WO2000027215A1

WO2000027215A1 - Methods for using a glucose isomerase in baking

Info

Publication number: WO2000027215A1
Application number: PCT/DK1999/000583
Authority: WO
Inventors: Ramiro Gutierrez Martinez; Tina Spendler; María Jesús GONZALES CALLEJO; Guillermo Badiola Rodriquez; Manuel Juan Gonzales Gil; Dionisio Casero Ramirez
Original assignee: Novozymes A/S
Priority date: 1998-11-05
Filing date: 1999-10-27
Publication date: 2000-05-18
Also published as: AU6326699A; WO2000027215A8

Abstract

The present invention relates to methods for preparing a dough, comprising incorporating into the dough a composition comprising an effective amount of one or more glucose isomerase(s) which improve one or more properties of the dough or a baked product obtained from the dough. The present invention also relates to methods for preparing a baked product. The present invention also relates to compositions comprising an effective amount of one or more glucose isomerase(s) for improving one or more properties of a dough and/or a baked product obtained from the dough. The present invention further relates to doughs or baked products and to pre-mixes for a dough.

Description

METHODS FOR USING A GLUCOSE ISOMERASE IN BAKING

Background of the Invention

Field of the Invention

The present invention relates to methods for preparing a dough and/or baked product with a glucose isomerase .

Description of the Related Art

The strength of a dough is an important aspect of baking for both small-scale and large-scale applications. A strong dough has a greater tolerance of mixing time, proofing time, and mechanical vibrations during dough transport, whereas a weak dough is less tolerant to these treatments. A strong dough with superior rheological and handling properties results from flour containing a strong gluten network. Flour with a low protein content or a poor gluten quality results in a weak dough. Dough "conditioners" are well known in the baking industry. The addition of conditioners to bread dough has resulted in improved machinability of the dough and improved texture, volume, flavour, and freshness (anti-staling) of the bread. Nonspecific oxidants, such as iodates, peroxides, ascorbic acid, potassium bromate and azodicarbonamide have a gluten strengthening effect. It has been suggested that these conditioners induce the formation of interprotein bonds which strengthen the gluten, and thereby the dough. However, the use of several of the currently available chemical oxidizing agents has been met with consumer resistance or is not permitted by regulatory agencies.

The use of enzymes as dough conditioners has been considered as an alternative to the chemical conditioners. A number of enzymes have been used recently as dough and/or bread improving agents, in particular, enzymes that act on components present in large amounts in the dough. Examples of such enzymes are found within the groups of amylases, proteases, glucose oxidases, and (hemi) cellulases, including pentosanases . Summary of the Invention

The problem to be solved by the present invention is to improve the properties of dough and/or baked products.

The solution is based on that the present inventors have identified that by adding a glucose isomerase enzyme to a dough the resulting bread obtained improved properties. See e . g. working example 1 herein ( vide infra) .

Accordingly, the present invention relates to methods for preparing a dough, comprising incorporating into the dough an effective amount of one or more glucose isomerase (s) .

The present invention also relates to methods for preparing a baked product .

The present invention also relates to compositions comprising an effective amount of one or more glucose isomerase (s) , for improving one or more properties of a dough and/or a baked product obtained from the dough, and a carrier and/or a baking ingredient .

The present invention also relates to doughs or baked products . The present invention further relates to pre-mixes for a dough comprising an effective amount of one or more glucose isomerase (s) , for improving one or more properties of a dough and/or a baked product obtained from the dough, and a carrier and/or a baking ingredient .

Detailed Description of the Invention

The present invention relates to methods for preparing a dough or a baked product comprising incorporating into the dough an effective amount of one or more glucose isomerase (s) which improve one or more properties of the dough or the baked product obtained from the dough relative to a dough or a baked product in which a glucose isomerase is not incorporated.

In the methods of the present invention, one or more glucose isomerase (s) are incorporated into the dough by adding the glucose isomerase (s) to the dough, to any single ingredient from which the dough is to be made, and/or to any mixture of dough ingredients from which the dough is to be made. In other words, the glucose isomerase (s) may be added in any step of the dough preparation and may be added in one, two, or more steps.

The term "effective amount" is defined herein as an amount of a glucose isomerase that is sufficient for providing a measurable effect on at least one property of interest of the dough and/or baked product .

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, which is improved by the action of a glucose isomerase relative to a dough or product in which a glucose isomerase is not incorporated. The improved property may include, but is not limited to, increased strength of the dough, increased elasticity of the dough, increased stability of the dough, reduced stickiness of the dough, improved extensibility of the dough, improved machinability of the dough, increased volume of the baked product, improved crumb structure of the baked product, improved softness of the baked product, improved flavour of the baked product, and/or improved antistaling of the baked product.

Preferably, one or more improved properties are selected from the group consisting of increased volume of the baked product, improved softness of the baked product, and improved antistaling of the baked product. See working example 1 herein

(vide infra) for an example of such improved properties obtained by using a glucose isomerase according to the invention.

The use of a glucose isomerase may result in an increased strength, stability, and/or reduced stickiness of the dough, resulting in improved machinability, as well as in an increased volume and improved crumb structure and softness of the baked product. The effect on the dough may be particularly advantageous when a poor quality flour is used. Improved machinability is of particular importance in connection with dough that is to be processed industrially.

The improved property may be determined by comparison of a dough and/or a baked product prepared with and without addition of a glucose isomerase in accordance with the methods of the present invention. Techniques which can be used to determine improvements achieved by use of the methods of present invention are described below in the Examples. Organoleptic qualities may be evaluated using procedures well established in the baking industry, and may include, for example, the use of a panel of trained taste-testers. The term "increased strength of the dough" is defined herein as the property of a dough that has generally more elastic properties and/or requires more work input to mould and shape .

The term "increased elasticity of the dough" is defined herein as the property of a dough which has a higher tendency to regain its original shape after being subjected to a certain physical strain.

The term "increased stability of the dough" is defined herein as the property of a dough that is less susceptible to mechanical abuse thus better maintaining its shape and volume.

The term "reduced stickiness of the dough" is defined herein as the property of a dough that has less tendency to adhere to surfaces, e . g. , in the dough production machinery, and is either evaluated empirically by the skilled test baker or measured by the use of a texture analyzer ( e . g. , TAXT2) as known in the art .

The term "improved extensibility of the dough" is defined herein as the property of a dough that can be subjected to increased strain or stretching without rupture. The term "improved machinability of the dough" is defined herein as the property of a dough that is generally less sticky and/or more firm and/or more elastic.

The term "increased volume of the baked product" is measured as the specific volume of a given loaf of bread (volume/weight) determined typically by the traditional rape seed displacement method.

The term "improved crumb structure of the baked product" is defined herein as the property of a baked product with finer and/or thinner cell walls in the crumb and/or more uniform/homogenous distribution of cells in the crumb and is usually evaluated empirically by the skilled test baker.

The term "improved softness of the baked product" is the opposite of "firmness" and is defined herein as the property of a baked product that is more easily compressed and is evaluated either empirically by the skilled test baker or measured by the use of a texture analyzer ( e . g. , TAXT2) as known in the art.

The term "improved flavour of the baked product" is evaluated as mentioned above by a trained test panel.

The term "improved antistaling of the baked product" is defined herein as the properties of a baked product that have a reduced rate of deterioration of quality parameters, e . g. , softness and/or elasticity, during storage. In a preferred embodiment, the one or more glucose isomerase (s) improve one or more properties of the dough or the baked product obtained from the dough. In another preferred embodiment, the one or more glucose isomerase (s) improve one or more properties of the dough and the baked product obtained from the dough.

In a preferred embodiment, the improved property is increased strength of the dough. In another preferred embodiment, the improved property is increased elasticity of the dough. In another preferred embodiment, the improved property is increased stability of the dough. In another preferred embodiment, the improved property is reduced stickiness of the dough. In another preferred embodiment, the improved property is improved extensibility of the dough. In another preferred embodiment, the improved property is improved machinability of the dough. In another preferred embodiment, the improved property is increased volume of the baked product.

In another preferred embodiment, the improved property is improved crumb structure of the baked product . In another preferred embodiment, the improved property is improved softness of the baked product. In another preferred embodiment, the improved property is improved flavour of the baked product. In another preferred embodiment, the improved property is improved antistaling of the baked product.

The term "dough" is defined herein as a mixture of flour and other ingredients firm enough to knead or roll . The dough may be fresh, frozen, pre-bared, or pre-baked. The preparation of frozen dough is described by Kulp and Lorenz in Frozen and Refrigerated Doughs and Batters . The term "baked product" is defined herein as any product prepared from a dough, either of a soft or a crisp character. Examples of baked products, whether of a white, light or dark type, which may be advantageously produced by the present invention are bread (in particular white, whole-meal or rye bread) , typically in the form of loaves or rolls, French baguette-type bread, pasta, pi^'ta bread, tortillas, tacos, cakes, pancakes, biscuits, cookies, pie crusts, steamed bread, and crisp bread, and the like. The glucose isomerase (s) may be any glucose isomerase which provides an improved property to a dough and/or to a baked product obtained from the dough.

Generally glucose isomerases are described as xylose isomerases. In regard to enzyme nomenclature they are classified as EC 5.3.1.5 and the term glucose isomerase is not found in the official enzyme nomenclature.

Accordingly, for the sake of clarity the term "glucose isomerase" is herein defined as an enzyme capable of performing isomerisation of a glucose to a fructose in the assay described immediately below.

Glucose isomerase activity assay:

The procedure of the assay is based on the polarimetric measurement of the rate of conversion of glucose to fructose, in a packed bed column, under below mentioned standard conditions.

One IGIU - Immobilized Glucose Isomerase Unit is defined as the amount of enzyme which converts glucose to fructose at an initial rate of 1 micromole per minute under the standard conditions specified below.

Standard conditions:

Substrate: glucose 45% w/w pH (inlet) : 7.5

Temperature: 60°C Mg2+: 99 mg/1 (1.0 g/1 of MgSo4 • 7 H20)

Ca2+: < 2 ppm

Activator, S02 : 100 ppm (0.18 g/1 of Na2S205)

Buffer, Na2C03: 2 mM Na2C03 Apparatus :

Thermostated water bath 2.5 x 20 cm glass columns Variable peristaltic pump Refractometer Polarimeter

Reagents : 539 g glucose (anhydrous), 1.0 g MgS0₄*7H₂0, 0.21 g Na₂C0₃, and 0.18 g Na₂S₂0₅ are dissolved in 700 ml of demineralized water by stirring and heating (max. 70°C) . After cooling to 25°C pH is adjusted to 7.50 by addition of 0.5 M H₂S0₄. Demineralized water is added to a volume of 1000 ml, or a weight of 1199 g. The substrate is deaerated. The exact glucose concentration is determined refractometrically.

1.0 g MgS0₄«7H₂0 is dissolved in 700 ml of demineralized water. Adjust the pH to 7.5 using IN NaOH, and dilute to 1000 ml with demineralized water.

Procedure :

Glass columns with perforated steel bottoms are immersed in a thermostated water bath at 60°C. A variable speed peristaltic pump supplying glucose susbstrate is connected to the top of the columns. The outlet goes to the collection system or into the drain.

Approximately 9.0 g of enzyme are weighed accurately into a 100 ml beaker. Glucose substrate is added ad 45 ml and stirred gently with a spoon every five minutes for 15 minutes, and thereafter occasionally for 45 minutes.

With 0.1% MgS0₄ solution the sample preparation is transferred quantitatively to the column. The enzyme granules are allowed to settle. Two cotton-plugs (0.7 - 0.75 g) soaked in 0.1% MgS0₄ solution are pushed into the column leaving spaces of 1-2 cm between top, plugs, and enzyme. This ensures proper heating and trapping of any dissolved gasses. The pump is adjusted to a flow giving a conversion of 0.40 < X < 0.45 during the analysis. The tube from the pump is connected to the top of the column, thereby avoiding air admission to the enzyme bed. The column is placed in the water bath (room temperature) , and the downward flow, giving the conversion mentioned above, is established. The water bath thermostat is then switched on (60°C) .

The first sample is collected 20-24 hours after start-up in order to check the conversion. If the conversion is less than 0.40 or more than 0.45, the flow rate is adjusted to bring the conversion into this range. The pH of the glucose is readjusted if necessary.

Two samples are collected 42-48 hours after start-up. These samples are used to determine the enzyme activity. If a flow rate adjustment is required, the column is allowed to re- equilibrate for at least two hours before determining the conversion. The flow rate is measured, and an effluent sample is collected. Approximately 0.5 meqv. 4N NaOH per 100 g of syrup is added in order to accelerate the attainment of mutarotation equilibrium. Samples are covered. The optical rotations of the glucose substrate and the effluent samples are determined by polarimetric measurement after minimum 10 minutes in the test tubes at 25°C.

Calculation:

The enzyme activity is expressed in IGIU/g, calculated by the formula:

Eq. 1 IGIU l g = 0.962— X_e - DS -In _ ^e herein

0.962 = unit conversion factor (g/h -> μmol/min)

F = syrup flow (g/h) W enzyme weight (g)

X_e = equilibrium conversion (0.507 at 60°C)

DS = substrate glucose calculated according to eq.5

(%w/w)

X = outlet syrup conversion calculated according to q. 6

A polarimeter is used for the determination of X

Wherein ia] _G specific rotation of glucose (53.5 at 20°C)

[ a] _F specific rotation of fructose (-95.9 at 20°C)

[ά] _s sample rotation

1 cuvette length (dm)

P substrate density (20°C)

Eq. 3 p = 0.00516 - DS + 0.9663

Applying the conversion equation (eq. 2) to the glucose substrate (X=0) gives:

100

Eq . 4 [a] sub = DS -

[c \_G Λ

[a] sub = glucose substrate rotation

Substituting for p (eq. 3) and solving the second order equation gives :

Eq . 5 [ ] .sub

DS = -93.63 + , 876727 + 362.24

100 Substituting [a]_suh (eq. 4) for DS ^■ p (eq. 2) gives

Preferably, a glucose isomerase used as described herein has an activity of at least 0.25 IGIU/g enzyme (dry matter); more preferably an activity of at least 1 IGIU/g enzyme (dry matter) , more preferably an activity of at least 10 IGIU/g enzyme (dry matter) , even more preferably an activity of at least 50 IGIU/g enzyme (dry matter) , and most preferably an activity of at least 100 IGIU/g enzyme (dry matter) .

Examples of glucose isomerases useful in the methods of the present inventions are defined by the Nomenclature Committee of the International Union of Biochemistry on the Nomenclature and Classification of enzymes and listed as enzyme subclass E.C. 5.3.1.5.

Suitable glucose isomerases from said enzyme subclass E.C. 5.3.1.5 include following enzymes which at the priority date of the present invention had following SWISS-PROT database AccNumber (AC) and ID number (i.e. P12851 (AC) , XYLA_ACTMI ( ID) ) :

PP1122885511, XXYYLLAA_AACCTTMMII ; P10654, XYLA_AMPSP; P12070 XYLA_ARTS7 P54272 XYLA_BACSP P54273, XYLA_BACST; P04788 XYLA_BACSU P29441 XYLA_CLOTS P00944, XYLA_ECOLI P44398 XYLA_HAEIN P29442 XYLA_KLEAE P29443, XYLA_LACBR; P21938 XYLA_LACPE P27157 XYLA_STAXY P24299, XYLA_STRAL; P50910, XYLA_STRDI P37031 XYLA_STRMR P15587, XYLA_STROLi P22857, XYLA_STRRO P24300 XYLA_STRRU PI9149, XYLA_STRSQ; P14405, XYLA_STRVN P09033 XYLA_STRVO P22842, XYLA_THEETj P45687, XYLA_THENE P30435 XYLA THESA P26997, XYLA_THETH; P19148, XYLA THETU The SWISS-PROT database is a widely known public database, which is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute.

A preferred glucose isomerase are a glucose isomerase obtained from the bacterial genera Streptoinyces, such as any of the ones described in US 4687742 (see column 2) .

More preferably, a glucose isomerase is a glucose isomerase obtained from a Streptomyces urinus . Suitable examples of such a glucose isomerase, obtained from a

Streptomyces murinus, are described in US 4687742 and a suitable commercial available glucose isomerase, obtained from a Streptomyces murinus, is Sweetzyme® T, Novo Nordisk A/S, Denmark .

In the methods of the present invention, any glucose isomerase may be used which possesses suitable enzyme activity in a pH and temperature range appropriate for making a dough and/or a baked product. It is preferable that the glucose isomerase (s) is active over broad pH and temperature ranges.

In a preferred embodiment, the glucose isomerase (s) has a pH optimum in the range of about 3 to about 10. In a more preferred embodiment, the glucose isomerase (s) has a pH optimum in the range of about 4.5 to about 8.5.

In another preferred embodiment, the glucose isomerase (s) has a temperature optimum in the range of about 5°C to about

100°C. In a more preferred embodiment, the glucose isomerase (s) has a temperature optimum in the range of about

25°C to about 75°C.

In the methods of the present invention, combinations of glucose isomerases may be used to improve one or more properties of the dough and/or baked product obtained from the dough.

The source of a glucose isomerase is not critical for improving one or more properties of a dough and/or a baked product. Accordingly, the glucose isomerase (s) may be obtained from any source such as a plant, microorganism, or animal. The glucose isomerase (s) is preferably obtained, e . g. , from a microbial source, such as a bacterium or a fungus, e . g. , a filamentous fungus or a yeast. The glucose isomerase (s) may be obtained from the organism in question by any suitable technique, and in particular by use of recombinant DNA techniques known in the art (c.f. Sambrook, J. et al . , 1989, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor, NY, USA) . The use of recombinant DNA techniques generally comprises cultivation of a host cell transformed with a recombinant DNA vector, consisting of the product gene of interest inserted between an appropriate promoter and terminator, in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may be of genomic, cDNA or synthetic origin or any mixture of these, and may be isolated or synthesized in accordance with methods known in the art. The enzyme may also be obtained from its naturally occurring source, such as a plant or organism, or relevant part thereof. Furthermore, the glucose isomerase (s) may be obtained from commercial suppliers.

When a glucose isomerase is added to dough intended for use in the preparation of baked products it may, due to its glucose to fructose isomerisation activity, cause an increase in the amount of fructose within the dough and thereby giving rise to a bread with improved properties as described above.

The glucose isomerase (s) is used in an amount sufficient to provide the desired effect, i.e., the improved properties in question. Thus, the dosage of the glucose isomerase (s) to be used in the methods of the present invention should be adapted to the nature and composition of the dough in question as well as to the nature of the glucose isomerase (s) to be used. The term "composition" is defined herein as a dough- improving and/or baked product-improving composition which, in addition to one or more glucose isomerase (s) , comprise one or more additional substances conventionally used in baking. The additional substance (s) may be other enzymes or chemical additives known in the art to be useful in dough preparation and/or baking.

The bread-improving and/or dough improving composition of the invention is generally included in the dough in an amount corresponding to 0.01-5%, in particular 0.1-3%. The glucose isomerase (s) is typically added in an amount corresponding to 0.01-100 mg enzyme protein per kg of flour, preferably 0.1-25 mg enzyme protein per kg of flour, more preferably 0.1-10 mg enzyme protein per kg of flour, and most preferably 0.5-5 mg enzyme protein per kg of flour. In terms of enzyme activity, the appropriate dosage of a given glucose isomerase for exerting a desirable improvement of dough and/or baked products will depend on the enzyme and the enzyme substrate in question. The skilled person may determine a suitable enzyme unit dosage on the basis of methods known in the art.

Preferably, the glucose isomerase (s) is typically added in an amount corresponding to 3-5000 IGIU/kg flour; more preferably the glucose isomerase (s) is typically added in an amount corresponding to 10-1000 IGIU/kg flour; and even more preferably the glucose isomerase (s) is typically added in an amount corresponding to 10-400 IGIU/kg flour.

The glucose isomerase (s) and/or additional enzymes to be used in the methods of the present invention may be in any form suitable for the use in question, e . g. , in the form of a dry powder, agglomerated powder, or granulate, in particular a non- dusting granulate, a liquid, in particular a stabilized liquid, or a protected enzyme . Granulates and agglomerated powders may be prepared by conventional methods, e . g. , by spraying the glucose isomerase (s) onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e . g. , a salt (such as NaCl or sodium sulfate) , a sugar (such as sucrose or lactose) , a sugar alcohol (such as sorbitol) , starch, rice, corn grits, or soy. The glucose isomerase (s) and/or additional enzymes may be contained in slow-release formulations. Methods for preparing slow-release formulations are well known in the art. Liquid enzyme preparations may, for instance, be stabilized by adding nutritionally acceptable stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established methods.

For inclusion in pre-mixes or flour it is advantageous that the glucose isomerase (s) is in the form of a dry product, e . g. , a non-dusting granulate, whereas for inclusion together with a liquid it is advantageously in a liquid form.

A substrate of the glucose isomerase in question may also be incorporated into the dough. The substrate may be incorporated into dough separately or together with the glucose isomerase of interest, optionally as constituent (s) of the bread-improving and/or dough-improving composition.

Alternatively, an enzyme which acts on a substance endogenous to the flour to produce a substrate for the of interest may also be incorporated in the dough. Furthermore, the substance and the enzyme which acts on the substance to produce a substrate for the glucose isomerase of interest may also be incorporated in the dough.

The specific amount of the substrate available for the glucose isomerase of interest will depend on a number of factors, such as the baking process used, the length of time for mixing, fermentation, proofing and/or baking, the quality of the yeast and/or flour used, as well as the activity of endogenous and exogenous enzymes present .

One or more additional enzymes may also be incorporated into the dough. The additional enzyme may be of any origin, including mammalian and plant, and preferably of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art.

In a preferred embodiment, the additional enzyme may be an amylase, such as an alpha-amylase (useful for providing sugars fermentable by yeast and retarding staling) , a beta-amylase, an amyloglucosidase, a cyclodextrin glucanotransferase, a peptidase, in particular, an exopeptidase (useful in flavour enhancement) , a transglutaminase, a lipase (useful for the modification of lipids present in the dough or dough constituents so as to soften the dough) , a phospholipase (useful for the modification of lipids present in the dough or dough constituents so as to soften the dough and improve gas retention in the dough) , a cellulase, a hemicellulase, in particular a pentosanase such as xylanase (useful for the partial hydrolysis of pentosans which increases the extensibility of the dough) , a protease (useful for gluten weakening in particular when using hard wheat flour), a protein disulfide isomerase, e . g. , a protein disulfide isomerase as disclosed in WO 95/00636, a glycosyltransferase, a peroxidase (useful for improving the dough consistency), a laccase, or an oxidase, e . g. , an aldose oxidase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, or an L-amino acid oxidase (useful in improving dough consistency) .

Most preferably, the additional enzyme may be an amyloglucosidase or a glucose oxidase. The xylanase is preferably of microbial origin, e . g. , derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of Aspergillus aculeatus, Aspergillus niger (cf. WO 91/19782), Aspergillus awamori (WO 91/18977), or Aspergillus tubigensis (WO 92/01793) , from a strain of Trichoder a, e . g. , Trichoderma reesei , or from a strain of Humicola, e. g. , Humicola insolens (WO 92/17573, the contents of which is hereby incorporated by reference) .

Commercially available amylases useful in the present invention are NOVAMYL™ (a Bacillus stearothermophilus maltogenic amylase, available from Novo Nordisk A/S, Denmark) , FUNGAMYL^® (an Aspergillus oryzae alpha-amylase, available from

Novo Nordisk A/S, Denmark) , and BAN™ (a Bacillus licheniformis alpha-amylase, available from Novo Nordisk A/S, Denmark) . A commercially available amyloglucosidase useful in the present invention is AMG™ (an Aspergillus niger amyloglucosidase, available from Novo Nordisk A/S, Denmark) . Other useful commercially available amylase products include GRINDAMYL™ A 1000 or A 5000 (available from Grindsted Products, Denmark) and

AMYLASE H or AMYLASE P (available from Gist-Brocades, The

Netherlands) . A commercially available glucose oxidase useful in the present invention is GLUZYME™ (an Aspergillus niger glucose oxidase, available from Novo Nordisk A/S, Denmark) . Commercially available proteases useful in the present invention are NEUTRASE™ (a Bacillus amyloliquefaciens endoprotease, available from Novo Nordisk A/S, Denmark) and GLUTENASE™ (available from Novo Nordisk A/S, Denmark) . Commercially available pentosanase useful in the present invention are PENTOPAN™ (a Humicola insolens pentosanase, available from Novo

Nordisk A/S, Denmark) and PENTOPAN™ MONO (a Thermomyces lanuginosus pentosanase, available from Novo Nordisk A/S, Denmark) . A commercially available lipase useful in the present invention is NOVOZYM^® 677 BG (a Thermomyces lanuginosus lipase, available from Novo Nordisk A/S, Denmark) .

When one or more additional enzyme activities are to be added in accordance with the methods of the present invention, these activities may be added separately or together with the glucose isomerase (s) , optionally as constituent (s) of the bread- improving and/or dough-improving composition. The other enzyme activities may be any of the enzymes described above and may be dosed in accordance with established baking practices.

In addition to the above-mentioned additional enzymes, a glucose isomerase may contain varying minor amounts of other enzymatic activities inherently produced by the producer organism in question. In addition, or as an alternative, to additional enzyme components, a conventionally used baking agent may also be incorporated into the dough. The baking agent may include proteins, such as milk powder (to provide crust colour) , gluten (to improve the gas retention power of weak flours) , and soy (to provide additional nutrients and improve water binding) ; eggs such (either whole eggs, egg yolks or egg whites) ; fat such as granulated fat or shortening (to soften the dough and improve the texture of the bread) ; an emulsifier (to improve dough extensibility and, to some extent, the consistency of the resulting bread); an oxidant, e . g. , ascorbic acid, potassium bromate, potassium iodate, azodicarbon amide (ADA) or ammonium persulfate (to strengthen the gluten structure) ; an amino acid, e . g. , L-cysteine (to improve mixing properties); a sugar; a salt, e . g. , sodium chloride, calcium acetate, sodium sulfate or calcium sulphate (to make the dough firmer) ; flour; and starch. Such components may also be added to the dough in accordance with the methods of the present invention.

Examples of suitable emulsifiers are mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, phospholipids, and lecithin. The dough and/or baked product prepared by a method of the present invention may be based on wheat meal or flour, optionally in combination with other types of meal or flour such as corn meal, corn flour, rye meal, rye flour, oat meal, oat flour, soy meal, soy flour, sorghum meal, sorghum flour, potato meal, or potato flour.

The handling of the dough and/or baking may be performed in any suitable manner for the dough and/or baked product in question, typically including the steps of kneading the dough, subjecting the dough to one or more proofing treatments, and baking the product under suitable conditions, i.e., at a suitable temperature and for a sufficient period of time. For instance, the dough may be prepared by using a normal straight dough process, a sour dough process, an overnight dough method, a low-temperature and long-time fermentation method, a frozen dough method, the Chorleywood Bread process, or the Sponge and Dough process .

From the above disclosure it will be apparent that the dough of the invention is generally a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways such as by adding sodium bicarbonate or the like, or by adding a leaven (fermenting dough) , but it is preferable that the dough be leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast). Any of the commercially available Saccharomyces cerevisiae strains may be employed.

The present invention also relates to the use of a glucose isomerase for the preparation of pasta dough, preferably prepared from durum flour or a flour of comparable quality. The dough may be prepared by use of conventional techniques and the glucose isomerase (s) used in a similar dosage as that described above. The glucose isomerase (s) may be any of the types described above. When used in the preparation of pasta, the glucose isomerase (s) results in a strengthening of the gluten structure, a reduction in the dough stickiness, and increased dough strength.

The present invention also relates to methods for preparing a baked product, comprising baking a dough obtained by a method of the present invention to produce a baked product . The baking of the dough to produce a baked product may be performed using methods well known in the art .

The present invention also relates to compositions comprising an effective amount of one or more glucose isomerase s, for improving one or more properties of a dough and/or a baked product obtained from the dough, and a carrier and/or a baking ingredient. The compositions may further comprise a substrate for the glucose isomerase (s) , one or more additional enzymes, one or more conventionally used baking agents, an enzyme which acts on a substance endogenous to the flour to produce a substrate for the glucose isomerase (s) of interest, and/or a substance and the enzyme which acts on the substance to produce a substrate for the glucose isomerase (s) . The present invention also relates to doughs and baked products, respectively, produced by the methods of the present invention.

The present invention further relates to a pre-mix, e . g. , in the form of a flour composition, for dough and/or baked products made from dough, in which the pre-mix comprises a glucose isomerase. The term "pre-mix" is defined herein to be understood in its conventional meaning, i . e . , as a mix of baking agents, generally including flour, which may be used not only in industrial bread-baking plants/facilities, but also in retail bakeries. The pre-mix may be prepared by mixing a glucose isomerase or a bread-improving and/or dough-improving composition of the invention comprising a glucose isomerase with a suitable carrier such as flour, starch, a sugar, or a salt. The pre-mix may contain other dough-improving and/or bread-improving additives, e . g. , any of the additives, including enzymes, mentioned above.

The present invention further relates to baking additives in the form of a granulate or agglomerated powder, which comprise the glucose isomerase (s) . The baking additive preferably has a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μm.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Materials and Methods

Preparation of White Bread (I)

The straight-dough bread-making method may be used according to AACC Method 10-lOB (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA). Basic regjpe

Wheat flour 100%

Salt 1.5% Yeast (fresh) 5.3%

Sugar 6.0%

Shortening 3.0%

Water optimum

All percentages are by weight relative to the wheat flour.

Procedure

1. Dough mixing (Hobart mixer) :

The mixing time and speed should be determined by the skilled baker so as to obtain an optimum dough consistency under the testing conditions used.

2. 1st punch ( e . g. , 52 minutes after start)

3. 2nd punch (e.g., 25 minutes later)

4. Molding and panning ( e . g. , 13 minutes later). 5. Proofing to desired height ( e . g. , 33 minutes at 32°C, 82% RH) 6. Baking ( e . g. , at 215°C for 24 minutes)

Preparation of White Bread (II) The sponge-dough bread-making method may be used according to AACC Method 10-11 (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).

Basic recipe for Sponge

Wheat flour 60%

Yeast (compressed) 36%

Yeast Food 2%

Water 36%

All percentages are by weight relative to the wheat flour.

Procedure 1. Add water to compressed yeast

2. Add yeast food in dry form with flour

3. Mix sponge (Hobart A-120; Hobart Corp., Troy OH, USA):

0.5 minute at 1^st speed 1 minute at 2^nd speed

The mixing time may be adjusted so as to obtain an optimum dough consistency under the testing conditions used.

4. Ferment in a fermentation cabinet: 4 hours at 30°C, 85% RH

Basic recipe for Dough

Wheat flour 40%

Water 24%

Sugar 5%

Shortening 3% Salt 2%

All percentages are by weight relative to the wheat flour.

Procedure 1. Add dough ingredients; begin mixer (1^st speed)

2. Add sponge in three approximately equal portions at 15, 25, and 35 seconds mixing time; total mixing time: 1 minute

3. At 2^nd speed, mix to obtain an optimum dough consistency

4. Ferment in a fermentation cabinet: 30 minutes at 30°C, 85% RH

5. Intermediate proof: 12-15 minutes in fermentation cabinet

6. Mold and final proof at 35.5°C, 92% RH

7. Bake: 25 minutes at 218 °C

Evaluation of Staling Properties of Bread

The degree of staling is determined on bread, e . g. , on day

1, 3, 7 and 9 after baking. Evaluation of staleness and texture can be done according to AACC method 74-09. The principles for determination of softness and elasticity of bread crumb are as follows:

1. A slice of bread is compressed with a constant speed in a texture analyser, measuring the force for compression in g.

2. The softness of the crumb is measured as the force at 25% compression (PI) .

3. The force at 40% compression (P2) and after keeping 40% compression constant for 30 seconds (P3) is measured. The ratio (P3/P2) is the elasticity of the crumb.

Preparation of White Layer Cake

The method may be used according to AACC Method 10-90 (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).

Basic reci e

Flour 100%

Sugar 140%

Shortening 50% Nonfat Dry Milk 12%

Dried Egg Whites 9%

Salt 3% Baking Powder and Water determined empirically

All percentages are by weight relative to the flour.

Procedure

1. Combine all dry ingredients and sift well 2. Add shortening and 60% of water

3. Mix at low speed for 0.5 minute in Hobart C-100 mixer

4. Mix at medium speed for 4 minutes

5. Add 50% of remaining water

6. Mix at low speed for 0.5 minute, scrape down and mix at medium speed for 2 minutes

7. Add remaining water, mix at low speed for 0.5 minute, scrape down and mix at medium speed for 2 minutes

8. Scale batter into each of two greased pans

9. Bake at 375°C or 350°C

Evaluation of Cakes

Cakes should be graded for volume and texture on the same day as baked according to AACC Method 10-90. The internal structure may be scored for the uniformity and size of cells as well as thickness of the walls; the grain; texture, such as moisture, tenderness and softness; crumb colour; and flavour.

Preparation of Cookies

Cookies may be prepared according to AACC Method 10-50D (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).

Basic recipe

Flour 225 g

Water 16 g

Dextrose Solution 33 g Bicarbonate of Soda 2.5 g

Salt 2.1 g

Sugar 130 g

Shortening 64 g

Procedure

1. Cream shortening, sugar, salt and soda on low speed 3 minutes using an electric mixer ( e . g. , Hobart C-100)

2. Add dextrose solution and distilled water

3. Mix at low speed for 1 minute 4. Mix at medium speed for 1 minute

5. Add all flour and mix at low speed for 2 minutes

6. Scrape dough from bowl and place six portions at well-spaced points on lightly greased cookie sheet

7. Flatten dough lightly 8. Cut dough with cookie cutter 9. Bake at 205°C for 10 minutes

Evaluation of Cookies

Cookie width should be measured after cooling 30 minutes and can be done by the method according to AACC Method 10-50D.

The width of each of the six cookies is measured in mm, then rotated 90° and remeasured to obtain the average width

(W) . An average thickness (T) may be obtained by measuring the cookies stacked on top of one another, then restacked in a different order. The spread factor is the ratio of W/T. However, the most sensitive and reliable estimate is the width measurement, and in some cases, thickness. Because the spread factor is a ratio of 2 empirically determined parameters, different values of W and T can result in the same W/T.

Preparation of Biscuits

Biscuits may be prepared according to AACC Method 10-31B (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).

Basic recipe

Flour 228 g Shortening 40 g

Milk Solution¹ 135 g

Bicarbonate of Soda² 3.4 g

Salt² 4.5 g

Monocalcium Phosphate² 130 g

^x50 g milk powder in 450 ml water

²omit if self-rising flour is used; use 240 g of self-rising flour Procedure 1. Sift together flour and other dry ingredients (bicarbonate of soda, salt and monocalcium phosphate, if used)

2. Add shortening to flour mixture

3. Mix, using electric mixer (e.g., Hobart, Kitchen Aid or equivalent) with timer control, at speed 1 for 15 seconds 4. Mix at speed 1 for 3 minutes

5. Add milk solution and mix at speed 1 for 15 seconds

6. Roll out dough using floured rolling pin

7. Cut dough with floured cutter

8. Place 8 dough pieces 4 cm apart on ungreased baking sheet . 9. Bake at 232°C for 10 minutes

Evaluation of Biscuits

Upon removal from oven, biscuits should be removed from the baking sheet and cooled for 30 minutes. Measurements of the eight biscuits can be made according to AACC Method 10-31B to obtain a total weight, a total diameter and a height at the top center of each biscuit.

Preparation of Pie Shells

Pie shells may be prepared according to AACC Method 10-60 (in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).

Basic recipe

Flour 100%

Shortening 60%

Salt 3.5% Water 30-64%

All percentages are by weight relative to the wheat flour, and all ingredients are at 10°C before mixing.

Procedure

1. Sift flour twice

2. Add shortening to flour and cut for 5 minutes using electric mixer ( e . g. , Hobart, Kitchen Aid or equivalent) with timer control, on low speed 3. Dissolve salt in a portion of water

4. Add salt solution to flour-shortening mixture, together with additional water if necessary

5. Mix at low speed for 2 minutes

6. Store dough at 10°C for 24 hours

Empty shells

7. Scale, press dough into ball

8. Roll dough, fold and roll again

9. Fold and roll a third time 10. Lay dough sheet over an inverted pie tin

11. Trim dough and prick with fork

12. Let dry for 30 minutes and cover with a second pan pressed down firmly 13. Bake at 218°C for 20-25 minutes, removing second pan after 10 minutes in the oven

Filled pies 7. Scale and roll bottom crust as outlined above for empty pie shell

8. Press dough sheet into pie tin and fill with either artificial fruit acid filling (water, corn starch, sugar and citric acid crystals) or true fruit filling (cling peaches, sugar corn starch and water)

9. Scale and roll dough once for top crust

10. Place over filling, trim and cut center lightly

11. Press edge over wetted edge of bottom crust

12. Bake at 218°C for about 30 minutes

Evaluation of Pie Crusts

Viscosity may be evaluated according to AACC Method 56-80.

Other parameters of empty and filled pie shells may be measured according to AACC Method 10-60 24 hours and 12 or 16 hours after baking, respectively. Pie crusts may be evaluated empirically for whether they are baked through; the edges have shrunk from edge of pan; blisters have appeared; the texture is flaky; the mouth-feel is tender; whether they are crisp or soft; the colour; and if the fruit filling has penetrated the crust.

Testing of Doughs and Breads

According to the methods of the present invention, the effect of adding a glucose isomerase may be tested in doughs and breads by using the following method:

Recipe:

Water 60%

Wheat Flour 100%

Yeast 4% Salt 1.5%

Sugar 1.5%

The wheat flour is of the type Meneba 964. Preparation of Breads Procedure

1. Dough mixing (Spiral mixer)

3 minutes at low speed 8 minutes at high speed

The mixing time may be adjusted by the skilled baker to obtain an optimum dough consistency under the testing conditions used.

2. 1st proof: 30°C - 80% RH, 20 minutes 3. Scaling and shaping;

4. Final proof: 32°C - 80% RH, 40 minutes;

5. Baking: 225°C, 20 minutes for rolls and 30 minutes for loaf.

Evaluation of Dough and Baked Products Dough and baked products made from the straight dough method described above may be evaluated as follows for loaf specific volume, dough stickiness, dough firmness, dough extensibility, dough elasticity, crumb structure, and gluten strength.

Loaf specific volume: The mean value of 4 loaves volume are measured using the traditional rape seed method. The specific volume is calculated as volume ml per g bread. The specific volume of the control (without enzyme) is defined as 100. The relative specific volume index is calculated as: specific vol. of 4 loaves

Specific vol. index = xlOO spec . vol . of 4 control loaves The dough stickiness, firmness, extensibility, elasticity and crumb structure may be evaluated relative to controls by the skilled test baker according to the following scale:

Dough stickiness: almost liquid 1 too sticky 2 sticky 3 normal 4 dry 5 too dry 6 Crumb structure: very poor 1 poor 2 non-uniform 3 uniform/good 4 very good 5

Douσh Firmness: extremely soft 1 too soft 2 soft/good 3 normal 4 firm 5 too firm 6

Douσh Extensibility: too short 1 short 2 normal 3 good 4 long 5 too long 6

Dough stability/Shock test: After the second proof a pan containing the dough is dropped from a height of 20 cm. The dough is baked and the volume of the resulting bread is determined.

Gluten Strengthening: The strengthening effect of a given dough conditioner on wheat flour dough or gluten dough may be measured by dynamic rheological measurements .These measurements are able to show the strength of a dough under oscillation. Both wheat flour dough and gluten dough are viscoelastic materials. In oscillatory measurements, the viscoelastic properties of a wheat dough and a gluten dough can be divided into two components, the dynamic shear storage modulus G' and the dynamic shear loss modulus G" . The ratio of the loss and the storage moduli is numerically equal to the tangent of the viscoelastic phase angle δ (Delta) . An increase in the storage modulus G' and a decrease in the phase angle δ indicate a stronger and more elastic dough.

Example 1:

Use of a glucose isomerase during the preparation of a White bread for improving the volume of the bread:

Preparation of the white bread was done as described above in section "preparation of whitebread (I)".

Enzymes used: a) AMG 1000 AGU/g (an amyloglucosidase) . AGU are standardised Novo Amylog.locosidase units per gram. The detailed analytical protocol is available on request to Novo Nordisk A/S, Denmark. b) Sweetzyme® T; 350 IGIU/g. (a glucose isomerase) . IGIU is standard glucose isomerase units. The detailed protocol is described above ( vide supra) . The Sweetzyme® T is an immobilised preparation. Consequently is was grounded before used as described below. The grounding was performed in a mortar until the enzyme preparation was substantially crushed. c) Fungamyl 2500 BG 2950 FAU/g. One FAU is the amount of enzyme which under standard conditions (i.e. at 37°C and pH 4.7) breaks down 5260 mg solid starch (Amylum solubile, Merck) per hour. A folder AF 9.1/3, describing this FAU assay in more details, is available upon request from Novo Nordisk A/S, Denmark, which folder is hereby included by reference.

All of said used enzymes are commercially available enzymes from Novo Nordisk A/S, Denmark.

Enzymes were added to the basic recipe according to following scheme :

Enzyme/dough 1 2 3

Fungamyl 25 25 25

FAU/kg flour

AMG 0 60 60

AGU/kg flour

Sweetzyme T O 0 70

IGIU/kg flour The loaf specific volume were determined as described above under section "Evaluation of Dough and Baked Products" and the results are shown in table I below.

Table I:

Dough Specific volume index

1 100

2 111

3 125

The results in table I clearly show that addition of a glucose isomerase (Sweetzyme T) significantly increased the loaf specific volume of the white bread.

The determination of softness and elasticity as described above under section "Evaluation of Staling properties of Bread" and the results are shown in table II below.

Table II:

FirmElastiness city

(PI) (P3/P2)

Dough/Day 0 1 4 0 1 4

1 188 451 1030 0.6873 0.6383 0.6304

2 184 439 940 0.6972 0.6388 0.5589

3 163 390 821 0.7091 0.6485 0.5804

The results in table II show that addition of a glucose isomerase (Sweetzyme T) decreases the firmness over time in the bread and the elasticity is retained.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended any of claims .

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

Claims :

1. A method for preparing a dough, comprising incorporating into the dough an effective amount of one or more glucose isomerase (s) .

2. The method of claim 1, wherein the one or more glucose isomerase (s) improve one or more properties of the dough.

3. The method of any of claims 1-2, wherein the glucose isomerase is obtained from a microbial source.

4. The method of any of claims 1-3, wherein the effective amount of the glucose isomerase is about 0.01 mg to about 100 mg per kilogram of dough.

5. The method of claim 4, wherein the effective amount of the glucose isomerase is about 0.1 mg to about 25 mg per kilogram of dough.

6. The method of claim 5, wherein the effective amount of the glucose isomerase is about 0.5 mg to about 5 mg per kilogram of dough.

7. The method of claim 6, wherein the effective amount of the glucose isomerase is about 1 mg to about 5 mg per kilogram of dough.

8. The method of any of claims 2-7, wherein the one or more improved properties are selected from the group consisting of increased strength of the dough, increased stability of the dough, reduced stickiness of the dough, improved machinability of the dough, increased volume of the baked product, improved crumb structure of the baked product, improved softness of the baked product, improved flavour of the baked product, and improved antistaling of the baked product.

9. The method of claim 8, wherein the improved property is increased volume of the baked product .

10. The method of claim 8, wherein the improved property is improved softness of the baked product.

11. The method of claim 8, wherein the improved property is improved antistaling of the baked product.

12. The method of any of claims 1-11, wherein the dough is obtained from one or more ingredients selected from the group consisting of wheat meal, wheat flour, corn meal, corn flour, durum flour, rye meal, rye flour, oat meal, oat flour, soy meal, soy flour, sorghum meal, sorghum flour, potato meal, and potato flour.

13. The method of any of claims 1-12, wherein the dough is fresh or frozen.

14. The method of any of claims 1-13, wherein the baked product is selected from the group consisting of a bread, a roll, a

French baguette-type bread, a pasta, a pita bread, a tortilla, a taco, a cake, a pancake, a biscuit, a cookie, a pie crust, steamed bread, and a crisp bread.

15. The method of any of claims 1-14, further comprising incorporating one or more additional enzymes selected from the group consisting of an amylase, a cellulase, a cyclodextrin glucanotransferase, a glycosyltransferase, a hemicellulase, a laccase, a lipase, an oxidase, a pentosanase, a peptidase, a peroxidase, a phospholipase, a protease, a protein disulfide isomerase, and a transglutaminase.

16. The method of any of claims 1-16, wherein the amylase is a maltogenic amylase obtainable from Bacillus stearothermophilus .

17. The method of any of claims 1-16, further comprising incorporating one or more additives selected from the group consisting of a protein, an emulsifier, a granulated fat, an oxidant, an amino acid, a sugar, a salt, a flour, and a starch.

18. A method for preparing a baked product, comprising baking a dough produced by the method of any of claims 1-17 to produce a baked product .

19. The method of claim 18, wherein the one or more glucose isomerase (s) improve one or more properties of the baked product .

20. A composition comprising an effective amount of one or more glucose isomeras(s) for improving one or more properties of a dough or a baked product obtained from the dough and a baking agent .

21. The composition of claim 20, wherein the one or more glucose isomerase (s) improve one or more properties of the dough and the baked product obtained from the dough.

22. The composition of any of claims 20-21, wherein the effective amount of the glucose isomerase is about 0.01 mg to about 100 mg per kilogram of dough.

23. The composition of any of claims 20-22, wherein the one or more improved properties are selected from the group consisting of increased strength of the dough, increased stability of the dough, reduced stickiness of the dough, improved machinability of the dough, increased volume of the baked product, improved crumb structure of the baked product, improved softness of the baked product, improved flavour of the baked product, and improved antistaling of the baked product.

24. The composition of any of claims 20-23, wherein the composition further comprises one or more additional enzymes selected from the group consisting of an amylase, a cellulase, a cyclodextrin glucanotransferase, a glycosyltransferase, a hemicellulase, a laccase, a lipase, an oxidase, a pentosanase, a peptidase, a peroxidase, a phospholipase, a protease, a protein disulfide isomerase, and a transglutaminase .

25. The composition of any of claims 20-24, wherein the composition further comprises one or more additives selected from the group consisting of a protein, an emulsifier, a granulated fat, an oxidant, an amino acid, a sugar, a salt, a flour, and a starch.

26. A dough product obtained from a dough prepared by the method of claim 1-17.

27. A baked product produced by the method of claim 18 or 19.

28. A pre-mix for a dough comprising an effective amount of one or more glucose isomerase (s) for improving one or more properties of a dough and/or a baked product obtained from the dough and a baking agent .

29. A baking additive in the form of a granulate or agglomerated powder, which comprises one or more glucose isomerase (s) .

30. The baking additive of claim 29, wherein more than 95% by weight of the baking additive has a particle size between about 25 and about 500 μm.