FIELD OF THE INVENTION

The present invention relates to methods for preparing steamed bread dough compositions and steamed breads using enzymes, enzymatic compositions for preparing steamed bread dough compositions and steamed breads, and to steamed bread dough compositions and steamed breads.

BACKGROUND OF THE INVENTION

In China, the traditional bread is referred to as "Mantou." Mantou is also referred to as "steamed bread" in Western cultures. In China, Mantou is often eaten as an alternative staple food to rice.

Mantou is made by steaming dough, and sometimes by deep frying dough. The dough is usually made from either wheat flour or rice flour. As a result of the very different preparation process as compared to processes used to produce Western-style breads, Mantou has very different properties then Western-style breads, e.g., Mantou does not have a brown outer crust traditionally associated with Western-style breads.

Enzymes are known in the art for use in extending the shelf-life (i.e., inhibiting or retarding staling) of bread. Enzymatic retardation of staling by means of alpha-amylases has been described, for example, in U.S. Pat. No 2,615,810, U.S. Pat. No. 3,026,205 and O. Silberstein, "Heat-Stable Bacterial Alpha-Amylase in Baking", Baker's Digest 38(4), August 1964, pp. 66-70 and 72.

In addition to alpha-amylases, other types of amylases are known in the art for retarding the staling of Western-style breads. For example, WO 1991/04669 discloses the use of maltogenic alpha-amylases to retard staling. An example of a maltogenic alpha- amylase is the enzyme commercially available from Novozymes A/S under the trade name NOVAMYL®. WO 2000/59307 describes the use of an exo-amylase which hydrolyzes starch to form mainly maltotriose to retard staling.

Although advances have been well developed in inhibiting staling in traditional Western-style bread, steamed breads provide additional and very different challenges for inhibiting staling. For example, the method of preparation of steamed breads (e.g., the steaming process used for heating) presents unique challenges for finding effective enzymatic solutions for inhibiting staling. In addition, the properties of the resulting steamed bread also present problems for finding effective solutions for inhibiting staling.

Examples of steamed bread methods using enzymatic compositions include CN

101088341 , disclosing a steamed bread process by adding into flour an aging resistance composition comprising 1 -7% weight% of an amylolytic enzyme, 3-7% weight of a hemicellulase, 3-7% weight of a lipase, 40-70% weight of an emulsifying agent, 15-40% weight of a hydrophilic colloid and 2-6% weight of an anti-caking agent.

KR 2006056652 discloses a method for producing rice flour-based dough for steamed bread by combining rice flour, wheat flour, live yeast, an alpha-maltogenic amylase.

CN 1795726 discloses a modifier for the flour of steamed bread in order to improve tension and the network gluten structure comprising glucose oxidase, amylase, pentosanase, steatolytic enzyme and starch proportionally.

CN 1759691 discloses a powdered enzyme for improving the quality of steamed bread using fungal alpha-amylase, xylanase, ascorbic acid, monoglycoside and soybean powder.

The use of enzymatic compositions to prevent staling of steamed bread products have been compromised, however, by negative side effects associated with the enzymatic treatment. For example, although shelf life extension may be obtained enzymatically in steamed bread, the enzymes often create problems in other equally important characteristics of the steamed breads, e.g., negatively impacting the crumb structure and/or mouth feel (in particular, gumminess ( stickiness) and resilience) of the steamed bread products.

Thus, although some advancement in extension of shelf-life of steamed bread have been made using enzymatic treatments, it is desirable to provide new solutions for inhibiting staling of steamed bread yet substantially maintain other organoleptic quality characteristics of the steamed bread products (in particular, in terms of crumb structure and mouth feel), and it is an object of the present invention to provide such solutions.

SUMMARY OF THE INVENTION

The present invention relates to methods for preparing dough used to prepare steamed breads and steamed bread enzymatic compositions. In accordance with the present invention, the shelf life (storage stability) of steamed bread can be extended by enzymatic treatment methods to dough used to prepare the steamed bread. The enzymatic treatment methods of the present invention retard (slow) the staling of steamed bread products, however, they substantially maintain other organoleptic quality characteristics of the steamed bread products (in particular, in terms of crumb structure and mouth feel). In particular, it has surprisingly been found that when the combination of a maltogenic alpha-amylase and a raw starch degrading enzyme are used in combination in dough used for producing steamed bread products, an anti-staling effect is obtained, however, the formation of stickiness or gumminess of the crumb of the steamed bread products is substantially avoided.

In a first aspect the present invention provides a method for preparing a steamed bread composition, comprising the step of making a dough used to prepare steamed bread with one or more maltogenic alpha-amylases, one or more raw starch degrading enzymes, and at least one lipolytic enzyme. The invention further provides embodiments comprising the step of steaming the dough to make steamed bread.

In a second aspect the present invention provides a steamed bread dough composition comprising one or more maltogenic alpha-amylases, one or more raw starch degrading enzymes and optionally at least one lipolytic enzyme.

In a third aspect the present invention provides steamed bread dough premix comprising one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes and flour selected from the group consisting of wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 provides an illustration of the measurements of hardness, cohesiveness, adhesiveness, resiliency, springiness, gumminess and chewiness using a texture profile analyzer.

Fig. 2 shows the anti-staling effectiveness of a maltogenic alpha-amylase in steamed bread. Fig. 3 shows the anti-staling effectiveness of a maltogenic alpha-amylase in steamed bread. Fig. 4 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

Fig. 5 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

Fig. 6 shows the anti-staling effectiveness of an example of a combination treatment of the present invention in comparison to treatment by the individual components.

Fig. 7 shows the anti-staling effectiveness of an example of a combination treatment of the present invention in comparison to treatment by the individual components.

Fig. 8 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

Fig. 9 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

Fig. 10 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

Fig. 1 1 shows the anti-staling effectiveness of an example of a combination treatment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Steamed Bread and Steamed Bread Dough Compositions

As used herein, "steamed bread" means bread and dough products prepared by steaming dough. Examples of steamed breads include North Chinese mantou, South Chinese mantou, and baozi, fagao, huajuan. The steamed breads may contain one or more additional ingredients, such, as meat (e.g., pork, beef, chicken or fish), vegetables (e.g., mushrooms, broccoli, and other green vegetables), fruits (e.g., dates and jujube), candies, cheese, and milk (or other dairy products), and combination thereof.

As used herein "steamed bread dough" means any dough used to prepare a steamed bread. The dough used to prepare a steamed bread product may be made from any suitable flour source, e.g., flour sourced from grains, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, potato flour and combinations thereof (e.g., wheat flour combined with one of the other flour sources; rice flour combined with one of the other flour sources).

As used herein, the term "steamed bread composition" may include a dough suitable for preparing steamed bread.

Any steamed bread process may be used to prepare the steamed bread. Methods for preparing steamed bread are well known in the art and include, for example, the "straight dough process" and the "sponge and dough process," non-limiting examples of which are provided under the "Materials and Methods" section below. The process of preparing steamed bread generally involves the sequential steps of dough making (with an optional proofing step), sheeting, shaping, proofing, and then steaming the dough, which steps are well known in the art. If the optional proofing step is used, preferably more flour is added and alkali may be added to neutralize acid produced or to be produced during the second proofing step. The dough is generally leavened by the addition of a suitable yeast culture, for example, a culture of Saccharomyces cerevisiae (baker's yeast) or a chemical leavening agent, as are well-known in the art.

In addition to preparing fresh steamed bread dough or steamed bread products, the present invention is directed to method for preparing a frozen steamed bread dough. The dough is frozen after preparation of the dough and treatment by the enzyme combinations of the present invention (i.e., prior to steaming). A frozen steamed bread dough may be advantageous for storage and/or distribution. An example of a method for preparing a frozen steamed bread dough includes the steps of making a dough (with an optional proofing), sheeting, shaping, proofing, and freezing the dough. The present invention is also directed to a frozen steamed bread dough comprising the enzyme combinations of the present invention.

The present invention may also be used to prepare other Chinese type of breads, including, e.g., baked flatbread and fried bread, in which instead of steaming, the dough is either baked or fried.

Industrial Processes The present invention is particularly useful for preparing steamed bread dough and steamed bread products in industrialized processes, that is, in which the dough used to prepare steamed bread and/or steamed bread products are prepared mechanically using automated or semi-automated equipment. The present invention provides significant advantages in that steamed bread can now be prepared using automated or semi-automated processes in which the steamed bread is stored for distribution and consumer use more than 24 hours after preparation yet substantially maintains the qualities of steamed bread prepared freshly on the same day.

The process of preparing steamed bread generally involves the sequential steps of dough making (and an optional proofing step), sheeting, shaping, proofing, steaming and packaging. If the optional proofing step is used, preferably more flour is added and alkali may be added to neutralize acid produced or to be produced during the second proofing step. In an industrial steamed bread production process according to the present invention, one or more of these steps, such as), sheeting, shaping, proofing, steaming and/or packaging, is/are performed using automated or semi-automated equipment.

Enzyme Combinations

The present invention is directed to methods and compositions for preparing dough used to prepare steamed breads and methods for preparing steamed breads by applying specific enzyme combinations to a dough used to prepare steamed breads. The enzyme combination comprises one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes.

Maltogenic alpha-amylase

As used herein, a "maltogenic alpha-amylase" (glucan 1 ,4-alpha-maltohydrolase, E.C. 3.2.1 .133) is an enzyme that is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. The enzymatic activity does not require a non-reducing end on the substrate and the primary enzymatic activity results in the degradation of amylopectin and amylose to maltose and longer maltodextrins.

An example of a maltogenic alpha-amylase is the amylase cloned from Bacillus stearothermophilus (amino acids 1 -686 of SEQ ID NO: 1 ) as described in EP 120 693 (NOVAMYL®). Other examples of maltogenic alpha-amylases are described in 2006/032281 and WO 1999/43794, and include, e.g., the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions F188L+ V336L+ T525A; the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions F188I+ Y422F+ I660V; the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions N1 15D+ F188L; the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions A30D+ K40R+ D261 G; the maltogenic alpha- amylase of amino acids 1 -686, but having substitutions T142A+ N327S+ K425E+ K520R+ N595I; the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions F188L+ D261 G+ T288P (OPTICAKE®); the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions K40R+ F188L+ D261 G+ A483T; and the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions T288K. These and many other maltogenic alpha- amylases are described in, e.g., WO 2006/032281 and WO 1999/43794. Commercial maltogenic alpha-amylases available include NOVAMYL® and OPTICAKE® 50BG (both available from Novozymes A/S). The maltogenic alpha-amylases are added into the dough in an amount effective to retard the staling of the steamed bread product. Examples of effective amounts include at least 25 MANU/kg flour to 1500 MANU/kg flour, such as, at least 50 MANU/kg flour to 1500 MANU/kg flour, at least 100 MANU/kg flour to 1500 MANU/kg flour, at least 200 MANU/kg flour to 1500 MANU/kg flour, or at least 500 MANU/kg flour to 1500 MANU/kg flour. One MANU (Maltogenic Amylase Novo Unit) may be defined as the amount of enzyme required to release one micromol 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 degree Celsius for 30 minutes. Effective amounts also include 5 to 100 ppm maltogenic alpha-amylase, such as, 20-100 ppm of NOVAMYL 10000 BG or 5-100 ppm of OPTICAKE 50 G.

Raw Starch Degrading Enzyme

As used herein, a "raw starch degrading enzymes" (also known as a raw starch hydrolyzing enzyme) refers to an enzyme or combination of enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. The gelatinization temperature of starch can range from 51 ^° C to 78 ^° C as the gelatinization initiation temperature can vary from about 51 ^° C to 68 ^° C. A raw starch degrading enzyme is an enzyme that can directly degrade raw starch granules under the following conditions: When wheat flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 52 ^° C -75 ^° C. When corn flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 62 ^° C -74 ^° C. When rye flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 55 ^° C -70 ^° C. When barley flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 53 ^° C -63 ^° C. When oat flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 55 ^° C -62 ^° C. When rice flour is used to make the dough, the raw starch degrading enzyme can directly degrade raw starch when the gelatinization temperature is 65 ^° C -75 ^° C. When sorghum flour is used to make the dough, the raw starch degrading enzyme can directly degrade starch when the gelatinization temperature is 70 ^° C -78 ^° C. When potato starch is used to make the dough, the raw starch degrading enzyme can directly degrade starch when the gelatinization temperature is 56 ^° C -69 ^° C.

In one embodiment, the raw starch degrading enzyme is defined as an enzyme that has a raw starch degrading index of at least 0.2, at least 0.3, at least, 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1 , at least 1.1 , at least 1 .2, at least 1 .3, at least 1.4, at least 1 .5, at least 1.6, at least 1.7, at least 1 .8, at least 1.9, at least 2, wherein the raw degrading index is a ratio of activity to degrade raw starch to activity to degrade gelatinized starch (Ra/Ga). Preferably, the raw starch degrading enzyme is defined as an enzyme that has a raw starch degrading index of higher than 1. The activity on gelatinized starch is measured by measuring the release of glucose produced by the enzyme on a 2% gelatinized (e.g., corn) starch reaction mixture. The activity is measured by the release of reducing sugars produced in 4mol per hour per mg of pure active enzyme. The same assay can then be used to measure the activity of the enzyme on raw starch, but substituting the 2% gelatinized (e.g., corn) starch by 2% of raw (e.g., corn) starch. In both assays, the temperature is 40 ^° C, the same pH and buffer solution is used and the incubation time is 6 hours, and is further described in the "Materials and Methods" section below.

Raw starch degrading enzymes are ubiquitous and produced by plants, animals, and microorganisms, such as, fungal, bacterial and yeast raw starch degrading enzymes. In embodiments, raw starch degrading enzymes include one or more glucoamylases. In another embodiment, raw starch degrading enzymes include one or more alpha-amylases. In yet another embodiment, raw starch degrading enzymes is a combination of one or more alpha-amylases and one or more glucoamylases. Sources of raw starch degrading enzymes, include enzymes obtained from Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii alpha-amylases. Example of such raw starch degrading enzymes include the raw starch degrading enzymes described in WO 2005/00331 1 , WO 2006/0692, WO 2006/060289 and WO 2004/080923.

In one embodiment, the raw starch degrading enzymes comprises an acid alpha- amylase. An "acid alpha-amylase" is an alpha-amylase (4-oD-glucan glucanohydrolase, E.C. 3.2.1.1 ) which when added in an effective amount has activity at a pH in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0.

A source of a raw starch degrading acid alpha-amylase is the acid alpha amylase from Aspergillus niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 1989/01969 (Example 3). The Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes A/S) which is hereby incorporated by reference. A suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is the product SP288 (SEQ ID NO:1 of U.S: Patent No. 7,244,597) (available from Novozymes A/S). Other sources of acid alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, such as, a strain of Rhizomucor pusillus (WO 2004/055178) or Meripilus giganteus. In yet another embodiment, the acid alpha-amylase is derived from Aspergillus kawachii and is disclosed by Kaneko et al. J. Ferment. Bioeng. 81 :292-298(1996) "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii'; and further as EMBL:#AB008370.

The raw starch degrading enzyme may also be a hybrid alpha-amylase comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (CD). A hybrid alpha- amylase may also comprise an alpha-amylase catalytic domain (CD), a starch binding domain (SBD), and a linker connecting the CD and SBD, as is known in the art. In an embodiment the catalytic domain is derived from a strain of Aspergillus kawachii. Examples of hybrid alpha-amylases include the ones disclosed in WO 2005/00331 1 , U.S. Patent Publication no. 2005/0054071 (Novozymes), and US Patent No. 7,326,548 (Novozymes) which is hereby incorporated by reference. Examples also include those enzymes disclosed in Table 1 to 5 of the examples in US Patent No. 7,326,548, and in U.S. Patent Publication no. 2005/0054071 (Table 3 on page 15), such as, an Aspergillus niger alpha-amylase catalytic domain (CD) with Aspergillus kawachii linker and starch binding domain (SBD).

Other acid alpha-amylase include the enzymes disclosed in WO 2004/020499 and WO 2006/06929 and the enzymes disclosed in WO 2006/066579 as SEQ ID NO:2 (hybrid A.niger alpha-amylase+CBM), SEQ ID NO:3, or SEQ ID NO:4 (JA129). Hybrid alpha- amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (referred to herein as RSDE-A, Novozymes A/S).

Raw starch degrading glucoamylases include certain Aspergillus glucoamylases, in particular A niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1 102), or variants thereof, such as those disclosed in WO 1992/00381 , WO 2000/04136 and WO 2001/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 1984/02921 , A. oryzae glucoamylase (Agric. Biol. Chem. (1991 ), 55 (4), p. 941 -949), or variants or fragments thereof. Other raw starch degrading glucoamylases include the glucoamylase derived from a strain of Athelia, preferably a strain of Athelia rolfsii (previously denoted Corticium rolfsii) (see U.S. Pat. No. 4,727,026 and (Nagasaka, Y. et al. (1998) "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 1999/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215), Trichoderma reesei glucoamylases disclosed as SEQ ID NO: 4 in WO 2006/060062, and the glucoamylase derived from Humicola grisea disclosed as SEQ ID NO: 3 in U.S. Ser. No. 10/992,187. Other raw starch degrading glucoamylases include glucoamylase derived from a strain of Trametes, preferably a strain of Trametes cingulata disclosed in WO 2006/069289 (which is hereby incorporated by reference). Other raw starch degrading glucoamylases include the glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 . Bacterial raw starch degrading glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 1986/01831 ).

Commercially available raw starch degrading enzyme compositions include SP288, SPIRIZYME® ULTRA, GOLDCRUST® and SACZYME® (available from Novozymes MS).

A raw starch degrading enzyme may comprise both glucoamylase and acid alpha- amylase, e.g. the Aspergillus niger glucoamylase and the A. niger acid alpha-amylase (SP288). Such products include ATTENUZYME®, and ATTENUZYME® FLEX (available from Novozymes A/S).

The raw starch degrading enzymes are added in an amount effective to improve the shelf life of the steamed bread products, and to prevent the negative organoleptic properties resulting from the treatment with the maltogenic alpha-amylase. An acid alpha-amylase, for example, may be added to the dough in an amount of 0.1 to 100 AFAU/kg flour, such as, 1 to 5 AFAU/kg flour, 0.5 to 3 AFAU/kg flour, and 0.3 to 2 AFAU/kg flour. Glucoamylases, for example, may be added to the dough in an amount of 0.2-70 AGU/kg flour, preferably 1 -50 AGU/kg flour, especially between 5-40AGU/kg flour.

When a raw starch degrading enzyme is used in combination with a maltogenic alpha-amylase, it is possible to use lower of amounts of maltogenic alpha-amylase, and in some embodiments, the dosage of the maltogenic alpha-amylase may be reduced by up to 40%. For example, although 25 ppm of OPTICAKE® 50BG is often preferred for obtaining an optimal shelf life extension, the present invention permits a reduction in OPTICAKE to, e.g., 15 ppm, when an effective amount of a raw starch degrading enzyme is used, e.g., 6.5 AGU of Trametes AMG. Thus, the present invention results in similar anti-staling effect using lower dosages of maltogenic alpha-amylases, while avoiding unwanted side effects associated with using higher dosages of maltogenic alpha-amylases (e.g., increased gumminess or stickiness).

Enzyme Treatment The enzyme combinations treatment are added to the steamed bread dough ingredients (i.e., and prior to steaming or freezing the dough), e.g., indirectly to the dough by adding it to flour used to prepare the dough or actually directly to the dough itself. Thus, for example, in one particular embodiment, the maltogenic alpha-amylase used to treat the steamed bread dough is a maltogenic alpha-amylase selected from the group consisting of the maltogenic alpha-amylase from Bacillus stearothermophilus (amino acids 1 -686 of SEQ ID NO: 1 ). In another embodiment, the maltogenic alpha-amylase is the maltogenic alpha-amylase of amino acids 1 -686, but having substitutions F188L+ D261 G+ T288P. These and other maltogenic alpha-amylases are used to treat the steamed bread dough in combination with a raw starch degrading enzymes, such as, e.g., a raw starch degrading enzyme from Talaromyces (e.g., Talaromyces emersonii), a raw starch degrading enzyme from Aspergillus (e.g., Aspergillus niger or Aspergillus oryzae), a raw starch degrading enzyme from Rhizomucor (e.g., Rhizomucor pusillus), a raw starch degrading enzyme from Athelia (e.g., Atheilia rolfsii), or a raw starch degrading enzyme from Trametes (e.g., Trametes cingulata), can combinations thereof. For example, the raw starch degrading enzymes may be selected from the group consisting of the amylase of SEQ ID NO:3 of WO 2006/066579, the amylase of SEQ ID NO:2 of WO 2006/066579, the Aspergillus niger acid alpha-amylase of SEQ ID NO:1 of U.S: Patent NO. 7,244,597, and/or the glucoamylase derived from Trametes cingulata as disclosed in WO 2006/069289, and combinations thereof.

The enzymes may be added to flour or dough in any suitable form, such as, e.g., in the form of a liquid, in particular a stabilized liquid, or it may be added to flour or dough as a substantially dry powder or granulate. Granulates may be produced, e.g. as disclosed in US Patent No. 4,106,991 and US Patent No. 4,661 ,452. Liquid enzyme preparations may, for instance, be stabilized by adding a sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well-known in the art. The enzyme combination treatment may be added to the steamed bread dough ingredients in any suitable manner, such as individual components (separate or sequential addition of the enzymes) or addition of the enzymes together in one step or one composition.

In addition to the enzyme combination treatment of the present invention, one or more additional enzymes may be added to the dough. Examples of such one or more additional enzymes include pentosanases, lipolytic enzymes (e.g., lipases, phospholipases, galactolipases), xylanases, proteases, transglutaminases, cellulytic (e.g, cellulases and hemicellulases), acyltransferases, protein disulfide isomerases, a pectinases, a pectate lyases, an oxidoreductases or oxidases (e.g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a hexose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase, and/or sulfurhydryl oxidase), non-raw starch degrading alpha- amylases and non-raw starch degrading glucoamylases. The one or more additional enzymes may be of any origin, including mammalian, plant, and preferably microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art. The one or more additional enzymes enzyme may be applied to the dough in any suitable form, such as, in liquid form or in dry or substantially dry form.

Particular combinations of enzymes include the enzyme combination treatment of the present invention (that is, one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes) combined with one or more enzymes selected from the group consisting of a lipases, phospholipase, galactolipase, xylanase, protease, oxidase, non-raw starch degrading amylase or non-raw starch degrading glucoamylase. As is known in the art, lipolytic enzymes may combine many different activities into one enzyme. An example of such lipolytic enzymes are the enzymes disclosed in WO 1998/26057 and WO 2000/32758 (Novozymes A/S).

The dough may also comprise other conventional ingredients, e.g., one or more emulsifiers. Emulsifiers serve to improve dough extensibility and may also be of some value for the consistency of the resulting steamed bread, making it easier to slice, as well as for its storage stability. Examples of suitable emulsifiers are mono- or diglycerides, polyoxyethylene stearates, diacetyl tartaric acid esters of monoglycerides, sugar esters of fatty acids, propylene glycol esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, lecithin or phospholipids.

Other conventional ingredients include 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, diluents such silica dioxide, starch of different origins.

Pre-Mixes

It will often be advantageous to provide the enzymes used in the treatment of the present invention in admixture with other ingredients used to improve the properties of steamed bread products. These are commonly known in the art as "pre-mixes," which usually comprise flour. Hence, in a further aspect, the present invention relates to a steamed bread pre-mix for improving the quality of dough used to prepare a steamed bread products or steamed bread products, which pre-mix comprises one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes in combination with one or more steam bread or dough ingredients, e.g., the ingredients described above. The pre-mix composition may be in liquid form or dry or substantially dry form.

In one embodiment, the present invention further relates to a steamed bread pre-mix comprising the enzyme combinations of the present invention and flour, such as, flour from grains, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, and combinations thereof. In another embodiment, the present invention relates to a steamed bread pre-mix comprising the enzyme combinations of the present invention and flour, such as, flour from grains, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, and combinations thereof, and one or more additional enzymes, as previously described.

Dough and Steamed Bread Properties

The steamed bread prepared by the methods and compositions of the present invention are used as anti-staling agents to improve the shelf life of the steamed bread product. The anti- staling effect (and improved shelf life) of a steamed bread product can be determined by a number of methods well known in the art.

Primarily anti-staling effectiveness is measured by the hardness (also referred to as "firmness" and the opposite of "softness") of the steamed bread product. Hardness can be measured using a texture profile analyzer. A standard method for measuring hardness is based on force-deformation of the steamed bread product. A force-deformation of the steamed bread products may be performed with a cylindrical probe with a maximum deformation of 40% of the initial height of the product at a deformation speed of 1 mm/second and waiting time between consecutive deformations of 3 seconds. Force is recorded as a function of time. Hardness is defined as the maximum peak force during first compression cycle.

Besides hardness, crumb structure and mouth feel (notably gumminess or stickiness and resilience) are also important quality parameters for steamed bread. Crumb structure is a term used to define the structure inside of the steamed bread, e.g., as seen when the steamed bread is cut into half. Gumminess or stickiness and resilience can be exemplified by actual effect in use, and is accordingly a tendency for a piece of steamed bread to get stuck to teeth while eating. Resilience is the opposite of compactness when one bites steamed bread. Although these properties may be measured using a texture profile analyzer, as described below, sensory evaluation is often a preferred way to measure these properties. An example of a sensory evaluation protocol is provided in the "Materials and Methods" section below.

Cohesiveness, resiliency and springiness may also be used to measured steam bread quality using force deformation, which properties can also be measured using a texture profile analyzer. A standard method for measure cohesiveness, resiliency and springiness is also based on force-deformation of the steamed bread product. Two consecutive deformations of the steam bread products is performed with a cylindrical probe with a maximum deformation of 40% of the initial height of the product at a deformation speed of 1 mm/second and waiting time between consecutive deformations of 3 seconds. Force is recorded as a function of time. Cohesiveness is calculated as the ratio between the area under the second deformation curve (downwards + upwards) and the area under the first deformation curve (downwards + upwards). Resiliency is calculated as the ratio between the area under the first downward curve and the first upward curve following deformation. Springiness is calculated as the ratio of the height of the decompression of the second deformation to the height of the decompression of the first deformation with 3 seconds waiting time between deformations.

Gumminess (discussed above) and chewiness have also been measured using a texture profile analyzer. Gumminess may be measured as the product of hardness times cohesiveness. Gumminess is a characteristic of semisolid food with a low degree of hardness and a high degree of cohesiveness.

Chewiness is defined as the product of gumminess times springiness (which also equals hardness times cohesiveness times springiness) and is therefore influenced by the change in any one of these parameters.

Hardness, cohesiveness, resiliency, springiness, gumminess (or stickiness), and chewiness, crumb structure may be compared to a control (steam bread product prepared under identical conditions but without the enzyme treatments of the present invention) and to other enzymatic treatments. Figure 1 provides an illustration of the measurements of hardness, cohesiveness, adhesiveness, resiliency, springiness, gumminess and chewiness using a texture profile analyzer as described above. These concepts and measurements are also described in Bourne, M. C, Food Texture and Viscosity. Concept and Measurement, Second Edition (2002). Other tests known in the art may be used to assess the shelf life and other organoleptic qualities of the steamed bread prepared by the methods and compositions of the present invention. The properties of the steamed bread may be referred to herein as organoleptic properties, which include anti-staling (hardness), crumb structure and mouth feel (e.g., gumminess and resiliency).

Storage/Shelf Life

In one embodiment, the present invention relates to a steamed bread having an improved shelf life at at least 24 hours after steaming. In another embodiment, the present invention relates to a steamed bread having an improved shelf life at at least 48 hours after steaming. In another embodiment, the present invention relates to a steamed bread having an improved shelf life at at least 72 hours after steaming. In another embodiment, the present invention relates to a steamed bread having an improved shelf life at at least 96 hours after steaming. In another embodiment, the present invention relates to a steamed bread having an improved shelf life at at least 120 hours after steaming. In another embodiment the present invention relates to a steamed bread having an improved shelf life at at least 144 hours after steaming. In yet another embodiment the present invention relates to a steamed bread having an improved shelf life at at least 178 hours after steaming.

In a further embodiment, the present invention relates to a steamed bread having a softness at 48 hours, 72 hours and/or 96 hours post steaming which is better than the softness of a steam bread which is prepared under the same conditions, but without treatment with one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes.

Shelf life can measured as follows: A steamed bread is prepared using enzyme compositions of the present invention (i.e., one or more maltogenic alpha-amylases and one or more raw starch degrading enzymes) and compared to a control steamed bread, that is, a steamed bread prepared in the same way but without enzyme compositions of the present invention. The steam bread is stored in a sealed plastic bag at 25 ^° C. After the storage period, (e.g., 72 hours), the hardness of the steamed bread is measured using a texture analyzer and compared to a control steamed bread stored under identical conditions. An improved shelf life is defined as a steamed bread which is less hard (i.e., softer) than the control as measured by the texture analyzer. Improved shelf life is the same as improved (better) softness.

In a preferred embodiment, the steamed bread is also compared to a control and other enzymes treatments in other quality parameters, such as, crumb structure and mouth feel (in particular, gumminess or stickiness and resilience). The steamed bread prepared by the combination treatment of the present invention and is analyzed at a time after steaming or during storage (e.g., 1 hour after steaming and/or 24 hours, 48 hours, 72 hours, 96 hours, etc. post steaming). The steamed bread prepared by the combination treatment of the present invention preferably has similar qualities in terms of improved gumminess (stickiness), resiliency and/or crumb structure as compared to the control at the comparison, such as, as measured using a texture analyzer and/or by sensory evaluation. The steamed bread prepared by the combination treatment of the present invention preferably has improved qualities in terms of improved gumminess (stickiness), resiliency and/or crumb structure as compared to treatment with maltogenic alpha-amylase alone, that is, without the combination with a raw starch degrading enzyme. The steamed bread may be prepared with other background enzymes. The above method may also be used to compare the effectiveness of one particular enzyme combination treatment of the present invention to another enzyme combination treatment of the present invention. Accordingly, in an embodiment, the steamed bread has improved gumminess characteristics at 1 hour and/or 24 hours post steaming as compared to steam bread prepared under the same conditions but without treatment with the raw starch degrading enzyme.

EXAMPLES Materials and Methods

The following are non-limiting examples of methods for producing steamed bread. Straight dough process

Preheat water to boiling before steaming. Put the fermented

6 Steaming dough in a steaming pot, and steam for 20min above the boiling water.

Open the lid of steamer 5min after steaming is finished in order to prevent shrinkage due to sudden pressure change.

7 Sampling

Take out the steamed bread from the pot, put in room temperature and cool for 1 hour.

After the steamed bread is taken out from the pot for 1 +/-

8 Evaluation 0.2hours, it is ready for whiteness evaluation & Volume, weight and other evaluation.

Sponge and Dough process

7 Proofing Put the dough on a protection paper and put them into

proofing machine for around 50 min under 35°C, RH80% ( the time can be prolonged if the dough is very hard and volume is too small or shortened if the dough become collapsed.

8 Steaming Preheat water to boiling before steaming. Put the fermented dough in a steaming pot, and steam for 20min above the boiling water.

9 Sampling Open the lid of steamer after 5min of steaming finished in order to prevent shrinkage cause by sudden pressure change. Take out the steamed bread from the pot, put in room temperature and cool 1 hours

10 Evaluation After the steamed bread been taken out from the pot for

1 +/-0.2hours, it is ready for whiteness evaluation & Volume, weight and other evaluation.

In the lab, a water temperature of 19-20 ^° C is used to make dough. Sample Evaluation

Samples are stored in a sealed plastic bag until evaluation. Before analysis, steamed bread is sliced transversely into 1 cm slice. The slice is put under a texture profile analyzer and analyzed for its hardness. The result is the average of duplicate analyses with different slices of the same steamed bread. The effect of maltogenic alpha-amylases and raw starch degrading enzymes were analyzed.

The enzymes were dissolved in water and added to flour just before mixing. All enzymes were dosed on the basis of flour weight, and steamed bread was prepared in a straight dough process as described above. In addition to the enzymes tested, other enzymes were also used as background enzymes in preparing the steamed breads, in particular: an alpha-amylase in a dosage of 9.6 ppm (Fungamyl® 2500, available from Novozymes A S); an xylanase in a dosage of 6 ppm (Pentopan® Plus available from Novozymes A/S): a lipase in a dosage of 3 ppm (Lipopan® S available from Novozymes A/S); and an enzyme having lipase/phospholipase/glycolipase activity in a dosage of 3 ppm (Lipopan® F available from Novozymes A/S). The "control" steamed bread was prepared with such other enzymes, but did not contain either a maltogenic alpha-amylase or a raw starch degrading enzyme.

Sensory Evaluation To perform sensory evaluation, a panel (e.g., at least 3 well-trained persons) is used to assess the qualities of the steamed bread. A piece of steamed bread is broken into two halves and the crumb of which is compared to that of a blank control (without enzyme). When tasting a small piece of crumb is taken, put into mouth and bitten carefully to evaluate its gumminess or stickiness and resilience. A 10-point system based on Table 1 may be used to score the quality parameters of interest with the score of the control being 5. The higher the score, the better the quality of steamed bread. For the sensory evaluation of fresh steamed bread, the steamed bread is cooled down after steaming at room temperature for 1 hr, and then sensory evaluation is performed. Sensory evaluation can also be performed at any other designated time, e.g., 24 hours post steaming, 48 hours post steaming, 72 hours post steaming, 96 hours post steaming, etc.

Table 1

Raw Starch Degrading Enzyme (Ra/Ga) Assay

A protocol to obtain a raw starch degrading enzyme index (Ra/Ga) value of the enzymes is as follows :

1 ) The assays are performed at a temperature of 40°C.

2) First, the pH profile of the enzyme is obtained on raw starch. The profile is obtained from the plotting of the % activity versus the pH. This optimum pH value is used in the assay.

3) Any type of starch may be used, such as, wheat, corn, barley, rice, etc. In an example, the raw starch used is corn starch. A 2% solution of raw starch is used. Alternatively, to obtain the gelatinized starch solution, a solution of raw starch is heated above the gelatinzation temperature for at least 60 minutes. In the case of corn, the solution of raw starch is heated to 70°C for at least 60 minutes.

4) The reaction solution contains the gelatinized starch (or raw starch) and a buffer. The composition of the buffer used in the assay depends on the pH optimum of the enzyme. The buffer composition and concentration must be identical for both the raw and gelatinized starch activity measurements.

5) The enzyme concentration used in the assay must be identical for both the raw and gelatinized starch activity measurements. 6) The enzyme activity is measured by determination of the reducing sugars in solution. Suitable methods are the following: The method of Bernfield for determining reducing sugars using dinitrosalicylic acid is described in Bernfield P., Methods Enzymology 1 ,149-158 (1955) and the method for determining reducing sugars with copper-bicinchoninate as described in Fox J. D. et al, Analytical Biochemistry 195,93-96 (1991 ) or in Waffenschmidt S. et al, Anal. Biochem. 165,337-340 (1987). Prior to the determination of reducing sugars, the solutions are boiled for 3 minutes and centrifugated to inactivate the enzyme.

7) The time for incubation to measure the enzyme activities is 6 hours.

8) The enzyme activity is expressed as the number reducing sugars produced per hour and per mg of pure active enzyme.

9) The activity on gelatinized starch is measured by measuring the release of glucose produced by the enzyme on a 2% gelatinized (e.g., corn) starch reaction mixture and the activity on raw starch is measured by measuring the release of glucose produced by the enzyme on a 2% raw (e.g., corn) starch reaction mixture. The activity is measured by the release of reducing sugars produced in 4mol per hour per mg of pure active enzyme.

Example 1

The anti-staling effectiveness of maltogenic alpha-amylases in steamed bread was assessed. Two different maltogenic alpha-amylases were tested in steamed bread:

MAA1 : (Novamyl® 10000 BG, Novozymes A/S); and

MAA2: (Opticake® 50BG, Novozymes A/S)

MAA1 was added in an amount of 55 ppm and MAA2 was added in an amount of 25 ppm. The steamed bread was prepared using the straight dough process as described above. Hardness of the steamed bread was measured using the texture profile analyzer at 24 hours, 48 hours and 72 hours after production.

Table 2

As illustrated in Figures 2 and 3, both maltogenic alpha-amylases provided very significant anti-staling improvement as compared to a control.

Example 2

The anti-staling effectiveness of a maltogenic alpha-amylases (Opticake® 50BG, Novozymes A/S) at two different dosages 25 ppm (MAA1 ) and 15 ppm (MAA2) was assessed in steamed bread and compared to a combination of treatment (Combi) of the same maltogenic alpha- amylase at dosage 15 ppm in combination with a raw starch degrading enzyme (RSDE-A, Novozymes A S). Hardness was measured as described in Example 1 .

Table 3

As shown in Figure 4, the combination treatment produced similar anti-staling effect as the higher dosage of the maltogenic alpha-amylase.

Example 3

An experiment was performed to compare the anti-staling effect of the combination of raw starch degrading enzyme and a maltogenic alpha-amylase as compared to a maltogenic alpha-amylase alone. In this example, steamed bread was prepared in a straight dough process using the combination of a raw starch degrading enzyme (RSDE-A, Novozymes A/S) at two different dosages 10 AFAU/kg (Combi 1 ) and 2 AFAU/kg Combi 2) and a maltogenic alpha-amylase (Opticake® 50BG, Novozymes A/S) at dosage of 15 ppm as compared to the use of the same maltogenic alpha-amylase (MAA) at dosage of 25 ppm. Hardness was measured as described in Example 1 .

Table 4

As shown in Figure 5, a raw starch degrading enzymes can substitute some of the maltogenic alpha-amylase without compromising anti-staling effectiveness.

Example 4

An experiment was performed to compare the anti-staling effect of raw starch degrading enzyme alone to a maltogenic alpha-amylase alone and then to the combination of a maltogenic alpha-amylase and a raw starch degrading enzyme. In this example, steamed bread was prepared in a straight dough process using:

a raw starch degrading enzyme (RSDE) (RSDE-A, Novozymes A/S) at a dosage of 20 AFAU/kg flour;

a maltogenic alpha-amylase (MAA) (Opticake® 50BG, Novozymes A/S) at dosage of 25 ppm; the combination (Combi) of a maltogenic alpha-amylase (MAA) (Opticake® 50BG, Novozymes A S) at a dosage of 15 ppm and raw starch degrading enzyme (RSDE-A, Novozymes A S) at a dosage of 5 AFAU/kg flour. Table 5

As shown in Figure 6, the use of a raw starch degrading enzyme alone had a poor anti- staling effect. A better anti-staling effect was seen with the maltogenic alpha-amylase alone and the combination with a maltogenic alpha-amylase and a raw starch degrading enzyme (RSDE-A, Novozymes A/S).

Example 5

An experiment was performed to compare the anti-staling effect of raw starch degrading enzymes alone to a maltogenic alpha-amylase alone and then to the combination of a malogenic alpha-amylase and a raw starch degrading enzyme. In this example, steamed bread was prepared in a straight dough process using:

a raw starch degrading enzyme (RSDE) (ATTENUZYME FLEX, Novozymes A/S) at dosage of 100 PPM;

a maltogenic alpha-amylase (MAA) (Opticake® 50BG, Novozymes A/S) at dosage of 25 ppm,

and combination (Combi) of a maltogenic alpha-amylase (Opticake® 50BG, Novozymes A/S) at a dosage of 15 ppm and a raw starch degrading enzyme (ATTENUZYME FLEX, Novozymes A/S) at a dosage of 60 PPM.

Table 6

As shown in Figure 7, the use of a raw starch degrading enzyme had a lower anti-staling effect as compared to either the anti-staling enzyme alone or the combination of anti-staling enzyme and the raw starch degrading enzyme.

Example 6 An experiment was performed to compare the anti-staling effectiveness of the combination of raw starch degrading enzyme with a maltogenic alpha-amylase as compared to a maltogenic alpha-amylase alone. In this example, steamed bread was prepared in a straight dough process using:

a combination raw starch degrading enzyme (SP288, available from Novozymes

A/S) at a dosage 20 AFAU/kg and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A S) dosage of 15 ppm (Combi 1 );

a combination raw starch degrading enzyme (SP288, available from Novozymes A/S) at a dosage 30 AFAU/kg and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) dosage of 15 ppm (Combi 2); and

maltogenic alpha-amylase (MAA) (OPTICAKE® 50BG, Novozymes A/S) alone at dosage of 25 ppm.

Hardness was measured as described in Example 1 . Table 7

As shown in Figure 8, the anti-staling effectiveness shows that the combination of a maltogenic alpha-amylase and raw starch degrading enzyme had a similar effect in anti- staling effectiveness as the optimal dosage of maltogenic alpha-amylase alone.

Example 7

An experiment was performed to compare the anti-staling effectiveness of the combination of raw starch degrading enzyme with a maltogenic alpha-amylase as compared to a maltogenic alpha-amylase alone. In this example, steamed bread was prepared in a straight dough process using:

a combination of a raw starch degrading enzyme (Trametes AMG, Novozymes A/S) at a dosage of 6.5 AGU/kg and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) at a dosage of 15 ppm (Combi 1 );

a combination of a raw starch degrading enzyme (Trametes AMG, Novozymes A/S) at a dosage of 6 10.7 AGU/kg and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) at a dosage of 15 ppm; and

a maltogenic alpha-amylase alone (MAA) (OPTICAKE® 50BG, Novozymes A/S) at dosage of 25 ppm.

Hardness was measured as described in Example 1 . Table 8

As shown in Figure 9, a raw starch degrading enzymes can substitute some of the maltogenic alpha-amylase without compromising anti-staling effectiveness.

Example 8

An experiment was performed to compare the anti-staling effectiveness of the combination of raw starch degrading enzyme with a maltogenic alpha-amylase as compared to a maltogenic alpha-amylase alone. In this example, steamed bread was prepared in a straight dough process using:

combination of a raw starch degrading enzyme (ATTENUZYME®, Novozymes A/S, which contains 90 FAU/g of SP288) at a dosage of 40ppm with a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) at a dosage of 15 ppm (Combi 1 );

combination of a raw starch degrading enzyme (SPIRIZYME ULTRA, Novozymes A/S, which contains 170 AGU/g of Trametes AMG and 1 1.4 AFAU/g of RSDE-A) at a dosage of 60 ppm and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) at a dosage of 15 ppm (Combi 2);

use of the maltogenic alpha-amylase (MAA) (OPTICAKE® 50BG, Novozymes A/S) alone at dosage of 25 ppm.

Hardness was measured as described in Example 1.

Table 6

As shown in Figure 10, a raw starch degrading enzymes can substitute some of the maltogenic alpha-amylase without compromising anti-staling effectiveness.

Example 9

An experiment was performed to compare the anti-staling effectiveness of the combination of raw starch degrading enzyme with a maltogenic alpha-amylase to a maltogenic alpha- amylase alone. In this example, steamed bread was prepared in a sponge and dough process using the combination (Combi) of a raw starch degrading enzyme (Trametes AMG, Novozymes A/S) at a dosage of 10.7 AGU/kg and a maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) at a dosage of 15 ppm, which was compared to the use of the maltogenic alpha-amylase (MAA) alone (OPTICAKE® 50BG, Novozymes) at dosage of 25 ppm. Hardness was measured as described in Example 1 .

Table 10

As shown in Figure 1 1 , the anti-staling effectiveness of the raw starch degrading enzyme and a lower amount of maltogenic alpha-amylase was similar to the maltogenic alpha-amylase alone.

Example 10

An experiment was performed to compare the effect on crumb structure of the combination treatment. A straight dough process was used to prepare the steamed bread as described in the "Material and Methods." The crumb structure was analyzed using sensory evaluation, using the protocol described above in the "Materials and Methods." Sensory evaluation was performed after cooling the steamed bread for 1 hour after steaming. In this experiment, the crumb structure of steamed bread was determined for the following enzyme compositions:

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 25 ppm (MAA 25)

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 15 ppm (MAA 15)

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 15 ppm plus the raw starch degrading enzyme composition (ATTENUZYME®,

Novozymes A/S) in an amount of 40 ppm (Combi 1 )

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 15 ppm plus the raw starch degrading enzyme composition (purified T. cingulata glucoamylase, Novozymes A/S) in an amount of 6.5 AGU/kg flour. (Combi 2)

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 15 ppm plus the raw starch degrading enzyme composition (RSDE-A, Novozymes A/S) in an amount of 10 AFAU/kg flour (Combi 3).

The results are shown below:

Table 11 control MAA 25 MAA 15 Combi 1 Combi 2 Combi 3

Crumb 5 4.5 4.8 4.8 5 4.8

As can be seen in Table 1 1 , the use of the raw starch degrading enzyme improved the crumb structure as compared to the use of 25 ppm of the maltogenic alpha-amylase, and in Combi 2 resulted in a crumb structure which was the same as the control. As previously shown in Example 2, superior anti-staling effect is obtained when a higher dosage of a maltogenic alpha-amylase is used (e.g., comparing 15 ppm of OPTICAKE® 50BG to 25 ppm of OPTICAKE® 50BG). Thus, the results show that the raw starch degrading enzymes can when used in combination with the maltogenic alpha-amylase enable the use of lower amounts of maltogenic alpha-amylase while still obtaining optimal anti-staling effect, but reducing the negative impact on crumb structure.

Example 11

An experiment was performed to compare the effect on mouth feel of the combination treatment. A straight dough process was used to prepare the steamed bread as described in the "Material and Methods." The mouth feel was analyzed using sensory evaluation, using the protocol described above in the "Materials and Methods." Sensory evaluation was performed after cooling the steamed bread for 1 hour after steaming. In this experiment, the mouth feel of steamed bread was determined for the following enzyme compositions:

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A S) in an amount of 25 ppm (MAA 25)

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A S) in an amount of 15 ppm plus the raw starch degrading enzyme composition (ATTENUZYME®, Novozymes A/S) in an amount of 40 ppm (Combi 1 )

maltogenic alpha-amylase (OPTICAKE® 50BG, Novozymes A/S) in an amount of 15 ppm plus the raw starch degrading enzyme composition (purified T. cingulata glucoamylase, Novozymes A/S) in an amount of 10 ppm (Combi 2).

The results are shown below:

Table 10

As can be seen in Table 12, the combination had improved mouth feel as compared to the use of 25 ppm of the maltogenic alpha-amylase.