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:
SEQUENCE LISTING AND DEPOSITED MICROORGANISMS  
       [0000]     Sequence Listing  
         [0001]     The present invention comprises a sequence listing.  
         [0000]     Deposit of Biological Material  
         [0002]     None.  
       FIELD OF THE INVENTION  
       [0003]     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  
       [0004]     U.S. Pat. No. 3,026,205 describes a process of producing baked confections and the products resulting therefrom by alpha-amylase.  
         [0005]     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  
       [0006]     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.  
         [0007]     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.  
         [0008]     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. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0000]     Maltogenic Alpha-Amylase and Sucrose Docking  
         [0009]     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.  
         [0010]     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.  
         [0011]     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 .  
         [0000]     Maltogenic Alpha-Amylase Assay  
         [0012]     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.  
         [0000]     Amino Acid Alterations  
         [0013]     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 Ångstroms 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.  
         [0014]     Further the following positions have been identified as relevant: I15, R81, T87, G88, L196, N371 or N375 of SEQ ID NO: 1.  
         [0015]     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.  
         [0016]     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
 
         [0017]     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.  
         [0018]     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.  
         [0019]     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.  
         [0020]     Examples of deletions are deletion of residue 191 or 192. An example of an insertion is Ala inserted between 192 and 193.  
         [0021]     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.  
         [0000]     Nomenclature for Amino Acid Alterations  
         [0022]     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.  
         [0000]     Properties of Anti-Staling Amylase for Use with Sucrose  
         [0023]     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.  
         [0024]     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).  
         [0025]     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.  
         [0026]     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.  
         [0027]     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.  
         [0028]     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.  
         [0029]     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.  
         [0000]     Dough and Dough-Based Edible Product  
         [0030]     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.  
         [0031]     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  
       [0032]     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:  
                                                                     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  
       [0033]     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:  
                                                   Alterations compared to   Sugar           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                      
 
         [0034]     The following variants are also considered of interest in the context of the present invention:  
                                         Alterations compared to SEQ ID NO: 1                                    115T, 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  
       [0035]     The following amylases were tested for thermostability and sugar tolerance: bacterial alpha-amylase from  B. amyloliquefaciens  (BAN™, product of Novozymes A/S), 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).  
         [0000]     Exo-Amylase Activity  
         [0036]     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.  
         [0000]     Thermostability  
         [0037]     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:  
                                                                                     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                      
 
         [0038]     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.  
         [0000]     Sucrose Tolerance  
         [0039]     The experiment was repeated in 10% sucrose solution. The results are expressed as residual activity in % of the initial activity without sucrose:  
                                                                                     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                      
 
         [0040]     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  
       [0041]     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.  
         [0042]     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.  
         [0043]     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.  
         [0044]     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.  
         [0045]     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.  
         [0046]     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.  
         [0047]     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%.  
         [0048]     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  
       [0049]     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.  
         [0050]     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.  
         [0051]     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.  
         [0052]     The addition of the Novamyl variant delayed the decrease in cohesiveness while BAN did not influence it at all.  
         [0053]     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.  
         [0054]     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.  
         [0055]     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  
       [0056]     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.  
         [0057]     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.  
         [0058]     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  
       [0000]     Recipe  
         [0059]     The following recipe was used:  
                                                             %                                        Tegral Allegro mix*   100           Pasteurized whole egg   50           Butter   50           Enzymes   According to trial. 0 or 25 mg/kg flour.                         *commercially available from Puratos NV/SA, Groot-Bijgaarden, Belgium             
 
 Procedure 
 
         [0060]     The ingredients were scaled into a mixing bowl and mixed using an industrial mixer (e.g. Bjørn 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.  
         [0061]     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.  
         [0062]     The cakes were evaluated on day 1, 7 and 14, two cakes were used at each occasions.  
         [0063]     The cohesiveness and hardness of the cakes was evaluated with Texture analyser and the water mobility was characterized by low field NMR.  
         [0064]     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.  
         [0065]     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 und Technologie 30, 178-183 (1997). The mobility of free water has been described in literature to correlate to moistness of bread crumb.  
         [0000]     Result  
         [0066]     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.  
         [0067]     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.  
                                 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                  
 
         [0068]     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.  
                                 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                  
 
         [0069]     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.  
                                                   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                  
 
         [0070]