Note: Descriptions are shown in the official language in which they were submitted.
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s FLAVOUR ENHANCER
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a flavour enhancer,
methods for preparing the flavour enhancer, to food and feed
compositions comprising the flavour enhancer and to the use
of the flavour enhancer.
is BACKGROUND OF THE INVENTION
Flavour enhancers enhance the existing flavour of a food
product. Two classes of well-known flavour enhancing
compounds are monosodium glutamate and 5'-ribonucleotides.
2o These flavour enhancing compounds are used as such, but are
also, separately or in combination, part of flavour enhancing
compositions.
Yeast extracts, for instance, which are prepared by
enzymatic degradation of yeast, contain the flavour enhancing
zs 5'-ribonucleotides guanosine-5'-monophosphate (5'-GMP) and
inosine-5'-monophosphate (5'-IMP}.
Hydrolysed vegetable proteins (HVPs), which are prepared
by acid or enzymatic hydrolysis of vegetable protein,
typically contain monosodium glutamate as their flavour
ao enhancing compound. This monosodium glutamate is derived from
the amino acids glutamic acid and glutamine released from the
protein during hydrolysis.
Flavour enhancers which do not contain substantial
amounts of at least either of the two classes of flavour
3s enhancing compounds are very scarce. As far as the inventors
know, the only disclosure of a flavour enhancer of this type
is in US Patent 5,077,062.
US Patent 5,077,062 describes a soy hydrolysate which is
prepared by hydrolysing at pH 6.6-7.2, 30-38°C for about two
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hours. The resulting hydrolysate contains no free amino
acids, is low in glutamate, and can be used as a flavour
enhancer. However, the described flavour enhancer enhances
fish flavours only.
s
BRIEF SUN~1ARY OF THE INVENTION
The present invention provides a flavour enhancer that
is low in monosodium glutamate, methods for preparing the
flavour enhancer, compositions comprising the flavour
enhancer, and uses of the flavour enhancer. A preferred
method for preparing the flavour enhancer as a soy protein
hydrolysate comprises: (i) forming an aqueous suspension of a
soy protein containing starting'material (e.g. soy flour, soy
~s protein isolate, soy beans, or soy bean flakes, meal or
grits, which preferably are defatted}; (ii} heating the
aqueous suspension for at least from about 1 minute to about
15 minutes at a temperature of from about 60°C to about 82°C;
(iii) incubating the suspension with a protease mixture
zo comprising endoprotease and exoprotease activity, to obtain
an amino acid level in the suspension of from about 20o to
about 55%; (iv) adjusting the pH and temperature of the
suspension to inactivate the endoprotease and exoprotease;
and (v) recovering the soy protein hydrolysate (e.g. by
zs concentrating and/or drying, or other appropiate means).
The present invention thus provides a flavour enhancer
which is low in monosodium glutamate, has no yeast-like after
taste, and enhances both meat, vegetable and dairy flavours.
This offers the advantage of a wide applicability. The
3o flavour enhancer can be used as such or, in a flavouring
composition, e.g. in combination with a flavouring agent.
Since the flavour enhancer according to the invention is
low in monosodium glutamate, it can also be used by
individuals who prefer to minimise their monosodium glutamate
as intake, e.g. due to a sensitivity to monosodium glutamate.
Although the present flavour enhancer is a soy
hydrolysate with its own characteristic taste, the taste of a
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food product comprising the soy hydrolysate as flavour
enhancer is not reminiscent of the soy used to prepare the
flavour enhancer. These and other objects and advantages of
the present invention, as well as additional inventive
s features, will be apparent from the description of the
invention set forth herein.
DETAILED DESCRIPTION OF THE INVENTION
io
The present invention provides, among other things, a soy
hydrolysate which is obtainable by:
(i) heating a suspension of defatted soy flour in water for
at least about 10 min at from about 65°C to about 82°C;
~s (ii) incubating the suspension with a mixture of endo- and
exo-proteases obtained from Aspergi~lus species at from about
40°C to about 60°C at a pH of about 4 to about 6 for a
sufficient time to obtain an amino acid level of 20o to 55%;
(iii) lowering the pH to between about 3.5 and about 4.5 and
ao increasing the temperature to from about 80°C to about 100°C
for a period of time ranging from about 10 minutes to about 4
hours; and
(iv) lowering the temperature to from about 25°C to about
40°C and, optionally, recovering the hydrolysate.
zs The starting material used for hydrolysis may contain
from about 50% to about 100% (w/w) soy protein, preferably
from about 50% to about 750. In a preferred embodiment of the
invention, defatted non-toasted soy flour (Cargill B.V., the
Netherlands) containing about 52% (w/w) soy protein,
3o maximally about 1.5% (w/w) fat, and from about 2 to about 4%
(w/w) fibres is used. However, the person skilled in the art
will understand that also other defatted soy protein
containing material, such as soy protein isolate or toasted
soy flour, may be used as starting material. Although soy
3s beans may be used as well, the result can be less
satisfactory, due to the oil present in these beans.
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Advantageously the viscosity may be reduced, e.g. to
facilitate further manipulation of the suspension; such
reduction in viscosity preferably may be obtained by enzyme
addition. Suitable enzyme preparations include Pescalase°
s (Gist-brocades, the Netherlands), B500~ (Gist-brocades, the
Netherlands) and Viscozyme~ (NOVO Nordisk, Denmark), or
enzyme preparations having similar activity.
Preferably Pescalase~ protease is used to reduce the
viscosity. Although other enzymes, like cellulase e.g.
present in Viscozyme~, are known to reduce viscosity,
surprisingly we found that a short incubation with a
protease, such as Pescalase° protease, sufficiently reduced
the viscosity. Far example, about 1 hour incubation at about
60'C with about 0.5 w/w % Pescalase~ protease sufficiently
~s reduces viscosity of the suspension comprising soy flour.
Hydrolysis is desirably preceded by a heat step in order
to inactivate disturbing native soy proteins, such as
glycosidases, that may interfere with the reaction or quality
of the end product. Heating the mixture for at least about 5
zo to 15 minutes, preferably at least about 7 minutes, at a
temperature of from about 65°C to about 82°C substantially
reduces the amount of off-flavours (or undesirable flavours)
produced by the degradation of isoflavones. In a preferred
embodiment, the mixture is heated for about 10 minutes at
as about 75°C. Notably while incubations at increased
temperature according to the invention desirably is carried
out for at least about 5 to about 15 minutes, incubation
times longer than 15 minutes can be employed as long as this
step does not produce (other) undesirable flavours.
ao The soy protein optimally is enzymatically hydrolysed by
mixtures of endoproteases and exoproteases. The ratio between
the activities for the endo- and exo- proteases may vary from
about 1 to 10, to about 10 to 1. To one skilled in the art it
will be clear that this ratio (as one of the options) can be
ss varied in order to provide for the desired amino acid level.
Commercially available mixtures of endo- and exo-protease
that can be used in the present invention are, for instance,
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Sumizyme°FP, Sumizyme°LP - proteases (both from Shin Nihon,
Japan), Flavourzyme° protease (Novo Nordisk A/S, Denmark) and
Protease M° Amano (Amano, Japan). Other comparable enzymes
having similar properties can be used as well. These endo-
s and exo-protease mixtures preferably are obtained from an
Aspergillus species, especially a species such as A.oryzae or
A.sojae, although enzymes from other Aspergillus species) or
indeed, other fungal species, similarly can be employed.
Mixtures of these proteases, e.g., with other proteases such
as for example Pescalase° protease which is a bacterial
endoprotease, may also be used. Furthermore it may be
advantageous to incubate several proteases either
concurrently or sequentially. The proteases will preferably
be incubated at optimum pH-conditions, or an average of the
~s optimum pH conditions for the various proteases employed in
conjunction. The pH is preferably controlled at a setpoint.
Alkali or acid i.s added to maintain the pH at the setpoint .
During hydrolysis the pH setpoint may be changed, e.g. when
another protease is added sequentially. Food grade alkalis
zo such as a . g . NaOH or KOH and food grade acids, such as a . g .
HC1, HZS04 may be used to maintain the pH at a given set
point. Enzyme concentrations may vary from about 0.2% to
about 4a (w/w}. Hydrolysis with mixtures of endo- and exo-
proteases obtained from Aspergillus species are preferably
is carried out at a pH of from about 4 to about 6, more
preferably at a pH of about 5.1, desirably at a temperature
of from about 40°C to about 60°C. Preferably, the temperature
is from about 55°C to about 60°C. Temperatures optimally
should not exceed about 60°C, because of the risk of
ao inactivating the enzymes in the mixture. However higher
protease hydrolysis temperatures can be used if thermostabile
or thermoresistant enzymes are employed.
According to one preferred embodiment, soy protein is
incubated with a 2% (w/w) mixture of endo- and exo-proteases
3s obtained from Aspergillus species (e. g. Sumizyme°FP protease)
at a pH of about 5.1 at about 55°C for about 15 hours. The
person skilled in the art will understand that to reach the
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same result in a shorter or longer period, the amount of
enzyme will need to be adjusted. Hydrolysis for much longer
than about 25 hours will increase the risk of contamination
(undesirable bacterial growth) which may cause the production
s of undesirable compounds that affect the taste or food
quality, and is therefore less preferable.
Optionally, in addition to exoproteases and
endoproteases, other enzymes are added as well, such as for
example glutaminases or cell wall degrading enzymes.
Furthermore it is optional to add microorganisms in order to
grow and ferment during hydrolysis. The microorganisms which
may be added are preferably food grade. Also mixtures of such
microorganisms can be used.
The microorganisms preferably are able to grow at a pH of
~s from about 4 to about 6, and grow at temperatures of from
about 40°C to about 60°C, more preferably at temperatures of
from about 50°C to about 60°C. Incubation at temperatures
from about 50°C or higher reduces the risk of contamination
with microorganisms that produce unwanted metabolites.
ao Particularly preferred according to the invention is the use
of bacteria such as Bacillus, e.g. a Bacillus
stearothermophilus species or Bacillus coagulans species,
including but not limited to a microorganism which has been
classified by DSM(Z) (Deutsche Sammlung vom Mikroorganismen
as and Zellkulturen GmbH (Braunschweig; Germany)) as a B.
coagulans (which was previously classified as a B.
stearothermophilus) and is known under number CBS 772.97
(deposited at Centraal Bureau voor Schimmelcultures (the
Netherlands) on 13 May 1997). B.coagulans CBS 772.97 is a
ao bacterium capable of growth under (semi-) anaerobic
conditions at temperatures of 55°C, and which during the
fermentation of sugars produces lactic acid.
Addition of B.coagulans CBS 772.97 (or another
microorganism) during proteolytic hydrolysis of the soy
35 protein results in an anaerobic fermentation. During the
anaerobic fermentation sugars are converted into acid, e.g.,
lactic acid. The free sugar content is therefore lowered
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during hydrolysis and this reduces formation of Maillard
compounds (from free sugars and free amino acids). The final
product therefore will contain less Maillard compounds and is
therefore less colored and less spicy or less roasted
s flavored.
So according to another preferred embodiment of the
invention, soy protein is incubated with a mixture of endo-
and exo-proteases at from about 50 to about 60°C for about 15
to about 25 hours in the presence of a fermenting, preferably
food grade, microorganism, which is more preferably a
B.stearothermophilus species or a B. coagulans species, most
preferably B.coagulans CBS 772.97. Incubation is carried out
under conditions such that a desirable amino acid level is
obtained as further described herein.
~s Due to the presence of a culture of a microorganism and
the use of relatively high temperature, the risk of
contamination is efficiently reduced. Therefore, longer
incubation is possible when, in addition to the proteolytic
enzyme mixtures of endo- and exo-proteases, a microorganism
zo is added to the soy protein suspension.
After hydrolysis (optionally combined with fermentation)
the pH of the hydrolysed suspension is lowered and the
temperature is raised so as to inactivate the enzymes. This
will also kill microorganisms which are present, such as
as Bacillus stearothermophilus (which belongs to the natural
microbial flora of untoasted soy flour) or Bacillus
coagulans. The pH is preferably adjusted to between about 3.5
and about 4.5, desirably to between about 3.8 and about 4.2.
This pH adjustment will result in precipitation of a part of
3o the proteins present in the hydrolysed suspension. The lower
the pH, the more protein will precipitate. This precipitated
protein will be seperated, e.g., by filtration or
centrifugation, from the final soluble soy protein
hydrolysate. Although this results in lower production yields
35 for the final product, the final product can now be used in
almost any desirable food or feed product, without producing
protein precipitate in these food or feed products. E.g. in
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clear beverages or other products this may be advantageous.
Optimally the temperature may be raised to from about 80°C to
about 100°C, for from about 10 minutes to about 4 hours,
preferably for from about 10 to about 30 minutes. In a
s preferred embodiment of the invention, hydrolysis is
terminated by incubating for about 15 minutes at a pH of
about 4.0 and a temperature of about 80°C.
Before, during or after concentrating and/or drying the
hydrolysate may be cooled. If the hydrolysate is subjected to
centrifugation or filtration, the hydrolysate is preferably
first cooled to from about 25°C to about 40°C before
centrifugation or filtration.
The product can be concentrated and/or dried in any
convenient way, such as spray-drying, freeze-drying,
~s fluidised-bed treatment, or a combination of these methods.
The person skilled in the art will understand that the method
chosen will depend on the formulation of the product. The
product may be formulated in any convenient way, e.g. as a
paste, liquid, emulsion, powder, flakes, extrudate,
zo granulate, or pellets. According to one preferred embodiment,
the product is formulated as a spray-dried powder.
The amino acid level (AAL) of the product obtained is
preferably from about 20% to about 55%. The 'amino acid
level' is defined as 'free amino acids/total amount of amino
as acids', wherein 'total amount of amino acids' - 'free amino
acids + amino acid freed after acid hydrolysis of remaining
protein material'. The amino acid level is expressed in terms
of percentages, whereas free amino acids and total amount of
amino acids are both expressed in ~mol/gram. In a preferred
3o embodiment of the invention, the amino acid level is from
about 25o to about 45%.
The product obtained may contain lactic acid, preferably
in amounts of from about 1 to about 20 w/w %, more preferably
in amounts of from about 2 to about 15 w/w %, most preferably
as in amounts of from about 3 to about 10 w/w %.
A typical soy potein hydrolysate according to the
invention comprises:
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_ g _
Carbohydrates about 5% to 10%
Protein about 45% to 55%
Lactate about 1% to 20%
Ash about 10% to 30%
s All percentages based on w/w dry matter. The protein fraction
comprises an amino acid level of from about 20% to about 55%.
The amount of monosodium glutamate on dry matter is below
about 4%, preferably below about 3%, more preferably below
about 2.5%. A typical soy protein hydrolysate according to
the invention comprises about 1.5% to about 2.5% (w/w dry
matter) monosodium glutamate.
The soy protein hydrolysate is substantially free of 5'-
IMP and 5'-GMP, which means that less than about 0.1% (w/w
dry matter) will be present in the soy protein hydrolysate.
as Preferably less than 0.01% (w/w dry matter) 5'-IMP and 5'-GMP
will be present in the soy protein hydrolysate.
Since the flavour enhancer according to the invention is
low in monosodium glutamate, it can also be used by
individuals who prefer or need to minimise their monosodium
ao glutamate intake. Since it is not a yeast extract, the
flavour enhancer according to the invention has no yeast-like
after taste.
Unlike the flavour enhancer disclosed in US Patent
5,077,062, the flavour enhancer according to the invention
z5 enhances a broad range of flavours. Therefore, it can be used
in meat applications, e.g., to enhance beef or poultry
flavour; vegetable applications, e.g., to enhance paprika,
carrot, mushroom, onion or garlic flavour; and dairy
applications, e.g., to enhance cheese or butter flavour;
3o bakery applications, e.g., to enhance the flavour of baked
products; and to enhance the flavour of beverages. It can be
added to food products whether fresh, frozen, vacuum
preserved or dried; processed or unprocessed; liquid or
solid; alcoholic or non-alcoholic; for human consumption or
as animal consumption. Food products to which it can be added
include but are not limited to basic bouillons, such as beef
stock, lobster stock, chicken stock, fish stock, vegetable
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stock, and the like; snacks, such as, e.g., cheese crackers,
crisps, and the like; sauces and dressings, such as cheese
sauce, brown gravy, curry sauce, garlic sauce, dip sauces,
dressings for salads and/or vegetables, and the like; soups,
s such as onion soup, beef noodle soup, and the like;
mayonnaise, halvanaise, margarine, butter and the like; baked
goods like croissants, bread, cake and crackers; ready to eat
meals; seasonings, such as paprika seasoning, and the like;
custard and whipped cream; chocolate flavoured products, like
cocoa flavoured beverages, e.g., chocolate flavoured soy
milk, or chocolate bars (to enhance the cocoa-flavour of
these products). According to one preferred embodiment, the
flavour enhancer is added to mushroom soup to enhance
mushroom flavour.
Although the flavour enhancer according to the invention
enhances the richness of the taste of meat-based foodstuffs,
the flavour enhancer is particularly suitable for enhancing
dairy-type flavour notes (like cheese), vegetable-type notes
zo (e. g, carrot, tomato, mushroom, onion) and spices (e. g.
pepper (pepper heat note enhancement), garlic).
A particularly new effect of the flavour enhancer according
to the invention is the prolonged flavour perception.
Addition of the flavour enhancer according to the invention
zs to a food product makes the food product's taste last longer
in the mouth (this is called the linger longer taste
effect) .
Furthermore creamy-tasting products taste more creamy and
will obtain a thicker mouthfeel when the flavour enhancer
3o according to the invention is added to the food product. The
use of the soy protein hydrolysate will enhance the
creaminess and mouthfeel of the food or feed product,
essentially without increasing the viscosity of these food or
feed compositions.
The flavour enhancer of the invention may be used as
such or in flavouring compositions, e.g. in combination with
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flavouring agents. In this context, the term 'flavouring
agent' is used to indicate a compound or a mixture of
compounds which is used to create a flavour which is not
present in a product. The flavour enhancer may further be
S used as a compound in the production of processed flavours.
Due to the high amount of free amino acids it may be used as
a source of amino acids in the production of processed or
reaction flavours.
The invention is further illustrated by the following
examples. Of course, the following examples are illustrative
only and should not be construed as in any way limiting the
scope of the invention.
is EXAMPLES
Methods
Amino acid analysis was carried out according to the
Picotag method of Waters (Milford MA, USA). The Picotag
method comprises a prederivatization step using
zo phenylisothiocyanate. HPLC analysis is performed on a Picotag
column using reversed phase chromatography. Total hydrolysis
of proteins was achieved by dry hydrolysis over 6N HC1, also
according to Waters. Amino acids hydrolysed by enzymatic
activity were determined in the supernatant. After
zs hydrolysis, the various samples were immediately centrifuged
in an Eppendorf table top centrifuge 5417 at 14000 rpm for 5
minutes after which the total supernatant was removed and
kept frozen at -20°C. Amino acid analysis took place
immediately after thawing the sample material.
- Carbohydrates were determined according to Anthrone,
J.H. Roe (1955) J.Biol.Chem. 212, 335-343.
Protein contents were determined according to Kjeldahl,
3s Approved methods of the American Association of Cereal
Chemists, Volume II, 1983 American Association of Cereal
Chemists, Inc., Method 46-09.
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Example 1 Preparation of the hydrolysate
450 g of defatted soy flour 200/80 (52% w/w protein,
Cargill B.V., the Netherlands) was suspended in 2.5 1 water
s at 20°C in the presence of 0.5% (w/w) Pescalase~ protease
(Gist-Brocades, the Netherlands). This suspension was heated
for 10 minutes at 75°C. After cooling to 55°C and adjusting
to pH 5.1, the suspension was hydrolysed for 15 hours using
20 (w/w) Sumizyme~FP protease (Shin Nihon, Japan). After
hydrolysis, this mixture was incubated at pH 4.0 and 80°C for
15 minutes to stop hydrolysis. After cooling to 40°C the
hydrolysate was obtained by centrifugation for 30 minutes at
2200 g. The pellet was washed twice with process water. The
resulting slurry was filtered at a pressure of 0.4 to 1 bar
~s using Dicalite 418 as a filter aid. After concentration by
rotary evaporation at 40°C, 50 mbar, the filtrate was spray-
dried (inlet temperature 130°C, outlet temperature 80°C). A
light coloured powder was obtained.
zo The taste of the obtained powder was evaluated by a
trained Expert Taste Panel of ten individuals.
to of the powder was solved in water of 60°C. The experts
evaluated both smell and taste of the to solution.
Remarks of the Expert Taste Panel:
25 smell: malty, sickly, floury, soy
taste: slightly bitter, slightly a-stringent, sickly,
soy, no umami.
Example 2 Vegetable soup
3o To 100 g of liquid mushroom soup 0.1-10 (w/w) of the
powder obtained in example 1 was added. An expert taste panel
found the soup to have an increased mushroom character, an
increased richness in flavour, more mushroom after taste and
generally more mouthfeel.
Example 3 Meat stock
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To 100 g of beef stock up to 0.3% (w/w) of the powder
obtained in example 1 was added. An expert taste panel found
the resulting stock to have increased beef flavour, more beef
after taste and an increased richness in flavour.
s
Example 4 Mayonnaise
To 100 g of mayonnaise 0.3% (w/w) of the powder obtained
in example 1 was added. An expert taste panel found the
resulting mayonnaise to have increased richness in flavour,
more freshness and creaminess.
Example 5 Paprika seasoning
A paprika seasoning was prepared by mixing
19 g salt '
~s 10 g tomato powder
19 g paprika powder
15 g dextrose
15 g f lour
7 g monosodium glutamate
zo 0.6 g Maxarome~ yeast extract (Gist-Brocades)
11 g powder obtained in example 1
All ingredients were blended until a homogeneous dust-on
flavour was obtained. To 100 g snacks (unsalted natural
zs crisps) 10 g of this dust-on flavour was added. An expert
taste panel found the resulting snack to have an enhanced
paprika flavour.
A reference seasoning mix which contained yeast extract
instead of the powder obtained in example.l was found by the
ao taste panel to have a more beefy character and a salty
impact. .
Example 6 Curry sauce
0.5% (w/w) powder obtained in example 1 was added to 100 g
as of a curry sauce. An expert taste panel found the resulting
curry sauce to have a more rounded character, enhanced spicy
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notes, particularly, the curry, ginger, pepper and chili
notes.
Example 7 Cheese crackers
s 8.0 g of the powder obtained in example 1 was added to the
following ingredients:
66.0 g cheese powder
2.5 g salt
10.0 g sugar
0.5 g maltodextrin
7.0 g malt flour
8.5 g whey powder
14.0 g ammonium
~s 1.4 g bicarbonate
0.5 g bisulphate
113.0 g water
69.0 g hydrogenated oil
381.8 g flour
zo
All ingredients were mixed for 140 seconds in a Hobart mixer
using a dough hook; kneaded for 80 seconds and rolled out to
1 mm thick sheets. The sheet was transferred to a baking
plate, cut in 5 x 5 cm squares and docked. The dough was
zs baked f or about 6 minutes in an oven at about 2 7 0 - 2 8 0 ° C .
An
expert taste panel found the cheese crackers prepared with
the powder obtained in example 1 to have more cheesy taste.
The cheese tasted more mature and obtained a more melted
cheese type character as well according to the taste panel.
Example 8 Preparation of the hydrolysate, using B. coagulans
CBS 772.97
351 g of defatted soy flour 200/80 (52o w/w protein, Cargill
B.V., the Netherlands) was suspended in 1.5 1 water at 60°C
in the presence of 0.5o Pescalase° protease (Gist-brocades,
the Netherlands). The enzyme was dosed as percentage of the
dry matter of the suspension. The temperature of the
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suspension was raised to 75°C in 3.5 hours. During cooling of
the suspension to 55°C, the pH was raised to 8.0 using KOH.
After adding 0.750 (weight/dry weight) Pescalase~ protease,
these conditions were maintained for 2 hours. Then the pH was
adjusted to 5.1 using HZS04, and an inoculum of B. coagulans
CBS 772.97 and 1% (weight/dry weight) Sumizyme~ FP protease
(Shin Nihon, Japan) were added to the mixture. The inoculum
of B. coagulans CBS 772.97 was made by culturing a frozen
culture of B. coagulans CBS 772.97 on a medium of glucose and
Gistex~ yeast extract (Gist-brocades) pH = 5 for 16 hours at
55°C. To the suspension about 5.103 cells per ml (final
concentration of cells in the suspension, after addition of
the cells to the suspension) were dosed. The mixture was
fermented and hydrolysed at constant pH and temperature for
~s 15 hours. The reaction was terminated by adding HzS04 to a pH
of 4 . 0 was reached and raising the temperature to 82 ° C in 2
hours. After cooling the suspension to 40°C, the non-
solubilized material was removed by centrifugation for 30 min
at 2200 g. The pellet was washed twice with water.
2o After a heatshock for 5 min at 95°C, the supernatant was
concentrated in a glass evaporator at 60°C and 120 - 150
mbar. Afterwards, the pH of the concentrate was adjusted to
5.1 and the material was spray dried.
Analysis of the obtained spray dried powder resulted in the
z5 following:
Protein (Kjeldahl) 48%
Carbohydrates (Anthrone)7.5%
Lactate 40
Ash 24%
3o The amino acid level was 43% and the powder contained 2.3%
monosodium glutamate.
Example 9 Mushroom soup prepared with the product of
Example 8
To 100 g of liquid mushroom soup 0.1-l0 (w/w) of
the powder obtained in example 8 was added. An expert taste
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panel found the soup to have an increased mushroom character,
an increased richness in flavour, more mushroom after taste
and generally more mouthfeel.
s Example 10 Cheese sauce
1.32 g of the powder obtained in Example 1 was
added to the following ingredients:
500 g Water (100 g cold, 400 g. boiling)
20.00 g Starch
17.67 g Maltodextrine
15.00 g Cheddar cheese powder
10.00 g Gouda cheese powder
10.00 g Wheat flour
~s 10.00 g Milk powder
5.00 g Cream powder
5.00 g Fat powder
4.50 g Salt
1.00 g Lactose
ao 0.50 g Milkprotein (EM6)
0.05 g Citric acid
0.02 g Caramel powder
0.06 g Turmeric (liquid)
0.15 g White pepper
as 0.40 g Onion powder
0.05 g Mace
0.05 g Nutmeg
0.05 g Laurel
0.50 g Guar
All ingredients (except water) were mixed until homogeneous.
Then water was added while stirring.
An Expert Taste Panel found the sauce to have an increased
age perception of the cheese, an increased salt and spice
3s perception, more creaminess, fuller mouthfeel and an extended
flavour release (linger longer) .
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WO 98/27828 . PCT/EP97/07293
- 17 -
Example 11 Medium fat margarine
To 100 g of medium fat margarine 0.30% (w/w) of the powder
obtained in Example 1 was added. An Expert Taste Panel found
the resulting medium fat margarine to have an increased
s flavour impact, an increased creaminess, improved fatty
butter taste and an extended flavour release (linger
longer°).
Example 12 Preparation of the hydrolysate, using B 500 and
Flavourzyme~ proteases
351 g of defatted soy flour, Nutrisoy~ 7B flour (53% w/w
protein, ADM, The Netherlands) was suspended in 1.5 1 water
at 60°C in the presence of 0.5% B 500 protease (Gist-
brocades, the Netherlands). The enzyme was dosed as
~s percentage of the dry matter of the suspension. The
temperature of the suspension was raised to 75°C in 3.5
hours. During cooling of the suspension to 55°C, the pH was
raised to 8.0 using KOH. After adding 0.75% (weight/dry
weight) B 500° protease, these conditions were maintained for
ao 2 hours . Then the pH was adj usted to 5 . 1 using HzS04 , and an
inoculum of B. coagulans CBS 772.97 and 1% (weight/dry
weight) Flavourzyme~ protease (Novo Nordisk A/S, Denmark)
were added to the mixture. The inoculum of B. coagulans CBS
772.97 was made by culturing a frozen culture of B. coagulans
zs CBS 772.97 on a medium of glucose and Gistex° yeast extract
(Gist-brocades) pH - 5 for 16 hours at 55°C. To the
suspension about 5.103 cells per ml (final concentration of
cells in the suspension, after addition of the cells to the
suspension) were dosed. The mixture was fermented and
ao hydrolysed at constant pH and temperature for 15 hours. The
reaction was terminated by adding H2S04 to a pH of 4.0 was
reached and raising the temperature to 82°C in 2 hours. After
= cooling the suspension to 40°C, the non-solubilized material
was removed by centrifugation for 30 min at 2200 g. The
3s pellet was washed twice with water.
After a heatshock for 5 min at 95°C, the supernatant was
concentrated in a glass evaporator at 60°C and 120 - 150
CA 02275423 1999-06-16
WO 98/27828 . PCT/EP97/07293
- 18 -
mbar. Afterwards, the pH of the concentrate was adjusted to
5.1 and the material was spray dried.
Example 13 Mushroom soup prepared With the product of
s Example 12
To 100 g of liquid mushroom soup 0.1-l0 (w/w) of the powder
obtained in example 12 was added. An expert taste panel found
the soup to have an increased mushroom character, an
increased richness in flavour, more mushroom after taste and
generally more mouthfeel.
Example 14 Croissants
~s Recipe for the production of 120 small croissants:
2000 g flour
1100 g water
100 g yeast
40 g salt
zo 60 g commercial bread improver
800 g fat
g soy protein hydrolysate obtained in Example 1
The dough was kneaded at 25°C
as moulding
resting time: 15 min. at -20°C
moulding
resting time 5 min. at -20°C
proof time 65 min. at 35°C
ao steaming 2 min. at 85°C
baking: 15 min. at 230-250°C
The croissant had a more cheese-like smell. The taste was
more fresh and roasted compared to the control croissants.
Example 15 Cream crackers
CA 02275423 1999-06-16
WU 98/27828 . PCT/EP97107293
- 19 -
Recipe:
984 g flour
330 g water
150 g soft type bakery fat
s 6 g salt
30 g dextrose
0.070 g cystein
7.5 g Soy protein hydrolysate obtained in Example 1
The dough was kneaded, moulded and baked for 6 min. at
270/280°C. The smell of these crackers changed from staled to
more fresh, due to the addition of the soy protein
hydrolysate. the taste changed from musty, floury to new,
roasted, nutty and cheese-like.
is
All references cited herein, including patents, patent
applications, and publications, are hereby incorporated in
their entireties by reference.
While this invention has been described with an emphasis
Zo upon preferred embodiments, it will be obvious to those of
ordinary skill in the art that variations of the preferred
embodiments can be used and that it is intended that the
invention can be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
as modifications encompassed within the spirit and scope of the
invention as defined by the following claims.
CA 02275423 1999-06-16
WO 98!27828 . PCT/EP97/07Z93
- 20 -
Intemstfonal Application No: PCT/
PC'T_~Rt 5
MICROORGANISMS
6 29
OpMend 3Aest In eonneetlen
with the mlcroeraenlset tefeered
to on pe~~ . Iln ~ of the
deeerlpten t
-
A. lOtw1'ltlICAT1011 01 Dt~OItT
s
Fwther deposit! aro IdeMMed
on an eddlflenel sASef D s
Nome of deooelary IneNttAlen
~
Centraal Bureau voor Schimmelcultures
(CBS)
Addme of deooeltlttr InlHtunen
(Includlne poeW code ena eoeM~)
o
Oosterstraat 1
P.O.Box 273
3740 AG BAARN (The Netherlands)
Dete el dpedt ~
Aeeeeelon Nunwer ~
13 Ma 1997 __ ~ CBS 772.97
. AD01Tt01lAL 11101CAT101i!
1 (trove blurt d not sppllubls).
This Infennetlon Is continued
on a eewrne atnches Meat a
We inform you that the availability
of the microorganism identified
above, referred to
Rule l3bis PCT, shall be effected
only by issue of a sample
to an expert nominated by
the requester until the publication
of the mention of grant of
the national patent or until
the date on which the application
has been refused or withdrawn
or is deemed to be
withdrawn.
C. 0tl10dATED lTATE! F0lt WMICII
IN01CAT10N! Altt OADt t (It
the Indteatlone sro net tot
NI desleneted Stales)
D. ltPAltATt FUIIIIIlIIlfld
OF IIIDICAT10N! ~ (leave blank
il not applicable)
The indications IistW beiew
will b wbnhttW to the International
9enw later s (Spedtp the penetal
nature of tM Indlwtlons e.p.,
Aeuselon Number of Deposit
")
t. Q Tills eheel wee retetved
wnh the Inlerne00na1 eppheatlon
when filed (to be chected
by IM recelvin0 Ofhta)
(Aethori:ed Oflka)
a The eaN of rocelpt (hour
tM aoplieant) bY tM Interhetional
Bureeu to
wet
(Author):ed OIReer)
Ferns PCTIROrt~1 (,lenuary lift)
CA 02275423 1999-06-16
WO 98/27828 . PCT/EP97/07293
- 21 -
$p/>\/ TT/ 1:
pear 14
HUDAPEST TREATY ON T1lLr LNTLNNA'rtoNAt,
RECOi.NITION OF TILE DEP~atn' of MIt:ROORGANI5MS
FOR THE PURPOSI:a OF DATF.NT PRL7C~DL)RE
(N'rIiRNATIONAL FORM
' Gist-brocades N.V. ascell~T Irt TttE CASE 9F AN ORIGINAL DEpOSI'f
Research do Development / Stamconserwcring t..u.a pursuant to uum ~ . i ~r c5w
POSttfuS 1 I1~ERNR'fTaNnr, nRPOttrTnrcv nsm~t t'fr
idwntiliad at thw bottom aL thii vun~
Nederland
IIyIIY aYllCI ar.:drca~ of du,:r~ai~;~Y
z. sasrrzlscassox oa rxs xzcxoo>waaxsaa
Idrrntit'icas:i:m rrfrrnr.~ Acr,aouiun :uunu~:L ~)lvYr1
~i~: .~ ~lo: lay tnw
DPOSITORs INTERNATIONAL DEF!'S:T'AI?'i
!.tl'fhIC:HIT"t:
Bacillus specirS DS31910 (ATL-2)CHS 772.97
z z . scxs>wrslzc o>ESCxztrrzox
a~/on lstopoasa raxo>xox:c
asaxox~Txo>,r
~Chrt mirrocr2anlam i~9cntitiYd
:::~t= I ~bcvw was ac=ompaniad
by:
a sci wnt i f i c dwac:r j pt
i.;r;
a propownr! raxonoml~ ~iur_.s::.ss.i:n
Imurk vft:h a cross where apr;,:ai".~:
t II . itlCEIPT atrD ><CCilT71>ITCE
This Intwrnat Tonal DNttuaslt.
lr; aca~pts the mi .-.raorflar:imsn
:dwnt.i t : ed und~wr I :itxrvy,
rn ieh wee
rwcwivad by it: on Tucsday,13
May 1997 ferule c.' ..-,ha
nt'a7in31 dwpor; t 1
IY . ItECE=PT O! REQtZEST loft
cotrvERsltION
Thw mieroorywniem idsntifi~ru
~.n.:;sr I a00v~r wen Cr~:~:iised
by :hir inr.rtldtiunul Dwpoartdr!
Authority On not applicable
r,'t,~w of iha rriyTit'.ai
cT~rpuarlt;) wA .,
sequwe to eonvart tYtse uriinai
.iwp~;>;1S to a dwposit unrl':
the Budapase Traaty Wur cwaivR:~
i;
it stn not applicable (dat0
oi' recw3pt of rar/uuyi for
convwrnAr~y
V . =Erli0.1f7tTZC>x7lL ntlOlITAItY
ADTEORZT!
N.~w : Centraalbureau vctor .; iunot ura ( s 1 o r >?~,raun
Schimr=Lelcultur~ I a 1 nevi no r tu: L~I)WlIC
:.~.
tCOtwwwnt the trrtuttust.ional
Depoait;st/
Autlu~rity or autitv ixwA ofti.i
11W
'
Addrwia: ~QSbtrStTilat ] ~
~~..
P.O. Hox Z73
3740 AG BAARN Mrs F.H. Snippe'~iaus . . Samsoii
The Netherlands Dacw: Monday, 7 July 1997
' Whwre Ruler 6. d (d1 appliwss. such .iy V iy thw derv 4n which tha rstat.uss
of i nt~t~ssat ions L
dwpowitary autlscrity wsa hr..:auirwd.
~'wt'm 9t':4 (aul~ Ooøtl CDS/'ilU'!
