysis of wheat proteins and produced soluble fraction containing a very high level of free amino acids, i.e., arginine, valine, leucine, and tryptophan.
  • the baking trials were then conducted in white pan bread to compare the bread with sugar added and bread without sugar added with respect to mixing time, dough, handling, proof time, crust colour, crumb structure, and flavour.
  • the fermentable syrup from Example 7 was then mixed with remaining 800 grams of wheat flour in addition to all ingredients listed in Table 5 and were then taken in a spiral mixer. It was then mixed to development for a final dough temperature of 26.5°C (80°F). Dough was scaled to 0.680 kg (24 oz) pieces. Dough was rounded, sheeted and moulded, and then placed into a proof box set at 40.5 to 43.3°C (105-1 10°F) and 85-90% relative humidity.
  • Loaves were proofed to 0.635 to 1 .27 cm (1 ⁇ 4 - 1 ⁇ 2 inches) above the pan lip (approximately 1 hour). Loaves were baked at 204.4°C (400°F) for 23 minutes to an internal temperature of 93.3-97.8°C (200-208°F). The results are summarized in Tables 10A and 10B.
  • a baking adjunct containing very high glucose was prepared by mixing 200 grams of wheat flour with 1 ,200 g water and incubated with only glucoamylase (Tr-GA GC321 at 5 GAU/g ds) at pH 5.6 and 60°C for 6 hours. Samples were withdrawn at 6 hours for HPLC.
  • a baking adjunct containing IMO was prepared by mixing 200 grams of wheat flour with 1 ,200 grams water and incubated with alpha amylase (0.3 AAU SPEZYME ® Xtra)/g ds and 5 TGU units of TRANSGLUCOSIDASE L-2000 /g ds of wheat flour at pH 5.2 and 60°C for 6 hours. Samples were withdrawn at 6 hours for HPLC.
  • alpha amylase 0.3 AAU SPEZYME ® Xtra
  • Sample 2 0.5% (w/w) DIMODAN ® distilled monoglyceride
  • Table 12A Effect of in situ glucose and IMO production under baking conditions without added sugar on the loaf volume of the bread after storage for 1 day at room temperature
  • the DIMODAN ® HS 150 used in Sample 2 is a softener commercially available from DuPont Nutrition Biosciences ApS comprising distilled monoglycerides made from edible, refined hydrogenated vegetable oil.
  • Sample 3 (using glucoamylase GC321) has a similar volume to the control, the comparative Sample 2 (DIMODAN ® HS 150) has a slightly lower volume, and Sample 4, where IMO was produced in situ using alpha amylase (SPEZYME ® Xtra) + 1 ,4-a-glucan 6-a- glucosyltransferase (TRANSGLUCOSIDASE L-2000) has a slightly higher volume.
  • SPEZYME ® Xtra alpha amylase + 1 ,4-a-glucan 6-a- glucosyltransferase

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Abstract

L'invention porte sur un procédé permettant d'améliorer les caractéristiques d'une pâte sans sucre ajouté. Le procédé consiste à traiter de la farine de céréales avec une enzyme pour produire une composition contenant des sucres fermentescibles (glucose, fructose ou maltose) et/ou des isomaltooligosaccharides. La composition peut être utilisée pour former un mélange de pâte : la mise en forme du mélange de pâte et la cuisson dudit mélange de pâte façonnée pour former des produits cuits au four.
PCT/US2017/033942 2016-05-23 2017-05-23 Procédé de cuisson et son procédé Ceased WO2017205337A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
CN108717093A (zh) * 2018-04-26 2018-10-30 胡贝贞 茶叶中蔗糖的离子色谱-串联质谱联用检测方法
EP3869978A4 (fr) * 2018-10-22 2022-11-23 DuPont Nutrition Biosciences ApS Enzymes pour brassage relevant du domaine technique du brassage impliquant des adjuvants
WO2023097202A1 (fr) * 2021-11-24 2023-06-01 Dupont Nutrition Biosciences Aps Production de bières fortement atténués
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
US12256743B2 (en) 2014-07-08 2025-03-25 Caravan Ingredients Inc. Sugar-producing and texture-improving bakery methods and products formed therefrom

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12256743B2 (en) 2014-07-08 2025-03-25 Caravan Ingredients Inc. Sugar-producing and texture-improving bakery methods and products formed therefrom
CN108717093A (zh) * 2018-04-26 2018-10-30 胡贝贞 茶叶中蔗糖的离子色谱-串联质谱联用检测方法
EP3869978A4 (fr) * 2018-10-22 2022-11-23 DuPont Nutrition Biosciences ApS Enzymes pour brassage relevant du domaine technique du brassage impliquant des adjuvants
WO2023097202A1 (fr) * 2021-11-24 2023-06-01 Dupont Nutrition Biosciences Aps Production de bières fortement atténués
JP2024543131A (ja) * 2021-11-24 2024-11-19 インターナショナル エヌ アンド エイチ デンマーク エーピーエス 高発酵度ビールの製造
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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