FIELD OF THE INVENTION

The present invention relates to a method of improving prop¬ erties of a dough and/or of a baked product made from dough, to a dough and baked product produced by the method, as well as a premix and a bread-improving or dough-improving composition useful for preparing dough.

BACKGROUND OF THE INVENTION

In the bread-making process it is known to add bread-improving and/or dough-improving additives to the bread dough, the action of which, inter alia, results in improved texture, volume, flavour and freshness of the bread as well as improved machinability of the dough.

In recent years a number of enzymes have been used as dough and/or bread improving agents, in particular enzymes which act on components present in large amounts in the dough. Examples of such enzymes are found within the groups of amylases, proteases and cellulases. Also pentosanases such as xylanase has been suggested for use in baking, cf., for instance, EP 0 493 850, EP 0 487 122, WO 92/01793, WO 91/18977, WO 91/19782 and WO 92/17573.

EP 0 396 162 discloses a bread improver which comprises cellulase, in particular xylanase, in combination with an oxidase or a peroxidase, the latter being exemplified as a horse radish peroxidase. The use of the horse radish peroxidase is shown to improve certain properties of the dough, but the bread prepared from this dough has a decreased volume as compared to that of the control baked without peroxidase addition.

DISCLOSURE OF THE INVENTION

It has now surprisingly been found that when a microbial peroxidase is used for the preparation of dough, it is possible not only to improve properties of the dough, but also prop- erties of the baked product prepared therefrom.

Accordingly, in a first aspect the present invention relates to a method of preparing a dough comprising adding an enzyme prep¬ aration to the dough and/or to any ingredient of the dough and/or to any mixture of the dough ingredients, in which method the enzyme preparation comprises a microbial peroxidase enzyme.

In the present context, the term "peroxidase" (EC 1.11.1) designates an enzyme catalyzing the conversion of a peroxide such as hydrogen peroxide into its basic constituents (e.g. H ₂ 0 ₂ into H ₂ 0 and 0 ₂ ) . The enzymatic activity of peroxidase (and other enzymes used herein) may be determined by standard assays, examples of which are described in the Materials and Methods section below.

In addition to the improved effects obtain by using a microbial peroxidase in the present invention, the fact that the peroxidase is of microbial origin has the further important advantage that, normally, the microbial enzyme is easier to produce on a large scale than a peroxidase of, e.g., plant origin. Furthermore, microbial peroxidases may generally be obtained in a higher purity than peroxidases of other origins, resulting in a lower amount of undesirable enzymatic side- activities.

The term "improved properties" as used about the effect obtained on dough and/or baked products made from dough by the method of the invention, includes any property which may be improved by the action of the microbial peroxidase, important examples of which are an increased volume, an improved fresh¬ ness (in terms of antistaling) and an improved structure and sofness of the baked product, as well as an increased dough

stability and thereby improved machinability of the dough (i.e. a less sticky dough) . The improved machinability is of particu¬ lar importance in connection with dough which is to be pro¬ cessed industrially. The improved properties may, of course, be evaluated by comparison with dough and/or baked products prepared without addition of peroxidase in accordance with the present invention.

The microbial peroxidase enzyme to be used in the method of the invention may be derived from bacteria or fungi (including filamentous fungi and yeasts) . Examples of suitable fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g. Fusarium , Humicola , Tricoderma , Myrothecium , Verticillum , Arthromyces, Caldariomyces , Ulocladi- um , Embellisia , Cladosporium or Dreschlera , in particular Fusarium oxysporum (DSM 2672) , Humicola xnsolens, Trichoderma resii , Myrothecium verrucana (IFO 6113), Verticillum alboatrum , Verticillum dahlie , Arthromyces ramosus (FERM P-7754) , Caldari¬ omyces fumago , Ulocladium chartarum , Embellisia allior Dre¬ schlera halodes; strains belonging to the subdivision Basidio- mycotina, class Basidiomycetes, e.g. Coprinus , Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371) , Coprinus macrorhizuε , Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus) , e.g. T . versicolor (e.g. PR4 28-A) ; or strains belonging to the subdivision Zygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particular Mucor hiemalis. Examples of suitable bacteria include strains of the order Actinomycetales, e.g. Streptomyceε εpheroideε (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium ; strains of Bacillus sp., e.g. Bacillus pumilus (ATCC 12905) , Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri , Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-ll) ; or strains of Myxococcus sp., e.g. M. virescens . Other potential sources of useful particular peroxidases are listed in Saunders B C, et al . , Peroxidase, London, 1964, pp. 41-43.

The peroxidase may be obtained from the microorganism in qu¬ estion by use of any suitable technique. For instance, a peroxidase preparation may be obtained by fermentation of a microorganism and subsequent isolation of a peroxidase containing preparation from the fermented broth or microor¬ ganism by methods known in the art, but more preferably by use of recombinant DNA techniques as known in the art. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector capable of expressing and carrying a DNA sequence encoding the peroxidase in question, in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture.

The dosage of the enzyme preparation to be used in the method of the present invention should be adapted to the nature and composition of the dough in question. Normally, the enzyme preparation is added in an amount corresponding to 500- 500,000 PODU/kg of flour. The Peroxidase Units (PODU) may be determined as described in the Materials and Methods section below.

A peroxidase activity below 500 PODU/kg of flour is believed to provide no substantial effect, while a peroxidase activity above 500,000 PODU/kg of flour is believed to result in an over-modification of the dough, e.g. a dough which is to rigid.

It is preferred that the enzyme preparation is added in an amount corresponding to 1,000-200,000 PODU/kg of flour, and in particular about 5,000-150,000 PODU/kg of flour such as 6,000- 150,000 PODU/kg of flour.

The enzyme preparation to be used in the method of the invention may comprise one or more additional enzyme activ- ities. Alternatively, one or more additional enzyme activities may be added separately from the enzyme preparation comprising the peroxidase.

Examples of other enzymes are a cellulase, a hemicellulase, a pentosanase (useful for the partial hydrolysis of pentosans which increases the extensibility of the dough) , a glucose oxidase (useful for strengthening the dough) , a lipase (useful for the modification of lipids present in the dough or dough constituents so as to soften the dough) , a laccase (useful for gluten strengthening) , a protease (useful for gluten weakening, in particular when using hard wheat flour) , a peptidase and/or an amylase, e.g. α-amylase (useful for providing sugars fermentable by yeast) .

The other enzymes are preferably of microbial origin and may be obtained by conventional techniques used in the art as men¬ tioned above.

The optional other enzyme activities may be dosed in accordance with established baking practice. As concerns laccase, which has not been used for baking prior to the present invention (as far as the inventor is aware) a suitable dosage is in the range of 5-100,000 Laccase Units (LACU) , when used in combination with peroxidase and optionally other enzymes.

In accordance with the invention, it is preferred that the peroxidase is used in combination with a xylanase and/or a glucose oxidase. The xylanase is preferably of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of Aεpergillus , in particular of A . aculeatus , A. niger (cf. WO 91/19782) , A. awamori (WO 91/18977) , or A . tubigensis (WO 92/01793) , or from a strain of Humicola , e.g. H. insolens (WO 92/17573, the contents of which is hereby incorporated by reference) . Pentopan® and Novozym 384® (both from Novo Nordisk A/S) are commercially available xylanase preparations produced by Trichoderma reesei .

Analogously, it is preferred that the glucose oxidase is of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. niger, or Penicillium.

When the peroxidase is used in combination with the xylanase and/or glucose oxidase in the method of the invention a suitable dosage of the peroxidase and xylanase and/or glucose oxidase is 1,000-100,000 PODU/kg of flour, 10-500 FXU/kg of flour and/or 20-1000 GODU/kg of flour such as 50-1000 GODU/kg of flour.

The xylanase activity FXU (Farbe-Xylanase-Units) and the glucose oxidase activity (GODU) may be determined by the procedure given in the Materials and Methods section below.

The other enzyme components may be added separately or may be present in the peroxidase preparation by either being added thereto or by being produced or recovered together with the peroxidase from the microbial source in question. Thus, any other components present in the enzyme preparation may be of a different or of the same origin as the peroxidase. Besides the above mentioned additional enzyme activities a microbially produced peroxidase preparation may contain varying minor amounts of other enzymatic activities inherently produced by the producer organism in question.

The enzyme preparation to be used in the method of the in¬ vention may be in any form suited for the use in question, e.g. in the form of a dry powder or granulate, in particular a non- dusting granulate, a liquid, in particular a stabilized liquid, or a protected enzyme. Granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661,452 (both to Novo Indus- tri A/S) , and may optionally be coated by methods known in the art. Liquid enzyme preparations may, for instance, be stabil¬ ized by adding nutritionally acceptable stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods. Pro¬ tected enzymes may be prepared according to the method dis¬ closed in EP 238,216.

Normally, for inclusion in pre-mixes or flour it is advan¬ tageous that the enzyme preparation 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.

In accordance with the invention, the enzyme preparation may be used in combination with conventional emulsifiers. Emulsifiers serve to improve dough extensibility and may also be of some value for the consistency of the resulting bread, making it easier to slice, as well as for its storage stability. 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.

As mentioned above the enzyme preparation is added to any mixture of dough ingredients, to the dough, or to any of the ingredients to be included in the dough, in other words the enzyme preparation may be added in any step of the dough preparation and may be added in one, two or more steps, where appropriate. However, the enzyme preparation should not be added together with any strong chemical or under conditions where the enzyme is inactivated.

The enzyme preparation may be added as such to the mixture from which the dough is made, or may, alternatively, be added as a constituent of a dough-improving and/or a bread-improving composition. The dough-improving and/or bread-improving composition may be any conventionally used composition, e.g. comprising one or more of the following constituents:

A milk powder (providing crust colour) , gluten (to improve the gas retention power of weak flours) , an emulsifier (such as mentioned above) , granulated fat (for dough softening and consistency of bread) , and oxidant (added to strengthen the gluten structure; e.g. ascorbic acid, potassium bromate, potassium iodate or ammonium persulfate) , an amino acid (e.g.

cysteine) , a sugar, and salt (e.g. sodium chloride, calcium acetate, sodium sulfate or calcium sulfate serving to make the dough firmer) .

Typically, the dough-improving and/or bread-improving composi- tion is added in an amount corresponding to about 0.1-5%, such as 0.1-3% of the added flour.

The handling of the dough and/or baking is 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 tempera¬ ture 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-tempera- ture and long-time fermentation method, a frozen dough method, the Chorleywood Bread process, and the Sponge and Dough process.

In a further aspect the present invention relates to a dough or a baked product prepared by the method of the present inven- tion. The dough and the baked product of the invention has improved qualities as defined above as compared with products which has not been prepared according to the invention.

The dough and/or baked product prepared by the method of the invention are normally based on wheat meal or flour, optionally in combination with other types of meal or flour such as corn flour, rye meal, rye flour, oat flour or meal, soy flour, sorghum meal or flour, or potato meal or flour.

However, it is contemplated that the method of the present invention will function equally well in the preparation of dough and baked products primarily based on other meals or flours, such as corn meal or flour, rye meal or flour, or any other types such as the types of meal or flour mentioned above.

In the present context, the term "baked product" is intended to include any product prepared from dough, either of a soft or a crisp character. Examples of baked products, whether of a white, light or dark type, which may advantageously be 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, pita bread, tacos, cakes, pan¬ cakes, biscuits, crisp bread and the like.

The dough of the invention may be of any of the types discussed above, and may be fresh or frozen.

From the above disclosure it will be apparent that the dough of the invention is normally 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 preferred to leaven the dough by adding a suitable yeast culture such as a culture of Saccharomyces cerevisiae (baker's yeast) . Any of the commercially available S . cereviciae strains may be employed.

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 comprising a microbial peroxidase. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g. any of the additives, including enzymes, mentioned above.

In a further aspect the present invention relates to a dough- improving and/or bread-improving composition comprising a microbial peroxidase enzyme.

The peroxidase to be used as a dough-improving and/or bread- improving agent is preferably a peroxidase as defined above, i.e. a peroxidase derivable from bacteria or fungi (including filamentous fungi or yeasts) , and in particular from a strain of Coprinus , e.g. C . cinerius (EP 179,486), a strain of

Myxococcus , a strain of Curvularia, or a strain of Myrothecium .

The peroxidase may be included in the dough-improving or bread- improving composition in an amount corresponding to 50-150,000 PODU/g of bread-improving and/or dough-improving composition.

Examples of suitable dough-improving and/or bread-improving compositions to which the peroxidase may be added are known in the art.

In a further aspect the invention relates to the use of a microbial peroxidase for improving properties of a dough and/or a baked product made therefrom. The type of peroxidase as well as the manner in which it may be used is described in detail above.

In a final aspect the invention relates to the use of peroxidase 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 peroxidase used in a similar dosage as that described above. The peroxidase is preferably of microbial origin, e.g. as disclosed herein. It is contemplated that when used in the preparation of pasta the peroxidase results in a strengthening of the gluten structure and thus a reduction in the dough stickiness and an increased dough strength.

METHODS

Enzymes

Peroxidase: Recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae (as described in WO 92/16634) . The peroxidase was found to be without any detectable amount of the following enzymatic activities: Glucose oxidase, amylase (bacterial or fungal) , xylanase, lipase, protease, or laccase activities.

Glucose oxidase: SP 358® (an A . niger glucose oxidase) avail¬ able from Novo Nordisk A/S, Denmark.

Xylanase: A xylanase produced by the Humicola xnsolens strain DSM 1800 available from the Deutsche Sammlung von Mikroor- 5 ganismen und Zellkulturen GmbH and further described in EP 507 723.

Determination of peroxidase activity (PODU)

Peroxidase activity is determined by an assay based on the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate)

10 (ABTS®) by hydrogen peroxide (as described by Bergmeyer H.U., Methods of enzymatic analysis, 3rd edition, vol. Ill, p. 286- 293, 1983, modified). The greenish-blue colour produced is photometered at 418 nm. The analytical conditions are 0.88 mM hydrogen peroxide, 1.67 mM ABTS, 0.1 M phosphate buffer, pH

157.0, 30°C incubated for 3 minutes.

1 peroxidase unit (PODU) is the amount of enzyme that catalyses the conversion of lmymol hydrogen peroxide per minute at these conditions.

Determination of xylanase activity (FXU)

20 The endo-xylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan (beech) substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) , pH 6.0. The incubation is performed at 50°C for 30 min. The background of non-degraded dyed

25 substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity.

The endoxylanase activity of the sample is determined rela¬ tively to an enzyme standard.

Determination of glucose oxidase activity (GODU)

Glucose oxidase activity is determined by use of an assay, in which a glucose oxidase containing sample is incubated with beta-D-glucose (16.2 g/1, 90 mM) in the presence of oxygen (30°C, 20 min.), whereby gluconolacton and hydrogen peroxide are formed. Subsequently, 2,2-Azino-di-3-ethylbenthiazolin)-6- sulphonate (ABTS) is oxidized with the hydrogen peroxide formed in the presence of peroxidase (No. P8125, Sigma (approx. 80 U/mg) (20 min., 30° C) , whereby a green-blue colour is formed. The sample is finally analysed photometrically at 418 nm, whereby the amount of peroxidase formed by the action of glucose oxidase may be determined by comparison to a hydrogen peroxide standard.

One Glucose Oxidase Unit (GODU) is defined as the amount of enzyme, which under standard conditions liberates 1 μ ol hydrogen peroxide per minute.

Preparation of bread

Basic recipe

Flour 1500 g Salt 22.5 g

Yeast 75 g

Water 810 g

The flour was wheat flour of the type termed "Reform" and "Prima", respectively, supplied by Havnemøllerne, Denmark. The type of flour used is indicated in the examples. The yeast was conventional baker's yeast.

Procedure

Dough mixing (Spiral mixer) 2 + 6 min.

The mixing time was determined and adjusted by a skilled baker so as to obtain an optimum dough consistence under the testing conditions used.

Dough temperature 27°C +/- 1 Resting, 30°C 15 min. Scaling

Resting 32-34°C, 80% RH 10 min. Forming

Proofing 32-34°C, 80% RH 45 min. for rolls

40 min. for loaves

Baking (steamed) 225°C 15 min. for rolls

30 min. for loaves

Evaluation of dough and baked products

Dough and baked products were evaluated as follows:

Volume index: The volume of 30 rolls 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 volume of 30 rolls

Specific vol. index = -*100 specific volume of 30 control rolls

hardness of sample

Hardness index = *100 hardness of control

The crumb structure is evaluated visually according to the following scale: non-uniform + uniform/good ++ very good +++

The softness of bread crumb is measured by a SMS-Texture Analyzer. A plunger with a diameter of 45 mm is pressed on the

middle of a 20 mm thick slice of bread, The force needed for the plunger to depress the crumb 5 mm with a speed of 2.0 mm/s is recorded and it is expressed as the crumb firmness. The lower the value, the softer is the crumb. Four slices of each bread are measured and the mean value is used.

EXAMPLE 1

Rolls were prepared by use of the flour Reform (available from Havnemøllerne A/S, Denmark) using the procedure described above. The following results were obtained:

Peroxidase was found to improve the dough consistency so that the dough became easier to handle. Furthermore, the dough stability was improved and the baked bread had an improved crumb structure and a softer crumb after storage than the control.

EXAMPLE 2

Rolls were prepared by use of the flour Prima (Havnemøllerne A/S, Denmark) using the procedures described above. The follwo- ing resluts were obtained:

From the above results it is evident that the combined use of xylanase and peroxidase results in a substantial volume improvement. Furthermore, it was observed that the combined use of peroxidase and xylanase results in a nice dough with an improved stability (as compared to the use of xylanase alone which results in a slightly sticky dough) . The baked bread had an improved crumb structure and a softer crumb after storage than the control.