AU2002301639C1 - A method of improving flour dough and flour dough compositions - Google Patents

Description

2 Field of the Invention The present invention relates to a method for preparing flour products such as flour doughs and cooked and/or baked products therefrom.

More in particular, the present invention relates to the field of food manufacturing, in particular to the preparation of improved bakery products and other farinaceous food products. Specifically, the invention concerns the use of a new combination for improving the stability and/or machineability of dough and/or improving the quality of baked and dried products made from such doughs.

In a preferred aspect, the present invention relates to: a method of improving the rheological and/or machineability properties of a flour dough and/or the quality of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent.

Teachings relating to this preferred aspect now follow.

TECHNICAL BACKGROUND AND PRIOR ART The "strength" or "weakness" of doughs is an important aspect of making farinaceous finished products from doughs, including baking. The "strength" or "weakness" of a dough is primarily determined by its content of protein and in particular the content and the quality of the gluten protein is an important factor in that respect.

Flours with a low protein content are generally characterised as "weak". Thus, the cohesive, extensible, rubbery mass which is formed by mixing water and weak flour will usually be highly extensible when subjected to stress, but it will not return to its original dimensions when the stress is removed.

Flours with a high protein content are generally characterised as "strong" or "hard" flours and the mass formed by mixing such a flour and water will be less extensible than the mass formed from a weak flour, and stress which is applied during mixing will be restored without breakdown to a greater extent than is the case with a dough mass formed from a weak flour. Strong flour is generally preferred in most baking contexts because of the superior rheological and handling properties of the dough and the superior form and texture qualities of the finished baked or dried products made from the strong flour dough.

Dough quality may be largely dependent on the type or types of flour present in the dough and/or the age of the flour or flours.

Doughs made from strong flours are generally more stable.

Stability of a dough is one of the most important characteristics of flour doughs. Within the bakery and milling industries it is known to use dough "conditioners" to strengthen the dough to increase its stability and strength. Such dough conditioners are normally nonspecific oxidising agents such as e.g. iodates, peroxides, ascorbic acid, K-bromate or azodicarbonamide and they are added to dough with the aims of improving the baking performance of flour to achieve a dough with

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improved stretchability and thus having a desirable strength and stability. The mechanism behind this effect of oxidising agents is that the flour proteins, in particular gluten contains thiol groups which, when they become oxidised, form disulphide bonds whereby the protein forms a more stable matrix resulting in a better dough quality and improvements of the volume and crumb structure of the baked products.

However, the use of several of the currently available non-specific oxidising agents is either objected to by consumers or is not permitted by regulatory bodies. Hence it has been attempted to find alternatives to these conventional flour and dough additives, and the prior art has inter alia suggested the use of oxidoreductases such as glucose oxidase, carbohydrate oxidase, glycerol oxidase and hexose oxidase for this purpose.

US 2,783,150 discloses the addition of glucose oxidase to flour to improve dough strength and texture and appearance of baked bread.

CA 2,012,723 discloses bread improving compositions comprising cellulolytic enzymes such as xylanases and glucose oxidase, the latter enzyme being added to reduce certain disadvantageous effects of the cellulolytic enzymes (reduced dough strength and stickiness) and it is disclosed that addition of glucose is required to obtain sufficient glucose oxidase activity.

JP-A-92-084848 suggests the use of a bread improving composition comprising glucose oxidase and lipase.

i As disclosed in WO 96/39851, the use of glucose oxidase as a dough improving additive has the limitation that this enzyme requires the presence of sufficient amounts of glucose as a substrate in order to be effective in a dough system and generally the glucose content in cereal flours is low. Therefore the absence of glucose in doughs or the low content thereof in doughs will be a limiting factor for the effectiveness of glucose oxidase as a dough improving agent.

WO 96/39851 discloses a method of improving the rheological properties of a flour dough and the quality of the finished product made from the dough comprising adding to the dough an oxidoreductase such as hexose oxidase (Hox).

Hexose oxidase (Hox) (D-hexose:0 2 -oxidoreductase, EC 1.1.3.5) is an enzyme which in the presence of oxygen is capable of oxidising D-glucose and several other reducing sugars including maltose, glucose, lactose, galactose, xylose, arabinose and cellubiose to their corresponding lactones with subsequent hydrolysis to the respective aldobionic acids. Accordingly, hexose oxidases differ from glucose oxidase which can only convert D-glucose, in that hexose oxidases can utilise a broader range of sugar substrates.

WO 94/04035 discloses a method of improving properties of a dough (with and without fat) and/or baked product made from dough by adding a lipase of microbial origin to the dough. The use of the microbial lipase resulted in an 6 increased volume and improved softness of the baked O product. Furthermore an antistaling effect was found.

z 00 EP 1 108 360 Al discloses a method of preparing a flour dough. The method comprises adding to the dough components an enzyme that under dough conditions is MO capable of hydrolysing a nonpolar lipid, a glycolipid and a phospholipid, or a composition containing said enzyme

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M and mixing the dough components to obtain the dough.

I0 C-i WO 02/03805 discloses that the addition to dough of a combination of two lipases with different substrate specificities. The combination produces a synergistic effect on the dough or on a baked product made from the dough. Optionally, an additional enzyme may be used together with the lipase Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

O We have surprisingly found that a combination of a Hox and Z an emulsifying agent results in particularly advantageous 00 0 properties in dough and dough products and/or in baked products therefrom. In particular the stability (e.g.

shock stability) and/or rheological decrease in stickiness) and/or machineability properties and/or the resultant volume of either the dough and/or baked products S(e.g. baked products with better crumb structure and/or homogeneity) is/are improved. Furthermore, the combination of the Hox and emulsifying agent results in an improvement in bread quality, in particular in respect of specific volume and/or crumb homogeneity, which is not a simple additive effect, but may reflect a synergistic effect of these types of enzymes.

i The invention further relates to the use of a Hox and an emulsifying agent to improve the rheological and/or machineability properties of dough.

The invention further relates to the use of a Hox and an emulsifying agent to improve the volume of a baked product made from a dough.

DETAILED ASPECTS In one aspect the invention provides a method of improving the rheological and/or machineability properties of a flour dough and/or the quality volume) of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent.

Factors which influence the rheological properties and/or the machineability include stickiness and extensibility.

In another aspect the invention provides a method of improving the rheological and/or machineability properties of a flour dough and/or the quality volume) of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent wherein the flour dough comprises at least one further dough additive or ingredient.

In another aspect the invention provides a method of improving the rheological and/or machineability properties of a flour dough and/or the quality volume) of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent wherein the product is selected from the group consisting of a bread product, a noodle product, a cake product, a pasta product and an alimentary paste product.

In another aspect the invention provides a method of improving the rheological and/or machineability properties of a flour dough and/or the quality volume) of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent wherein at least one further enzyme is added to the dough ingredients, dough additives or the dough.

In another aspect the invention provides a dough improving composition comprising a Hox and an emulsifying agent.

In another aspect the invention provides a dough improving composition comprising a Hox and an emulsifying agent wherein the flour dough comprises at least one further dough additive or ingredient.

In another aspect the invention provides use of a dough improving composition comprising a Hox and an emulsifying agent in the manufacture of a product made from dough wherein the product is selected from the group consisting of a bread product, a noodle product, a cake product, a pasta product and an alimentary paste product.

In another aspect the invention provides a dough improving composition comprising a Hox and an emulsifying agent wherein at least one further enzyme is added to the dough ingredients, dough additives or the dough.

In another aspect the invention provides use of a dough improving composition comprising a Hox and an emulsifying agent wherein said composition improves the rheological and/or machineability properties of flour dough.

In another aspect the invention provides use of a dough improving composition comprising a Hox and an emulsifying agent wherein said composition improves the volume of a baked product made from a flour dough.

In another aspect the invention provides a dough for addition to a sponge wherein said dough comprises a Hox and an emulsifying agent.

In another aspect the invention provides a dough for addition to a sponge wherein said dough comprises a Hox and an emulsifying agent and wherein the dough comprises at least one further dough additive or ingredient.

Dough Preparation In accordance with the invention, the dough is prepared by admixing flour, water, the dough improving composition and optionally other ingredients and additives. The dough improving composition can be added together with any dough ingredient including the flour, water or optional other ingredients or additives. The dough improving composition can be added before the flour or water or optional other ingredients and additives. The dough improving composition can be added after the flour or water, or optional other ingredients and additives. The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products.

The dough improving composition can be added as a liquid preparation or in the form of a dry powder composition either comprising the composition as the sole active component or in admixture with one or more other dough ingredients or additive.

Dough The dough of the invention generally comprises wheat meal or wheat flour and/or other types of meal, flour or starch such as corn flour, corn starch, maize flour, rice flour, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch.

The dough of the invention may be fresh, frozen, or partbaked.

The dough of the invention can be a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by adding chemical leavening agents, sodium bicarbonate or by adding a leaven (fermenting dough), but it is preferred to leaven the dough by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g.

a commercially available strain of S. cerevisiae.

The dough may also comprise other conventional dough ingredients, proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg white); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

The dough may comprise fat such as granulated fat or shortening.

The dough may further comprise a further emulsifier such as mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxethylene stearates, or lysolecithin.

The invention also provides a pre-mix comprising flour together with the combination as described herein. The pre-mix may contain other dough-improving and/or breadimproving additives, e.g. any of the additives, including enzymes, mentioned herein.

Preferably the flour dough comprises a hard flour.

The term "hard flour" as used herein refers to flour which has a higher protein content such as gluten than other flours and is suitable for the production of, for example, bread. The term "hard flour" as used herein is synonymous with the term "strong flour".

A preferred flour is wheat flour. However doughs comprising flour derived from, for example, maize, corn, oat, barley, rye, durra, rice, soy, sorghum and potato are also contemplated.

Preferably the flour dough comprises a hard wheat flour.

Rheological properties The phrase "rheological properties" as used herein relates to the physical and chemical phenomena described herein which in combination will determine the performance of flour doughs and thereby also the quality of the resulting products.

The phrase "machineability of a flour dough" as used herein refers to the improved manipulation by machinery of the dough. The dough is less sticky compared to the dough without the addition of the combination.

In a further embodiment, the invention relates to improvement of the rheological characteristics of the dough including that the gluten index in the dough is increased by at least relative to a dough without addition of a combination, the gluten index is determined by means of a Glutomatic 2200 apparatus.

The phrase "rheological properties" as used herein refers to the effects of dough conditioners on dough strength and stability as the most important characteristics of flour doughs. According to American Association of Cereal Chemists (AACC) Method 36-01A the term "stability" can be defined as "the range of dough time over which a positive response is obtained and that property of a rounded dough by which it resists flattening under its own weight over a course of time". According to the same method, the term "response" is defined as "the reaction of dough to a known and specific stimulus, substance or set of conditions, usually determined by baking it in comparison with a control" As it is mentioned herein, it is generally desirable to improve the baking performance of flour to achieve a dough with improved stretchability and thus having a desirable strength and stability by adding oxidising agents which cause the formation of protein disulphide bonds whereby the protein forms a more stable matrix resulting in a better dough quality and improvements of the volume and crumb structure of baked products.

The effect of the combination on the rheological properties of the dough can be measured by standard methods according to the International Association of Cereal Chemistry (ICC) and the American Association of Cereal Chemistry (AACC) including the amylograph method (ICC 126), the farinograph method (AACC 54-21) and the extensigraph method (AACC 54-10) The AACC method 54-10 defines the extensigraph in the following manner: "the extensigraph records a load-extension curve for a test piece of dough until it breaks. Characteristics of loadextension curves or extensigrams are used to assess general quality of flour and its responses to improving agents". In effect, the extensigraph method measures the relative strength of a dough. A strong dough exhibits a higher and, in some cases, a longer extensigraph curve than does a weak dough.

In a preferred embodiment of the invention, the resistance to extension of the dough in terms of the ratio between the resistance to extension (height of curve, B) and the extensibility (length of curve, i.e. the B/C ratio as measured by the AACC method 54-10 is increased by at least relative to that of an otherwise similar dough not containing a combination. In more preferred embodiments, the resistance to extension is increased by at least such as at least 50% and in particular by at least 100%.

It has been found that the addition of the composition of the present invention to bakery product doughs results in bakery products such as yeast leavened and chemically leavened products in which the specific volume is increased relative to an otherwise similar bakery product.

In this context, the expression "specific volume" is used to indicate the ratio between volume and weight of the product. It has been found that, in accordance with the method described herein, the specific volume can be increased significantly such as by at least preferably by at least 20%, including by at least preferably by at least 40% and more preferably by at least The present invention is highly suitable for improving the rheological and/or machineability properties and/or quality volume) of the finished products (products made from the dough) of conventional types of yeast leavened bread products based on wheat flour, such as loaves and rolls. The present invention is also suitable for improving the rheological properties of doughs containing chemical leavening agents (baking powder) and the quality volume) of products made from such doughs. Such product include as examples breads, sponge cakes and muffins.

Noodles In one interesting aspect, the invention is used to improve the rheological and/or machineability properties of doughs intended for noodle products including "white noodles" and "Chinese noodles" and to improve the textural qualities of the finished noodle products. A typical basic recipe for the manufacturing of noodles comprises the following ingredients: wheat flour 100 parts, salt parts and water 33 parts. Furthermore, glycerol is often added to the noodle dough. The noodles are typically prepared by mixing the ingredients in an appropriate mixing apparatus followed by rolling out the noodle dough using an appropriate noodle machine to form the noodle strings which are subsequently air dried.

The quality of the finished noodles is assessed inter alia by their colour, cooking quality and texture. The noodles should cook as quickly as possible, remain firm after cooking and should preferably not loose any solids to the cooking water. On serving the noodles should preferably have a smooth and firm surface not showing stickiness and provide a firm "bite" and a good mouthfeel.

Furthermore, it is important that the white noodles have a light colour.

Since the appropriateness of wheat flour for providing noodles having the desired textural and eating qualities may vary according to the year and the growth area, it is usual to add noodle improvers to the dough in order to compensate for sub-optimal quality of the flour.

Typically, such improvers will comprise dietary fibre substances, vegetable proteins, emulsifiers and hydrocolloids such as e.g. alginates, carrageenans, pectins, vegetable gums including guar gum and locust bean gum, and amylases, and glycerol.

It is therefore an important aspect of the invention that the composition according to the invention is useful as a noodle-improving agent optionally in combination with glycerol and other components currently used to improve the quality of noodles. Thus, it is contemplated that noodles prepared in accordance with the method herein will have improved properties with respect to colour, cooking and eating qualities including a firm, elastic and nonsticky texture and consistency.

Alimentary paste product In a further useful embodiment, the dough which is prepared by the method according to the invention is a dough for preparing an alimentary paste product. Such products which include as examples spaghetti and macaroni are typically prepared from a dough comprising main ingredients such as flour, eggs or egg powder and/or water. After mixing of the ingredient, the dough is formed to the desired type of paste product and air dried.

It is contemplated that the addition of the combination to paste dough, optionally in combination with its substrate, will have a significant improving effect on the extensibility and stability hereof resulting in finished paste product having improved textural and eating qualities.

Bread In the invention the improvement of the theological properties of the dough include that the resistance to extension of the dough in terms of the ratio between resistance to extension (height of curve, B) and the extensibility (length of curve, i.e. the B/C ratio, as measured by the AACC method 54-10 is increased by at least 10% relative to that of an otherwise similar dough that does not comprise the combination and wherein the improvement of the quality of the finished product made from the dough is that the average pore diameter of the crumb of the bread made from the dough is reduced by at least 10%, relative to a bread which is made from a bread dough without addition of the combination.

In a further embodiment, the invention, implies that the improvement of the quality of the product made from the dough consists in that the pore homogeneity of the crumb of the bread made from the dough is increased by at least relative to a bread which is made from a bread dough without addition of the combination. The pore homogeneity of bread is conveniently measured by means of an image analyser composed of a standard CCD-video camera, a video digitiser and a personal computer with WinGrain software.

Using such an analyzer, the results of pore diameter in mm and pore homogeneity can be calculated as an average of measurements from 10 slices of bread. The pore homogeneity is expressed in of pores that are larger than 0.5 times the average of pore diameter and smaller than 2 times the average diameter.

Preferably, the dough is a yeast leavened dough.

Although, it is preferred to use the method of the present invention for the manufacture of yeast leavened bread products such as bread loaves, rolls or t.oast bread, the use of the method for any other type of dough and dough based products such as noodle and pasta products and cakes, the quality of which can be improved by the addition of the combination according to the present invention, is also contemplated.

Preferably the method comprises a further step that the dough is baked to obtain a baked product.

Preferably, when the dough is a bread dough, the method comprises as a further step that the dough is baked to obtain a baked product. One particularly desired property of baked bread products is a high specific volume as defined in the examples. Accordingly, the addition of the combination of the invention preferably results in an increase of the specific volume of the baked product that is at least 10%, relative to a baked product made under identical conditions except that the enzyme is not added.

More preferably, the increase of the specific volume is at least 20% such as at least 30%, e.g. at least Alternatively, the dough is a dough selected from the group consisting of a pasta dough, a noodle dough, and a cake dough or batter.

The phrase "quality of the product" as used herein refers to the final and stable volume and/or crust colour and/or texture and taste.

The term "product made from dough" as used herein refers to a bread product such as in the form of loaves or rolls, french baguette type bread, pita bread, tacos and crisp bread. Preferably the term refers to cakes, pan-cakes, biscuits. More preferably the term refers to pasta. More preferably the term refers to noodles. More preferably the term refers to alimentary paste product.

Enzyme amount Preferably the or each enzyme is added in an amount from 1-1000 ppm, preferably 25-500 ppm, more preferably 50-300 ppm.

Hox The term "Hox" as used herein refers to Hexose oxidase (Dhexose:0 2 -oxidoreductase, EC WO 96/39851 discloses some of the sources of Hox. WO 96/40935 discloses a method of producing Hox by recombinant DNA technology. US6251626 discloses hexose oxidase sequences.

The Hox may be isolated and/or purified from natural sources or it may be prepared by use of recombinant DNA techniques.

The Hox may be isolated from red algae such as Iridophycus flaccidum and Chondrus crispus, Euthora cristata.

The Hox may be a variant or derivative of a natural Hox.

The Hox, or the variant or derivative of a natural Hox, is capable of oxidising maltose in the dough.

Preferably the Hox is added in a substantially pure and/or substantially isolated form.

Emulsifying agent The emulsifying agent may be an emulsifier per se or an agent that generates an emulsifier in situ.

Conventional emulsifiers used in making flour dough products include as examples monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, and lecithins e.g. obtained from soya.

Examples of emulsifying agents that can generate an emulsifier in situ include enzymes.

Preferably the emulsifying agent is a lipase.

Lipase The term "lipase" as used herein refers to enzymes which are capable of hydrolysing carboxylic ester bonds to release carboxylate (EC Examples of lipases include but are not limited to triacylglycerol lipase (EC galactolipase (EC 3.1.1.26), phospholipase (EC 3.1.1.32).

The lipase may be isolated and/or purified from natural sources or it may be prepared by use of recombinant DNA techniques.

Preferably the lipase is selected from the group comprising triacylglycerol lipase, a galactolipase, phospholipase.

More preferably the lipase(s) may be one or more of: triacylglycerol lipase (EC phospholipase A2 (EC galactolipase (EC 3.1.1.26), phospholipase Al (EC 3.1.1.32), lipoprotein lipase A2 (EC 3.1.1.34).

The lipase may be a variant or derivative of a natural lipase.

For some aspects, preferably the lipase is a phospholipase (including a variant phospholipase) Preferably the lipase is added in a substantially pure and/or substantially isolated form.

Lipases that are useful in the present invention can be derived from a bacterial species, a fungal species, a yeast species, an animal cell and a plant cell. Whereas the enzyme may be provided by cultivating cultures of such source organisms naturally producing lipase, it may be more convenient and cost-effective to produce it by means of genetically modified cells such as it is described WO 9800136. The term "derived" may imply that a gene coding for the lipase is isolated from a source organism and inserted into a host cell capable of expressing the gene.

WO 02/03805 teaches some of the sources of lipases. The lipases that are taught therein are incorporated herein by reference.

For some aspects of the present invention the lipase may be Lipopan F (supplied by Novozymes) or a variant thereof.

Further dough additives or ingredients Typically, further dough additives or ingredients (components) include conventionally used dough additives or ingredients such as salt, sweetening agents such as sugars, syrups or artificial sweetening agents, lipid substances including shortening, margarine, butter or an animal or vegetable oil, glycerol and one or more dough additives such as emulsifying agents, starch degrading enzymes, cellulose or hemicellulose degrading enzymes, proteases, non-specific oxidising agents such as those mentioned above, flavouring agents, lactic acid bacterial cultures, vitamins, minerals, hydrocolloids such as alginates, carrageenans, pectins, vegetable gums including e.g. guar gum and locust bean gum, and dietary fibre substances.

The further dough additive or ingredient can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or the dough improving composition. The further dough additive or ingredient can be added before the flour, water, optional other ingredients and additives or the dough improving composition. The further dough additive or ingredient can be added after the flour, water, optional other ingredients and additives or the dough improving composition.

The further dough additive or ingredient may conveniently be a liquid preparation. However, the further dough additive or ingredient may be conveniently in the form of a dry composition.

Preferably the further dough additive or ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, glycerol, margarine, butter, butterfat and milk fat.

Preferably the further dough additive or ingredient is at least 1% the weight of the flour component of dough. More preferably, the further dough additive or ingredient is at least preferably at least preferably at least 4%, preferably at least preferably at least 6%.

If the additive is a fat, then typically the fat may be present in an amount of from 1 to typically 1 to 3%, more typically about 2%.

Further enzyme Further enzymes that may be used may be selected from the group consisting of a xylanase, a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, an oxidoreductase and a lipase.

Among starch degrading enzymes, amylases are particularly useful as dough improving additives. Other useful starch 7 degrading enzymes which may be added to a dough composition include glucoamylases and pullulanases.

The term "xylanase" as used herein refers to xylanases (EC 3.2.1.32) which hydrolyse xylosidic linkages.

The ,further enzyme can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or the dough improving composition. The further enzyme can be added before the flour, water, and optionally other ingredients and additives or the dough improving composition. The further enzyme can be added after the flour, water, and optionally other ingredients and additives or the dough improving composition.

The further enzyme may conveniently be a liquid preparation. However, the composition may be conveniently in the form of a dry composition.

In some aspects of the present invention it may be found that some enzymes of the dough improving composition of the invention are capable of interacting with each other under the dough conditions to an extent where the effect on improvement of the rheological and/or machineability properties of a flour dough and/or the quality of the product made from dough by the enzymes is not only additive, butthe effect is-synergistic.

In relation to improvement of the product made from dough (finished product), it may be found that the combination results in a substantial synergistic effect in respect to crumb homogeneity as defined herein. Also, with respect to the' specific volume of baked product a synergistic effect may be found.

Nucleotide sequence The enzyme need not be a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.

The nucleotide sequence of the present invention may be prepared using recombinant DNA techniques (i.e.

recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

Amino acid sequences The enzyme may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

Variants/homologues/derivatives The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme of the present invention or of any nucleotide sequence encoding such an enzyme. Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate homology between two or more sequences.

The invention will now be described by way of illustration in the following non-limiting examples and with reference to the following figures Figure 1 which is a photographic image of a bread; Figure 2 which is a photographic image of a bread; and Figure 3 which is a photographic image of a bread.

EXAMPLES

Definitions All PANODAN T products contain DATEM (Di-acetyl tartaric acid ester of monoglycerides) and are obtained from Danisco A/S.

PANODAN

T M 521: DATEM containing bacterial xylanase and fungal amylase TS-E 662 TM (obtained from Danisco A/S) is a product containing hexose oxidase (Hox) (EC 1.1.1.5) from Chondrus chrispus expressed in Hansenula polymorpha.

TS-E 680 TM (obtained from Danisco A/S) is a product containing fungal xylanase (EC 3.2.1.8) from Aspergillus niger.

TS-E 861 T (obtained from Danisco A/S) is a product containing fungal xylanase (EC 3.2.1.8) from Aspergillus niger, lipase (EC 3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillus oryzae, and hexose oxidase (EC 1.1.1.5) from Chondrus crispus expressed in Hansenula polymorepha.

GRINDAMYLTM H 640 (obtained from Danisco A/S): contains bacterial xylanase Grindamyl T H 121 (obtained from Danisco A/S) is a fungal xylanase (EC 3.2.1.8) from Aspergillus niger.

i GrindamylTM EXEL 16 (obtained from Danisco A/S) is lipase (EC 3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillus oryzae.

Grindamyl T M EXEL 66 (obtained from Danisco A/S) is a mixture of lipase (EC 3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillus oryzae and a fungal xylanase (EC 3.2.1.8) from Aspergillus niger.

Lipopan FTM (Lipopan F BG) (obtained from Novozymes) is according to its producer (Novozymes) a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism. According to its producer, Lipopan F has inherent activity toward phospholipids, glycolipids and triglycerides.

Recipes/Procedures High Volume Tweedy Recipe Product Name Gram ppm Ijsvogel flour 3000 Water 58 Salt Compressed yeast 180 Ascorbic acid Procedure Dough temperature: 29 0 C (dough temp. flour temp.

4°C water temp.) Mixing: 55 WH no vacuum Resting: 5 min. at room temperature Scaling: 500 g (bread), 1350 g (rolls) Resting: 5 min. at room temperature Moulding: Puma I 13 II 18 (bread), Fortuna 3/17/7 (rolls), Glimek (moulding machine) 1:4, 2:3, 3:12, 4:14 Proofing: 70 min. at 43°C, 70% RH. (bread), 50 min.

at 340C, 85% RH. (rolls) Baking: BAGO, 35 min. 5 min. with the steamer open at 2200C, 12 sec. steam (bread), 17 min. at 220°C, 17 sec. steam (rolls) Turkish Batard Recipe Product name Gram ppm Ijsvogel flour 2000 Water 57,00 Compressed yeast Salt Ascorbic acid Procedure: Flour temperature: 15-17 °C (for trials storage day before use at 15 OC) Mixing: 35 min. After 25 min. add salt After 30 min. add yeast Dough temp.: 23-250C Resting: 30 min. Bulk rest on table (table 22 0 C

RH)

Scaling 300 g. pieces Rounding: By hand 1 Resting: 25 min. on table (table 22 0 C 80% start clock when scaling starts Molding Glimek: 1:5, 2:4, 3:15, 4:10 in innerpos.

Proofing: 60 min. 90 min. for this trial at 300C 85% RH Shock test Baking: 20 min. in Bagol 25 min. in Bago2 the last 5 min. is with the damper open for both ovens.

Bagol: 2500C start temp. 5 sec. steam with damper open. Oven temp. down to 2300C at once. Close damper after 1 min.

Bago2: 2750C start temp. 8 sec. steam with damper open. Oven temp. down to 2600C at once. Close damper after 1i min.

Crispy Rolls Recipe Product Name Gram ppm Danish silver flour 2000 Water 58 Compressed yeast 120 Salt 32 Sugar 32 Ascorbic acid Procedure Mixing: Diosna 2 5 min. (depending on flour) Dough temperature: 26°C Scaling: 1350 g Resting: 10 min. at 300C in heating cabinet Moulding: Fortuna 3/17/7 Proofing: 45 min alternatively 90 min at 34°C,

RH.

Baking: 18 min. at 2200C, 8 sec. steam (Bago-oven), 7 sec. steam (Wachtel-oven) (MIWE program 28) (0.35 litre steam, 15 min. at 20000C, min. at 2200°C) US Toast Here a sponge as a pre-mix is prepared, to all of which is then added the dough.

Total flour amount: 1.800,000 g.

Recipe Gr US Flour 900.000 g 50.000% Sponge Water 900.000 g 50.000% Dry 23.400 g 1.300% Yeast Yeast Food 5.400 g 0.300% Enzyme 0.054 g 0.003% Complex ADA 0.036 g 0.002% 1

US

Dough: Flour Water Dry Yeast Sugar Salt Shortening (fat) Sod.Prop.

Dimodan (P100/B) Asc. Acid.

900.000 g 234.000 g 25.200 g 153.000 g 43.200 g 36.000 g 50.000% 13.000% 1.400% 8.500% 2.400% 2.000% 0.450% 0.500% 8.100 SDM-T 9.000 0.072 c 0.004% (=7,200 g to 1000 ml.

Take 10 ml. from the solution) Datem 22-CA- S685 H640

TS-E

662 4.500 300 20 100 0.2500%

PPM

PPM

PPM

Care has to be taken with the water amount added from asc. acid solution and other water based solutions ex. enzymes.

The extra added water amount should be be withdrawn from the water amount on the Dough-side of the recipe.

The enzyme complex is a mix of alpha amylase and amyloclucosidase.

DIMODAN SDM-T (P100/B) (obtained from Danisco A/S) is a distilled monoglyceride.

Procedure: For the Sponge: Water Temp.: 25 0

C

Hobart mixer Step 1, 1min.

Step 2, Imin.

Step 3, Imin.

Fermentation: 2h 15min. 40 oC 80% RH (relative humidity) 45min. in freezer.

For the Dough: Mix all ingredients together Diosna-Mixer: Speed 1, 120 secs Speed 2, 450 secs (or 28 degrees dough temp.) On table rest 5 min.

Weigh out the breads at 450 g pr. bread rest 5 min.

Glimek (moulding machine) adjustments: 1, 2, 14, 11 9 cm read on outer position.

Fermentation: lh 10min. 45 degrees Celsius 90% RH Bake-off: Start temp.= 250 degrees Celsius in 25 min.

Insert the breads and adjust bake-off temperature to 200 degrees Celsius at once.

Baking trials In each trial the dough characteristic, stickiness and all over bread score have been evaluated. The dough characteristic is a total of three different parameters: dough extensibility evaluated just after mixing and again after resting and stickiness after resting. Each parameter has been evaluated by bakers on a scale from 1 where 10 are the best. The score in the examples are a total of these different scores.

Stickiness evaluation has been subjectively evaluated by bakers just after mixing on a scale from 1 to 10, where is the best, meaning non sticky.

All over bread score is a total of an evaluation made on bread crust, -crumb, possible capping and all over energy of the bread. Again each parameter is evaluated on a scale from 1 10, where 10 is the best.

EXAMPLE 1: Testing alternatives in Tweedy bread (UK procedure) The breads were rested for 70 min each and after a full proofing, each bread was shock treated in order to evaluate the shock resistance and thereby the dough stability.

In the baking trials, both pure enzyme solutions and combinations of DATEM and enzymes were tested as alternative to Lipopan F.

Baking trials 4969-29 Specific Shocked Dough Dough All Test volume, volume, charact- sticki over ccm/g ccm/g eristic -ness bread score 0.4% 5.6 4.64 15 4 29 PANODAN GB 0.2% 5.75 4.92 14 4 PANODAN GB, 100 ppm GRINDAMYL H121, 100 ppm TS-E 662 100 ppm TS-E 5.57 4.47 14 4 662, 100 ppm GRINDAMYL H 121, 100 ppm GRINDAMYL EXEL 16 ppm Lipopan F 5.7 4.6 13 4 29 0.2% PANODAN GB, 5.88 4.6 14 4 27 ppm Lipopan F ppm Lipopan 5.65 4.78 14 4 29 F, 100 ppm TS-E 662, 100 ppm GRINDAMYL H 121 ppm Lipopan 5.79 4.82 13 4 29 F, 100 ppm TS-E 662, 100 ppm GRINDAMYL H 121 From the results it can be concluded that PANODAN GB results in a better crust of the product and a product.

The combination of PANODAN GB in combination with xylanase and hexose oxidase yields a beneficial effect.

When using DATEM and/or HOX in combination with GRINDAMYL EXEL 66 the volume is increased significantly and the crust is considerably improved. The test with 0.1% PANODAN GB 100 ppm GRINDAMYL EXEL 66 and 100 ppm TS-E 662 (HOX), gave a significantly good result at the same level as 0.4% PANODAN GB. Use of DATEM clearly gives a significantly positive effect on the crust as compared to pure enzyme solutions.

EXAMPLE 2: Testing alternatives in Turkish batard Baking trials 7258-2 Specific Dough Dough All over Test volume, charact- sticki- bread ccm/g eristic ness score Lipopan F, 60 5.01 14 4 33 ppm TS-E 680 ppm Lipopan F 3.78 15 5 32 100 ppm TS-E 861* 5.03 16 5 44 *A combination of fungal xylanase, degrading lipase and hexose oxidase.

1,3 triglyceride Both from the specific volume in the table as well as the pictures shown in Figures 1 3 it can be concluded that TS-E 861 performs better.

EXAMPLE 3: Testing alternatives in Crispy Rolls: The rolls were fermented at two different fermentation times 45 and 90 min in order to stress the system and thereby give a better picture of the dough strengthening effect of the products. In general it can be said that min of fermentation for a small crispy roll is quite long.

Baking test: 4969-28 Specific Specific Dough Dough All over Test volume volume charact- stickines bread min, 90 min, eristic* s score ccm/g ccm/g 0.3% PANODAN 7.15 8.48 14 5 A2020 ppm Lipopan F 6.83 8.1 14 4 26 100 ppm TS-E 662, 6.98 8.98 14 5 27 100 ppm GRINDAMYL H 121, 100 ppm GRINDAMYL EXEL 16 From the results it can be seen that use of the combination of xylanase, 1,3 triglyceride degrading lipase and hexose oxidase produces beneficial results.

In short fermentation times (45 min.) at certain concentrations PANODAN A2020 and Lipopan F gave comparable volume results. However, 0.3% PANODAN A2020 showed better results with regard to crispiness of the crust and a better dough stability in general. We found that Lipopan F often gave a slightly more "wet" crust.

Using HOX in combination with GRINDAMYL EXEL 66 and PANODAN 660 results in an increase in dough stability.

With prolonged fermentation times (90 min.) all buns become relatively unstable. At some concentrations PANODAN A2020 does, however, give the best result.

EXAMPLE 4: Testing alternatives in US Toast Test of Lipopan F in a US sponge and dough using flour from Mexico hard wheat type. The breads have all been fully proofed and after that each bread have been shock treated in order to evaluate the shock resistance and thereby the dough stability.

Baking trials 7230-1: Test Specific volume, ccm/g Shocked volume, ccm/g Test PANODAN 521 6.88 5.47 ppm Lipopan F 6.16 5.36 ppm Lipopan F 6.44 5.30 ppm Lipopan F 6.28 5.52 0.25% PANODAN 521, 7.15 5.74 ppm GRINDAMYL H 640, 100 ppm TS-E 662 From these tests it is clear that the use of Hox results in a far better dough stability and consequently an increase of volume.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention may be apparent to those skilled in the 39 art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims (37)

1. 2. The method according to claim 1 wherein said S 10 emulsifying agent is a purified lipolytic enzyme from C< Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism.
2. 3. The method according to claim 1 or 2 wherein the Hox is isolated from red algae.
3. 4. The method according to claim 3 wherein the red algae is Iridophycus flaccidum, Chondrus crispus or Euthora cristata. The method according to any of the preceding claims wherein the flour dough comprises at least one further dough additive or ingredient.
4. 6. The method according to claim 5 wherein the further dough additive or ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, butterfat, glycerol and milk fat.
5. 7. The method according to any of the preceding claims wherein the flour dough comprises a hard flour.
6. 8. The method according to any of the preceding claims wherein the product is a bread product. 583527_2.doc 41 Cl 9. The method according to any of the preceding claims Swherein at least one further enzyme is added to the dough Z ingredients, dough additives or the dough. 00
7. 10. The method according to claim 9 wherein the further enzyme is selected from the group consisting of a C xylanase, a cellulase, a hemicellulase, a starch degrading IO w- enzyme, a protease, a lipoxygenase, an oxidoreductase and a lipase. C S11. The method according to claim 10 wherein the further enzyme is a xylanase and/or an amylase.
8. 12. A dough improving composition comprising a Hox and an emulsifying agent wherein said emulsifying agent has triacylglycerol lipase activity.
9. 13. The dough improving composition according to claim 12 wherein said emulsifying agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism.
10. 14. The dough improving composition according to claim 12 or 13 wherein the Hox is isolated from red algae. The dough improving composition according to claim 14 wherein the red algae is Iridophycus flaccidum, Chondrus crispus or Euthora cristata.
11. 16. The dough improving composition according to any one of claims 12-15 wherein the flour dough comprises at least one further dough additive or ingredient.
12. 17. The dough improving composition according to claim 16 wherein the further dough additive or ingredient is 583527_2.doc 42 selected from the group consisting of a vegetable oil, a 0 vegetable fat, an animal fat, shortening, butterfat, -Z glycerol and milk fat. 00
13. 18. The dough improving composition according to claim 16 wherein the further dough additive or ingredient is a hard wheat flour.
14. 19. The dough improving composition according to any one of claims 12 to 18 wherein at least one further enzyme is C-I added to the dough ingredients, dough additives or the dough. The dough improving composition according to claim 19 wherein the further enzyme is selected from the group consisting of a xylanase, a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, an oxidoreductase and a lipase.
15. 21. Use of a dough improving composition according to claim 20 wherein the further enzyme is a xylanase and/or an amylase.
16. 22. The dough improving composition according to any one of claims 12 to 21 in the manufacture of a product made from dough wherein the product is a bread product.
17. 23. Use of a dough improving composition according to any one of claims 12 to 21 wherein said composition improves the rheological and/or machineability properties of flour dough.
18. 24. Use of a dough improving composition according to any one of claims 12 to 21 wherein said composition improves the volume of a baked product made from a flour dough. 583527 2.doc 43 C 25. Use of a combination comprising a Hox and an Semulsifying agent wherein said emulsifying agent has Z triacylglycerol lipase activity in the manufacture of a OO dough wherein said combination improves the rheological and/or machineability properties of the flour dough and/or the quality of the product made from the dough. IND \O
19. 26. Use according to claim 25 wherein said emulsifying M^ agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism.
20. 27. A method of improving the rheological and/or machineability properties of a flour dough and/or the quality of the product made from the dough, comprising adding to the dough a combination comprising a Hox, an emulsifying agent and a further dough additive or ingredient wherein said further dough additive or ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, butterfat, glycerol and milk fat and wherein said further dough additive or ingredient is present in an amount from 1 to 5% by the weight of the flour component of the dough; and wherein said emulsifying agent has triacylglycerol lipase activity.
21. 28. The method according to claim 27 wherein the emulsifying agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism.
22. 29. The method according to claim 27 or 28 wherein the Hox is isolated from red algae. 583527_2.doc 44 The method according to claim 29 wherein the red O algae is Iridophycus flaccidum, Chondrus crispus or Z Euthora cristata. 00 O0
23. 31. The method according to any one of claims 27 to wherein the flour dough comprises at least one further dough additive or ingredient.
24. 32. The method according to claim 31 wherein the further 0 10 dough additive or ingredient is a hard flour. (N
25. 33. The method according to any one of claims 27 to 32 wherein the product is a bread product.
26. 34. The method according to any one of claims 27 to 33 wherein at least one further enzyme is added to the dough ingredients, dough additives or the dough. The method according to claim 34 wherein the further enzyme is selected from the group consisting of a xylanase, a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, an oxidoreductase and a lipase.
27. 36. The method according to claim 35 wherein the further enzyme is a xylanase and/or an amylase.
28. 37. A dough comprising a dough improving composition wherein said dough improving composition comprises a Hox, an emulsifying agent and a further dough additive or ingredient wherein said further dough additive or ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, butterfat, glycerol and milk fat and wherein said further dough additive or ingredient is present in an amount of from 1 to 5% by the weight of the flour component of the 583527_2.doc N dough; and wherein said emulsifying agent has O triacylglycerol lipase activity. 00 0 38. The dough according to claim 37 wherein the emulsifying agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a r genetically modified Aspergillus oryzae microorganism. C 39. The dough according to claim 37 or 38 wherein the Hox S 10 is isolated from red algae. The dough according to claim 39 wherein the red algae is Iridophycus flaccidum, Chondrus crispus or Euthora cristata.
29. 41. The dough according to any one of claims 37 to wherein the dough improving composition comprises at least one further dough additive or ingredient.
30. 42. The dough according to claim 41 wherein the further dough additive or ingredient is a hard wheat flour.
31. 43. The dough according to any one of claims 37 to 42 wherein at least one further enzyme is added to the dough ingredients, dough additives or the dough.
32. 44. The dough according to claim 43 wherein the further enzyme is selected from the group consisting of a xylanase, a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, an oxidoreductase and a lipase. The dough according to claim 44 wherein the further enzyme is at least xylanase and/or at least an amylase. 583527_2.doc 46 N 46. The dough according to any one of claims 37 to O wherein said dough comprise a further emulsifier. z 00 47. A dough improving composition for use in the preparation of a dough according to any one of claims 37 to 46 wherein said dough improving composition comprises a C Hox, an emulsifying agent and a further dough additive or \O ingredient wherein said further dough additive or Cc ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, Sbutterfat, glycerol and milk fat; and wherein said emulsifying agent has triacylglycerol lipase activity.
33. 48. The dough improving composition according to claim 47 wherein the emulsifying agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism.
34. 49. Use of a dough improving composition according to claim 47 in the manufacture of a product made from dough wherein the product is a bread product. Use of a dough improving composition according to claim 47 wherein said composition improves the rheological and/or machineability properties of flour dough.
35. 51. Use of a dough improving composition according to claim 47 wherein said composition improves the volume of a baked product made from a flour dough.
36. 52. Use of a combination comprising a Hox, an emulsifying agent and a further dough additive or ingredient wherein said further dough additive or ingredient is selected from the group consisting of a vegetable oil, a vegetable fat, an animal fat, shortening, butterfat, glycerol and milk
37. 5835272.doc 47 CI fat and wherein said further dough additive or ingredient Sis present in an amount of from 1 to 5% by the weight of Z the flour component of the dough, and wherein said 00 combination improves the rheological and/or machineability properties of the flour dough and/or the quality of the product made from the dough; and wherein said emulsifying C agent has triacylglycerol lipase activity. \O IND S53. The use according to claim 52 wherein the emulsifying agent is a purified lipolytic enzyme from Fusarium oxysporum produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism. 54. A method or dough improving composition in accordance with the present invention and substantially as described herein. 583527_2.doc