Abstract:
A xylanase from  Bacillus halodurans  can increase the shelf life of baked products. More specifically, the xylanase in combination with a maltogenic amylase further improves the softness of bread crumb without having detrimental effects on elasticity.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a process for preparing a dough-based product and to a dough composition. 
       BACKGROUND OF THE INVENTION 
       [0002]    JP 2001-245665A (Oji Paper) describes a xylanase from  Bacillus halodurans  and its amino acid sequence. H. Takami et al.,  Nucl. Acid Res.  28 (21), 4317 (2000) describes the complete genome sequence of  B. halodurans ; it is included in the TrEMBL database with the accession number Q9KEF3. 
         [0003]    WO 0039289 describes the use of a xylanase from  Bacillus subtilis  for preparing dough. 
       SUMMARY OF THE INVENTION 
       [0004]    The inventors have found that a xylanase from  Bacillus halodurans  can increase the shelf life of baked products. More specifically, the xylanase in combination with a maltogenic amylase further improves the softness of bread crumb without having detrimental effects on elasticity. 
         [0005]    Accordingly, the invention provides a process for preparing a dough-based product, comprising adding a xylanase with a high identity to SEQ ID NO: 2 to a dough, leavening, and heating the dough. More specifically, the xylanase is a polypeptide which has at least 85% identity to the amino acid sequence as shown in positions 1-182 of SEQ ID NO: 2 or is encoded by a DNA sequence which can hybridize at 41° C. to the complementary strand of nucleotides 142-687 of SEQ ID NO: 1. The xylanase may be a polypeptide having an amino acid sequence which can be obtained from the mature polypeptide of SEQ ID NO: 2 by substitution, deletion, and/or insertion of one or more amino acids or be encoded by a polynucleotide having a sequence that can be derived from SEQ ID NO: 1 by substitution, deletion, and/or insertion of one or more nucleotides. 
         [0006]    The invention further provides a dough composition which comprises flour together with the xylanase and a dough and/or bread-improving additive comprising the xylanase in the form of a granulate or agglomerated powder. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Genomic DNA Source 
       [0007]    A donor strain  B. halodurans  C-125 containing the xylanase gene Q9KEF3 is obtainable from Japan Collection of Microorganisms (JCM), RIKEN (The Institute of Physical and hemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan with accession number CM 9153. 
         [0008]    It is also available under accession number BAA-125 from American Type Culture Collection (ATCC) or LGC Promochem, Queens Road, Teddington, Middlesex TW11 0LY, UK. 
       Xylanase 
       [0009]    The xylanase used in the invention is a polypeptide which has at least 85% identity to the amino acid sequence as shown in positions 1-182 of SEQ ID NO: 2 or is encoded by a DNA sequence which can hybridize at 41° C. to the complementary strand of nucleotides 142-687 of SEQ ID NO: 1. The xylanase can be produced as described in the examples or in JP 2001-245665A (Oji Paper). 
       Alignment and Identity 
       [0010]    The polypeptide and polynucleotide of the invention may have identities to the disclosed sequences of at least 85%, particularly at least 90%, e.g. at least 95%. 
         [0011]    For purposes of the present invention, the alignments and identities of the protein sequences are analysed by Vector NTI-program (Invitrogen Corporation). The alignments are created using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994). Alignment Parameters used for polypeptide alignments are: penalty for the first residue in a gap 10, penalty for additional residues in a gap 0.1, no penalty for gaps introduced at the end of a sequence 
       Hybridization 
       [0012]    Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involve presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5×SSC (Sambrook et al. 1989), 5×Denhardt&#39;s solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)  Anal. Biochem.  132:6-13),  32 P-dCTP-labeled (specific activity&gt;1×10 9  cpm/μg) probe for 12 hours at approx. 45° C. The filter is then washed two times for 30 minutes in 0.1×SSC, 0.5% SDS at a temperature of 30° C., 35° C., 41° C., 45° C. or 50° C. Molecules to which the oligonucleotide probe hybridizes under these conditions may be detected using a x-ray film. 
         [0000]    Dough and/or Bread-Improving Additive 
         [0013]    The xylanase may be provided as a dough and/or bread improving additive in the form of a granulate or agglomerated powder. The dough and/or bread improving additive may have a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μm. 
         [0014]    Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the amylase onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy. 
       Flour Composition or Dough 
       [0015]    The flour composition may particularly comprise wheat flour. It may a dry mixture comprising flour and the xylanase, particularly in the form of the additive described above. The flour composition may also be a dough, which may be fresh, frozen or par-baked. It may be a laminated dough. 
         [0016]    The xylanase may be added to the flour composition or dough at a dosage of 0.1-10 mg enzyme protein per kg of flour, particularly 0.2-5 mg/kg. 
         [0017]    The dough may also comprise other conventional dough ingredients, e.g. proteins, such as milk powder and gluten; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. The dough may comprise fat (triglyceride) such as granulated fat or shortening. 
       Additional Enzyme 
       [0018]    Optionally, one or more additional enzymes may be added to the dough together with the xylanase of the invention. The additional enzyme may be an amylase, a lipolytic enzyme (e.g. as described in WO 9953769) or a second xylanase. 
         [0019]    The amylase may be an exo-acting maltogenic alpha-amylase. An example is a maltogenic alpha-amylase from  B. stearothermophilus  strain NCIB 11837, available from Novozymes A/S under the tradename Novamyl®; described in WO 9104669 and having the amino acid sequence shown as SEQ ID NO: 1 of U.S. Pat. No. 6,162,628A1. Another example is a Novamyl variant, e.g. as described in WO 9943794. The maltogenic amylase may be added at a dosage of 100-1000 MANU per kg flour (MANU activity unit defined in WO 9104669). 
       Dough-Based Product 
       [0020]    The invention provides a method for preparing a dough-based product by leavening the dough and heating it, e.g. by baking or steaming. The dough may be leavened e.g. by adding chemical leavening agents or yeast, usually  Saccharomyces cerevisiae  (baker&#39;s yeast). The product may be of a soft or a crisp character, either of a white, light or dark type. Examples are steamed or baked bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls. 
       EXAMPLES 
     Example 1 
     Production of Xylanase 
     Microbial Strain 
       [0021]      B. subtilis  PL1801. This strain is the  B. subtilis  DN1885 with disrupted apr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from  Bacillus brevis . J. Bacteriol., 172, 4315-4321). 
         [0022]    Competent  B. subtilis  cells were prepared and transformed as described by Yasbin, R. E., Wilson, G. A. and Young, F. E. (1975) Transformation and transfection in lysogenic strains of  Bacillus subtilis : evidence for selective induction of prophage in competent cells. J. Bacteriol, 121:296-304. 
       General Molecular Biology Methods 
       [0023]    Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for  Bacillus ”. John Wiley and Sons, 1990). 
         [0024]    Enzymes for DNA manipulations were used according to the specifications of the suppliers (e.g. restriction endonucleases, ligases etc. are obtainable from New England Biolabs, Inc.). 
       Media 
       [0025]    TY (as described in Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995). LB agar (as described in Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995). LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0. BPX media is described in EP 0 506 780 (WO 91/09129). 
         [0000]    Expression of Putative Xylanase Q9KEF3 from  Bacillus halodurans  in  Bacillus subtilis    
         [0026]    In order to express the xylanase gene in  B. subtilis  the gene was cloned into the plasmid expression vector pMOL944 (WO 00/75344 A1). It was cloned in such a way that the part of the gene encoding the mature enzyme was fused to the signal peptide from the amylase gene on the expression vector. 
       Propagation of the Donor Strain. 
       [0027]    The strain  Bacillus halodurans  C-125 was propagated in liquid TY medium adjusted to approx. pH 9 by addition of 5% Na 2 CO 3 . After 18 hours of incubation at 37° C. and 300 rpm the cells were harvested and genomic DNA was isolated by the method below. 
       Genomic DNA Preparation 
       [0028]    The  Bacillus halodurans  strain was propagated in liquid media as described above. The cells were harvested and genomic DNA was isolated by the method described by Pitcher et al. Chromosomal DNA from  Bacillus halodurans  was isolated (C-125). The xylanase gene was amplified by PCR using Primers 1 and 2 (SEQ ID NO: 3 and 4). 
         [0029]    Restriction sites SacII and NotI are present at nucleotides 11-16 of SEQ ID NO: 3 and 13-20 of SEQ ID NO: 4. Nucleotides 19-47 of SEQ ID NO: 3 are identical to nucleotides 142-170 of SEQ ID NO: 1. Nucleotides 21-51 of SEQ ID NO: 4 are complementary to nucleotides 747-777 of SEQ ID NO: 1. 
         [0030]    This creates a DNA fragment of approx. 700 bp. The fragment was cut by restriction enzymes SacII and NotI. Plasmid vector pMOL944 was isolated and cut by restriction enzymes SacII and NotI. The vector fragment (approx. 4.8 kb) and the PCR fragment was ligated and the ligation mixture was used to transform competent  B. subtilis  PL1801 cells. Selection for transformants was performed on LB agar plates containing Kanamycin (10 micro-g/ml). The resulting strain PL3522-3 showed increased xylan clearing zones as compared to the non-transformed host when plated on LB agar plates containing 0.2% AZCL-xylan (Megazyme) and Kanamycin (10 micro-g/ml). In order to produce the xylanase enzyme the strain PL3522-3 was inoculated in BPX medium at 30 C and 300 rpm for 4 days. The xylanase can be extracted from the supernatant of the culture medium. 
       Example 2 
     Effect of Xylanase on Freshness of Bread 
       [0031]    Bread were baked according to the sponge &amp; dough method. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                   
               
               
                 Recipes 
               
             
          
           
               
                   
                 % on flour basis 
               
               
                   
                   
               
             
          
           
               
                   
                 Sponge 
                   
               
               
                   
                 Soya oil 
                 2.5 
               
               
                   
                 SSL 
                 0.38 
               
               
                   
                 Yeast 
                 5 
               
               
                   
                 Wheat flour 
                 60 
               
               
                   
                 Water 
                 62 
               
               
                   
                 Dough 
               
               
                   
                 Ascorbic acid 
                 optimized for each flour 
               
               
                   
                 ADA 
                 20 ppm 
               
               
                   
                 Salt 
                 2 
               
               
                   
                 Syrup 
                 7 (dry substance) 
               
               
                   
                 Water 
                 to be optimized for each flour 
               
               
                   
                 Wheat flour 
                 40 
               
               
                   
                 Calcium propionate 
                 0.25 
               
               
                   
                   
               
             
          
         
       
     
       Sponge 
       [0032]    Scaling of ingredients, addition of yeast, water, flour, SSL and oil into mixer bowl Mixing 90 rpm for 1 minutes, 150 rpm for 4 minutes. The sponge was weighted, the temperature was measured and the sponge was placed in a bowl˜fermentation 3 hours at 27 C, 86% RH 
       Dough 
       [0033]    Addition of ingredients and the sponge into the mixer bowl. The sponge and ingredients were mixed together 90 rpm for 9 minutes 
         [0034]    The temperature was measured, dough characteristics were evaluated, the dough was scaled into smaller pieces of 435 g each. 
         [0035]    The dough rests on the table for 10 minutes 
         [0036]    Doughs were sheeted and molded. 
         [0037]    Fermentation for 55 minutes at 42 C and 86% RH. 
         [0038]    Bread were baked at 200 C for 22 minutes 
         [0039]    Enzymes were dosed at 1 mg of xylanase of the invention per kg of flour together with 400 MANU/kg of Novamyl. A control was made with 400 MANU/kg of Novamyl alone. 
         [0040]    Bread were stored at room temperature until analysis. 
         [0041]    Texture and water migration by NMR were measured on day 7, 14 and 21. A small sensory evaluation of softness and moistness was performed on day 21. 
       Results 
       [0042]    Firmness of the loaves was measured as described in WO 9953769. The results were as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Xylanase 
                   
                   
                   
               
               
                 Novamyl 
                 of 
                   
                   
                   
               
               
                 dosage 
                 invention 
                 Firmness after 
                 Firmness after 
                 Firmness after 
               
               
                 MANU/kg 
                 mg/kg 
                 7 days g 
                 14 days g 
                 21 days g 
               
               
                   
               
             
             
               
                 400 
                 1 
                 427 
                 560 
                 758 
               
               
                 400 
                 0 
                 481 
                 576 
                 836 
               
               
                   
               
             
          
         
       
     
         [0043]    Elasticity of the loaves was measured as described in U.S. Pat. No. 6,162,628. The results were as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Xylanase 
                   
                   
                   
               
               
                 Novamyl 
                 of 
                   
                   
                   
               
               
                 dosage 
                 invention 
                 Elasticity after 
                 Elasticity after 
                 Elasticity after 
               
               
                 MANU/kg 
                 mg/kg 
                 7 days % 
                 14 days % 
                 21 days % 
               
               
                   
               
             
             
               
                 400 
                 1 
                 52.7 
                 49.2 
                 46.2 
               
               
                 400 
                 0 
                 52.7 
                 50.1 
                 46.0 
               
               
                   
               
             
          
         
       
     
         [0044]    The data show that the xylanase of the invention has a significant effect on firmness in combination with Novamyl. Elasticity is only slightly reduced 
         [0045]    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 results were as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Xylanase 
                 Free water 
                 Free water 
                 Free water 
               
               
                   
                 of 
                 after 7 days 
                 after 14 days 
                 after 21 days 
               
               
                 Novamyl dosage 
                 invention 
                 micro- 
                 micro- 
                 micro- 
               
               
                 MANU/kg 
                 mg/kg 
                 seconds 
                 seconds 
                 seconds 
               
               
                   
               
             
             
               
                 400 
                 1 
                 8139 
                 7354 
                 6719 
               
               
                 400 
                 0 
                 8067 
                 7169 
                 6541 
               
               
                   
               
             
          
         
       
     
         [0046]    The amount of free water has been described in literature to correlate to moistness of bread crumb. The data show that the xylanase is able to improve moistness measured by NMR when dosed on top of Novamyl. 
         [0047]    The ranking from the small sensory evaluation of softness and moistness on day 21 showed that bread crumb made with the xylanase of the invention together with Novamyl was perceived as more moist than bread made with Novamyl alone.