Note: Descriptions are shown in the official language in which they were submitted.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
METHOD OF PREPARING A DOUGH OR A BAKED PRODUCT MADE FROM A DOUGH, WITH
ADDITION
OF LIPOLYTIC ENZYMES
TECHNICAL FIELD
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
WO 94/04035, EP 109244, EP 585988, WO 98/26057, WO 98/45453, WO
99/53769, 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
o triacylglycerol lipase, phospholipase and galactolipase.
SUMMARY OF THE INVENT10N
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
5 volume of the baked product and/or a better shape retention during baking.
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.
2o 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
25 Lipolytic enzyme
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
so 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.
~ Galactolipase activity (EC 3.1.1.26), i.e. hydrolytic activity on carboxylic
ester
bonds in galactolipids such as DGDG (digalactosyl diglyceride). The
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
2
galactolipase activity (digalactosyl diglyceride hydrolyzing activity or
DGDGase
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.
~ Phospholipase activity (A1 or A2, EC 3.1.1.32 or 3.1.1.4), i.e. hydrolytic
activity
s 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 7
or 2.
~ Triacylglycerol lipase activity (EC 3.1.1.3), i.e. hydrolytic activity for
carboxylic
o ester bonds in triglycerides, e.g. 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
~5 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 butter fat. Suitably, this ratio may be above
3.
Each lipolytic enzyme may have a narrow specificity with activity for one of
the
2o 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.
A lipolytic enzyme is considered to be a galactolipase if it has a higher
activity
on galactolipids than on phospholipids and triglycerides. Similarly, it is
considered to
25 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.
The enzyme combination may comprise three or more lipolytic enzymes, e.g.
3o comprising a galactolipase, a phospholipase and a triacylglycerol lipase.
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
35 baked product.
Sources of lipolytic enzymes
The lipolytic enzymes may be prokaryotic, particularly bacterial, e.g. from
Pseudomonas or Bacillus. Alternatively, the lipolytic enzymes may be
eukaryotic, e:g.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
3
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.
Some particular examples of lipolytic enzymes follow:
~ Phospholipase from bee or snake venom or from mammal pancreas, e.g.
porcine pancreas.
o ~ 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. subtilissimus, Neurospora, N,
crassa,
Rhizomucor, R. pusillus, Rhizopus, R. arrhizus, R, japonicus, R. stolonifer,
Sclerotinia,
S. libertiana, Trichophyton, T. rubrum, VVhetzelinia, W sclerotiorum,
Bacillus, 8.
~ s megaterium, B, subtilis, Citrobacter, C. freundii, Enterobacter, E.
aerogenes, E.
cloacae Edwardsiella, E. tarda, Erwinia, E. herbicola, Escherichia, E. coli,
Klebsiella, K.
pneumoniae, Proteus, P. vulgaris, Providencia, P. stuartii, Salmonella, S.
typhimurium,
Serratia, S. liguefasciens, S. marcescens, Shigella, S. flexneri,
Streptomyces, S.
violeceoruber, Yersinia, or Y. enterocolitica.
20 ~ Lipase from Thermomyces lanuginosus (Humicola lanuginosa) (EP
305216, US 5869438).
~ Lipase/phospholipase from Fusarium oxysporum (WO 98/26057 ).
~ Lysophospholipases from Aspergillus niger and A. oryzae (WO 0127251 ).
~ Phospholipase A1 from Aspergillus oryzae (EP 575133, JP-A 10-155493).
25 ~ Lysophospholipase from F. venenatum (WO 00/28044).
~ Phospholipase B from A. oryzae (US 6146869).
~ Lipase from A. tubigensis (WO 9845453).
~ Lipase from F, solani (US 5990069).
~ Lipolytic enzyme from F. culmorum (US 5830736).
o ~ Phospholipase from Hyphozyma (US 6127137).
~ Lipolytic enzymes described in PCTIDK 01/00448.
~ Lipolytic enzymes described in DK PA 2001 00304.
~ 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,
s5 6 and 13, such as variants of lipase from Thermomyces lanuginosus (also
called
Humicola lanuginosa).
The lipolytic enzymes may have a temperature optimum in the range of 30-
90°C, e.g. 30-70°C.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
4
Synergistic effect
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, e.g. in
the case of over-proofing or over-mixing.
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 walls, more rounded cells), of the baked
product,
~o Additionally or alternatively, there may be a synergistic effect on dough
properties, e.g.
a less soft dough, higher elasticity, lower extensibility.
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
~ 5 effect than each enzyme used separately.
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
20 2.0 mg of enzyme B.
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
25 greater than with 1.5 mg of enzyme A or B alone.
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.
3o 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.
A lipolytic enzyme with phospholipase activity may be used at a dosage of
s5 200-5000 LEUIkg of flour, e.g. 500-2000 LEU/kg. The LEU activity unit is
described in
WO 99/53769.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
A lipolytic enzyme with triacylglycerol lipase activity may be used at a
dosage
of 20-1000 LUlkg of flour, particularly 50-500 LU/kg. The LU method is
described in
WO 2000/32758.
Additional enzyme
5 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
cellulase, a hemicellulase, in particular a pentosanase such as xylanase, a
protease, a
o protein disulfide isomerase, e.g., a protein disulfide isomerase as
disclosed in WO
95/00636, a glycosyltransferase, a branching enzyme (1,4-a-glucan branching
enzyme), a 4-a-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.
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.
The amylase may be a fungal or bacterial alpha-amylase, e.g. from Bacillus,
particularly 8. 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).
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
91 /19782), 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).
The amyloglucosidase may be from Aspergillus, particularly A, oryzae.
The glucose oxidase may be a fungal glucose oxidase, particularly from
Aspergillus niger.
so The protease may be a neutral protease from Bacillus amyloliquefaciens.
Anti-staling amylase
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
s5 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.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
6
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
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.
o Enzyme preparation
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
~5 the particles in the range from ~5 to 500 ~,m.
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 NaCI or sodium sulfate), a sugar
(such as
2o sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn
grits, or soy.
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.
25 Dough
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.
30 The dough of the invention may be fresh, frozen or par-baked.
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
35 culture, such as a culture of Saccharomyces cerevisiae (baker's yeast),
e.g. a
commercially available strain of S. cerevisiae.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
7
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.
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
~ o fat.
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
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.
2o MATERIALS AND METHODS
Enzyme activity assays
Phospholipase activity (PHLU)
Phospholipase activity is measured as the release of free fatty acids from
lecithin. 500 p1 4% L-alpha-phosphatidylcholine (plant lecithin from Avanti),
5 mM
CaCl2 in 50 mM NaOAc, pH 5 is added to 50 p1 enzyme solution diluted to an
appropriate concentration in water. The samples are incubated for 10 min at 30
°C and
the reaction stopped at 95 °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 GmbH; 25
p1
reaction mixture is added 250 p1 Reagent A and incubated 10 min at 37
°C. Then 500
so p1 Reagent B is added and the sample is incubated again, 10 min at 37
°C. The
absorption is measured at 550 nm. Substrate and enzyme blinds (preheated
enzyme
samples (10 min at 95 °C) + substrate) are included. Oleic acid is used
as a fatty acid
standard. 1 PHLU equals the amount of enzyme capable of releasing 1 Nmol of
free
fatty acid/min at these conditions.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
Plate assay for ~hospholipase activity
A) 50 ml 2% agarose in purified water is melted/stirred for 5 minutes and
cooled to 60 - 63°C.
B) 50 ml 2% plant L-alpha-Phosphatidylcholine 95% in 0,2M NaOAc, 10 mM
CaCl2, pH 5,5 at 60°C in 30 min. is blended in 15 sec. with
ultrathorax.
Equal volumes of 2% agarose and 2% Lecithin (A and B) are mixed. 250 p1 4
mg/ml crystal violet in purified water is added as indicator. The mixture is
poured into
appropriate petri dishes (e.g. 30 ml in 14cm O dish), and appropriate holes
are made
in the agar (3-5 mm) for application of enzyme solution.
o The enzyme sample is diluted to a concentration corresponding to OD2ao = 0.5
and 10 microliter is applied into holes in the agarose/lecithin-matrix. Plates
are
incubated at 30°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 aalactolipase and triacylglycerol lipase activity
Plate assays are carried out as for the phospholipase assay, except that
digalactosyl diglyceride (DGDG) or olive oil is used instead of L-alpha-
Phosphatidylcholine.
Baking methods
Sponge dough
2o A liquid sponge is prepared by mixing 34.8 parts of water, 60 parts of
flour and
1.5 parts . of instant yeast, and fermenting for 3 hours at 24°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-lactylate), 2 parts ofi salt, 6
parts of sugar and
water and ascorbic acid as required.
European strai. hq t dou~ih procedure
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
so to reach a suitable dough consistency.
Shape factor (shape retention
The shape factor is taken as the ratio between the height and diameter of
rolls
after baking (average of 10 rolls). A higher value indicates a better shape
retention.
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
9
Douah softness
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
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
o 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.
Douah elasticit rL
Elasticity is a measure of the degree to which a dough tends to recover its
5 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.
2o EXAMPLES
Example 1: Synergistic effect of phospholipase and galactolipase on dough
stabilization
Lipolytic enzyme combinations were tested in a European Straight dough
procedure as described above. Fungal alpha-amylase (Fungamyl Super MA, 40 ppm)
25 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.).
Combinations with the following lipolytic enzymes were tested: Variant 39 was
tested in combination with variant 91 or with Aspergillus oryzae
phospholipase.
so Variants 39 and 91 are variants of the Thermomyces lanuginosus lipase
according to
WO 2000/32758.
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
35 Of a pure phospholipase and an enzyme with high DGDG activity. The plate
assays
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
described above showed that each enzyme wasx specific with little or no
activity for
the two other substrates.
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
5 enzyme in question, and combinations were tested as indicated, with each
enzyme at
33, 50 or 67 % of optimal dosage.
Rolls Pan bread
Lipolytic enzyme Specific volume Specific volume
(ml/g) Shape factor (ml/g)
Variant 91 7 66 5.75
52 0
(20 LU/kg) . .
Variant 39 7 0 5.77
42 65
(250 LU/kg) . .
Variant 91 (50 7 66 5.94
%) + 57 0
variant 39 (33%) . .
The results demonstrate that the combination of Variant 91 with Variant 39,
1 o added at 50 % and 33 % respectively of optimal dosage, improves the
specific volume
for both the rolls and the pan bread compared to the each enzyme added
separately at
optimum dosage.
The results regarding volume and stability improvement from the combination
of Aspergillus oryzae phospholipase with Variant 39 are listed in the table
below.
Rolls Pan bread
Lipolytic enzyme Specific Specific volume
Shape factor
volume (ml/g) (ml/g)
A. oryzae
Phospholipase 6,27 0,57 4,96
0,1 mglkg
Variant 39 (250 6,40 0,60 5,18
LUlkg)
A. oryzae
Phospholipase (33 7,31 0,68 5,80
%)
+ variant 39 (67%)
The combination of A. oryzae Phospholipase and Variant 39 at 33 % and 67
%, respectively, of optimal dosage increases the specific volume considerably
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
11
compared to each enzyme added separately at optimum dosage. The combination
also has a positive contribution to the shape factor of the rolls.
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 rolls and bread containing up to thrice
the
dosages of the enzymes added separately.
Example 2: Synergistic effect of triacylgtycerol lipase and phospholipase on
dough stabilization
A phospholipase A2 from porcine pancreas was tested in combination with a
0 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.
Each dough was split info roils and pan bread. The rolls were proofed for 70
~5 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.
Rolls Pan bread
Sp. Vol (mllg) Shape factor Sp. Vol (ml/g)
Phospholipase 6 56 5.60
24 0
(3mg) . .
Triacylglycerol 6 58 5.43
43 0
lipase (1000LU) . .
Phospholipase
+
triacylglycerol 6.88 0.60 5.93
lipase (50%/50%)
2o 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.
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
25 separately in a stressed system.
Example 3: Synergistic effect on dough properties and loaf volume
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
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
12
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).
Variant 6: SPIRR +G91A +D96W +E99K +G263Q +L264A +1265T +G266D
+T267A +L269N +270AGGFS
Loaves were prepared according to the invention by adding Variant 6 (25
LU/kg flour) and FoL (500 LUlkg flour) to the dough. For comparison, loaves
were
baked without iipolytic 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
o method is described in WO 2000132758.
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
(Fungamyl
Super MA, 50 ppm). The oxidation system was 50 ppm ascorbic acid. In addition
to
5 subjective evaluations, crumb softness and elasticity were measured 24 hours
after
baking. The trial was repeated once.
Dough evaluations. Evaluations of the dough at the sheeter are shown below.
The dough scores for the two trials were identical.
Lipolytic None FoL Variant FoL +
enzyme 6 Variant
6
Softness 5.5 4.5 4.5 4.0
Extensibility5.0 4.5 4.5 4.0
Elasticity 5.0 5.5 5.5 6.0
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.
The specific volume data from the tested loaves are shown below.
2~ Reproducibility between the 2 days was high.
FoL +
Lipolytic enzymeNone FoL Variant Variant
6 6
Specific Volume,6 6.15 6.15 6.3
0
cclgram .
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
13
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.
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):
~ Variant 32: 91 A +D96W +E99K +G263Q +L264A +1265T +G266D +T267A
+L269N + 270AGGFS
o ~ Variant 60: G91A +D96W +E99K +G263Q +L264A +1265T +G266S
+T267A +L269N + 270AGGFS
~ Variant 13: D96F +G266S
Each combination was tested in a European straight dough procedure, as
described above. The results were compared to each enzyme used atone.
Lipolytic
~5 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.
2o 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
lipolytic
enzymes as stabilizers.
The results from over-proofing are shown below:
Rolls Pan bread
Lipolytic enzyme Sp. Vol. (ml/g)Shape factor Sp. Vol. (mllg)
added
70 min 70 min 80 min
Variant 32 (200LU) 7.78 0.67 6.16 ,
Variant 13 (500LU) 7.24 0.66 5.69
Variant 60 (100LU) 7.02 0.67 5.69
Variant 32+Variant 8.26 0.71 6.24
13,
50%
CA 02412533 2002-12-11
WO 02/03805 PCT/DKO1/00472
14
Variant 32+Variant 8.03 0.69 6.30
13,
40%
Variant 32+Variant 7.87 0.69 6.39
60,
50%
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.
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
1o each enzyme used alone.
