U.S. patent application number 11/738838 was filed with the patent office on 2007-09-06 for preparation of dough or baked products.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Gitte Budolfsen, Luise Christiansen, Todd Forman, Tina Spendler.
Application Number | 20070207247 11/738838 |
Document ID | / |
Family ID | 26068851 |
Filed Date | 2007-09-06 |
United States Patent
Application |
20070207247 |
Kind Code |
A1 |
Budolfsen; Gitte ; et
al. |
September 6, 2007 |
PREPARATION OF DOUGH OR BAKED PRODUCTS
Abstract
The addition to dough of a combination of two lipolytic enzymes
with different substrate specificities produces a synergistic
effect on the dough or on a baked product made from the dough,
particularly a larger loaf volume of the baked product and/or a
better shape retention during baking.
Inventors: |
Budolfsen; Gitte;
(Frederiksberg, DK) ; Christiansen; Luise;
(Kobenhavn V, DK) ; Forman; Todd; (Raleigh,
NC) ; Spendler; Tina; (Herlev, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
26068851 |
Appl. No.: |
11/738838 |
Filed: |
April 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10332164 |
Jan 3, 2003 |
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PCT/DK01/00472 |
Jul 6, 2001 |
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11738838 |
Apr 23, 2007 |
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60217051 |
Jul 10, 2000 |
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Current U.S.
Class: |
426/549 ;
426/442 |
Current CPC
Class: |
C12N 9/16 20130101; C12N
9/18 20130101; C12Y 301/01032 20130101; A21D 8/042 20130101; C12Y
301/01026 20130101; C12Y 301/01003 20130101; C12N 9/20 20130101;
C11B 3/003 20130101; C12Y 301/01004 20130101 |
Class at
Publication: |
426/549 ;
426/442 |
International
Class: |
A23B 9/16 20060101
A23B009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 2000 |
DK |
2000 01054 |
Claims
1. A method for increasing the loaf volume or improving the shape
retention of a baked product made from dough, comprising adding to
the dough: a) a phospholipase and a galactolipase, or b) a
phospholipase and a triacylglycerol lipase, or c) a galactolipase
and a triacylglycerol lipase
2. A method of preparing a dough or a baked product made from
dough, comprising adding to the dough. a) a galactolipase and b) a
phospholipase or a triacylglycerol lipase.
3. A method of preparing a dough or a baked product made from
dough, comprising adding to the dough a combination of two
lipolytic enzymes wherein the combination produces a synergistic
effect on the loaf volume or shape retention of the baked
product.
4. The method of the preceding claim wherein the two lipolytic
enzymes are: a) a phospholipase and a galactolipase, or b) a
phospholipase and a triacylglycerol lipase, or c) a galactolipase
and a triacylglycerol lipase.
5. A method of preparing a dough or a baked product made from
dough, which comprises adding to the dough: a) a phospholipase and
a galactolipase, or b) a phospholipase and a triacylglycerol
lipase, or c) a galactolipase and a triacylglycerol lipase and
which does not comprise adding a maltogenic alpha-amylase to the
dough.
6. A composition comprising: a) a galactolipase and b) a
phospholipase or a triacylglycerol lipase.
7. A composition comprising a combination of two lipolytic enzymes
wherein the combination produces a synergistic effect on the loaf
volume or shape retention of a baked product made from dough
including the composition.
8. The composition of claim 7 wherein the two lipolytic enzymes
are: a) a phospholipase and a galactolipase, or b) a phospholipase
and a triacylglycerol lipase, or c) a galactolipase and a
triacylglycerol lipase.
9. A composition which comprises. a) a phospholipase and a
galactolipase, or b) a phospholipase and a triacylglycerol lipase,
or c) a galactolipase and a triacylglycerol lipase, and which does
not comprise a maltogenic alpha-amylase.
10. The composition of claim 1 which comprises flour.
11. A method of preparing a dough or a baked product made from
dough, comprising: a) determining substrate specificities of at
least two lipolytic enzymes, b) selecting two lipolytic enzymes
which are: i) a phospholipase and a galactolipase, or ii) a
phospholipase and a triacylglycerol lipase, or iii) a galactolipase
and a triacylglycerol lipase. c) for each enzyme, determining an
effective dosage in the dough to increase the loaf volume or
improve the shape retention of a baked product made from the dough,
d) adding to the dough a combination of the first and the second
lipolytic enzyme wherein each enzyme is added in an amount of 33-67
of the effective dosage.
12. A method for producing a lipolytic enzyme preparation,
comprising: a) determining the substrate specificities of at least
two lipolytic enzymes, b) selecting two lipolytic enzymes which
are: i) a phospholipase and a galactolipase, or ii) a phospholipase
and a triacylglycerol lipase, or iii) a galactolipase and a
triacylglycerol lipase. c) making baked products from doughs with
addition of the two lipolytic enzymes separately and in combination
d) determining the loaf volumes or the shape retention of the baked
products, e) selecting two lipolytic enzymes having a synergistic
effect, and f) producing the enzyme preparation comprising a
combination of the two lipolytic enzymes.
13. A method of preparing a dough or a baked product made from
dough, comprising: a) selecting two lipolytic enzymes, b) for each
enzyme, determining an effective dosage in the dough to increase
the loaf volume or improve the shape retention of a baked product
made from the dough, c) adding to the dough a combination of the
first and the second lipolytic enzyme wherein each enzyme is added
in an amount of 33-67% of the effective dosage.
14. The method of claim 13 wherein the two lipolytic enzymes are:
a) a phospholipase and a galactolipase, or b) a phospholipase and a
triacylglycerol lipase, or c) a galactolipase and a triacylglycerol
lipase.
15. The composition of claim 10, wherein the flour which comprises
a dough or a premix.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/332.164 filed on Jan. 3 2003 which claims priority of 35
U.S.C, 371 national application of PCT/DKO1/00472 filed Jul. 6,
2001, which claims priority or the benefit under 35 U.S.C. 119 of
Danish application no. PA 2000 01054 filed Jul. 6, 2000 and U.S.
provisional application No. 60/217,051 filed Jul. 10, 2000, the
contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods of preparing a
dough or a baked product made from dough by use of lipolytic
enzymes, and to compositions for use therein.
BACKGROUND
[0003] WO 94104035, EP 109244, EP 585988, WO 98/26057, WO 98/45453,
WO 99153769, WO 00/32758 and EP 575133 describe the addition of
various lipolytic enzymes to dough in the preparation of bread,
e.g. enzymes with activities such as triacylglycerol lipase,
phospholipase and galactolipase.
SUMMARY OF THE INVENTION
[0004] The inventors have found that the addition to dough of a
combination of two lipolytic enzymes with different substrate
specificities produces a synergistic effect on the dough or on a
baked product made from the dough, particularly a larger loaf
volume of the baked product and/or a better shape retention during
baking.
[0005] Accordingly, the invention provides a method of preparing a
dough or a baked product made from dough, comprising adding a
combination of two lipolytic enzymes to the dough. The invention
also provides a composition comprising a combination of two
lipolytic enzymes.
[0006] The combination may comprise at least two lipolytic enzymes
selected from the group consisting of galactolipase, phospholipase
and triacylglycerol lipase. Thus, the combination may comprise a
galactolipase+a phospholipase, a phospholipase+a triacylglycerol
lipase or a triacylglycerol lipase+a galactolipase.
DETAILED DESCRIPTION OF THE INVENTION
Lipolytic Enzyme
[0007] The invention uses a combination of lipolytic enzymes, i.e.
enzymes which are capable of hydrolyzing carboxylic ester bonds to
release carboxylate (EC 3.1.1). The enzyme combination includes at
least two of the following three: a galactolipase, a phospholipase
and a triacylglycerol lipase, i.e. enzymes predominantly having
activity for a galactolipids, a phospholipid, and a triglyceride,
respectively. The activities may be determined by any suitable
method, e.g. by assays known in the art or described later in this
specification. [0008] Galactolipase activity (EC 3.1.1.26), i.e.
hydrolytic activity on carboxylic ester bonds in galactolipids such
as DGDG (digalactosyl diglyceride). The galactolipase activity
(digalactosyl diglyceride hydrolyzing activity or DGOGase activity)
may be determined, e.g. by the plate assay in this specification or
by the monolayer assay 1 or 2 in WO 2000/32758. [0009]
Phospholipase activity (A1 or A2, EC 3.1.1.32 or 3,1.1,4), i.e.
hydrolytic activity towards one or both carboxylic ester bonds in
phospholipids such as lecithin. The phospholipase activity may be
determined by the plate assay in this specification or by an assay
WO 2000/32758, e.g. the PHLU, LEU, monolayer or plate assay 1 or 2.
[0010] Triacylglycerol lipase activity (EC 3.1.1.3): i.e.
hydrolytic activity for carboxylic ester bonds in triglycerides,
egg. 1,3-specific activity, particularly on long-chain
triglycerides such as olive oil. The activity on long-chain
triglycerides (olive oil) and short-chain triglycerides
(tributyrin) may be determined by the SLU and LU methods (described
in WO 00/32758), respectively, or by the plate assay described in
this specification. The enzyme may have a substrate specificity for
hydrolyzing long-chain fatty acyl groups rather than short-chain
groups, expressed e.g. as a high ratio of activities on olive oil
and tributyrin, e.g. the ratio SLU/LU. Favorably, this may reduce
the development of off-odor in dough containing milk lipids such as
buffer fat. Suitably, this ratio may be above 3.
[0011] Each lipolytic enzyme may have a narrow specificity with
activity for one of the three substrates and little or no activity
for the other two, or it may have a broader specificity with
predominant activity for one substrate and less activity for the
other two substrates.
[0012] A lipolytic enzyme is considered to be a galactolipase if it
has a higher activity on galactolipids than on phospholipids and
triglycerides. Similarly, R is considered to be a phospholipase or
a triacylglycerol lipase if it has a higher activity for that
substrate than for the other two. The comparison may be done, e.g.,
by the plate assay in this specification using the three
substrates; the largest clearing zone indicating the predominant
activity.
[0013] The enzyme combination may comprise three or more lipolytic
enzymes, e.g. comprising a galactolipase, a phospholipase and a
triacylglycerol lipase.
[0014] The enzyme combination may have low activity on partially
hydrolyzed lipids such as digalactosyl monoglyceride (DGMG),
lysophospholipids (LPL) and mono- and diglycerides (MG, DG).
Favorably, this may lead to accumulation of such partially
hydrolyzed lipids in the dough and may improve the properties of
the dough and/or the baked product.
Sources of Lipolytic Enzymes
[0015] The lipolytic enzymes may be prokaryotic, particularly
bacterial, e.g. from Pseudomonas or Bacillus. Alternatively, the
lipolytic enzymes may be eukaryotic, e.g. from fungal or animal
sources. Fungal lipolytic enzymes may be derived, e.g. from the
following genera or species: Thermomyces, particularly T.
lanuginosus (also known as Humicola lanuginosa); Humicola,
particularly H. insolens; Fusarium, particularly F. oxysporum, F.
solani, and F. heterosporum; Aspergillus, particularly A.
tubigensis, A. niger. and A. oryzae, Rhizomucor; Candida,
particularly C. antarctica; Penicillium, particularly P.
camembertii; Rhizopus particularly Rhizopus oryzae, or Absidia.
[0016] Some particular examples of lipolytic enzymes follow: [0017]
Phospholipase from bee or snake venom or from mammal pancreas, e.g.
porcine pancreas. [0018] Phospholipase of microbial origin, e.g.
from filamentous fungi, yeast or bacteria, such as the genus or
species Aspergillus, A. niger, Dictyostelium, D. discoideum, Mucor,
M. javanicus, M. mucedo, M. stubtilissimus, Neurospora, N. crassa,
Rhizomucor, P. pusillus, Rhizopus, R. arrhizus, R. japonicus, R.
stolonifer, Sclenotinia, S. libertiana, Trichophyton, T. rubrum.
Whetzelinia, W. sclerotiorum, Bacillus, B. megaterium, B. subtilis,
Citrobacter, C. freundii, Enterobacter, E. aerogenes, E. cloacae
Edwaedsiella, E. tarda, Erwinia, E. herbicola, Escherichia, E.
coli, Klebsiella, K. pneumoniae, Proteus, P. vulgaris, Providencia,
P. stuartii, Salmonella, S. typhimurium, Serratia, S.
liquefasciens, S. marcescens, Shigella, S. flexneri, Streptomyces,
S. violeceoruber, Yersinia, or Y. enterocolitica. [0019] Lipase
from Thermomyces lanuginosus (Humicola lanuginosa) (EP 305216, U.S.
Pat. No. 5,869,438). [0020] Lipase/phospholipase from Fusarium
oxysporum (WO 98/26057). [0021] Lysophospholipases from Aspergillus
niger and A. oryzae (WO 01 27251). [0022] Phospholipase A1 from
Aspergillus oryzae (EP 575133, JP-A 10-155493). [0023]
Lysophospholipase from F. venenatum (WO 00128044). [0024]
Phospholipase B from A. oryzae (U.S. Pat. No. 6,146,869). [0025]
Lipase from A. tubigensis (WO 9845453). [0026] Lipase from F.
solani (U.S. Pat. No. 5,990,069). [0027] Lipolytic enzyme from F.
culmorum (U.S. Pat. No. 5,830,736). [0028] Phospholipase from
Hyphozyma (U.S. Pat. No. 6,127,137). [0029] Lipolytic enzymes
described in PCT/DK 01/00448. [0030] Lipolytic enzymes described in
DK PA 2001 00304. [0031] A variant obtained by altering the amino
acid sequence a lipolytic enzyme, e.g. one of the above, e.g. as
described in WO 2000/32758, particularly Examples 4, 5. 6 and 13,
such as variants of lipase from Thermomyces lanuginosus (also
called Humicola lanuginosa).
[0032] The lipolytic enzymes may have a temperature optimum in the
range of 30-90.degree. C., e.g. 30-70.degree. C.
Synergistic Effect
[0033] The combination of the two lipolytic enzymes has a
synergistic effect on dough made with the combination or on a baked
product made from the dough, particularly improved dough
stabilization, i.e. a larger loaf volume of the baked product
and/or a better shape retention during baking, particularly in a
stressed system, edgy in the case of over-proofing or
over-mixing.
[0034] Additionally or alternatively, the synergistic effect on the
baked product may include a lower initial firmness and/or a more
uniform and fine crumb, improved crumb structure (finer crumb,
thinner cell wails, more rounded cells), of the baked product.
Additionally or alternatively, there may be a synergistic effect on
dough properties, e.g. a less soft dough, higher elasticity, lower
extensibility.
[0035] Synergy may be determined by making doughs or baked products
with addition of the first and the second lipolytic enzyme
separately and in combination, and comparing the effects; synergy
is indicated when the combination produces a better effect than
each enzyme used separately.
[0036] The comparison may be made between the combination and each
enzyme alone at double dosage (on the basis of enzyme protein or
enzyme activity). Thus, synergy may be said to occur if the effect
of 0.5 mg of enzyme A+1.0 mg of enzyme B is greater than the effect
with 1.0 mg of enzyme A and also greater than the effect with 2.0
mg of enzyme B.
[0037] Alternatively, the comparison may be made with equal total
enzyme dosages (as pure enzyme protein), if the effect with the
combination is greater than with either enzyme alone, this may be
taken as an indication of synergy. As an example synergy may be
said to occur if the effect of 0.5 mg of enzyme A+1.0 mg of enzyme
B is greater than with 1.5 mg of enzyme A or B alone.
[0038] Suitable dosages for the enzymes may typically be found in
the range 0.01-10 mg of enzyme protein per kg of flour,
particularly 0.1-5 mg/kg, e.g. 0.2-1 mg/kg. Suitable dosages for
each of the two enzymes in the combination may be found by first
determining a suitable dosage for each enzyme alone (e.g. the
optimum dosage, i.e. the dosage producing the greatest effect) and
using 30-67% (e.g. 33-50%, particularly 50%) of that dosage for
each enzyme in the combination. Again, if the effect with the
combination is greater than with either enzyme used separately,
this is taken as an indication of synergy.
[0039] A lipolytic enzyme with phospholipase activity may be used
at a dosage of 200-5000 LEU/kg of flour, e.g. 500-2000 LEU/kg. The
LEU activity unit is described in WO 99/53769.
[0040] A lipolytic enzyme with triacylglycerol lipase activity may
be used at a dosage of 20-1000 LU/kg of flour, particularly 50-500
LU/kg. The LU method is described in WO 2000/32758,
Additional Enzyme
[0041] Optionally, an additional enzyme may be used together with
the lipolytic enzymes. The additional enzyme may be an amylase,
particularly an anti-staling amylase, an amyloglucosidase, a
cyclodextrin glucanotransferase, or the additional enzyme may be a
peptidase in particular an exopeptidase, a transglutaminase, a
celluluse, a hemicellulase, in particular a pentosanase such as
xylanase, a protease, a protein disulfide isomerase, e.g., a
protein disulfide isomerase as disclosed in WO 95100636, a
glycosyltransferase, a branching enzyme (1,4-glucan branching
enzyme). a 4-glucanotransferase (dextrin glycosyltransferase) or an
oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a
pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a
carbohydrate oxidase.
[0042] The additional enzyme may be of any origin, including
mammalian and plant, and preferably of microbial (bacterial, yeast
or fungal) origin and may be obtained by techniques conventionally
used in the art.
[0043] The amylase may be a fungal or bacterial alpha-amylase, e.g.
from Bacillus, particularly B. licheniformis or B.
amyloliquefaciens, or from Aspergillus, particularly A. oryzae, a
beta-amylase, e.g. from plant (e.g. soy bean) or from microbial
sources (e.g. Bacillus).
[0044] The xylanase is preferably of microbial origin, e.g. derived
from a bacterium or fungus, such as a strain of Aspergillus, in
particular of A. aculeatus, A. niger (cf. WO 91119782), A. awamori
(WO 91/18977), or A. tubigensis (WO 92/01793), from a strain of
Trichoderma, e.g. T. reesei, or from a strain of Humicola, e.g. H.
insolens (WO 92/17573).
[0045] The amyloglicosidase may be from Aspergillus, particularly
A. oryzae.
[0046] The glucose oxidase may be a fungal glucose oxidase,
particularly from Aspergillus niger.
[0047] The protease may be a neutral protease from Bacillus
amyloliquefaciens.
Anti-Stating Amylase
[0048] The method or the composition of the invention may include
addition of an anti-staling amylase. In particular, a galactolipase
and a phospholipase may be used together with an anti-staling
amylase, as described in WO 99/53769. The anti-staling amylase is
an amylase that is effective in retarding the staling (crumb
firming) of baked products, particularly a maltogenic
alpha-amylase, e.g. from Bacillus stearothermophilus strain NCIB
11837.
[0049] Alternatively, the method or composition of the invention
may be made without addition of an anti-staling amylase. In
particular, a lipase and a phospholipase may be used without
addition of an anti-staling amylase or without addition of a
maltogenic alpha-amylase.
Composition Comprising Lipolytic Enzymes
[0050] The present invention provides a composition comprising a
combination of two lipolytic enzymes as described above. The
composition may be an enzyme preparation for use as a baking
additive. The composition may also comprise flour and may be a
dough or a premix.
Enzyme Preparation
[0051] The composition may be an enzyme preparation comprising a
combination of lipolytic enzymes, for use as a baking additive in
the process of the invention. The enzyme preparation may
particularly be in the form of a granulate or agglomerated powder,
e.g. with a narrow particle size distribution with more than 95%
(by weight) of the particles in the range from 25 to 500 .mu.m.
[0052] Granulates and agglomerated powders may be prepared by
conventional methods, e.g. by spraying the enzymes 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.
[0053] The enzyme preparation may also be supplied as a liquid
formulation, particularly a stabilized liquid. Liquid enzyme
preparations may, for instance, be stabilized by adding a polyol
such as propylene glycol, a sugar or sugar alcohol, lactic acid or
boric acid according to established methods.
Dough
[0054] The dough of the invention generally comprises wheat meal or
wheat flour and/or other types of meal, flour or starch such as
corn flour, corn starch, rye meal, rye flour, oat flour, oat meal,
soy flour, sorghum meal, sorghum flour, potato meal, potato flour
or potato starch.
[0055] The dough of the invention may be fresh, frozen or
par-baked.
[0056] The dough of the invention is normally a leavened dough or a
dough to be subjected to leavening. The dough may be leavened in
various ways, such as by adding chemical leavening agents, e.g.,
sodium bicarbonate or by adding a leaven (fermenting dough), but it
is preferred to leaven the dough by adding a suitable yeast
culture, such as a culture of Saccharomyces cerevisiae (baker's
yeast), e.g. a commercially available strain of S. cerevisiae.
[0057] The dough may also comprise other conventional dough
ingredients, e.g. proteins, such as milk powder, gluten, and soy,
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.
[0058] The dough may comprise fat (triglyceride) such as granulated
fat or shortening, but the invention is particularly applicable to
a dough where less than 1% by weight of fat (triglyceride) is
added, and particularly to a dough which is made without addition
of fat.
[0059] The dough may further comprise an emulsifier such as mono-
or diglycerides, diacetyl tartaric acid esters of mono- or
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic acid esters of monoglycerides, acetic acid
esters of monoglycerides, polyoxyethylene stearates, or
lysolecithin.
Pre-Mix
[0060] The invention also provides a pre-mix comprising flour
together with two lipolytic enzymes as described above. The pre-mix
may contain other dough-improving and/or bread-improving additives,
e.g. any of the additives, including enzymes, mentioned above.
Materials and Methods
Enzyme Activity Assays
Phospholipase Activity (PHLU)
[0061] Phospholipase activity is measured as the release of free
fatty acids from lecithin. 500 .mu.l 4% L-alpha-phosphatidylcholine
(plant lecithin from Avanti), 5 mM CaCl.sub.2 in 50 mM NaOAc. pH 5
is added to 50 .mu.l enzyme solution diluted to an appropriate
concentration in water. The samples are incubated for 10 min at
30.degree. C. and the reaction stopped at 95.degree. C. for 5 min
prior to centrifugation (5 min at 7000 rpm). Free fatty acids are
determined using the NEFA C kit from Wako Chemicals GmbHb 25 .mu.l
reaction mixture is added 250 .mu.l Reagent A and incubated 10 min
at 37.degree. C. Then 500 .mu.l Reagent B is added and the sample
is incubated again, 10 min at 37.degree. C. The absorption is
measured at 550 nm. Substrate and enzyme blinds (preheated enzyme
samples (10 min at 95.degree. C.)+substrate) are included. Oleic
acid is used as a fatty acid standard. 1 PHLU equals the amount of
enzyme capable of releasing 1 .mu.mol of free fatty acid/min at
these conditions.
Plate Assay for Phospholipase Activity
[0062] A) 50 ml 2% agarose in purified water is melted/stirred for
5 minutes and cooled to 60-63.degree. C.
[0063] B) 50 ml 2% plant L-alpha-Phosphatidylcholine 95% in 0.2M
NaOAc, 10 mM CaCl.sub.2, pH 5.5 at 60.degree. C. in 30 min, is
blended in 15 sec. with ultrathorax.
[0064] Equal volumes of 2% agarose and 2% Lecithin (A and B) are
mixed. 250 .mu.l 4 mg/ml crystal violet in purified water is added
as indicator. The mixture is poured into appropriate petri dishes
(eg. 30 ml in 14 cm O dish), and appropriate holes are made in the
agar (3-5 mm) for application of enzyme solution.
[0065] The enzyme sample is diluted to a concentration
corresponding to OD.sub.280=0.5 and 10 microliter is applied into
holes in the agarose/lecithin-matrix. Plates are incubated at
30.degree. C. and reaction zones in the plates are identified after
20-24 hours incubation, and the size of the clearing zone indicates
the phospholipase activity
Plate Assays for Galactolipase and Triacylglycerol Lipase
Activity
[0066] Plate assays are carried out as for the phospholipase assay
except that digalactosyl diglyceride (DGDG) or olive oil is used
instead of L-alpha-Phosphatidyicholine.
Baking Methods
Sponge Dough
[0067] A liquid sponge is prepared by mixing 34.8 pans of water, 60
parts of flour and 1.5 pans of instant yeast, and fermenting for 3
hours at 24.degree. C. A dough is then prepared by mixing the
liquid sponge with 22.93 parts of water, 40 parts of flour, 0.5
part of instant yeast, 11.26 parts of 42 high-fructose corn syrup,
0.25 part of calcium propionate, 2 parts of oil and 2 parts of
salt, 50 ppm of ascorbic acid 50 parts of wheat flour, 0.5 part of
SSL (sodium stearoyl-2-tactytate), 2 pats of salt, 6 parts of sugar
and water and ascorbic acid as required.
European Straight Dough Procedure
[0068] A dough is prepared by mixing 100 parts (by weight) of wheat
flour, 4 parts of yeast, 1.5 parts of salt and 1.5 parts of sugar
with water and ascorbic acid as required to reach a suitable dough
consistency.
Shape Factor (Shape Retention)
[0069] The shape factor is taken as the ratio between the height
and diameter of roles after baking (average of 10 rolls). A higher
value indicates a better shape retention.
Dough Softness
[0070] Softness is a measure of the degree to which, or ease with
which, a dough will compress or resist compression. A sensory
evaluation is done by a trained and skilled baker feeling and
squeezing the dough. The results are expressed on a scale from 0
(little softness) to 10 (very soft) with the control (dough without
enzyme addition) taken as 5.
Dough Extensibility
[0071] Extensibility is a measure of the degree by which a dough
can be stretched without tearing. A sensory evaluation is done by a
trained and skilled baker pulling a piece of kneaded dough (about
30 g) and judging the extensibility. The results are expressed on a
scale from 0 (Short/low extensibility) to 10 (long /high
extensibility) with the control (dough without enzyme addition)
taken as 5.
Dough Elasticity
[0072] Elasticity is a measure of the degree to which a dough tends
to recover its original shape after release from a deforming force.
It is evaluated by rolling a piece of dough (about 30 g) to a size
of about 10 cm, and having a trained and skilled baker carefully
pulling at opposite ends to judge the resistance and elasticity.
The results are expressed on a scale from 0 (low/weak
elasticity/recovery) to 10 (high/strong elasticity/recovery) with
the control (dough without enzyme addition) taken as 5.
EXAMPLES
Example 1
Synergistic Effect of Phospholipase and Galactolipase on Dough
Stabilization
[0073] Lipolytic enzyme combinations were tested in a European
Straight dough procedure as described above. Fungal alpha-amylase
(Fungamyl Super Mass., 40 ppm) and an oxidation system (ascorbic
acid, 30 ppm) were added to the dough system. Each dough was split
into rolls and pan bread. Over-proofing (indicating a stressed
system) was carried out for the rolls (70 min.) and the pan bread
(80 min.).
[0074] Combinations with the following lipolytic enzymes were
tested. Variant 39 was tested in combination with variant 91 or
with Aspergillus oryzae phospholipase. Variants 39 and 91 are
variants of the Thermomyces lanuginosus lipase according to WO
2000/32758.
[0075] The combination of variants 91 and 39 was selected because
of the high phospholipase activity and the high galactolipase
activity, respectively. The Aspergillus oryzae phospholipase and
variant 39 combination were chosen due to the combination of a pure
phospholipase and an enzyme with high DGDG activity. The plate
assays described above showed that each enzyme wasx specific with
little or no activity for the two other substrates.
[0076] The lipolytic enzymes were added according to the table
below. The tests with a single enzyme were conducted with a dosage
found to be optimum for the enzyme in question, and combinations
were tested as indicated, with each enzyme at 33, 50 or 67% of
optimal dosage. TABLE-US-00001 Rolls Pan bread Specific Specific
volume Shape volume Lipolytic enzyme (ml/g) factor (ml/g) Variant
91 (20 LU/kg) 7.52 0.66 5.75 Variant 39 (250 LU/kg) 7.42 0.65 5.77
Variant 91 (50%) + variant 39 (33%) 7.57 0.66 5.94
[0077] The results demonstrate that the combination of Variant 91
with Variant 39 added at 50% and 33% respectively of optimal dosage
irmproves the specific volume for both the rolls and the pan bread
compared to the each enzyme added separately at optimum dosage.
[0078] The results regarding volume and stability improvement from
the combination of Aspergillus oryzae phospholipase with Variant 39
are listed in the table below TABLE-US-00002 Rolls Pan bread
Specific Specific volume Shape volume Lipolytic enzyme (ml/g)
factor (ml/g) A. oryzae Phospholipase 0.1 mg/kg 6.27 0.57 4.96
Variant 39 (250 LU/kg) 6.40 0.60 5.18 A. oryzae Phospholipase (33%)
+ 7.31 0.68 5.80 variant 39 (67%)
[0079] The combination of A. oryzae Phospholipase and Variant 39 at
33% and 67%. respectively, of optimal dosage increases the specific
volume considerably compared to each enzyme added separately at
optimum dosage. The combination also has a positive contribution to
the shape factor of the rolls.
[0080] Both the results described above show that the combination
of a phospholipase and a galactolipase improves the volume and
stability (shape factor) of the rolls and bread compared to the
roots and bread containing up to three the dosages of the enzymes
added separately.
Example 2
Synergistic Effect of Triacylglycerol Lipase and Phospholipase on
Dough Stabilization
[0081] A phospholipase A2 from porcine pancreas was tested in
combination with a 1,3-specific triacylglycerol lipase from
Thermomyces lanuginosus in the European straight dough procedure as
described above. The results were compared to each enzyme used
alone in dosages considered to be optimal. The enzyme combination
was made with 50% of optimal dosage of each of the enzymes.
[0082] Each dough was split into rolls and pan bread. The rolls
were proofed for 70 minutes (over proofing) and the pan bread was
proofed for 80 minutes (over proofing). The over proofing was
carried out to stress the system in order to test the stabilizing
effect of the enzymes. TABLE-US-00003 Rolls Pan bread Sp. Vol Shape
Sp. Vol (ml/g) factor (ml/g) Phospholipase (3 mg) 6.24 0.56 5.60
Triacylglycerol lipase (1000 LU) 6.43 0.58 5.43 Phospholipase +
triacylglycerol 6.88 0.60 5.93 lipase (50%/50%)
[0083] The two enzymes were found to be very specific, i.e. the
triacylglycerol lipase has very little activity on phospholipid and
galactolipids, and the phospholipase has very little activity on
triglycerides and galactolipids.
[0084] The results show that when the phospholipase and the
triacylglycerol lipase are combined they give a better volume and
shape factor than each of the enzyme separately in a stressed
system.
Example 3
Synergistic Effect on Dough Properties and Loaf Volume
[0085] The combination of Lipase/phospholipase from Fusarium
oxysporum (FoL) and Variant 6 on dough and bread was evaluated.
Variant 6 is a variant of the Thermomyces lanuginosus lipase with
the following amino acid alterations (SPIRR indicates a peptide
extension at the N-terminal, and 270AGGFS indicates a peptide
extension at the C-terminal).
[0086] Variant 6.
SPIRR+G91A+D96W+E:99K+G263Q+L264A+I265T+G266D+T267A+L269N+270AGGFS
[0087] Loaves were prepared according to the invention by adding
Variant 6 (25 LU/kg flour) and FoL (500 LU/kg flour) to the dough.
For comparison, loaves were baked without lipolytic enzymes, with
FoL alone (1000 LU/kg) or Variant 6 alone (50 LU/kg) which were
found to be the optimal dosages for the enzymes. The LU assay
method is described in WO 2000/32758.
[0088] The standard sponge dough WPB formula was used as described
above, with the hydrated distilled MG and SSL eliminated to avoid
masking effects on the enzyme. Loaves contained 2% soy oil as well
as fungal amylase and pentosanase (Fungamyi Super Mass., 50 ppm).
The oxidation system was 50 ppm ascorbic acid. In addition to
subjective evaluations, crumb softness and elasticity were measured
24 hours after baking. The trial was repeated once.
[0089] Dough evaluations. Evaluations of the dough at the sheeter
are shown below. The dough scores for the two trials were
identical. TABLE-US-00004 Lipotytic enzyme FoL + None FoL Variant 6
Variant 6 Softness 5.5 4.5 4.5 4.0 Extensibility 5.0 4.5 4.5 4.0
Elasticity 5.0 5.5 5.5 6.0
[0090] The results show that the combination of FoL and Variant 6
yielded dough that was less soft, less extensible and more elastic
than either enzyme alone at double dosage.
[0091] The specific volume data from the tested loaves are shown
below. Reproducibility between the 2 days was high. TABLE-US-00005
Lipolytic enzyme FoL + None FoL Variant 6 Variant 6 Specific
Volume, cc/gram 6.0 6.15 6.15 6.3
[0092] The results demonstrate that the combination of two
lipolytic enzymes gives a larger loaf volume than either enzyme
alone at double dosage.
Example 4
Synergistic Effect on Dough Stabilization
[0093] Variant 32 was tested in combination with Variant 13 and
Variant 60. The variants are variants of the Thermomyces
lanuginosus lipase with the following amino acid alterations (where
SPIRR indicates a peptide extension at the N-terminal, and 270AGGFS
indicates a peptide extension at the C-terminal):
[0094] a Variant 32:
91SA+D96W+E99K+G263Q+L264A+I265T+G266D+T267A+L269N+270AGGFS
[0095] Variant 60:
G91A+D96W+E99K+G263Q+L264A+I265T+G266S+T267A+L269N+270AGGFS
[0096] Variant 13: D96F+G266S
[0097] Each combination was tested in a European straight dough
procedure, as described above. The results were compared to each
enzyme used alone. Lipolytic enzymes were added as shown in the
table below. The tests with a single enzyme were conducted with a
dosage considered optimum for that enzyme, and the enzyme
combinations were tested with each enzyme at 50% of the optimum
dosage. The combination of Variant 32 and Variant 13 was also
tested with each enzyme at 40% of the optimum dosage, i.e. 20%
lower dosage.
[0098] Each dough was split into rolis and pan bread. The rolls
were proofed for 70 minutes (over proofing), and the pan bread was
proofed for 80 minutes (over proofing). The over proofing was
carried out to stress the system, in order to test the lipolytic
enzymes as stabilizers.
[0099] The results from over-proofing are shown below:
TABLE-US-00006 Rolls Pan bread Sp. Vol. Shape Sp. Vol. (ml/g)
factor (ml/g) Lipolytic enzyme added 70 min 70 min 80 min Variant
32 (200 LU) 7.78 0.67 6.16 Variant 13 (500 LU) 7.24 0.66 5.69
Variant 60 (100 LU) 7.02 0.67 5.69 Variant 32 + Variant 13, 50%
8.26 0.71 6.24 Variant 32 + Variant 13, 40% 8.03 0.69 6.30 Variant
32 + Variant 60, 50% 7.87 0.69 6.39
[0100] The results show that when the bread is stressed
(over-proofed), the combinations of Variant 32 with Variant 13 or
Variant 60 clearly give an improved volume and shape compared to
each enzyme used alone, particularly Variant 32+Variant 13, even at
reduced dosage. The results reveal that when bread is stressed
(over proofed), the combinations show a significantly improved
effect on volume and shape factor.
[0101] Furthermore, it was observed that the combination of Variant
32 and Variant 13 at 40% of optimum dosage provided a more uniform
and fine crumb compared to each enzyme used alone.
Sequence CWU 1
1
1 1 5 PRT Artificial Sequence Synthetic 1 Ser Pro Ile Arg Arg 1
5
* * * * *