TITLE: DOUGH WITH FRUCTAN AND FRUCT AN-DEGRADING ENZYME

FIELD OF THE INVENTION

The present invention relates to a dough comprising at least one fructose-containing polysaccharide and at least one enzyme capable of degrad ing the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose; it also relates to a method of making said dough, a baked product made from said dough, a method of making a baked product, and to uses of an enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose in making a dough or a baked product. More particularly, it relates to such a process where the bread has an increased softness even after several days of storage.

BACKGROUND OF THE INVENTION

It has been described in literature that flour, especially rye flour, can contain up to 4% of fructose-containing polysaccharides (Fretzdorff, B. Welge, N. Abbau von getreideeigenen Fructanen wahrend der Herstellung von Roggenvollkornbrot, Getreide Mehl und Brot, 57, 2003, 3, 147-151 ; as well as Nardi, S. et. al, Nutritional benefits of developing cereals for functional foods, Cereal Research Communications, 2003, 31 , Nos 3-4, 445-453). This was also confirmed in a presentation at the 2004 American Association of Cereal Chemists "AACC" conference in San Diego about rye dietary fibre and its influence on the baking activity of rye given by Hansen of the University of Copenhagen. It was said that rye flour contains a lot of fibre, 14.7 - 20.9%, and also 4.5 - 6.4% of fructan, both of them influencing the overall water absorption and giving structure to rye bread as there is no gluten present.

Fructozyme ^® L is a fructan degrading enzyme product that is produced by submerged fermentation of Aspergillus niger CBS 594.94 (patent deposit). Fructozyme ^® L is a current product in the starch industry (Novozymes A/S). This enzyme product has endo- (inulinase; EC 3.2.1.7) as well as exo-activity (fructan beta-fructosidase; EC 3.2.1.80), which means that it degrades its substrate fructan (inulin or levan) into short chained fructo-oligosaccharides (FOS) and fructose.

SUMMARY OF THE INVENTION

The inventors found that increased softness can be achieved in baked products made from dough comprising fructans by adding an enzyme to the dough that is capable of degrading the fructans into short-chained fructo-oligosaccharide and fructose. Accordingly, the invention provides in a first aspect a dough comprising at least one fructose-containing polysaccharide and at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose.

In a second aspect the invention provides a baked product made from a dough as defined in the first aspect.

A third aspect of the invention relates to a pre-mix for dough comprising at least one enzyme capable of degrading fructose-containing polysaccharide into short-chained fructo- oligosaccharide and fructose.

A fourth aspect relates to a method of preparing a dough comprising at least one fructose-containing polysaccharide, said method comprising adding to the dough at least one enzyme capable of degrading fructose-containing polysaccharide into short-chained fructo- oligosaccharide and fructose. In a fifth aspect the invention relates to a method of preparing a baked product, said method comprising baking a dough as defined in the first aspect or a dough prepared as defined in the fourth aspect.

A final aspect of the invention relates to a use of an enzyme capable of degrading fructose-containing polysaccharides into short-chained fructo-oligosaccharides and fructose in the production of a dough or a baked product made from dough comprising at least one fructose-containing polysaccharide.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the softness of rye/wheat bread containing varying dosages of lnulinase (1-80ppm) versus a control bread with no inulinase. Addition of inulinase gave consistently softer bread than the control. 20ppm of Inulinase showed a significant improved softness compared to the control at all 4 measurements (day 1 , 3, 7 and 9).

Figure 2 shows that there was no difference in terms of overall elasticity between the control and any dosage of inulinase used in the breads of figure 1. Only on day 7 is the control significantly more elastic than bread containing various dosages of inulinase.

Figure 3 shows the procedure for softness and elasticity measurements with a Texture Analyzer.

DETAILED DESCRIPTION OF THE INVENTION Dough

The dough of the invention generally comprises rye flour or rye meal and/or other types of meal, flour or starch such as wheat flour, wheat meal, corn flour, corn starch, 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 par-baked.

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 chemical

leavening agents, e.g., 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, e.g.: proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg whites); 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 (triglyceride) such as granulated fat or shortening, but the invention is particularly applicable to a dough where less than 1 % by weight of fat (triglyceride) is added, and particularly to a dough which is made without addition of fat.

The dough may further comprise an emulsifier such as 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, or lysolecithin,.

Accordingly, the first aspect of the invention relates to a dough comprising at least one fructose-containing polysaccharide and at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose. In a preferred embodiment, the dough of the first aspect comprises rye flour; preferably 10% (w/w) or more of the total flour content is rye flour, preferably at least 15 %, 20%, 25%, 30%, 35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or preferably at least 95% (w/w) of the flour is rye flour.

In another preferred embodiment of the first aspect of the invention, the at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo- oligosaccharide and fructose is an enzyme composition comprising inulinase, EC 3.2.1.7, and/or fructan beta-fructosidase, EC 3.2.1.80; preferably the at least one enzyme is an enzyme composition comprising an inulinase produced by submerged fermentation of Aspergillus niger. The enzyme may have exo- and/or endo activity on the fructose-containing polysaccahride, preferably the enzymes has both exo and endo activity on the fructose- containing polysaccahride (such as, FRUCTOZYME L, available from NOVOZYMES A/S, Denmark).

It is preferred that the dough of the first aspect of the invention comprises up to 500 ppm of the at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose; preferably up to 400 ppm, 300 ppm, 200 ppm, 100 ppm, 80 ppm, 40 ppm, 20 ppm, or more preferably up to 10 ppm of the at least one enzyme.

Another aspect of the invention relates to a method of preparing a dough comprising at least one fructose-containing polysaccharide, said method comprising adding to the dough at least one enzyme capable of degrading fructose-containing polysaccharide into short- chained fructo-oligosaccharide and fructose; preferably the dough comprises rye flour; and preferably 10% (w/w) or more of the total flour content of the dough is rye flour, preferably at least 15 %, 20%, 25%, 30%, 35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or preferably at least 95% (w/w) of the flour is rye flour.

In a preferred embodiment of the invention, the at least one enzyme is an enzyme composition comprising inulinase, EC 3.2.1.7, and/or fructan beta-fructosidase, EC 3.2.1.80; more preferably the at least one enzyme is an enzyme composition comprising an inulinase produced by submerged fermentation of Aspergillus niger.

Yet another preferred embodiment relates to the method of the above aspect, wherein up to 500 ppm is added to the dough of the at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose; preferably up to 400 ppm, 300 ppm, 200 ppm, 100 ppm, 80 ppm, 40 ppm, 20 ppm, or more preferably up to

10 ppm is added to the dough of the at least one enzyme.

Additional enzyme

Optionally, an additional enzyme may be used together with the fructan degrading enzyme. The additional enzyme may be an amylase, such as an a alpha-amylase, a maltogenic amylase, amyloglucosidase, a beta-amylase, a cyclodextrin glucanotransferase, or the additional enzyme may be a peptidase, in particular an exopeptidase, a transglutaminase, a lipolytic enzyme, a cellulase, a hemicellulase, in particular a pentosanase such as xylanase, a protease, a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95/00636, a glycosyltransferase, a branching enzyme (1 ,4- alpha-glucan branching enzyme), a 4-alpha-glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase.

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.

The maltogenic amylase may be derived from Bacillus stearothermiphilus as described in EP 494233 or a variant thereof as described in WO 99/43794.

The lipolytic enzyme may have lipase activity (EC 3.1.1.3), phospholipase A1 activity, phospholipase A2 activity and/or galactolipase activity.

Baked product

The process of the invention may be used for any kind of baked product prepared from dough, either of a soft or a crisp character, either of a white, light or dark type. Examples are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls,

French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and the like.

Accordingly, the second aspect of the invention relates to a baked product made from a dough as defined in the first aspect.

A preferred embodiment relates to the baked product of the second aspect, which has an increased softness after 3 days or more, when compared to an otherwise identical control bread or baked product without the at least one enzyme capable of degrading the at least one polysaccharide into short-chained fructo-oligosaccharide and fructose, as determined herein; preferably after 5 days or more; 7 days or more; or 9 days or more.

In another aspect, the invention relates to a method of preparing a baked product, said method comprising baking a dough as defined in the first aspect or a dough prepared as defined in any of the above-mentioned aspects.

P re-mix

The present invention further relates to a pre-mix comprising flour together with at least one fructan degrading enzyme. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g. any of the additives, including enzymes, mentioned above.

Accordingly, the third aspect relates to a pre-mix for dough comprising at least one enzyme capable of degrading fructose-containing polysaccharide into short-chained fructo- oligosaccharide and fructose.

A preferred embodiment of the third aspect relates to a premix, wherein the at least one enzyme is an enzyme composition comprising inulinase, EC 3.2.1.7, and/or fructan beta- fructosidase, EC 3.2.1.80; more preferably the at least one enzyme is an enzyme composition comprising an inulinase produced by submerged fermentation of Aspergillus niger.

Enzyme preparation The invention provides an enzyme preparation comprising a fructan degrading enzyme, for use as a baking additive in the process of the invention. The enzyme preparation is preferably in the form of a granulate or agglomerated powder. It preferably has a narrow particle size distribution with more than 95 % (by weight) of the particles in the range from 25 to 500 microns. Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the amylase 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 NaCI or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.

An aspect of the invention relates to a use of an enzyme or enzyme composition capable of degrading fructose-containing polysaccharides into short-chained fructo- oligosaccharides and fructose in the production of a dough or a baked product made from dough comprising at least one fructose-containing polysaccharide; preferably the enzyme is an enzyme composition comprising inulinase, EC 3.2.1.7, and/or fructan beta-fructosidase, EC 3.2.1.80; more preferably the enzyme is an enzyme composition comprising an inulinase produced by submerged fermentation of Aspergillus niger.

EXAMPLES

Example 1 - Inulinase in wheat/rye bread (50% rye flour)

Mixed rye/wheat sourdough bread

Mixed wheat/rye dough was prepared as outlined below, containing 50% lntermill Rye flour (Ash 1370) May 04 and Wheat flour (Ash 720) July 04. All dough contained 70ppm Pentopan ^® 500 and 60ppm ascorbic acid based on wheat flour weight.

All ingredients are added together; they are then mixed to optimum development; the first fermentation takes place at 24°C/15min; the dough is divided into 40Og (free-standing) / 50Og - 70Og (lidded); rounded/rested; molded/panned; proofed: Bread 50-80 min. 32- 34°C/80%rH; and finally baked.

Recipe and procedure:

Note: to compensate for variation in ingredient quality, minor changes maybe implemented, based on the bakers expertise.

^• Instant sour dough (Fermiplus from Agrano) is dosed based on the required S7kg rye flour. Every new batch need to be analyzed first of its acid content and pH.

Water addition as well as proofing time various depends on the rye flour content.

Work flow:

• Enzymes are added directly to the water in the mixing bowls.

• Add rest of the ingredients.

• Start the three Fork mixers at the same time and mix gently all ingredients to a dough.

• Directly after dough is finished mixing, measure the dough temperature and do the sensory dough evaluation.

• Target dough temp. 26.5°C (+/- 0.5 ⁰ C)

• After 10min. of resting time, scale 4x 40Og for open top pan bread. Please consider the weight changes when making lidded pan bread. Scale for lidded pans: 50Og for 30% rye, 60Og for 50% rye and 70Og for 70% rye.

• Round scaled dough pieces up by hand and let it rest for 5min. (covert).

Mold the dough pieces into basked size ropes, and set into the, with rye flour floured basked or directly in the greased baking pans.

Place into the fermentation cabinet for 1 hour at 32-34°C + 80%rH.

Only the free standing breads are baked with oven steam.

Volume measurements and sensory evaluations are done as outlined below after the baked product has cooled.

Setting of equipment:

The breads were stored at 24°C and measured after 1 , 3, 7 and 9 days. The baking tests were repeated to enable statistical LSD analysis. The LSD value indicates the significance according to statistical calculation, meaning that if the difference between two treatments is greater than the LSD this difference is significant.

Softness and elasticity measurement of mixed wheat/ rye bread was carried out using a modified version of AACC method 74_09 (see below).

Moistness was evaluated by moistness perception test after 9 days of storage through a sensory panel (5 persons) according to RnD procedures using a two-sided Directional

Difference Test (see below).

The term "internal bread characteristics" is defined as crumb structure, which is a subjective evaluation of uniformity, color, cell wall thickness, cell form and grain of crumb by experienced bakers. The term "external bread characteristics" is defined as crust color.

Dough parameters:

In terms of dough stickiness and softness there was no difference between the control dough and doughs containing 1 to 80ppm of inulinase. Dough containing 5% fructose showed increased stickiness and less extensibility as well as elasticity compared to a control and the doughs containing Inulinase.

External and internal bread characteristics:

No influence of inulinase regardless of the dosage (1-80ppm) was seen in terms of changes in crust colour, internal or external bread characteristics compared to a control. Only the bread containing 5% fructose seemed to show a darker crust colour.

Shelf life - crumb texture:

As seen in figure 1 , in terms of softness bread containing varying dosages of Inulinase (1-80ppm) gave consistently softer bread than the control. 20ppm of Inulinase showed a significant improved softness compared to the control at all 4 measurements (day 1 , 3, 7 and 9).

As seen in figure 2, there was no difference in terms of overall elasticity between the control and any dosage of Inulinase used. Only on day 7 is the control significantly more elastic than bread containing various dosages of Inulinase.

The bread containing various dosages of Inulinase and the bread with 5% of fructose seemed to have a more moist crumb compared to the control. This was noticed because during texture analysis these slices stuck to the piston after the measurement. The bread was also evaluated by hand after 9 days of storage. It was difficult to conclude whether there was a difference in moistness between the control, the breads with increasing dosages of inulinase and bread containing 5% of fructose.

Example 2. Bread volume measurement

Volume measurements are taken after baked product has cooled.

^■ Baked goods are weighed with a balance (analytical equipment) and recorded.

^■ Volume of baked goods is measured by seed displacement operated with local manual equipment (mechanical hardware) and recorded in ml or cc (cubic centimetre). Beside the seed displacement, laser equipment such as TexVol can be used to measure the volume of baked goods, here; the results are recorded in the equipment software.

^■ The specific volume of baked goods is calculated by dividing the volume in cubic centimetres by the weight in grams (ml/g or cc/g).

^■ Calculation example:

Volume = 1400ml Weight =300g

1400 / 300 = 4.66ml/g Specific volume = 4.66ml/g

Example 3. Softness and elasticity measurement with texture analyzer The following protocol is a modified version of the AACC method 74-09. Scope

An analytical method to measure pan bread/loaf crumb softness and elasticity. Compared to the AACC method this modified method causes less standard deviation within one loaf of bread. Absolute values for softness and elasticity are different to the AACC method, while relative values and tendencies are comparable.

Procedure

After baking and cooling, baked goods are sealed in moisture-proof plastic bags. Specific days of measurement respective length of bread storage and sample preparation are carried out according to the individual baking procedures.

In order to avoid the influence of different volumes on softness, the volume of bread loaves may not differ by more than ±3% respectively lidded pans are used.

Softness (in grams) is defined as the force needed to press a probe 6.25 mm into a crumb of a 25 mm thick slice of bread (P1 ). The Elasticity in % is expressed as: (P3/P2) ^* 100%, wherein

P1 = The force needed to press a probe 6.25 mm into the bread crumb (see Figure 3). P2 = The maximum force needed to press the probe 7 mm into the crumb (see Figure 3). P3 = The force needed to keep the probe at 7 mm for 30 seconds (see Figure 3).

Equipment

1. Electric knife or bread slicing machine

2. Texture Analyzer: Stable Micro System TA.XT2 Software: XT.RA Dimension (SMS) Acrylic probe (40 mm) with edges blunted to remove sharpness.

Setup - Texture Analyzer

Measure force in compression: Option Hold until time

Force units Grams

Test speed 1 mm/sec

Distance 7 mm

Time 32 sec

Method - Texture Analyzer

Force vs. time:

Force threshold 5 grams

Result file Graph

Peaks 2

Ratios 1

Contact force 5 grams

Macro - Texture Analyzer P1 Peak at 6.25 mm

P2 Peak at 7mm Hold at 7 mm for 30 sec

P3 Anchor after 30 sec at 7 mm

P3/P2 Elasticity as a ratio in %

Example 4. Sensory analysis

The evaluation is done subjective (not analytic) and is based on experience of the trained responsible employs (Baker / Lab technician; At the annual yearly s Baker s meeting an independent calibration trial is done in order to insure compaiablo sensory iosults trough ai! Coioal Food customer solutions baking laboratories

Note" not all of the defecations points are to be found in each test tπal the points are chosen from this list as they apply to tho various baking procedures.

Parameters of dough and baked product evaluation.

Every test tπa! contains a reference to which the rest of the test samples are compared to.

This moans, tho evaluation score is always relative to tho reference, the roferenco can various from test set-up to test set-up due to different flour quality or added ingredients

The assessment area is between 1 and 10. whereas the reference Is always set with Ih e number 5

Dough evaluation:

Stickiness: O Littk 5 Reference 10 Very

Softness: O Less 5 Reference 10 More

Extensibility: O Low (short) 5 Reference 10 High (k 3ng)

Elasticity: O Low (weak) 5 Reference 10 High (strong)

Baked qoods external:

Crust Color: O Light 5 Reference 10 Dark

Crispiness: O Low 5 Reference 10 High

Crust resilience O Low 5 Reference 10 High

Sυmmetry O Low/fiat 5 Reference 10 High/round

Break O None 5 Reference 10 Large

Shred O Smooth 5 Reference 10 Ragged

Crust characteristics O Bad 5 Reference 10 Good

Pan flow O Less 5 Reference 10 More

Bloom: O Blind 5 Reference 10 Strong

Baked goods internal:

Uniformity: 0 Less 5 Reference 10 More

Grain: 0 Open 5 Reference 10 Fine

Cell VVaIh 0 Thick 5 Reference 10 Thin

Cell Form: 0 Round 5 Reference 10 Elongate

Crumb Color; 0 Dark/Grey 5 Reference 10 Light

(Bleached)

Average of internal crumb 0 Open grains 5 Reference 10 Uniform, with characteristics: with thick, round fine, thin, ceils elongated ceils