U.S. patent application number 12/964189 was filed with the patent office on 2011-12-15 for method of preparing a dough-based product.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Lars Beier, Esben Peter Friis, Henrik Lundquist.
Application Number | 20110305795 12/964189 |
Document ID | / |
Family ID | 36090369 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110305795 |
Kind Code |
A1 |
Beier; Lars ; et
al. |
December 15, 2011 |
METHOD OF PREPARING A DOUGH-BASED PRODUCT
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.
Inventors: |
Beier; Lars; (Lyngby,
DK) ; Friis; Esben Peter; (Herlev, DK) ;
Lundquist; Henrik; (Malmo, SE) |
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
36090369 |
Appl. No.: |
12/964189 |
Filed: |
December 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11575644 |
Mar 20, 2007 |
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PCT/DK05/00602 |
Sep 23, 2005 |
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12964189 |
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60614826 |
Sep 30, 2004 |
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Current U.S.
Class: |
426/18 ;
426/549 |
Current CPC
Class: |
C07K 2299/00 20130101;
A21D 8/042 20130101; C12N 9/2411 20130101; C12Y 302/01133 20130101;
C12N 9/2417 20130101; A21D 13/60 20170101 |
Class at
Publication: |
426/18 ;
426/549 |
International
Class: |
A21D 2/26 20060101
A21D002/26; A21D 8/04 20060101 A21D008/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 2004 |
DK |
PA 2004 01458 |
Claims
1. 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: a)
has an amino acid sequence which is at least 70% identical to SEQ
ID NO: 1, and b) compared to SEQ ID NO: 1 comprises an amino acid
alteration which is substitution or deletion of or insertion
adjacent to I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188,
T189, D190, P191, A192, F194, L196, D329, N371, D372, P373, N375 or
R376.
2. The method of claim 1 wherein the alteration is substitution
with a larger or smaller amino acid residue.
3. The method of claim 1 wherein the alteration is insertion of 1-4
amino acid residues at the N- or C-side of the specified
residue.
4. The method of claim 1 wherein the polypeptide comprises a
substitution I15T/S/V/L, R18K, K44R/S/T/Q/N, N86Q/S/T, T87N/Q/S,
G88A/S/T, Y89W/F/H, H90W/FN/R/K/N/Q/M, W93Y/F/M/E/G/V/T/S,
F188H/L/I/T/G/V, D190E/Q/G, A192G/S/T/Q/R, F194S/LN, L196F,
N371K/R/FN/Q or D372E/Q/S/T/A, a deletion of 191 or 192 or an
insertion of Ala after 192.
5. The method of claim 1 wherein the polypeptide has the amino acid
sequence of SEQ ID NO: 1 with one of the following sets of
alterations: TABLE-US-00010 Alterations compared to SEQ ID NO: 1
D261G, T288P F188L, D261G, T288P T288P Y89F, D261G, T288P N86V,
F188L, D261G, T288P Y89F, F188L, D261G, T288P Y89H, F188L, D261G,
T288P N86T, F188L, D261G, T288P F194S, D261G, T288P L196F D261G,
T288P, D372V Q184H, N187D, F194Y D190G N86G, Y89M, F188L, D261G,
T288P F188L, D190G, D261G, T288P A192Q, D261G, T288P, S446A F188H
P191* A192* A192*, G193* *192aA N86K, F252L, D261G, T288P F194Y,
L225S, D261G, T288P F194L, D261G, T288P F194S, D261G, T288P, P642Q
D261G, T288P, N375S F188T F188G F188V A192R, F194L, D261G, T288P,
G469R A192G, D261G, T288P Y89F, D261G, T288P, I290V, N375S
6. A polypeptide which: a) has amylase activity which is less
inhibited by sucrose than the amylase activity of SEQ ID NO: 1, b)
has an amino acid sequence which is at least 70% identical to SEQ
ID NO: 1, and c) compared to SEQ ID NO: 1 comprises an amino acid
alteration which is substitution or deletion of or insertion
adjacent to K44, Y89, H90, Y92, W93, D329, D372, P373 or R376 or an
alteration N86T/VG/K, T87N/Q, H90W/FN/R/M, F1881/L/T/G/V, D190G,
P191*, A192G/Q/R, F194S/LN, L196F, L225S, F252L, D261G, T288P,
I290V, N371K/R/FN/Q, D372E/Q/S/T/A/V, N375S, S446A, G469R, S578G or
P642Q.
7. The polypeptide of claim 6 wherein the alteration is
substitution with a larger or smaller amino acid residue.
8. The polypeptide of claim 6 which comprises insertion of 1-4
amino acid residues at the N- or C-side of the specified
residue.
9. The polypeptide of claim 6 which comprises a substitution
K44R/S/T/Q/N, Y89W/F/H, W93Y/F/M/E/G/V/T/S, D261G, T288P.
10. The polypeptide of claim 6 which has the amino acid sequence of
SEQ ID NO: 1 with one of the following sets of alterations:
TABLE-US-00011 F194L, S578G N171S, S175C, F194L, V215D, Q449R L196F
Y89F, D261G, T288P D261G, T288P Q184H, N187D, F194Y D190G Y89F,
D261G, T288P A192*, G193* N86V, F188L, D261G, T288P Y89F, F188L,
D261G, T288P Y89H, F188L, D261G, T288P N86T, F188L, D261G, T288P
F194S, D261G, T288P D261G, T288P, D372V D190G N86G, Y89M, F188L,
D261G, T288P F188L, D190G, D261G, T288P A192Q, D261G, T288P, S446A
N86K, F252L, D261G, T288P F194Y, L225S, D261G, T288P F194L, D261G,
T288P F194S, D261G, T288P, P642Q D261G, T288P, N375S A192R, F194L,
D261G, T288P, G469R A192G, D261G, T288P Y89F, D261G, T288P, I290V,
N375S F188T F188G F188V
11. A method of preparing a polypeptide, comprising a) providing a
parent polypeptide having an amino acid sequence, and having
maltogenic alpha-amylase activity, b) selecting an amino acid
residue in the sequence corresponding to I15, R18, K44, N86, T87,
G88, Y89, H90, Y92, W93, F188, T189, D190, P191, A192, F194, L196,
D329, N371 D372, P373, N375 or R376 in SEQ ID NO: 1, c)
substituting or deleting the selected residue or inserting one or
more residues adjacent to the selected residue to obtain an altered
amino acid sequence, d) preparing an altered polypeptide having the
altered amino acid sequence, e) testing the amylase activity and
the sugar tolerance of the altered polypeptide, and f) selecting a
polypeptide which has amylase activity and has higher sucrose
tolerance than the parent polypeptide.
12. A method of constructing a polypeptide, comprising: a)
providing a parent maltogenic alpha-amylase having an amino acid
sequence and a three-dimensional structure, b) docking a sucrose
molecule in the structure, c) selecting an amino acid residue
having a C-alpha atom located <10 .ANG. from an atom in the
docked sucrose molecule, d) substituting or deleting the selected
residue or inserting one or more residues adjacent to the selected
residue to obtain an altered amino acid sequence, e) docking a
sucrose molecule in a structure of the polypeptide having the
altered sequence and calculating the binding energy for the
sucrose, and f) selecting a polypeptide wherein the binding energy
and/or binding configuration is changed.
13. A method of preparing dough or a dough-based edible product,
comprising adding an exo-amylase to dough wherein the dough
comprises at least 10% sucrose by weight, and the exo-amylase has
amylase activity in the presence of 10% sucrose by weight which is
more than 20% of the activity without sucrose.
14. The method of the preceding claim wherein the exo-amylase
retains at least 20% (particularly at least 40%) activity after 30
minutes incubation at 85.degree. C. at pH 5.7 (50 mM Na-acetate, 1
mM CaCl.sub.2) without substrate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/575,644 filed on Mar. 20, 2007 which is a 35 U.S.C. 371
national application of PCT/DK2005/000602 filed Sep. 23, 2005,
which claims priority or the benefit under 35 U.S.C. 119 of Danish
application no. PA 2004 01458 filed Sep. 24, 2004 and U.S.
provisional application No. 60/614,826 filed Sep. 30, 2004, the
contents of which are fully incorporated herein by reference.
SEQUENCE LISTING AND DEPOSITED MICROORGANISMS
Sequence Listing
[0002] The present invention comprises a sequence listing.
Deposit of Biological Material
[0003] None.
FIELD OF THE INVENTION
[0004] The present invention relates to the use of anti-staling
amylases in the preparation of dough or dough-based edible products
with a high sucrose content.
BACKGROUND OF THE INVENTION
[0005] U.S. Pat. No. 3,026,205 describes a process of producing
baked confections and the products resulting therefrom by
alpha-amylase.
[0006] WO 9104669 describes the use of a maltogenic alpha-amylase
to retard the staling of baked products such as bread; the
maltogenic alpha-amylase described therein is commercially
available under the tradename Novamyl.RTM. (product of Novozymes
A/S). U.S. Pat. No. 6,162,628 describes Novamyl variants and their
use for the same purpose. Three-dimensional structures of Novamyl
are published in U.S. Pat. No. 6,162,628 and in the Protein Data
Bank (available at http://www.rcsb.org/pdb/) with identifiers 1QHO
and 1QHP.
SUMMARY OF THE INVENTION
[0007] The inventors have found that a high sucrose content dough
(such as cake dough) tends to inhibit the activity of an
anti-staling amylases such as Novamyl, making it less effective to
prevent the staling of dough-based products with high sucrose
content such as cakes. They have found that a good anti-staling
effect in cakes can be achieved by using a carefully selected
anti-staling amylase with certain properties, and they have
identified such amylases.
[0008] By analyzing a 3D structure of Novamyl, the inventors
further found that sucrose may inhibit by binding in the active
site. They have found that sucrose docks into the active site of
Novamyl differently from the substrate or inhibitor in published
models 1QHO and 1QHP, and they have used this finding to design
sucrose-tolerant variants.
[0009] Accordingly, the invention provides a method of preparing
dough or a dough-based edible product (e.g. a baked product) by
adding a sucrose-tolerant anti-staling amylase. It also provides
novel sucrose tolerant variants of a maltogenic alpha-amylase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the cartesian coordinates for the sucrose atoms
in this binding configuration, using the coordinate system of the
x-ray structure 1QHO.pdb.
DETAILED DESCRIPTION OF THE INVENTION
Maltogenic Alpha-Amylase and Sucrose Docking
[0011] A maltogenic alpha-amylase (EC 3.2.1.133) having more than
70% identity (particularly more than 80% or 90%, such as at least
95% or 96% or 97% or 98% or 99%) with the Novamyl sequence shown as
SEQ ID NO: 1 may be used as the parent enzyme for designing sucrose
tolerant variants. Amino acid identity may be calculated as
described in U.S. Pat. No. 6,162,628.
[0012] For Novamyl (SEQ ID NO: 1), a 3D structure including a
substrate or inhibitor as described in U.S. Pat. No. 6,162,628 or
in the Protein Data Bank with the identifier 1QHO or 1QHP may be
used. Alternatively, a Novamyl variant may be used, such as a
variant described in U.S. Pat. No. 6,162,628 or in this
specification, e.g. the variant F188L+D261G+T288P. A 3D structure
of a variant may be developed from the Novamyl structure by known
methods, e.g. as described in T. L. Blundell et al., Nature, vol.
326, p. 347 ff (26 Mar. 1987); J. Greer, Proteins: Structure,
Function and Genetics, 7:317-334 (1990); or Example 1 of WO
9623874.
[0013] The inventors found that sucrose may inhibit Novamyl by
binding in the active site. Docking of sucrose into the active site
of Novamyl (using the software GOLD version 2.1.2, Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK
and the protein part of the x-ray structure 1QHO.pdb) reveals a
specific binding configuration as unique to sucrose. The cartesian
coordinates for the sucrose atoms in this binding configuration,
using the coordinate system of the x-ray structure 1QHO.pdb are
given in FIG. 1.
Maltogenic Alpha-Amylase Assay
[0014] The activity of a maltogenic alpha-amylase may be determined
using an activity assay such as the MANU method. One MANU
(Maltogenic Amylase Novo Unit) is defined as the amount of enzyme
required to release one micro-mole of maltose per minute at a
concentration of 10 mg of maltotriose substrate per ml in 0.1 M
citrate buffer at pH 5.0, 37.degree. C. for 30 minutes.
Amino Acid Alterations
[0015] The amino acid sequence of a maltogenic alpha-amylase may be
altered to decrease the sucrose inhibition. The inventors found
that the alteration may be made at an amino acid residue having at
least one atom within 4 .ANG.ngstroms from any of the sucrose atoms
when the sucrose molecule is docked in the 3D structure of the
maltogenic alpha-amylase. Using the Novamyl structure 1QHO and the
sucrose docking in FIG. 1, the following Novamyl residues are
within 4 .ANG.: K44, N86, Y89, H90, Y92, W93, F188, T189, D190,
P191, A192, F194, D372, P373, R376.
[0016] Further the following positions have been identified as
relevant: I15, R81, T87, G88, L196, N371 or N375 of SEQ ID NO:
1.
[0017] The alteration may be a substitution or deletion of one or
more of the selected residues, or one or more residues
(particularly 1-4 residues or 5-6 residues) can be inserted
adjacent to a selected residue.
[0018] The substitution may be with a smaller or larger residue. A
substitution to increase the size of the residue may diminish the
space obtained by the docked sucrose molecule thereby preventing
the binding of sucrose. Amino acid residues are ranked as follows
from smallest to largest: (an equal sign indicates residues with
sizes that are practically indistinguishable):
G<A=S=C<V=T<P<L=I=N=D=M<E=Q<K<H<R<F<Y<W
[0019] 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.
[0020] 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.
[0021] 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/FN/R/K/N/Q/M, W93Y/F/M/E/G/V/T/S, F188H/L/I/T/G/V, D190E/Q/G,
A192S/T, F194S/LN, L196F, N371K/R/FN/Q, D372E/Q/S/T/A and
N375S/T/D/E/Q.
[0022] Examples of deletions are deletion of residue 191 or 192. An
example of an insertion is Ala inserted between 192 and 193.
[0023] The polypeptide may include other alterations compared to
Novamyl (SEQ ID NO: 1), e.g. alterations to increase the
thermostability as described in U.S. Pat. No. 6,162,628.
Nomenclature for Amino Acid Alterations
[0024] In this specification, an amino acid substitution is
described by use of one-letter codes, e.g. K44R. Slashes are used
to indicate alternatives, e.g. K44R/S/T/Q/N to indicate
substitution of K44 with R or S etc. P191* indicates a deletion of
P191. *192aA indicates insertion of one Ala after A192. Commas are
used to indicate multiple alterations in the sequence, e.g.
F188L,D261G,T288P to indicate a variant with three
substitutions.
Properties of Anti-Staling Amylase for Use with Sucrose
[0025] 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.
[0026] 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.degree. 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).
[0027] An exo-amylase for use in high-sucrose dough may have a
specified sugar tolerance. Compared to its activity in the absence
of sucrose, the amylase may have more than 20% activity at 10%
sugar, more than 10% activity at 20% sucrose, or more than 4%
activity at 40% sucrose. The sugar tolerance may be determined as
described in the examples.
[0028] 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.degree. C. at pH 5.7 (50 mM Na-acetate, 1 mM
CaCl.sub.2) without substrate.
[0029] 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.
[0030] 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.
[0031] The exo-amylase may have maltogenic alpha-amylase activity
(EC 3.2.1.133). The exo-amylase may be the amylase described in DK
PA 2004 00021, or it may be a Novamyl variant described in this
specification.
Dough and Dough-Based Edible Product
[0032] 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.
[0033] The dough is generally heat treated, e.g. by baking or deep
frying to prepare an edible product such as cakes including pound
cake, yellow and white layer cakes, cakes containing chocolate and
cocoa products, sponge cakes, angel food cake, fruit cakes and
foam-type cakes and doughnuts.
EXAMPLES
Example 1
Sucrose Tolerance of Novamyl Variants
[0034] The amylase activity of a number of polypeptides were tested
by incubation with Phadebas tablets (product of Pharmacia.RTM.) for
15 minutes at 60.degree. C. in the presence of sucrose at various
concentrations (in % by weight). The results are expressed in % of
the result without sugar:
TABLE-US-00001 Alterations compared to 0% 20% SEQ ID NO: 1 sucrose
10% sucrose sucrose 40% sucrose None 100 13 6 1.5 F188L, D261G,
T288P 100 27.5 14.5 6 F194S 100 31.5 18.5 7.5 L196F 100 69 42 23
D190G 100 65 43 21
Example 2
Sucrose Tolerance of Novamyl Variants
[0035] A number of polypeptides were tested as in Example 1. The
results are expressed as activity with 10% sucrose in % of the
activity without sucrose:
TABLE-US-00002 Sugar Alterations compared to SEQ ID NO: 1 tolerance
None 15 D261G, T288P 24 F188L, D261G, T288P 35 T288P 56 Y89F,
D261G, T288P 42 N86V, F188L, D261G, T288P 37 Y89F, F188L, D261G,
T288P 38 Y89H, F188L, D261G, T288P 50 N86T, F188L, D261G, T288P 49
F194S, D261G, T288P 47 L196F 65 D261G, T288P, D372V 62 Q184H,
N187D, F194Y 47 D190G 66 N86G, Y89M, F188L, D261G, T288P 47 F188L,
D190G, D261G, T288P 68 A192Q, D261G, T288P, S446A 46 F188H 49 P191*
42 A192* 51 A192*, G193* 67 *192aA 44 N86K, F252L, D261G, T288P 49
F194Y, L225S, D261G, T288P 49 F194L, D261G, T288P 54 F194S, D261G,
T288P, P642Q 60 D261G, T288P, N375S 58 F188T 37 F188G 36 F188V 41
A192R, F194L, D261G, T288P, G469R 60 A192G, D261G, T288P 41 Y89F,
D261G, T288P, I290V, N375S 60
[0036] The following variants are also considered of interest in
the context of the present invention:
TABLE-US-00003 Alterations compared to SEQ ID NO: 1 I15T, N86K,
P191S, D261G, T288P I15T, P191S, D261G, T288P I15T, P191S, Y258F,
D261G, T288P, N375S, Y549C, Q648H I15T, G153R, P191S, D261G, T288P,
N371K, K645R
Example 3
Sucrose Tolerance and Thermostability of Amylases
[0037] The following amylases were tested for thermostability and
sugar tolerance: bacterial alpha-amylase from B. amyloliquefaciens
(BAN.TM., product of Novozymes A/S), fungal alpha-amylase from A.
oryzae (Fungamyl.RTM., product of Novozymes A/S), maltogenic
alpha-amylase having the sequence of SEQ ID NO: 1 (Novamyl.RTM.,
product of Novozymes A/S), a Novamyl variant having SEQ ID NO: 1
with the substitutions F188L+D261G+T288P, and bacterial
alpha-amylase from B. licheniformis (Termamyl.RTM., product of
Novozymes A/S).
Exo-Amylase Activity
[0038] The five amylases were tested for exo-amylase activity as
described above. The results show that Novamyl and the Novamyl
variant had exo-amylase activity by this test, and the other three
did not.
Thermostability
[0039] Each amylase was incubated at 85.degree. C. at pH 5.7 (50 mM
Na-acetate, 1 mM CaCl.sub.2) without substrate, and the amylase
activity was measured after 0, 15, 30 and 60 minutes heat
treatment. The results are expressed as residual activity in % of
the initial activity:
TABLE-US-00004 0 15 30 60 BAN 100 3 1 0 Fungamyl 100 0 0 0 Novamyl
100 51 29 13 Novamyl variant 100 64 48 54 Termamyl 100 100 71
85
[0040] The results show that the Novamyl variant and Termamyl were
not deactivated by the heat-treatment. BAN and Fungamyl lose all
their activity after 15 min while Novamyl loses it gradually with
heat-treatment time.
Sucrose Tolerance
[0041] The experiment was repeated in 10% sucrose solution. The
results are expressed as residual activity in % of the initial
activity without sucrose:
TABLE-US-00005 0 15 30 60 BAN 93 2 1 0 Fungamyl 31 0 0 0 Novamyl 7
6 1 3 Novamyl variant 21 19 14 16 Termamyl 116 112 97 82
[0042] The results show that BAN and Termamyl were not inhibited by
sugar while Fungamyl and the Novamyl variant were somewhat
inhibited, and Novamyl was heavily inhibited by sugar. The
combination of sugar and heat-treatment shows that the Novamyl
variant and Termamyl could be active during baking of cakes.
Termamyl and the Novamyl variant fulfill the criterion for
thermostability and sugar tolerance used in this invention.
Example 4
Preparation of Sponge Cake with Amylase
[0043] Sponge cakes were made with addition of amylase as follows:
BAN (0.83. 8.3 or 83 mg/kg flour), Novamyl (1.3 or 13 mg/kg flour)
or the Novamyl variant used in Example 1 (1, 10 10 or 100 mg/kg
flour). A control cake was made without amylase.
[0044] The cakes were baked according to the High Ratio Sponge
Sandwich Cake (HRSSC) method. After baking, the cakes were cooled
down for 60-120 minutes, and the cakes were stored at room
temperature in sealed plastic bags filled with nitrogen until
analysis. The cakes were evaluated on day 1, 3, 7 or 23.
[0045] Texture profile analysis (TPA) was performed as described in
Bourne M. C. (2002) 2. ed., Food Texture and Viscosity: Concept and
Measurement. Academic Press. The results showed that the increase
in hardness was slower with increasing dosage of the Novamyl
variant. The addition of BAN or Novamyl had only a slight effect,
and only at the highest dosage.
[0046] The cohesiveness of the cakes decreased with storage time.
The addition of the Novamyl variant delayed this decrease. The
addition of BAN or Novamyl had a slight effect, and only at the
highest dosage.
[0047] Water mobility was characterized by low field NMR. The
addition of the Novamyl variant and BAN increased the mobility,
indicating that the two amylases were able to keep the cakes more
moist. Novamyl had virtually no effect.
[0048] A small sensory evaluation of softness and moistness was
performed on day 13 for the 3 cakes with the Novamyl variant and
the control cake. The cakes were evaluated regarding three
parameters; Firmness, Moistness and preferability. The control was
the firmest, driest and least preferred. The higher dosage of the
Novamyl variant, the less firm (softer), moister and better
liked.
[0049] A large panel sensory evaluation was performed on day 13. It
was a paired comparison test where a control cake was compare to
the cake with the Novamyl variant at the highest dosage. A
30-member panel was asked two questions (1) Which cake is moister
and (2) which cake is fresher. All panel members agreed on that the
cake with the Novamyl variant was moister and fresher. The
preference was significant at a significance level above
99.999%.
[0050] To summarize, the data show that the Novamyl variant had
anti-staling properties and was able to improve moistness
perception and moistness measured by NMR. The two other amylases
had only a slight effect.
Example 5
High-Ratio Unit Cakes
[0051] Cakes were made with addition of amylase as follows: BAN
(0.83. 8.3 or 83 mg/kg flour) or the Novamyl variant used in
Example 1 (1, 10 or 100 mg/kg flour). A control cake was made
without amylase.
[0052] Cakes were baked according to the High ratio unit cake
(HRUC) method. After baking, the cakes were cooled down for 60-120
minutes, and the cakes were stored at room temperature in sealed
plastic bags filled with Nitrogen until analysis. The cakes were
evaluated on day 7, 20 and 34 by the same methods as in the
previous example.
[0053] The increase in hardness was slower with the Novamyl variant
at the highest dosage. The addition of BAN to the cake resulted in
a low volume and a doughy cake which gave poor results in hardness
measurements.
[0054] The addition of the Novamyl variant delayed the decrease in
cohesiveness while BAN did not influence it at all.
[0055] The Novamyl variant and BAN were able to keep the cake more
moist than the control. This increase in mobility of the free water
could partly be explained by the cakes with BAN and the Novamyl
variant being able to retain the moisture content.
[0056] A small sensory evaluation on day 34 showed that the cake
with the Novamyl variant at the highest dosage was clearly better
than the control cake; it was more moist and it was less
crumbly.
[0057] Over-all, there was an anti-staling effect of the Novamyl
variant at the high dosage, similar to the effect on sponge cakes
in the previous example. The staling of HRUC cakes was slower than
Sponge cakes but it was still evident that the Novamyl variant had
an anti-staling effect. The anti-staling effect was seen with
texture analysis, NMR and sensory evaluation. BAN showed
anti-staling effects in HRUC but it was sensitive to over-dosage
which resulted in cake collapse and a doughy cake.
Example 6
Sponge Cake
[0058] Sponge cakes were made with addition of the amylase of DK PA
2004 00021 at dosages 0.5, 1, 2, 5 and 20 mg/kg flour and a control
cake without amylase.
[0059] Texture and NMR was measured on day 1, 7 and 13. The
addition of the amylase reduced the increase in firmness,
especially at the highest dosage. The amylase also had a beneficial
effect on the mobility of water which was correlated with the
moistness of the cake.
[0060] A blind sensory ranking evaluation performed on day 14
showed a ranking according to the dosage, the higher dosage the
more soft and moist cake. The most preferred cake was the one with
the highest dosage.
Example 7
Baking Procedure Tegral Allegro Cake
Recipe
[0061] The following recipe was used:
TABLE-US-00006 % Tegral Allegro mix* 100 Pasteurized whole 50 egg
Butter 50 Enzymes According to trial. 0 or 25 mg/kg flour.
*commercially available from Puratos NV/SA, Groot-Bijgaarden,
Belgium
Procedure
[0062] The ingredients were scaled into a mixing bowl and mixed
using an industrial mixer (e.g. Bjorn A R 5 A Varimixer) with a
suitable paddle speed. 300 g of the dough was poured into forms.
The cakes are baked in a suitable oven (e.g. Sveba Dahlin deck
oven) for 45 min. at 180.degree. C. The cakes were allowed to cool
down at room temperature for 1 hour.
[0063] The volume of the cakes was determined when the cakes had
cooled down using the rape seed displacement method. The cakes were
packed under nitrogen in sealed plastic bags and stored at room
temperature until analysis.
[0064] The cakes were evaluated on day 1, 7 and 14, two cakes were
used at each occasions.
[0065] The cohesiveness and hardness of the cakes was evaluated
with Texture analyser and the water mobility was characterized by
low field NMR.
[0066] 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.
[0067] 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
[0068] 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.
[0069] The cohesiveness of the cakes decreased with storage time.
The addition of variants of SEQ ID NO: 1 delayed this decrease as
can be seen in Table 1.
TABLE-US-00007 TABLE 1 Change in Cohesiveness [gs/gs] with storage
time of cakes with 25 mg protein enzyme per kg flour Enzyme Day 1
Day 7 Day 14 No enzyme 0.44 0.35 0.32 Seq ID No: 1 0.43 0.38 0.36
F188L, D261G, T288P 0.46 0.42 0.41 Y89F, D261G, T288P 0.45 0.43
0.39 N86G, Y89M, F188L, D261G, T288P 0.44 0.42 0.38 T288P 0.44 0.40
0.41 F194S, D261G, T288P 0.47 0.43 0.42 D261G, T288P, D372V 0.46
0.43 0.37 A192Q, D261G, T288P, S446A 0.44 0.42 0.39 A192R, F194L,
D261G, T288P, G469R 0.47 0.44 0.42 A192G, D261G, T288P 0.46 0.42
0.39 N86K, F252L, D261G, T288P 0.45 0.41 0.39 F194L, D261G, T288P
0.45 0.42 0.42 F194S, D261G, T288P, P642Q 0.44 0.40 0.39 Y89F,
D261G, T288P, I290V, N375S 0.43 0.42 0.40
[0070] The free water mobility is correlated with the moist
perception of the cake crumb, it decreases with time. The addition
of the Novamyl variants increased the mobility compared to the
control, indicating that the amylases were able to keep the cakes
more moist. Results are listed in Table 2.
TABLE-US-00008 TABLE 2 Change in free water mobility [micros] with
storage time of cakes with 25 mg protein enzyme per kg flour Enzyme
Day 1 Day 7 Day 14 No enzyme 7077 5111 4175 Seq ID No: 1 6990 5460
4583 F188L, D261G, T288P 7216 5624 4656 Y89F, D261G, T288P 7085
6044 5151 N86G, Y89M, F188L, D261G, T288P 7493 5349 5120 T288P 7458
5785 4858 F194S, D261G, T288P 7746 6373 5325 D261G, T288P, D372V
7417 5517 4525 A192Q, D261G, T288P, S446A 7357 5714 5041 A192R,
F194L, D261G, T288P, G469R 7549 5536 no data A192G, D261G, T288P
7546 5815 no data N86K, F252L, D261G, T288P 7349 5295 4775 F194L,
D261G, T288P 7773 6803 5750 F194S, D261G, T288P, P642Q 8152 5969
4971 Y89F, D261G, T288P, I290V, N375S 7753 6175 4811
[0071] The hardness of the cakes increased with storage time. The
addition of variants of SEQ ID NO: 1 delayed this increase in
hardness as can be seen in Table 3.
TABLE-US-00009 TABLE 3 Change in hardness [g] with storage time of
cakes with 25 mg protein enzyme per kg flour Enzyme Day 1 Day 7 Day
14 No enzyme 647 1060 1408 Seq ID No: 1 677 997 1171 F188L, D261G,
T288P 683 951 1167 Y89F, D261G, T288P 649 998 1160 N86G, Y89M,
F188L, D261G, T288P 630 844 1194 T288P 719 1101 1098 F194S, D261G,
T288P 672 943 1061 D261G, T288P, D372V 593 962 1344 A192Q, D261G,
T288P, S446A 680 931 1159 A192R, F194L, D261G, T288P, G469R 720 987
1209 A192G, D261G, T288P 707 1024 1102 N86K, F252L, D261G, T288P
678 955 1248 F194L, D261G, T288P 648 895 1050 F194S, D261G, T288P,
P642Q 674 1028 1316 Y89F, D261G, T288P, I290V, N375S 602 731 827
Sequence CWU 1
1
11686PRTBacillus stearothermophilus 1Ser Ser Ser Ala Ser Val Lys
Gly Asp Val Ile Tyr Gln Ile Ile Ile1 5 10 15Asp Arg Phe Tyr Asp Gly
Asp Thr Thr Asn Asn Asn Pro Ala Lys Ser 20 25 30Tyr Gly Leu Tyr Asp
Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp Gly 35 40 45Gly Asp Leu Glu
Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln Leu 50 55 60Gly Val Thr
Thr Ile Trp Leu Ser Pro Val Leu Asp Asn Leu Asp Thr65 70 75 80Leu
Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg Asp 85 90
95Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp Thr
100 105 110Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val
Asp Phe 115 120 125Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp
Ser Thr Phe Ala 130 135 140Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr
Tyr Met Gly Asn Tyr Phe145 150 155 160Asp Asp Ala Thr Lys Gly Tyr
Phe His His Asn Gly Asp Ile Ser Asn 165 170 175Trp Asp Asp Arg Tyr
Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro Ala 180 185 190Gly Phe Ser
Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala Gln 195 200 205Tyr
Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Ala Asp Gly 210 215
220Leu Arg Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys
Ser225 230 235 240Leu Ala Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe
Leu Val Gly Glu 245 250 255Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn
His Leu Glu Lys Val Arg 260 265 270Tyr Ala Asn Asn Ser Gly Val Asn
Val Leu Asp Phe Asp Leu Asn Thr 275 280 285Val Ile Arg Asn Val Phe
Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu 290 295 300Asn Asn Met Val
Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn305 310 315 320Leu
Ile Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val 325 330
335Asn Ser Asn Lys Ala Asn Leu His Gln Ala Leu Ala Phe Ile Leu Thr
340 345 350Ser Arg Gly Thr Pro Ser Ile Tyr Tyr Gly Thr Glu Gln Tyr
Met Ala 355 360 365Gly Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro
Ala Phe Asp Thr 370 375 380Thr Thr Thr Ala Phe Lys Glu Val Ser Thr
Leu Ala Gly Leu Arg Arg385 390 395 400Asn Asn Ala Ala Ile Gln Tyr
Gly Thr Thr Thr Gln Arg Trp Ile Asn 405 410 415Asn Asp Val Tyr Ile
Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu 420 425 430Val Ala Ile
Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu 435 440 445Gln
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu 450 455
460Leu Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser
Phe465 470 475 480Thr Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr
Ser Thr Ser Ala 485 490 495Ser Ala Pro Gln Ile Gly Ser Val Ala Pro
Asn Met Gly Ile Pro Gly 500 505 510Asn Val Val Thr Ile Asp Gly Lys
Gly Phe Gly Thr Thr Gln Gly Thr 515 520 525Val Thr Phe Gly Gly Val
Thr Ala Thr Val Lys Ser Trp Thr Ser Asn 530 535 540Arg Ile Glu Val
Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val545 550 555 560Lys
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile 565 570
575Leu Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro
580 585 590Pro Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile
Pro Glu 595 600 605Leu Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val
Asn Asn Ala Gln 610 615 620Gly Pro Leu Leu Ala Pro Asn Tyr Pro Asp
Trp Phe Tyr Val Phe Ser625 630 635 640Val Pro Ala Gly Lys Thr Ile
Gln Phe Lys Phe Phe Ile Lys Arg Ala 645 650 655Asp Gly Thr Ile Gln
Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr 660 665 670Pro Thr Gly
Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn 675 680 685
* * * * *
References