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 
       [0001]    The present invention comprises a sequence listing. 
       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, the structures are included herein by reference. 
         [0006]    WO 2006/032281 describes methods of preparing a dough-based product with a high sucrose content using anti-staling amylases. 
       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. A selection of particularly interesting Novamyl® (SEQ ID NO:1) variants were identified comprising the two specific substitutions D261G and T288P in combination with at least one other amino acid alteration, preferably at least two other amino acid alterations, or three other amino acid alterations, or most preferably in combination with at least four other amino acid alterations. 
         [0009]    Accordingly, in a first aspect the invention provides a method of preparing a dough or a dough-based edible product, comprising adding a polypeptide to the dough, wherein the dough comprises at least 10% sucrose by weight, and the polypeptide has an amino acid sequence which is at least 70% identical to SEQ ID NO: 1, and compared to SEQ ID NO: 1 comprises the two substitutions D261G and T288P and at least one additional amino acid alteration which is substitution or deletion of or insertion adjacent to Y89, W93, P191, F194, Y360, or N375. 
         [0010]    In a second aspect, the invention provides polypeptides, which have amylase activity less inhibited by sucrose than the amylase activity of SEQ ID NO: 1, which have an amino acid sequence which is at least 70% identical to SEQ ID NO: 1, and which when compared to SEQ ID NO: 1 comprises the two substitutions D261G and T288P and at least one additional amino acid alteration, which is substitution or deletion of or insertion adjacent to Y89, W93, P191, F194, Y360, or N375. The invention also provides methods of producing such novel sucrose tolerant polypeptide variants of a maltogenic alpha-amylase. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0011]    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 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Maltogenic Alpha-Amylase and Sucrose Docking 
       [0012]    A maltogenic alpha-amylase (EC 3.2.1.133) having more than 70% identity, such as, at least 75%, (particularly more than 80% identity, such as at least 85%, or 90% identity, 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. 
         [0013]    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, which is used as a reference enzyme in the examples below. 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 if (26 Mar. 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317-334 (1990); or Example 1 of WO 9623874. 
         [0014]    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 1 QHO.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 
       [0015]    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 
       [0016]    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 residues are within 4 Å: K44, N86, Y89, H90, Y92, W93, F188, T189, D190, P191, A192, F194, D372, P373, R376. 
         [0017]    Further the following two positions of SEQ ID NO: 1 have been identified as relevant: Y360 and N375. 
         [0018]    Particularly preferred embodiments of the invention are amino acid alterations in the following 2-6 positions compared to Novamyl® (SEQ ID NO: 1) in combination with the two specific substitutions D261G and T288P: 
         [0019]    Y89+W93 
         [0020]    Y89+P191 
         [0021]    Y89+F194 
         [0022]    Y89+Y360 
         [0023]    Y89+N375 
         [0024]    W93+P191 
         [0025]    W93+F194 
         [0026]    W93+Y360 
         [0027]    W93+N375 
         [0028]    P191+F194 
         [0029]    P191+Y360 
         [0030]    P191+N375 
         [0031]    F194+Y360 
         [0032]    F194+N375 
         [0033]    Y360+N375 
         [0034]    Y89+W93+P191 
         [0035]    Y89+W93+F194 
         [0036]    Y89+W93+Y360 
         [0037]    Y89+W93+N375 
         [0038]    Y89+P191+F194 
         [0039]    Y89+P191+Y360 
         [0040]    Y89+P191+N375 
         [0041]    Y89+F194+Y360 
         [0042]    Y89+F194+N375 
         [0043]    Y89+Y360+N375 
         [0044]    W93+P191+F194 
         [0045]    W93+P191+Y360 
         [0046]    W93+P191+N375 
         [0047]    W93+F194+Y360 
         [0048]    W93+F194+N375 
         [0049]    W93+Y360+N375 
         [0050]    P191+F194+Y360 
         [0051]    P191+F194+N375 
         [0052]    P191+Y360+N375 
         [0053]    F194+Y360+N375 
         [0054]    Y89+W93+P191+F194 
         [0055]    Y89+W93+P191+Y360 
         [0056]    Y89+W93+P191+N375 
         [0057]    Y89+W93+F194+Y360 
         [0058]    Y89+W93+F194+N375 
         [0059]    Y89+W93+Y360+N375 
         [0060]    Y89+P191+F194+Y360 
         [0061]    Y89+P191+F194+N375 
         [0062]    Y89+P191+Y360+N375 
         [0063]    Y89+F194+Y360+N375 
         [0064]    W93+P191+F194+Y360 
         [0065]    W93+P191+F194+N375 
         [0066]    W93+P191+Y360+N375 
         [0067]    W93+F194+Y360+N375 
         [0068]    P191+F194+Y360+N375 
         [0069]    Y89+W93+P191+F194+Y360 
         [0070]    Y89+W93+P191+F194+N375 
         [0071]    Y89+W93+P191+Y360+N375 
         [0072]    Y89+W93+F194+Y360+N375 
         [0073]    Y89+P191+F194+Y360+N375 
         [0074]    W93+P191+F194+Y360+N375 
         [0075]    Y89+W93+P191+F194+Y360+N375 
         [0076]    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. 
         [0077]    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): 
         [0078]    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≦Y≦W 
         [0079]    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. 
         [0080]    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. 
         [0081]    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/GN/T/S, F188H/L/I/T/G/V, D190E/Q/G, P191S/N, A192S/T, F194S/L/Y, L196F, Y360F/I/N, N371K/R/F/Y/Q, D372E/Q/S/T/A and N375S/T/D/E/Q. 
         [0082]    Most preferred embodiments of amino acid alterations in the above-listed preferred positions are the following substitutions, alone or in combination: 
         [0083]    Y89F, W 
         [0084]    W93F, Y 
         [0085]    P191S, N 
         [0086]    F194Y, S, L 
         [0087]    Y360F, I, N 
         [0088]    N375S, T 
         [0089]    Examples of deletions are deletion of residue 191 or 192. An example of an insertion is Ala inserted between 192 and 193. 
         [0090]    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. 
         [0091]    Particularly preferred embodiments of the invention are the following amino acid alterations compared to Novamyl® (SEQ ID NO: 1), all of which are tested in the examples below: 
         [0092]    Y89F, D261G, T288P, I290V, N375S 
         [0093]    F194Y, D261G, T288P, N375S 
         [0094]    I15T, P191S, D261G, T288P, N375S, S640I 
         [0095]    Y89F, P191S, D261G, T288P 
         [0096]    I15T, Y89F, P191S, D261G, T288P 
         [0097]    Y89F, F194Y, D261G, T288P 
         [0098]    Y89F, D261G, T288P, N375S 
         [0099]    Y89F, P1915, F194Y, D261G, T288P 
         [0100]    Y89F, P1915, D261G, T288P, N3755 
         [0101]    Y89F, P191S, D261G, T288P, Y360N 
         [0102]    Y89F, P191S, D261G, T288P, Y360F 
         [0103]    Y89F, W93Y, P191S, D261G, T288P 
         [0104]    Y89F, W93F, P191S, D261G, T288P 
         [0105]    Y89F, P1915, F194Y, D261G, T288P, N3755 
       Nomenclature for Amino Acid Alterations 
       [0106]    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 
         [0107]    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. 
         [0108]    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). 
         [0109]    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° A activity at 10% sugar, more than 10% activity at 20% sucrose, or more than 4% activity at 40° A sucrose. The sugar tolerance may be determined as described in the examples. 
         [0110]    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. 
         [0111]    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. 
         [0112]    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. 
         [0113]    The exo-amylase may have maltogenic alpha-amylase activity (EC 3.2.1.133). The exo-amylase may be the amylase described in WO 2005/066338, or it may be a Novamyl® variant described in this specification. 
       Dough and Dough-Based Edible Product 
       [0114]    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. 
         [0115]    Examples of emulsifiers include mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxethylene stearates, or lysolecithin. Conventional emulsifiers used in making flour dough products include as examples monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, and lecithins e.g. obtained from soya. The emulsifying agent may be an emulsifier per se or an agent that generates an emulsifier in situ. Examples of emulsifying agents that can generate an emulsifier in situ include enzymes, such as, lipase or phospholipases. 
         [0116]    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. 
         [0117]    Optionally, one or more additional enzymes may be used together with the anti-staling amylase of the present invention in preparing dough and dough-based edible products. The additional enzyme may be a starch degrading enzyme, such as, another amylase (e.g., an alpha-amylase, beta-amylase and/or a glucoamylase) or pullulanase, a cyclodextrin glucanotransferase, a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a phospholipase, a cellulase, a hemicelluase, a protease, a glycosyltransferase, a branching enzyme (1,4-alpha-glucan branching enzyme), an oxidoreductase or oxidase (e.g., a monosaccharide oxidase, such as, glucose oxidase, hexose oxidase, galactose oxidase or pyranose oxidase). Sources of these additional enzymes are well known in the art. 
         [0118]    The additional enzyme may be of any origin, including mammalian and plant, and preferably of microbial (bacterial, yeast or fungal) origin. For example, the amylase may be fungal or bacterial, e.g., an alpha-amylase from  Bacillus , e.g.  B. lichenifornis  or  B. amyloliquefaciens , a beta-amylase, e.g. from plant (e.g. soy bean) or from microbial sources (e.g.  Bacillus ), a glucoamylase, e.g. from  A. niger , or a fungal alpha-amylase, e.g. from  A. oryzae.    
         [0119]    The hemicellulase may be a pentosanase, e.g. a xylanase which may be of microbial origin, e.g. derived from a bacterium or fungus, such as a strain of  Aspergillus , in particular of  A. aculeatus, A. niger, A. awamori , or  A. tubigensis , from a strain of  Trichoderma , e.g.  T. reesei , or from a strain of  Humicola , e.g.  H. insolens.    
         [0120]    The protease may be from  Bacillus , e.g.  B. amyloliquefaciens.    
         [0121]    The lipase may be derived from a strain of  Thermomyces  ( Humicola ),  Rhizomucor, Candida, Aspergillus, Rhizopus , or  Pseudomonas , in particular from  T. lanuginosus  ( H. ianuginosa ),  Rhizomucor miehei, C. antarctica, A niger, Rhizopus delemar, Rhizopus arrhizus  or  P. cepacia.    
         [0122]    The phospholipase may have phospholipase A1 or A2 or lysophospholipase activity; it may or may not have lipase activity. It may be of animal origin, e.g. from pancreas, snake venom or bee venom, or it may be of microbial origin, e.g. from filamentous fungi, yeast or bacteria, such as  Aspergillus  or  Fusarium , e.g.  A. niger, A. oryzae  or  F. oxysporum . Also the variants described in WO 0032578 may be used. 
         [0123]    The oxidoreductase may be a peroxidase, a laccase or a lipoxygenase. The glucose oxidase may be derived from a strain of  Aspergillus  or  Penicillium , particularly  A. niger, P. notatum, P. amagasakiense  or  P. vitale . The hexose oxidase may be one described in EP 833563. The pyranose oxidase may be one described in WO 9722257, e.g. derived from Trametes, particularly  T. hirsuta . The galactose oxidase may be one described in WO 0050606. 
       EXAMPLES 
     Example 1 
     Baking Procedure Tegral Allegro Cake 
       [0124]    Baking examples on the following Novamyl® variants are included in this example: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Variants, Mutations, Report number and Study number 
               
             
          
           
               
                 Variant 
                 Substitutions 
               
               
                   
               
               
                 Ref 
                 F188L, D261G, T288P 
               
               
                 A 
                 Y89F, D261G, T288P, I290V, N375S 
               
               
                 B 
                 F194Y, D261G, T288P, N375S 
               
               
                 C 
                 I15T, P191S, D261G, T288P, N375S, S640I 
               
               
                 D 
                 Y89F, P191S, D261G, T288P 
               
               
                 E 
                 I15T, Y89F, P191S, D261G, T288P 
               
               
                 F 
                 Y89F, F194Y, D261G, T288P 
               
               
                 G 
                 Y89F, D261G, T288P, N375S 
               
               
                 H 
                 Y89F, P191S, F194Y, D261G, T288P 
               
               
                 I 
                 Y89F, P191S, D261G, T288P, N375S 
               
               
                 J 
                 Y89F, P191S, D261G, T288P, Y360N 
               
               
                 K 
                 Y89F, P191S, D261G, T288P, Y360F 
               
               
                 L 
                 Y89F, W93Y, P191S, D261G, T288P 
               
               
                 M 
                 Y89F, W93F, P191S, D261G, T288P 
               
               
                 N 
                 Y89F, P191S, F194Y, D261G, T288P, N375S 
               
               
                   
               
             
          
         
       
     
       Recipe 
       [0125]    The following recipe was used: 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 % 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Tegral 
                 100  
               
               
                   
                 Allegro mix* 
               
               
                   
                 Pasteurized 
                 50 
               
               
                   
                 whole egg 
               
               
                   
                 Butter 
                 50 
               
               
                   
                 Enzymes 
                 According to trial. In the 
               
               
                   
                   
                 range 0-25 mg protein 
               
               
                   
                   
                 enzyme/kg flour. 
               
               
                   
                   
               
               
                   
                 *commercially available from Puratos NV/SA, Groot-Bijgaarden, Belgium 
               
             
          
         
       
     
       Procedure 
       [0126]    The ingredients were scaled into a mixing bowl and mixed using an industrial mixer (e.g. Bjørn/Bear AR 5 A Varimixer®) with a suitable paddle speed. 300 g of the dough was poured into forms. The cakes were 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. 
         [0127]    The volumes of the cakes were 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. 
         [0128]    The cakes were evaluated on day 1, 7 and 14, two cakes were used at each occasion, and three slices of cakes were analyzed from each cake. 
         [0129]    The cohesiveness and hardness of the cakes was evaluated using the texture profile analysis (TPA) with TA-XTplus texture analyzer and the water mobility was characterized by low field NMR. 
         [0130]    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. 
         [0131]    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 
       [0132]    The volume of the cakes can be found in table 2, the volume of cakes without any enzyme is set to 100%. 
         [0133]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Volume of cakes as a function of added enzyme 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 0 mg/kg 
                 5 mg/kg 
                 10 mg/kg 
                 25 mg/kg 
               
               
                   
                   
               
               
                   
                 Ref 
                 100 
                   
                 98 
                 97 
               
               
                   
                 A 
                 100 
                   
                 95 
                 97 
               
               
                   
                 C 
                 100 
                 96 
                 96 
               
               
                   
                 E 
                 100 
                 98 
                 100 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 0 mg/kg 
                 3 mg/kg 
                 4 mg/kg 
                 5 mg/kg 
               
               
                   
                   
               
               
                   
                 M 
                 100 
                 94 
                 97 
                 95 
               
               
                   
                 B 
                 100 
                 95 
                 94 
                 93 
               
               
                   
                 D 
                 100 
                 95 
                 96 
                 96 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 0 mg/kg 
                 3 mg/kg 
                 6 mg/kg 
                 9 mg/kg 
                 12 mg/kg 
               
               
                   
                   
               
               
                   
                 F 
                 100 
                 99 
                 99 
                 101 
                 103 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 0 mg/kg 
                 2 mg/kg 
                 4 mg/kg 
                 6 mg/kg 
                 8 mg/kg 
               
               
                   
                   
               
               
                   
                 D 
                 100 
                 96 
                 97 
                 94 
               
               
                   
                 H 
                 100 
                 97 
                 93 
                 94 
               
               
                   
                 N 
                 100 
                 95 
                 95 
                 95 
               
               
                   
                 J 
                 100 
                 93 
                 93 
                 92 
               
               
                   
                 K 
                 100 
                 94 
                 92 
                 90 
               
               
                   
                 I 
                 100 
                 94 
                 97 
                 96 
               
               
                   
                 L 
                 100 
                 99 
                 98 
                 97 
               
               
                   
                 G 
                 100 
                   
                 93 
                 92 
                 94 
               
               
                   
                   
               
             
          
         
       
     
         [0134]    The cohesiveness of the cakes decreased with storage time. The addition of variants of Novamyl® delayed this decrease as can be seen in Table 3. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Change in Cohesiveness of cakes with time measured with 
               
               
                 the TPA method 
               
             
          
           
               
                   
                 Dosage 
               
               
                   
                   
               
             
          
           
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.46 
                 0.36 
                 0.32 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.45 
                 0.42 
                 0.39 
               
               
                   
                   
                 25 mg/kg 
                 0.45 
                 0.42 
                 0.42 
               
               
                   
                 A 
                 10 mg/kg 
                 0.44 
                 0.40 
                 0.38 
               
               
                   
                   
                 25 mg/kg 
                 0.43 
                 0.42 
                 0.40 
               
               
                   
                 No enzyme 
                 — 
                 0.52 
                 0.43 
                 0.39 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.56 
                 0.49 
                 0.44 
               
               
                   
                   
                 25 mg/kg 
                 0.57 
                 0.52 
                 0.49 
               
               
                   
                 H 
                  2 mg/kg 
                 0.56 
                 0.49 
                 0.45 
               
               
                   
                   
                  4 mg/kg 
                 0.58 
                 0.51 
                 0.48 
               
               
                   
                   
                  6 mg/kg 
                 0.56 
                 0.51 
                 0.48 
               
               
                   
                 N 
                  2 mg/kg 
                 0.54 
                 0.47 
                 0.45 
               
               
                   
                   
                  4 mg/kg 
                 0.57 
                 0.53 
                 0.47 
               
               
                   
                   
                  6 mg/kg 
                 0.56 
                 0.52 
                 0.48 
               
               
                   
                 D 
                  2 mg/kg 
                 0.57 
                 0.48 
                 0.45 
               
               
                   
                   
                  4 mg/kg 
                 0.55 
                 0.50 
                 0.46 
               
               
                   
                   
                  6 mg/kg 
                 0.55 
                 0.49 
                 0.46 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 15 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.52 
                 0.42 
                 0.36 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.54 
                 0.48 
                 0.44 
               
               
                   
                   
                 25 mg/kg 
                 0.56 
                 0.50 
                 0.46 
               
               
                   
                 J 
                  2 mg/kg 
                 0.54 
                 0.46 
                 0.41 
               
               
                   
                   
                  4 mg/kg 
                 0.53 
                 0.47 
                 0.42 
               
               
                   
                   
                  6 mg/kg 
                 0.54 
                 0.49 
                 0.44 
               
               
                   
                 K 
                  2 mg/kg 
                 0.54 
                 0.46 
                 0.40 
               
               
                   
                   
                  4 mg/kg 
                 0.53 
                 0.48 
                 0.43 
               
               
                   
                   
                  6 mg/kg 
                 0.53 
                 0.48 
                 0.43 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.50 
                 0.47 
                 0.43 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.54 
                 0.51 
                 0.48 
               
               
                   
                   
                 25 mg/kg 
                 0.55 
                 0.55 
                 0.52 
               
               
                   
                 I 
                  2 mg/kg 
                 0.53 
                 0.51 
                 0.48 
               
               
                   
                   
                  4 mg/kg 
                 0.54 
                 0.53 
                 0.50 
               
               
                   
                   
                  6 mg/kg 
                 0.51 
                 0.50 
                 0.49 
               
               
                   
                 L 
                  2 mg/kg 
                 0.52 
                 0.49 
                 0.47 
               
               
                   
                   
                  4 mg/kg 
                 0.54 
                 0.51 
                 0.49 
               
               
                   
                   
                  6 mg/kg 
                 0.53 
                 0.52 
                 0.51 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 6 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.46 
                 0.38 
                 0.35 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.49 
                 0.43 
                 0.42 
               
               
                   
                   
                 25 mg/kg 
                 0.48 
                 0.44 
                 0.44 
               
               
                   
                 E 
                  5 mg/kg 
                 0.47 
                 0.42 
                 0.39 
               
               
                   
                   
                 10 mg/kg 
                 0.47 
                 0.41 
                 0.41 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.43 
                 0.33 
                 0.28 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.45 
                 0.38 
                 0.35 
               
               
                   
                   
                 25 mg/kg 
                 0.45 
                 0.40 
                 0.37 
               
               
                   
                 G 
                  4 mg/kg 
                 0.46 
                 0.36 
                 0.34 
               
               
                   
                   
                  6 mg/kg 
                 0.45 
                 0.40 
                 0.37 
               
               
                   
                   
                  8 mg/kg 
                 0.44 
                 0.38 
                 0.37 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 3 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.45 
                 0.41 
                 0.37 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.50 
                 0.47 
                 0.45 
               
               
                   
                   
                 25 mg/kg 
                 0.51 
                 0.49 
                 0.48 
               
               
                   
                 D 
                  3 mg/kg 
                 0.48 
                 0.46 
                 0.43 
               
               
                   
                   
                  4 mg/kg 
                 0.50 
                 0.48 
                 0.45 
               
               
                   
                   
                  5 mg/kg 
                 0.50 
                 0.47 
                 0.45 
               
               
                   
                 B 
                  3 mg/kg 
                 0.50 
                 0.48 
                 0.45 
               
               
                   
                   
                  4 mg/kg 
                 0.51 
                 0.49 
                 0.47 
               
               
                   
                   
                  5 mg/kg 
                 0.52 
                 0.49 
                 0.47 
               
               
                   
                 M 
                  3 mg/kg 
                 0.50 
                 0.48 
                 0.45 
               
               
                   
                   
                  4 mg/kg 
                 0.50 
                 0.47 
                 0.45 
               
               
                   
                   
                  5 mg/kg 
                 0.51 
                 0.49 
                 0.46 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 0.42 
                 0.34 
                 0.31 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.44 
                 0.40 
                 0.38 
               
               
                   
                   
                 25 mg/kg 
                 0.44 
                 0.43 
                 0.40 
               
               
                   
                 F 
                  3 mg/kg 
                 0.43 
                 0.39 
                 0.34 
               
               
                   
                   
                  6 mg/kg 
                 0.43 
                 0.40 
                 0.35 
               
               
                   
                   
                  9 mg/kg 
                 0.43 
                 0.40 
                 0.35 
               
               
                   
                   
                 12 mg/kg 
                 0.43 
                 0.43 
                 0.36 
               
               
                   
                 No enzyme 
                 — 
                 0.40 
                 0.32 
                 0.28 
               
               
                   
                 Ref 
                 10 mg/kg 
                 0.42 
                 0.38 
                 0.35 
               
               
                   
                   
                 25 mg/kg 
                 0.44 
                 0.40 
                 0.40 
               
               
                   
                 C 
                  5 mg/kg 
                 0.43 
                 0.36 
                 0.33 
               
               
                   
                   
                 10 mg/kg 
                 0.42 
                 0.36 
                 0.34 
               
               
                   
                   
               
             
          
         
       
     
         [0135]    The hardness of the cakes increased with storage time. The addition of variants of Novamyl® delayed this increase in hardness as can be seen in Table 4. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Change in Hardness [g] of cakes with time measured 
               
               
                 with the TPA method 
               
             
          
           
               
                   
                 Dosage 
               
               
                   
                   
               
             
          
           
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 586 
                 875 
                 1093 
               
               
                   
                 Ref 
                 10 mg/kg 
                 597 
                 768 
                 955 
               
               
                   
                   
                 25 mg/kg 
                 603 
                 732 
                 957 
               
               
                   
                 A 
                 10 mg/kg 
                 612 
                 801 
                 953 
               
               
                   
                   
                 25 mg/kg 
                 602 
                 731 
                 827 
               
               
                   
                 No enzyme 
                 — 
                 515 
                 811 
                 1046 
               
               
                   
                 Ref 
                 10 mg/kg 
                 438 
                 651 
                 762 
               
               
                   
                   
                 25 mg/kg 
                 530 
                 687 
                 869 
               
               
                   
                 H 
                  2 mg/kg 
                 521 
                 809 
                 892 
               
               
                   
                   
                  4 mg/kg 
                 573 
                 773 
                 908 
               
               
                   
                   
                  6 mg/kg 
                 523 
                 702 
                 797 
               
               
                   
                 N 
                  2 mg/kg 
                 516 
                 751 
                 898 
               
               
                   
                   
                  4 mg/kg 
                 484 
                 668 
                 872 
               
               
                   
                   
                  6 mg/kg 
                 565 
                 753 
                 821 
               
               
                   
                 D 
                  2 mg/kg 
                 556 
                 794 
                 1015 
               
               
                   
                   
                  4 mg/kg 
                 524 
                 715 
                 953 
               
               
                   
                   
                  6 mg/kg 
                 521 
                 716 
                 876 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 15 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 484 
                 738 
                 956 
               
               
                   
                 Ref 
                 10 mg/kg 
                 518 
                 753 
                 976 
               
               
                   
                   
                 25 mg/kg 
                 559 
                 714 
                 868 
               
               
                   
                 J 
                  2 mg/kg 
                 576 
                 823 
                 1058 
               
               
                   
                   
                  4 mg/kg 
                 549 
                 761 
                 908 
               
               
                   
                   
                  6 mg/kg 
                 601 
                 798 
                 1092 
               
               
                   
                 K 
                  2 mg/kg 
                 584 
                 889 
                 1081 
               
               
                   
                   
                  4 mg/kg 
                 551 
                 811 
                 993 
               
               
                   
                   
                  6 mg/kg 
                 564 
                 782 
                 935 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 760 
                 1064 
                 1397 
               
               
                   
                 Ref 
                 10 mg/kg 
                 748 
                 943 
                 1175 
               
               
                   
                   
                 25 mg/kg 
                 747 
                 937 
                 1101 
               
               
                   
                 I 
                  2 mg/kg 
                 821 
                 1085 
                 1394 
               
               
                   
                   
                  4 mg/kg 
                 829 
                 1089 
                 1315 
               
               
                   
                   
                  6 mg/kg 
                 756 
                 947 
                 1071 
               
               
                   
                 L 
                  2 mg/kg 
                 795 
                 1033 
                 1363 
               
               
                   
                   
                  4 mg/kg 
                 808 
                 1062 
                 1164 
               
               
                   
                   
                  6 mg/kg 
                 770 
                 1020 
                 1160 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 6 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 602 
                 984 
                 1172 
               
               
                   
                 Ref 
                 10 mg/kg 
                 636 
                 897 
                 1035 
               
               
                   
                   
                 25 mg/kg 
                 627 
                 851 
                 1018 
               
               
                   
                 E 
                  5 mg/kg 
                 660 
                 987 
                 1176 
               
               
                   
                   
                 10 mg/kg 
                 644 
                 894 
                 1023 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 480 
                 966 
                 1284 
               
               
                   
                 Ref 
                 10 mg/kg 
                 593 
                 961 
                 1106 
               
               
                   
                   
                 25 mg/kg 
                 535 
                 843 
                 1079 
               
               
                   
                 G 
                  4 mg/kg 
                 692 
                 985 
                 1336 
               
               
                   
                   
                  6 mg/kg 
                 637 
                 1095 
                 1287 
               
               
                   
                   
                  8 mg/kg 
                 616 
                 1074 
                 1214 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 3 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 595 
                 827 
                 981 
               
               
                   
                 Ref 
                 10 mg/kg 
                 641 
                 829 
                 1020 
               
               
                   
                   
                 25 mg/kg 
                 589 
                 657 
                 809 
               
               
                   
                 D 
                  3 mg/kg 
                 599 
                 766 
                 987 
               
               
                   
                   
                  4 mg/kg 
                 678 
                 801 
                 1009 
               
               
                   
                   
                  5 mg/kg 
                 652 
                 847 
                 1018 
               
               
                   
                 B 
                  3 mg/kg 
                 627 
                 767 
                 973 
               
               
                   
                   
                  4 mg/kg 
                 639 
                 780 
                 923 
               
               
                   
                   
                  5 mg/kg 
                 578 
                 768 
                 960 
               
               
                   
                 M 
                  3 mg/kg 
                 679 
                 772 
                 1014 
               
               
                   
                   
                  4 mg/kg 
                 601 
                 814 
                 1005 
               
               
                   
                   
                  5 mg/kg 
                 646 
                 789 
                 957 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 616 
                 1038 
                 1255 
               
               
                   
                 Ref 
                 10 mg/kg 
                 682 
                 912 
                 1155 
               
               
                   
                   
                 25 mg/kg 
                 640 
                 844 
                 1077 
               
               
                   
                 F 
                  3 mg/kg 
                 683 
                 843 
                 1131 
               
               
                   
                   
                  6 mg/kg 
                 626 
                 831 
                 1131 
               
               
                   
                   
                  9 mg/kg 
                 644 
                 785 
                 947 
               
               
                   
                   
                 12 mg/kg 
                 571 
                 903 
                 992 
               
               
                   
                 No enzyme 
                 — 
                 469 
                 724 
                 952 
               
               
                   
                 Ref 
                 10 mg/kg 
                 474 
                 672 
                 956 
               
               
                   
                   
                 25 mg/kg 
                 515 
                 661 
                 873 
               
               
                   
                 C 
                  5 mg/kg 
                 546 
                 800 
                 1050 
               
               
                   
                   
                 10 mg/kg 
                 546 
                 753 
                 993 
               
               
                   
                   
               
             
          
         
       
     
         [0136]    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 5. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Change in Free water mobility [μs] of cakes with time 
               
               
                 measured with low field NMR 
               
             
          
           
               
                   
                 Dosage 
               
               
                   
                   
               
             
          
           
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 7703 
                 5339 
                 4138 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7755 
                 5670 
                 4434 
               
               
                   
                   
                 25 mg/kg 
                 7502 
                 5751 
                 4366 
               
               
                   
                 A 
                 10 mg/kg 
                 7809 
                 6124 
                 4839 
               
               
                   
                   
                 25 mg/kg 
                 7753 
                 6175 
                 4811 
               
               
                   
                 No enzyme 
                 — 
                 6464 
                 5577 
                 4618 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7009 
                 5507 
                 4831 
               
               
                   
                   
                 25 mg/kg 
                 7109 
                 5897 
                 5004 
               
               
                   
                 H 
                  2 mg/kg 
                 6819 
                 5521 
                 4743 
               
               
                   
                   
                  4 mg/kg 
                 7171 
                 5906 
                 5157 
               
               
                   
                   
                  6 mg/kg 
                 7340 
                 6196 
                 5131 
               
               
                   
                 N 
                  2 mg/kg 
                 7034 
                 5697 
                 4955 
               
               
                   
                   
                  4 mg/kg 
                 6897 
                 6024 
                 5067 
               
               
                   
                   
                  6 mg/kg 
                 7196 
                 6184 
                 5368 
               
               
                   
                 D 
                  2 mg/kg 
                 6936 
                 5753 
                 4910 
               
               
                   
                   
                  4 mg/kg 
                 7169 
                 5787 
                 5031 
               
               
                   
                   
                  6 mg/kg 
                 6876 
                 5983 
                 5228 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 15 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 7358 
                 5537 
                 4385 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7606 
                 5735 
                 4830 
               
               
                   
                   
                 25 mg/kg 
                 7456 
                 5648 
                 4977 
               
               
                   
                 J 
                  2 mg/kg 
                 7502 
                 5638 
                 4818 
               
               
                   
                   
                  4 mg/kg 
                 7575 
                 5663 
                 5081 
               
               
                   
                   
                  6 mg/kg 
                 7853 
                 6093 
                 5193 
               
               
                   
                 K 
                  2 mg/kg 
                 7714 
                 5649 
                 4845 
               
               
                   
                   
                  4 mg/kg 
                 7999 
                 5766 
                 5022 
               
               
                   
                   
                  6 mg/kg 
                 8029 
                 6291 
                 5312 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 7004 
                 4887 
                 4266 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7354 
                 5331 
                 4567 
               
               
                   
                   
                 25 mg/kg 
                 7489 
                 5486 
                 4856 
               
               
                   
                 I 
                  2 mg/kg 
                 7569 
                 5374 
                 4766 
               
               
                   
                   
                  4 mg/kg 
                 7596 
                 5241 
                 4925 
               
               
                   
                   
                  6 mg/kg 
                 7710 
                 5673 
                 5110 
               
               
                   
                 L 
                  2 mg/kg 
                 7495 
                 5101 
                 4647 
               
               
                   
                   
                  4 mg/kg 
                 7433 
                 5228 
                 4821 
               
               
                   
                   
                  6 mg/kg 
                 7577 
                 5342 
                 4988 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 6 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 7137 
                 5308 
                 4130 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7523 
                 6554 
                 4751 
               
               
                   
                   
                 25 mg/kg 
                 7449 
                 5808 
                 4778 
               
               
                   
                 E 
                  5 mg/kg 
                 7387 
                 6227 
                 4725 
               
               
                   
                   
                 10 mg/kg 
                 7288 
                 5670 
                 4786 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 6954 
                 5037 
                 4152 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7307 
                 5384 
                 4278 
               
               
                   
                   
                 25 mg/kg 
                 7461 
                 5323 
                 4440 
               
               
                   
                 G 
                  4 mg/kg 
                 7240 
                 5254 
                 4435 
               
               
                   
                   
                  6 mg/kg 
                 7077 
                 5371 
                 4659 
               
               
                   
                   
                  8 mg/kg 
                 7207 
                 5634 
                 4893 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 3 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 6322 
                 5101 
                 4458 
               
               
                   
                 Ref 
                 10 mg/kg 
                 6037 
                 5452 
                 4769 
               
               
                   
                   
                 25 mg/kg 
                 6882 
                 5515 
                 5304 
               
               
                   
                 D 
                  3 mg/kg 
                 6604 
                 5446 
                 4952 
               
               
                   
                   
                  4 mg/kg 
                 6373 
                 5466 
                 4550 
               
               
                   
                   
                  5 mg/kg 
                 6460 
                 5507 
                 4891 
               
               
                   
                 B 
                  3 mg/kg 
                 7126 
                 5896 
                 5334 
               
               
                   
                   
                  4 mg/kg 
                 7134 
                 5774 
                 5341 
               
               
                   
                   
                  5 mg/kg 
                 7117 
                 6014 
                 5203 
               
               
                   
                 M 
                  3 mg/kg 
                 7054 
                 5917 
                 5189 
               
               
                   
                   
                  4 mg/kg 
                 6449 
                 5362 
                 4833 
               
               
                   
                   
                  5 mg/kg 
                 7057 
                 5501 
                 5173 
               
               
                   
                   
               
               
                   
                   
                   
                 Day 1 
                 Day 7 
                 Day 14 
               
               
                   
                   
               
               
                   
                 No enzyme 
                 — 
                 7285 
                 4791 
                 3766 
               
               
                   
                 Ref 
                 10 mg/kg 
                 7410 
                 5437 
                 4542 
               
               
                   
                   
                 25 mg/kg 
                 7367 
                 4959 
                 4511 
               
               
                   
                 F 
                  3 mg/kg 
                 7114 
                 5462 
                 4593 
               
               
                   
                   
                  6 mg/kg 
                 7457 
                 5698 
                 4648 
               
               
                   
                   
                  9 mg/kg 
                 7298 
                 5309 
                 4293 
               
               
                   
                   
                 12 mg/kg 
                 7528 
                 5938 
                 4599 
               
               
                   
                 No enzyme 
                 — 
                 6722 
                 4344 
                 3477 
               
               
                   
                 Ref 
                 10 mg/kg 
                 6816 
                 4761 
                 3769 
               
               
                   
                   
                 25 mg/kg 
                 7008 
                 5109 
                 3923 
               
               
                   
                 C 
                  5 mg/kg 
                 6606 
                 5623 
                 4265 
               
               
                   
                   
                 10 mg/kg 
                 6746 
                 4782 
                 3690