Abstract:
Dough with a high sucrose content (such as cake dough) tends to inhibit the activity of an anti-staling amylase such as Novamyl, making it less effective to prevent the staling of dough-based products with high sucrose content such as cakes. A good anti-staling effect in cakes can be achieved by using a carefully selected anti-staling amylase with certain properties. 
     Analysis of a 3D structure of Novamyl shows that sucrose may inhibit by binding in the active site. Sucrose docks into the active site of Novamyl differently from the substrate or inhibitor in published models 1QHO and 1QHP. This finding is used to design sucrose-tolerant variants.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of U.S. application Ser. No. 13/742,998 filed on Jan. 16, 2013 (now allowed), which is a continuation of U.S. application Ser. No. 12/964,189 filed Dec. 9, 2010 (abandoned), which is a divisional of U.S. application Ser. No. 11/575,644 filed on Mar. 20, 2007 (abandoned), which is a 35 U.S.C. 371 national application of PCT/DK2005/000602 filed Sep. 23, 2005, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2004 01458 filed Sep. 24, 2004 and U.S. provisional application No. 60/614,826 filed Sep. 30, 2004, the contents of which are fully incorporated herein by reference. 
     
    
     SEQUENCE LISTING AND DEPOSITED MICROORGANISMS 
     Sequence Listing 
       [0002]    The present invention comprises a sequence listing. 
       Deposit of Biological Material 
       [0003]    None. 
       FIELD OF THE INVENTION 
       [0004]    The present invention relates to the use of anti-staling amylases in the preparation of dough or dough-based edible products with a high sucrose content. 
       BACKGROUND OF THE INVENTION 
       [0005]    U.S. Pat. No. 3,026,205 describes a process of producing baked confections and the products resulting therefrom by alpha-amylase. 
         [0006]    WO 9104669 describes the use of a maltogenic alpha-amylase to retard the staling of baked products such as bread; the maltogenic alpha-amylase described therein is commercially available under the tradename Novamyl® (product of Novozymes A/S). U.S. Pat. No. 6,162,628 describes Novamyl variants and their use for the same purpose. Three-dimensional structures of Novamyl are published in U.S. Pat. No. 6,162,628 and in the Protein Data Bank (available at http://www.rcsb.org/pdb/) with identifiers 1QHO and 1QHP. 
       SUMMARY OF THE INVENTION 
       [0007]    The inventors have found that a high sucrose content dough (such as cake dough) tends to inhibit the activity of an anti-staling amylases such as Novamyl, making it less effective to prevent the staling of dough-based products with high sucrose content such as cakes. They have found that a good anti-staling effect in cakes can be achieved by using a carefully selected anti-staling amylase with certain properties, and they have identified such amylases. 
         [0008]    By analyzing a 3D structure of Novamyl, the inventors further found that sucrose may inhibit by binding in the active site. They have found that sucrose docks into the active site of Novamyl differently from the substrate or inhibitor in published models 1QHO and 1QHP, and they have used this finding to design sucrose-tolerant variants. 
         [0009]    Accordingly, the invention provides a method of preparing dough or a dough-based edible product (e.g. a baked product) by adding a sucrose-tolerant anti-staling amylase. It also provides novel sucrose tolerant variants of a maltogenic alpha-amylase. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIG. 1  shows the cartesian coordinates for the sucrose atoms in this binding configuration, using the coordinate system of the x-ray structure 1QHO.pdb. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Maltogenic Alpha-Amylase and Sucrose Docking 
       [0011]    A maltogenic alpha-amylase (EC 3.2.1.133) having more than 70% identity (particularly more than 80% or 90%, such as at least 95% or 96% or 97% or 98% or 99%) with the Novamyl sequence shown as SEQ ID NO: 1 may be used as the parent enzyme for designing sucrose tolerant variants. Amino acid identity may be calculated as described in U.S. Pat. No. 6,162,628. 
         [0012]    For Novamyl (SEQ ID NO: 1), a 3D structure including a substrate or inhibitor as described in U.S. Pat. No. 6,162,628 or in the Protein Data Bank with the identifier 1QHO or 1QHP may be used. Alternatively, a Novamyl variant may be used, such as a variant described in U.S. Pat. No. 6,162,628 or in this specification, e.g. the variant F188L+D261G+T288P. A 3D structure of a variant may be developed from the Novamyl structure by known methods, e.g. as described in T. L. Blundell et al., Nature, vol. 326, p. 347 ff (26 Mar. 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317-334 (1990); or Example 1 of WO 9623874. 
         [0013]    The inventors found that sucrose may inhibit Novamyl by binding in the active site. Docking of sucrose into the active site of Novamyl (using the software GOLD version 2.1.2, Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK and the protein part of the x-ray structure 1QHO.pdb) reveals a specific binding configuration as unique to sucrose. The cartesian coordinates for the sucrose atoms in this binding configuration, using the coordinate system of the x-ray structure 1QHO.pdb are given in  FIG. 1 . 
       Maltogenic Alpha-Amylase Assay 
       [0014]    The activity of a maltogenic alpha-amylase may be determined using an activity assay such as the MANU method. One MANU (Maltogenic Amylase Novo Unit) is defined as the amount of enzyme required to release one micro-mole of maltose per minute at a concentration of 10 mg of maltotriose substrate per ml in 0.1 M citrate buffer at pH 5.0, 37° C. for 30 minutes. 
       Amino Acid Alterations 
       [0015]    The amino acid sequence of a maltogenic alpha-amylase may be altered to decrease the sucrose inhibition. The inventors found that the alteration may be made at an amino acid residue having at least one atom within 4 Angstroms from any of the sucrose atoms when the sucrose molecule is docked in the 3D structure of the maltogenic alpha-amylase. Using the Novamyl structure 1QHO and the sucrose docking in  FIG. 1 , the following Novamyl residues are within 4 Å: K44, N86, Y89, H90, Y92, W93, F188, T189, D190, P191, A192, F194, D372, P373, R376. 
         [0016]    Further the following positions have been identified as relevant: I15, R81, T87, G88, L196, N371 or N375 of SEQ ID NO: 1. 
         [0017]    The alteration may be a substitution or deletion of one or more of the selected residues, or one or more residues (particularly 1-4 residues or 5-6 residues) can be inserted adjacent to a selected residue. 
         [0018]    The substitution may be with a smaller or larger residue. A substitution to increase the size of the residue may diminish the space obtained by the docked sucrose molecule thereby preventing the binding of sucrose. Amino acid residues are ranked as follows from smallest to largest: (an equal sign indicates residues with sizes that are practically indistinguishable):
       G&lt;A=S=C&lt;V=T&lt;P&lt;L=I=N=D=M&lt;E=Q&lt;K&lt;H&lt;R&lt;F&lt;Y&lt;W       
 
         [0020]    The substitution may also be such as to eliminate contacts with the sucrose molecule, in particular by moving or removing potential sites of hydrogen bonding or Van der Waals interactions. 
         [0021]    The substitution may particularly be with another residue of the same type where the type is negative, positive, hydrophobic or hydrophilic. The negative residues are D,E, the positive residues are K/R, the hydrophobic residues are A,C,F,G,I,L,M,P,V,W,Y, and the hydrophilic residues are H,N,Q,S,T. 
         [0022]    Some particular examples of substitutions are I15T/S/V/L, R18K, K44R/S/T/Q/N, N86Q/S/T, T87N/Q/S, G88A/S/T, Y89W/F/H, H90W/F/Y/R/K/N/Q/M, W93Y/F/M/E/G/V/T/S, F188H/L/I/T/G/V, D190E/Q/G, A192S/T, F194S/L/Y, L196F, N371K/R/F/Y/Q, D372E/Q/S/T/A and N375S/T/D/E/Q. 
         [0023]    Examples of deletions are deletion of residue 191 or 192. An example of an insertion is Ala inserted between 192 and 193. 
         [0024]    The polypeptide may include other alterations compared to Novamyl (SEQ ID NO: 1), e.g. alterations to increase the thermostability as described in U.S. Pat. No. 6,162,628. 
       Nomenclature for Amino Acid Alterations 
       [0025]    In this specification, an amino acid substitution is described by use of one-letter codes, e.g. K44R. Slashes are used to indicate alternatives, e.g. K44R/S/T/Q/N to indicate substitution of K44 with R or S etc. P191 * indicates a deletion of P191. *192aA indicates insertion of one Ala after A192. Commas are used to indicate multiple alterations in the sequence, e.g. F188L,D261G,T288P to indicate a variant with three substitutions. 
       Properties of Anti-Staling Amylase for Use With Sucrose 
       [0026]    The amylase for use in high-sucrose dough may be selected so as to have mainly exo-amylase activity. More specifically, the amylase hydrolyzes amylose so that the average molecular weight of the amylose after 0.4-4% hydrolysis is more than 50% (particularly more than 75%) of the molecular weight before the hydrolysis. 
         [0027]    Thus, the amylase may hydrolyze amylose (e.g. wheat amylose or synthetic amylose) so that the average molecular weight of the amylose after 0.4-4% hydrolysis (i.e. between 0.4-4% hydrolysis of the total number of bonds) is more than 50% (particularly more than 75%) of the value before the hydrolysis. The hydrolysis can be conducted in a 1.7% amylose solution by weight at suitable conditions (e.g. 10 minutes at 60° C., pH 5.5), and the molecular weight distribution before and after the hydrolysis can be determined by HPLC. The test may be carried out as described in C. Christophersen et al., Starch 50 (1), 39-45 (1998). 
         [0028]    An exo-amylase for use in high-sucrose dough may have a specified sugar tolerance. Compared to its activity in the absence of sucrose, the amylase may have more than 20% activity at 10% sugar, more than 10% activity at 20% sucrose, or more than 4% activity at 40% sucrose. The sugar tolerance may be determined as described in the examples. 
         [0029]    The exo-amylase may have optimum activity in the pH range 4.5-8.5. It may have sufficient thermostability to retain at least 20% (particularly at least 40%) activity after 30 minutes incubation at 85° C. at pH 5.7 (50 mM Na-acetate, 1 mM CaCl 2 ) without substrate. 
         [0030]    The exo-amylase may be added to the dough in an amount corresponding to 1-100 mg enzyme protein per kg of flour, particularly 5-50 mg per kg. 
         [0031]    The exo-amylase may be non-liquefying. This can be determined by letting the exo-amylase act on a 1% wheat starch solution until the reaction is complete, i.e. addition of fresh enzyme causes no further degradation, and analyzing the reaction products, e.g. by HPLC. Typical reaction conditions are e.g. 0.01 mg enzyme per ml starch solution for 48 hours. The exo-amylase is considered non-liquefying if the amount of residual starch after the reaction is at least 20% of the initial amount of starch. 
         [0032]    The exo-amylase may have maltogenic alpha-amylase activity (EC 3.2.1.133). The exo-amylase may be the amylase described in DK PA 2004 00021, or it may be a Novamyl variant described in this specification. 
       Dough and Dough-Based Edible Product 
       [0033]    The dough may have a sucrose content above 10% by weight, particularly above 20% or 30%, e.g. 30-40%. The flour content is typically 25-35% by weight of total ingredients. The dough may be made by a conventional cake recipe, typically with cake flour, sugar, fat/oil and eggs as the major ingredients. It may include other conventional ingredients such as emulsifiers, humectants, gums, starch and baking powder. It generally contains such ingredients as soft wheat flour, milk or other liquids, sugar, eggs, chemical leaveners, flavor extracts and spices, as well as others that may or may not include shortening. 
         [0034]    The dough is generally heat treated, e.g. by baking or deep frying to prepare an edible product such as cakes including pound cake, yellow and white layer cakes, cakes containing chocolate and cocoa products, sponge cakes, angel food cake, fruit cakes and foam-type cakes and doughnuts. 
       EXAMPLES 
     Example 1 
     Sucrose Tolerance of Novamyl Variants 
       [0035]    The amylase activity of a number of polypeptides were tested by incubation with Phadebas tablets (product of Pharmacia®) for 15 minutes at 60° C. in the presence of sucrose at various concentrations (in % by weight). The results are expressed in % of the result without sugar: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Alterations compared to 
                 0% 
                 10% 
                 20% 
                 40% 
               
               
                 SEQ ID NO: 1 
                 sucrose 
                 sucrose 
                 sucrose 
                 sucrose 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 None 
                 100 
                 13 
                 6 
                 1.5 
               
               
                 F188L, D261G, T288P 
                 100 
                 27.5 
                 14.5 
                 6 
               
               
                 F194S 
                 100 
                 31.5 
                 18.5 
                 7.5 
               
               
                 L196F 
                 100 
                 69 
                 42 
                 23 
               
               
                 D190G 
                 100 
                 65 
                 43 
                 21 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Sucrose Tolerance of Novamyl Variants 
       [0036]    A number of polypeptides were tested as in Example 1. The results are expressed as activity with 10% sucrose in % of the activity without sucrose: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Sugar 
               
               
                   
                 Alterations compared to SEQ ID NO: 1 
                 tolerance 
               
               
                   
                   
               
             
             
               
                   
                 None 
                 15 
               
               
                   
                 D261G, T288P 
                 24 
               
               
                   
                 F188L, D261G, T288P 
                 35 
               
               
                   
                 T288P 
                 56 
               
               
                   
                 Y89F, D261G, T288P 
                 42 
               
               
                   
                 N86V, F188L, D261G, T288P 
                 37 
               
               
                   
                 Y89F, F188L, D261G, T288P 
                 38 
               
               
                   
                 Y89H, F188L, D261G, T288P 
                 50 
               
               
                   
                 N86T, F188L, D261G, T288P 
                 49 
               
               
                   
                 F194S, D261G, T288P 
                 47 
               
               
                   
                 L196F 
                 65 
               
               
                   
                 D261G, T288P, D372V 
                 62 
               
               
                   
                 Q184H, N187D, F194Y 
                 47 
               
               
                   
                 D190G 
                 66 
               
               
                   
                 N86G, Y89M, F188L, D261G, T288P 
                 47 
               
               
                   
                 F188L, D190G, D261G, T288P 
                 68 
               
               
                   
                 A192Q, D261G, T288P, S446A 
                 46 
               
               
                   
                 F188H 
                 49 
               
               
                   
                 P191* 
                 42 
               
               
                   
                 A192* 
                 51 
               
               
                   
                 A192*, G193* 
                 67 
               
               
                   
                 *192aA 
                 44 
               
               
                   
                 N86K, F252L, D261G, T288P 
                 49 
               
               
                   
                 F194Y, L225S, D261G, T288P 
                 49 
               
               
                   
                 F194L, D261G, T288P 
                 54 
               
               
                   
                 F194S, D261G, T288P, P642Q 
                 60 
               
               
                   
                 D261G, T288P, N375S 
                 58 
               
               
                   
                 F188T 
                 37 
               
               
                   
                 F188G 
                 36 
               
               
                   
                 F188V 
                 41 
               
               
                   
                 A192R, F194L, D261G, T288P, G469R 
                 60 
               
               
                   
                 A192G, D261G, T288P 
                 41 
               
               
                   
                 Y89F, D261G, T288P, I290V, N375S 
                 60 
               
               
                   
                   
               
             
          
         
       
     
         [0037]    The following variants are also considered of interest in the context of the present invention: 
         [0000]    
       
         
               
             
               
               
             
           
               
                   
               
               
                 Alterations compared to SEQ ID NO: 1 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 I15T, N86K, P191S, D261G, T288P 
               
               
                   
                 I15T, P191S, D261G, T288P 
               
               
                   
                 I15T, P191S, Y258F, D261G, T288P, N375S, Y549C, Q648H 
               
               
                   
                 I15T, G153R, P191S, D261G, T288P, N371K, K645R 
               
               
                   
                   
               
             
          
         
       
     
       Example 3 
     Sucrose Tolerance and Thermostability of Amylases 
       [0038]    The following amylases were tested for thermostability and sugar tolerance: bacterial alpha-amylase from  B. amyloliquefaciens  (BAN™, product of Novozymes NS), fungal alpha-amylase from  A. oryzae  (Fungamyl®, product of Novozymes A/S), maltogenic alpha-amylase having the sequence of SEQ ID NO: 1 (Novamyl®, product of Novozymes A/S), a Novamyl variant having SEQ ID NO: 1 with the substitutions F188L+D261G+T288P, and bacterial alpha-amylase from  B. licheniformis  (Termamyl®, product of Novozymes A/S). 
       Exo-Amylase Activity 
       [0039]    The five amylases were tested for exo-amylase activity as described above. The results show that Novamyl and the Novamyl variant had exo-amylase activity by this test, and the other three did not. 
       Thermostability 
       [0040]    Each amylase was incubated at 85° C. at pH 5.7 (50 mM Na-acetate, 1 mM CaCl 2 ) without substrate, and the amylase activity was measured after 0, 15, 30 and 60 minutes heat treatment. The results are expressed as residual activity in % of the initial activity: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 0 
                 15 
                 30 
                 60 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 BAN 
                 100 
                 3 
                 1 
                 0 
               
               
                   
                 Fungamyl 
                 100 
                 0 
                 0 
                 0 
               
               
                   
                 Novamyl 
                 100 
                 51 
                 29 
                 13 
               
               
                   
                 Novamyl variant 
                 100 
                 64 
                 48 
                 54 
               
               
                   
                 Termamyl 
                 100 
                 100 
                 71 
                 85 
               
               
                   
                   
               
             
          
         
       
     
         [0041]    The results show that the Novamyl variant and Termamyl were not deactivated by the heat-treatment. BAN and Fungamyl lose all their activity after 15 min while Novamyl loses it gradually with heat-treatment time. 
       Sucrose Tolerance 
       [0042]    The experiment was repeated in 10% sucrose solution. The results are expressed as residual activity in % of the initial activity without sucrose: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 0 
                 15 
                 30 
                 60 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 BAN 
                 93 
                 2 
                 1 
                 0 
               
               
                   
                 Fungamyl 
                 31 
                 0 
                 0 
                 0 
               
               
                   
                 Novamyl 
                 7 
                 6 
                 1 
                 3 
               
               
                   
                 Novamyl variant 
                 21 
                 19 
                 14 
                 16 
               
               
                   
                 Termamyl 
                 116 
                 112 
                 97 
                 82 
               
               
                   
                   
               
             
          
         
       
     
         [0043]    The results show that BAN and Termamyl were not inhibited by sugar while Fungamyl and the Novamyl variant were somewhat inhibited, and Novamyl was heavily inhibited by sugar. The combination of sugar and heat-treatment shows that the Novamyl variant and Termamyl could be active during baking of cakes. Termamyl and the Novamyl variant fulfill the criterion for thermostability and sugar tolerance used in this invention. 
       Example 4 
     Preparation of Sponge Cake with Amylase 
       [0044]    Sponge cakes were made with addition of amylase as follows: BAN (0.83. 8.3 or 83 mg/kg flour), Novamyl (1.3 or 13 mg/kg flour) or the Novamyl variant used in Example 1 (1, 10 or 100 mg/kg flour). A control cake was made without amylase. 
         [0045]    The cakes were baked according to the High Ratio Sponge Sandwich Cake (HRSSC) method. After baking, the cakes were cooled down for 60-120 minutes, and the cakes were stored at room temperature in sealed plastic bags filled with nitrogen until analysis. The cakes were evaluated on day 1, 3, 7 or 23. 
         [0046]    Texture profile analysis (TPA) was performed as described in Bourne M. C. (2002) 2. ed., Food Texture and Viscosity: Concept and Measurement. Academic Press. The results showed that the increase in hardness was slower with increasing dosage of the Novamyl variant. The addition of BAN or Novamyl had only a slight effect, and only at the highest dosage. 
         [0047]    The cohesiveness of the cakes decreased with storage time. The addition of the Novamyl variant delayed this decrease. The addition of BAN or Novamyl had a slight effect, and only at the highest dosage. 
         [0048]    Water mobility was characterized by low field NMR. The addition of the Novamyl variant and BAN increased the mobility, indicating that the two amylases were able to keep the cakes more moist. Novamyl had virtually no effect. 
         [0049]    A small sensory evaluation of softness and moistness was performed on day 13 for the 3 cakes with the Novamyl variant and the control cake. The cakes were evaluated regarding three parameters; Firmness, Moistness and preferability. The control was the firmest, driest and least preferred. The higher dosage of the Novamyl variant, the less firm (softer), moister and better liked. 
         [0050]    A large panel sensory evaluation was performed on day 13. It was a paired comparison test where a control cake was compare to the cake with the Novamyl variant at the highest dosage. A 30-member panel was asked two questions (1) Which cake is moister and (2) which cake is fresher. All panel members agreed on that the cake with the Novamyl variant was moister and fresher. The preference was significant at a significance level above 99.999%. 
         [0051]    To summarize, the data show that the Novamyl variant had anti-staling properties and was able to improve moistness perception and moistness measured by NMR. The two other amylases had only a slight effect. 
       Example 5 
     High-Ratio Unit Cakes 
       [0052]    Cakes were made with addition of amylase as follows: BAN (0.83. 8.3 or 83 mg/kg flour) or the Novamyl variant used in Example 1 (1, 10 or 100 mg/kg flour). A control cake was made without amylase. 
         [0053]    Cakes were baked according to the High ratio unit cake (HRUC) method. After baking, the cakes were cooled down for 60-120 minutes, and the cakes were stored at room temperature in sealed plastic bags filled with Nitrogen until analysis. The cakes were evaluated on day 7, 20 and 34 by the same methods as in the previous example. 
         [0054]    The increase in hardness was slower with the Novamyl variant at the highest dosage. The addition of BAN to the cake resulted in a low volume and a doughy cake which gave poor results in hardness measurements. 
         [0055]    The addition of the Novamyl variant delayed the decrease in cohesiveness while BAN did not influence it at all. 
         [0056]    The Novamyl variant and BAN were able to keep the cake more moist than the control. This increase in mobility of the free water could partly be explained by the cakes with BAN and the Novamyl variant being able to retain the moisture content. 
         [0057]    A small sensory evaluation on day 34 showed that the cake with the Novamyl variant at the highest dosage was clearly better than the control cake; it was more moist and it was less crumbly. 
         [0058]    Over-all, there was an anti-staling effect of the Novamyl variant at the high dosage, similar to the effect on sponge cakes in the previous example. The staling of HRUC cakes was slower than Sponge cakes but it was still evident that the Novamyl variant had an anti-staling effect. The anti-staling effect was seen with texture analysis, NMR and sensory evaluation. BAN showed anti-staling effects in HRUC but it was sensitive to over-dosage which resulted in cake collapse and a doughy cake. 
       Example 6 
     Sponge Cake 
       [0059]    Sponge cakes were made with addition of the amylase of DK PA 2004 00021 at dosages 0.5, 1, 2, 5 and 20 mg/kg flour and a control cake without amylase. 
         [0060]    Texture and NMR was measured on day 1, 7 and 13. The addition of the amylase reduced the increase in firmness, especially at the highest dosage. The amylase also had a beneficial effect on the mobility of water which was correlated with the moistness of the cake. 
         [0061]    A blind sensory ranking evaluation performed on day 14 showed a ranking according to the dosage, the higher dosage the more soft and moist cake. The most preferred cake was the one with the highest dosage. 
       Example 7 
     Baking Procedure Tegral Allegro Cake 
     Recipe 
       [0062]    The following recipe was used: 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 % 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Tegral Allegro mix* 
                 100 
               
               
                   
                 Pasteurized whole 
                 50 
               
               
                   
                 egg 
               
               
                   
                 Butter 
                 50 
               
               
                   
                 Enzymes 
                 According to trial. 
               
               
                   
                   
                 0 or 25 mg/kg flour. 
               
               
                   
                   
               
               
                   
                 *commercially available from Puratos NV/SA, Groot-Bijgaarden, Belgium 
               
             
          
         
       
     
       Procedure 
       [0063]    The ingredients were scaled into a mixing bowl and mixed using an industrial mixer (e.g. Bjorn AR 5 A Varimixer) with a suitable paddle speed. 300 g of the dough was poured into forms. The cakes are baked in a suitable oven (e.g. Sveba Dahlin deck oven) for 45 min. at 180° C. The cakes were allowed to cool down at room temperature for 1 hour. 
         [0064]    The volume of the cakes was determined when the cakes had cooled down using the rape seed displacement method. The cakes were packed under nitrogen in sealed plastic bags and stored at room temperature until analysis. 
         [0065]    The cakes were evaluated on day 1, 7 and 14, two cakes were used at each occasions. 
         [0066]    The cohesiveness and hardness of the cakes was evaluated with Texture analyser and the water mobility was characterized by low field NMR. 
         [0067]    The Texture profile analysis (TPA) was performed as described in Bourne M. C. (2002) 2. ed., Food Texture and Viscosity: Concept and Measurement. Academic Press. 
         [0068]    The mobility of free water was determined as described by P. L. Chen, Z. Long, R. Ruan and T. P. Labuza, Nuclear Magnetic Resonance Studies of water Mobility in Bread during Storage. Lebensmittel Wissenschaft and Technologie 30, 178-183 (1997). The mobility of free water has been described in literature to correlate to moistness of bread crumb. 
       Result 
       [0069]    Compared to cakes with no addition of enzymes the volume of the cakes is not affected by the addition of the reference enzyme (SEQ ID NO.: 1) nor by the addition of variants hereof, i.e. the cakes did not collapse upon addition of enzyme. 
         [0070]    The cohesiveness of the cakes decreased with storage time. The addition of variants of SEQ ID NO: 1 delayed this decrease as can be seen in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Change in Cohesiveness [gs/gs] with storage time of 
               
               
                 cakes with 25 mg protein enzyme per kg flour 
               
             
          
           
               
                 Enzyme 
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
               
               
                 No enzyme 
                 0.44 
                 0.35 
                 0.32 
               
               
                 Seq ID No: 1 
                 0.43 
                 0.38 
                 0.36 
               
               
                 F188L, D261G, T288P 
                 0.46 
                 0.42 
                 0.41 
               
               
                 Y89F, D261G, T288P 
                 0.45 
                 0.43 
                 0.39 
               
               
                 N86G, Y89M, F188L, D261G, T288P 
                 0.44 
                 0.42 
                 0.38 
               
               
                 T288P 
                 0.44 
                 0.40 
                 0.41 
               
               
                 F194S, D261G, T288P 
                 0.47 
                 0.43 
                 0.42 
               
               
                 D261G, T288P, D372V 
                 0.46 
                 0.43 
                 0.37 
               
               
                 A192Q, D261G, T288P, S446A 
                 0.44 
                 0.42 
                 0.39 
               
               
                 A192R, F194L, D261G, T288P, G469R 
                 0.47 
                 0.44 
                 0.42 
               
               
                 A192G, D261G, T288P 
                 0.46 
                 0.42 
                 0.39 
               
               
                 N86K, F252L, D261G, T288P 
                 0.45 
                 0.41 
                 0.39 
               
               
                 F194L, D261G, T288P 
                 0.45 
                 0.42 
                 0.42 
               
               
                 F194S, D261G, T288P, P642Q 
                 0.44 
                 0.40 
                 0.39 
               
               
                 Y89F, D261G, T288P, I290V, N375S 
                 0.43 
                 0.42 
                 0.40 
               
               
                   
               
             
          
         
       
     
         [0071]    The free water mobility is correlated with the moist perception of the cake crumb, it decreases with time. The addition of the Novamyl variants increased the mobility compared to the control, indicating that the amylases were able to keep the cakes more moist. Results are listed in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Change in free water mobility [micros] with storage time of 
               
               
                 cakes with 25 mg protein enzyme per kg flour 
               
             
          
           
               
                 Enzyme 
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
               
               
                 No enzyme 
                 7077 
                 5111 
                 4175 
               
               
                 Seq ID No: 1 
                 6990 
                 5460 
                 4583 
               
               
                 F188L, D261G, T288P 
                 7216 
                 5624 
                 4656 
               
               
                 Y89F, D261G, T288P 
                 7085 
                 6044 
                 5151 
               
               
                 N86G, Y89M, F188L, D261G, T288P 
                 7493 
                 5349 
                 5120 
               
               
                 T288P 
                 7458 
                 5785 
                 4858 
               
               
                 F194S, D261G, T288P 
                 7746 
                 6373 
                 5325 
               
               
                 D261G, T288P, D372V 
                 7417 
                 5517 
                 4525 
               
               
                 A192Q, D261G, T288P, S446A 
                 7357 
                 5714 
                 5041 
               
               
                 A192R, F194L, D261G, T288P, G469R 
                 7549 
                 5536 
                 no data 
               
               
                 A192G, D261G, T288P 
                 7546 
                 5815 
                 no data 
               
               
                 N86K, F252L, D261G, T288P 
                 7349 
                 5295 
                 4775 
               
               
                 F194L, D261G, T288P 
                 7773 
                 6803 
                 5750 
               
               
                 F194S, D261G, T288P, P642Q 
                 8152 
                 5969 
                 4971 
               
               
                 Y89F, D261G, T288P, I290V, N375S 
                 7753 
                 6175 
                 4811 
               
               
                   
               
             
          
         
       
     
         [0072]    The hardness of the cakes increased with storage time. The addition of variants of SEQ ID NO: 1 delayed this increase in hardness as can be seen in Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Change in hardness [g] with storage time of cakes with 
               
               
                 25 mg protein enzyme per kg flour 
               
             
          
           
               
                 Enzyme 
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
               
             
          
           
               
                 No enzyme 
                 647 
                 1060 
                 1408 
               
               
                 Seq ID No: 1 
                 677 
                 997 
                 1171 
               
               
                 F188L, D261G, T288P 
                 683 
                 951 
                 1167 
               
               
                 Y89F, D261G, T288P 
                 649 
                 998 
                 1160 
               
               
                 N86G, Y89M, F188L, D261G, T288P 
                 630 
                 844 
                 1194 
               
               
                 T288P 
                 719 
                 1101 
                 1098 
               
               
                 F194S, D261G, T288P 
                 672 
                 943 
                 1061 
               
               
                 D261G, T288P, D372V 
                 593 
                 962 
                 1344 
               
               
                 A192Q, D261G, T288P, S446A 
                 680 
                 931 
                 1159 
               
               
                 A192R, F194L, D261G, T288P, G469R 
                 720 
                 987 
                 1209 
               
               
                 A192G, D261G, T288P 
                 707 
                 1024 
                 1102 
               
               
                 N86K, F252L, D261G, T288P 
                 678 
                 955 
                 1248 
               
               
                 F194L, D261G, T288P 
                 648 
                 895 
                 1050 
               
               
                 F194S, D261G, T288P, P642Q 
                 674 
                 1028 
                 1316 
               
               
                 Y89F, D261G, T288P, I290V, N375S 
                 602 
                 731 
                 827