BACKGROUND OF THE INVENTION It is well known that the softness of bread deteriorates during storage from the time of baking to the time of consumption. The term staling is used to describe such undesirable changes in the properties of the bread. Staling results in an increase of the firmness of the crumb, a decrease of the elasticity of the crumb, and changes in the crust, which becomes tough and leathery.

Enzymatic retardation of staling by means of various amylases has been de- scribed. Thus, US 2,615,810; US 3,026,205 and O. Silberstein,"Heat-Stable Bacterial Alpha-Amylase in Baking", Baker's Digest 38 (4), Aug. 1964, pp. 66-70 and 72, de- scribe the use of alpha-amylase. WO 91/04669 (Novo Nordisk) describes the use of a <BR> <BR> <BR> maltogenic alpha-amylase from Bacillus stearothermophilus. It is also known to use ß- amylase to retard staling.

It is also known to add a phospholipase to dough. Thus, US 4,567,046 and EP 171,995 (both to Kyowa Hakko) disclose that the addition of phospholipase A en- hances the properties of dough and bread, including retardation of the staling.

M. R. Kweon et al., Journal of Food Science, 59 (5), 1072-1076 (1994) dis- close the effect of 2-4 % by weight of phospholipid hydrolysate together with an an- tistaling amylase on the retrogradation of starch in bread.

SUMMARY OF THE INVENTION The inventors confirmed that the addition of an anti-staling amylase reduces the rate of crumb firming during storage for 1-7 days after baking, but they found that there is a need to improve the softness in the initial period after baking, particularly the first 24 hours after baking. They further found that this can be achieved by using a phospholipase, so that bread made by the combined use of an anti-staling amylase and a phospholipase has improved softness, both when eaten on the same day and when stored for several days after baking. There is no significant change in the taste or smell of the baked product.

Accordingly, The invention provides a process for preparing a dough or a baked product prepared from the dough which comprises adding to the dough an anti-

staling amylase and a phospholipase. The invention also provides a dough and a pre- mix comprising these ingredients.

DETAILED DESCRIPTION OF THE INVENTION Anti-staling amylase The anti-staling amylase used in the invention may be any amylase that is effective in retarding the staling (crumb firming) of baked products.

The amylase preferably has a temperature optimum in the presence of starch in the range of 30-90°C, preferably 50-80°C, particularly 55-75°C, e. g. 60-70°C. The temperature optimum may be measured in a 1 % solution of soluble starch at pH 5.5.

The anti-staling amylase may be an endo-amylase, preferably a bacterial endo-amylase, e. g. from Bacillus. A preferred example is a maltogenic alpha-amylase (EC 3.2.1.133), e. g. from Bacillus. A maltogenic alpha-amylase from B. stearothermo- philus strain NCIB 11837 is commercially available from Novo Nordisk A/S under the tradename Novamyl @. It is further described in US 4,598,048 and US 4,604,355 and in C. Christophersen et al., Starch, vol. 50, No. 1,39-45 (1997).

Other examples of anti-staling endo-amylases are bacterial alpha-amylases, derived e. g. from Bacillus, particularly B. licheniformis or B. amyloliquefaciens.

The anti-staling amylase may be an exo-amylase such as-amylase, e. g. from plant (e. g. soy bean) or from microbial sources (e. g. Bacillus).

The anti-staling amylase is added in an effective amount for retarding the staling (crumb firming) of the baked product. The amount of anti-staling amylase will typically be in the range of 0.01-10 mg of enzyme protein per kg of flour, e. g. 1-10 mg/kg. A maltogenic alpha-amylase is preferably added in an amount of 50-5000 MANU/kg of flour, e. g. 100-1000 MANU/kg. One MANU (Maltogenic_mylase Novo Unit) may be defined as the amount of enzyme required to release one pmol of maltose per minute at a concentration of 10 mg of maltotriose (Sigma M 8378) substrate per ml of 0.1 M citrate buffer, pH 5.0 at 37 °C for 30 minutes.

Phospholipase The phospholipase may have A, or A2 activity to remove fatty acid from the phospholipid and form a lyso-phospholipid. It may or may not have lipase activity, i. e. activity on triglycerides. The phospholipase preferably has a temperature optimum in the range of 30-90°C, e. g. 30-70°C.

The phospholipase may be of animal origin, e. g. from pancreas (e. g. bovine or porcine pancreas), snake venom or bee venom. Alternatively, the phospholipase may be of microbial origin, e. g. from filamentous fungi, yeast or bacteria, such as the

genus or species Aspergillus, A. niger, Dictyostelium, D. discoideum, Mucor, M. javanicus, M. mucedo, M. subtilissimus, Neurospora, N. crassa, Rhizomucor, R. pu- sillus, Rhizopus, R. arrhizus, R. japonicus, R. stolonifer, Sclerotinia, S. libertiana, Trichophyton, T. rubrum, Whetzelinia, W sclerotiorum, Bacillus, B. megaterium, B. subtilis, Citrobacter, C. freundii, Enterobacter, E. aerogenes, E. cloacae Edwardsiella, E. tarda, Erwinia, E. herbicola, Escherichia, E. coli, Klebsiella, K. pneumoniae, Pro- teus, P. vulgaris, Providencia, P. stuartii, Salmonella, S. typhimurium, Serratia, S. liq- uefasciens, S. marcescens, Shigella, S. flexneri, Streptomyces, S. violeceoruber, Yersinia, or Y. enterocolitica. A preferred phospholipase is derived from a strain of Fusarium, particularly F. oxysporum, e. g. from strain DSM 2672, as described in co- pending PCT/DK 97/00557.

The phospholipase is added in an amount which improves the softness of the bread during the initial period after baking, particularly the first 24 hours. The amount of phospholipase will typically be in the range of 0.01-10 mg of enzyme protein per kg of flour (e. g. 0.1-5 mg/kg) or 200-5000 LEU/kg of flour (e. g. 500-2000 LEU/kg).

A phospholipase with lipase activity is preferably added in an amount corre- sponding to an lipase activity of 20-1000 LU/kg of flour, particularly 50-500 LU/kg.

One LU (Lipase Unit) is defined as the amount of enzyme required to release 1 pLmol butyric acid per minute at pH 7.0; with Gum Arabic as emulsifier and tributyrin as substrate.

Phospholipase activity (LEU) In the LEU assay, the phospholipase activity is determined from the ability to hydrolyze lecithin at pH 8.0,40°C. The hydrolysis reaction can be followed by titration with NaOH for a reaction time of 2 minutes. The phospholipase from porcine pancreas has an activity of 510 LEU/mg (taken as standard), and the phospholipase from Fusarium oxysporum has an activity of 1540 LEU/mg.

Phospholipid The phospholipase may act on phospholipid provided by flour in the dough, so the separate addition of a phospholipid is not required. However, the softening ef- fect may be increased by adding a phospholipid, preferably in an amount of 0.05-20 g/kg of flour, e. g. 0.1-10 g/kg. The phospholipid may be a diacyl-glycero-phospholipid, such as lecithin or cephalin.

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, 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 com- mercially 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 shorten- ing, 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, poly- glycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin, but the invention is par- ticularly applicable to a dough which is made without addition of emulsifiers (other than optionally phospholipid).

Additional enzyme Optionally, an addition enzyme may be used together with the anti-staling amylase and the phospholipase. The additional enzyme may be a second amylase, such as an amyloglucosidase, a beta-amylase, a cyclodextrin glucanotransferase, or the addition enzyme may be a peptidase, in particular an exopeptidase, a transglu- taminase, a lipase, 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-a- glucan branching enzyme), a 4-a-glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, e. g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxi- dase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase.

The addition 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 xylanase is preferably of microbial origin, e. g. derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger (cf. WO 91/19782), A. awamori (WO 91/18977), or A. tubigensis (WO 92/01793), from a strain of Trichoderma, e. g. T. reesei, or from a strain of Humicola, e. g. H. insolens (WO 92/17573, the contents of which is hereby incorporated by reference). PentopanO and Novozym 3840 (both from Novo Nordisk A/S) are commercially available xylanase preparations produced by Trichoderma reesei.

The amyloglucosidase may be an A. niger amyloglucosidase (such as AMG, available from Novo Nordisk A/S, Denmark). Other useful amylase products include GrindamylO A 1000 or A 5000 (available from Grindsted Products, Denmark) and Amylase H or Amylase (E) P (available from Gist-Brocades, The Netherlands).

The glucose oxidase may be a fungal glucose oxidase, in particular an Asper- gillus niger glucose oxidase (such as Gluzyme (D, available from Novo Nordisk A/S, Denmark).

The protease may in particular be NeutraseS (available from Novo Nordisk A/S, Denmark).

The lipase may be derived from a strain of Thermomyces (Humicola), Rhi- zomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, in particular from <BR> <BR> <BR> Thermomyces lanuginosus (Humicola lanuginosa), Rhizomucor miehei, Candida ant- arctica, Aspergillus niger, Rhizopus delemar or Rhizopus arrhizus or Pseudomonas cepacia. In specific embodiments, the lipase may be Lipase A or Lipase B derived from Candida antarctica as described in WO 88/02775, or the lipase may be derived from Rhizomucor miehei as described in EP 238,023, or Humicola lanuginosa de- scribed in EP 305,216, or Pseudomonas cepacia as described in EP 214,761 and WO 89/01032.

Baked product The process of the invention may be used for any kind of baked product pre- pared 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.

Pre-mix The present invention further relates to a pre-mix comprising flour together with an anti-staling amylase, a phospholipase and a phospholipid. The pre-mix may contain other dough-improving and/or bread-improving additives, e. g. any of the addi- tives, including enzymes, mentioned above.

Enzyme preparation The invention provides an enzyme preparation comprising an anti-staling amylase and a phospholipase, for use as a baking additive in the process of the in- vention. The enzyme preparation is preferably in the form of a granulate or agglomer- ated 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, um.

Granulats 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.

EXAMPLES Example 1 Bread was baked with anti-staling amylase, phospholipase and phospholipid.

As reference, bread was also baked without one or more of these ingredients.

The phospholipid was lecithin at a dosage of 10 g/kg. The phospholipase was from Fusarium oxysporum used at a dosage of 50,250 or 500 LU/kg, corresponding to or 0.04,0.19 or 0.38 mg/kg. The anti-staling amylase was a maltogenic alpha- amylase from B. stearothermophilus (Novamyl) at a dosage of 750 MANU/kg (1 mg/kg). All dosages in the Examples were based on kg of flour.

Doughs were prepared according to a standard European straight dough pro- cedure with 50 g yeast per kg of flour and 40 ppm of ascorbic acid. The doughs were scaled to 350 g and baked in lidded pans.

The crumb firmness was measured using a texture analyzer TA-XT2 from Stable Micro Systems. Texture was measured according to a modified ACCA method (American Cereal Chemists'Association). These measurements were made after 0 days (approximately 2 hours after baking) and again after 1,2 and 7 days storage (wrapped in double plastic bags and stored at 22°C).

The results are shown as firmness versus additive and storage time: Additives Phospho-2 hours 1 day 2 days 7 days lipase dosage (LU/kg) Invention: Anti-staling 50 316 417 517 868 amylase + phos-250 279 371 455 790 pholipase + 500 248 324 410 752 phospholipid Reference: None (control) 0 296 875 1207 2162 Antistaling 0 469 563 801 1083 amylase Phospholipid + 50 208 470 782 1560 phospholipase 250 231 467 721 1424 500 233 420 649 1303

Example 2 A baking test was made as in Example 1, but with dosages of 0.5 mg/kg of the phospholipase (770 LEU/kg) and 1 g/kg of the phospholipid. The results are given as firmness after storage, and for comparison the firmness is also expressed in % of the control. Additives 2 hours 5 hours 12 20 day 2 day 3 hours hours Invention: Antistaling 181 195 223 241 277 303 amylase + phos- (78%) (65%) (51%) (46%) (34%) (32%) pholipase + phos- pholipid Reference: None (control) 233 302 434 526 824 959 (100%) (100%) (100%) (100%) (100%) (100%) Antistaling 372 468 518 482 547 637 amylase (160%) (155%) (119%) (92%) (66%) (66%) Phospholipid + 144 144 212 258 364 482 phospholipase (62%) (47%) (49%) (49%) (44%) (50%)

Example 3 A baking test was made as in Examples 1 and 2, using a different phospholi- pase. The phospholipase was from porcine pancreas at a dosage of 2 mg/kg (1020 LEU/mg). The dosages of the anti-staling amylase and the phospholipid were as in Example 2, and the results are presented as in Example 2: Additives 2 hours 5 12 20 day 2 day 3 hours hours hours Invention: Antistaling 342 411 420 431 485 559 amylase + phos- (122%) (103%) (80%) (73%) (52%) (48%) pholipase + phospholipid Reference: None (control) 281 398 524 588 937 1157 (100%) (100%) (100%) (100%) (100%) (100%) Antistaling 409 490 514 526 625 673 amylase (146%) (123%) (98%) (89%) (67%) (58%) Phospholipid + 218 260 367 472 668 906 phospholipase (76%) (65%) (70%) (80%) (71 %) (78%)

The results of Examples 1-3 show that the addition of anti-staling amylase retards the crumb firming during storage, but increases the initial firmness compared to the control without additives. The addition of phospholipid + phospholipase ac- cording to the invention is effective in avoiding the increased initial firmness and fur- ther reduces the rate of crumb firming during storage, compared to the anti-staling amylase alone.

Example 4 Bread loaves were baked with and without phospholipid (lecithin) as indicated below. The phospholipase was F. oxysporum used at a dosage of 1 mg/kg (1540 LEU/kg). The anti-staling amylase and the baking conditions were as described in Ex- ample 1. The results are given as firmness after storage: Firmness PhospholipidAnti-stalingPhospholipase 2 hours 1 day 3 days amylase mg/kg g/kg MAN U/kg Control 0 0 0 294 687 1179 750 1 10 200 229 277 750 1 2 167 218 287 Invention 750 1 1 167 232 305 750 1 0.5 189 269 333 750 1 0.1 196 260 381 750 1 0 199 264 372

The results show that addition of anti-staling amylase and phospholipase clearly improve the softness, both initial softness (2 hours) and softness after storage (3 days). The softening effect can be further improved by addition of phospholipid.

The optimum dosage appears to be about 1 mg/kg of phospholipid.