Note: Descriptions are shown in the official language in which they were submitted.
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A method for the separation of flour
The present invention relates to the field of flour separa-
tion. The invention discloses a method for the separation of
s flour, in particular wheat flour, compositions for the separa-
tion of flour and the use thereof. Further the invention re-
lates to a fraction consisting essentially of gluten, and a
product comprising said fraction.
to Background of the invention
The industrial implications of flour and the products ob-
tained from its separation are receiving increasing attention.
Flour may be separated into fractions of starch, gluten and fi-
bres. In the separation of flour the very nature of the flour
is protein, i.e., gluten, as being water insoluble presents a
challenge desirable to overcome.
Gluten consists primarily of the proteins, glutenin and
gliadin. Upon hydration and during processing gliadin and glu-
tenin interact to form a network. Proteins of wheat flour form
zo a network with disulphide bridges (S-S-bridges). The network is
stronger, the more sulphur bridges are present. The network en-
traps carbon dioxide formed during fermentation creating the
characteristic elasticity of the wheat flour dough. The elastic
properties of gluten are due to the glutenin fraction and the
2s viscous properties come from the gliadin fraction. It is there-
fore a fact that the quality of the flour for the purpose of
baking is highly dependent on the amount of gluten contained in
the flour. Gluten may be added to flour of poor quality to im-
prove the baking properties of the flour.
3o Prior art attempts to separate wheat flour have been made.
U8 4,217,414, US 3,951,938 and UK 2 032 245 all describe exam-
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ples of methods for the separation of wheat flour and the re-
covery of wheat gluten. Here the separation of wheat flour into
gluten and starch fractions are performed by the means of me-
chanical processing. The mechanical separation methods de-
scribed in the references are centrifugation, screening, de-
canting or milling.
The application of mechanical techniques of separation pro
vided for fractions of gluten and starch. However, to obtain
less contaminated fractions effort was directed toward improv
to ing the purity of the fractions.
The addition of enzymes to the flour or dough proved to be
a successful way of achieving an improved separation. The added
enzymes are capable of reacting with the flour and thereby im-
prove the separation quality of the gluten.
In the prior art numerous references disclose such use of
gluten improving enzymes. Among those are Weegels et al. (Wee-
gels, P.L., Marseille, J.P., and Hamer, R.J., 1992,
Starch/starke 44, 2, pp. 44-48) who describe the use of enzymes
as a processing aid in the separation of wheat flour into
2o starch and gluten. The enzymes mentioned are lipase, hemicellu-
lase and cellulase.
Further, Christophersen et al. (Christophersen, C., Ander-
sen, E., Jakobsen, T. S., and Wagner, P., Starch/Starke, 1997,
49, pp. 5-12) describe the successful use of a xylanase to im-
z5 prove the yield of gluten and starch, without apparent negative
effects on the gluten quality.
Formerly it has not been possible to obtain gluten frac-
tions from prior art processes of flour separation having a
high content of pure protein, such as gluten fractions consist-
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ing essentially of gluten, having a very low content of starch
or fibres of the xylan or arabinoxylan type.
Summary
s It is an object of the present invention to provide for a
method for the separation of flour, in particular wheat flour,
into one gluten fraction and at least one other fraction, com-
prising the steps of:
a) mixing the flour and a liquid to obtain a dough,
to b) separating the dough into a fraction comprising gluten and
at least one other fraction,
c) recovering at least the gluten fraction, characterized in
that an oxidoreductase is added at any of steps a), b) or
c) .
is In another aspect the invention relates to a method for the
separation of flour into one gluten fraction and at least one
other fraction, comprising the steps of:
a) mixing the flour and a liquid and at least one oxidoreduc-
tale enzyme obtaining a dough,
2o b) separating the dough into a fraction comprising gluten and
at least one other fraction,
c) recovering at least the gluten fraction.
It should be mentioned that the method can also be carried
out as described above where the oxidoreductase(s) in question
z5 is(are) added to the (dry) flour. If the oxidoreductase(s) in
question has(have) been added to the flour the oxidoreduc-
tase (s) has (have) time to react with O2 (e.g. , from the air or
by addition of pure 02) to provide a flour composition with the
desired gluten quality.
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The present invention permits the separation of gluten from
any quality of flour, in particular wheat flour, even from
flour of poor quality, such as flour used for fodder.
Thus advantages of the invention may include improved
yields, more pure gluten and/or higher quality of gluten.
By the present method an improved separation of flour, in
particular wheat flour, is obtained. The improvement in the
separation of the flour increases the yield of gluten and
starch fractions by providing a more efficient method of sepa-
to ration. Further according to the invention the separated gluten
is of a higher quality, (i.e. less contaminated with other
flour components, such as starch or fibres) than gluten frac-
tions obtained according to the prior art.
Further the invention relates to a composition for the
separation of flour, in particular wheat flour, into one gluten
fraction, and at least one other fraction, comprising at least
one oxidoreductase enzyme.
The compositions of the invention may be used for the sepa-
ration of flour, in particular wheat flour.
2o In a further aspect the invention relates to a flour compo-
sition comprising any of the oxidoreductases mentioned below.
The gluten fraction obtained may be added to flour to in-
crease the gluten content, such as to enhance poor quality
flour, and thereby improving the baking quality of the flour.
Drawings
Fig. 1 shows an example of the steps of a process on wheat
flour for preparation of a dough, starch extraction and the
separation on sieves.
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Fig. 2 shows an example of the steps of a process on wheat
flour for preparation of a homogenized batter (thinned dough)
and separation by the means of a decanter centrifuge.
s Detailed description of the invention
Due to the significance of the industrial applications of
flour, in particular wheat flour, much attention is given to
the development of separation methods for flour. The present
invention reveals a method for the improved separation of
to flour, in particular wheat flour, into starch and gluten by us-
ing oxidoreductase enzymes, whereby gluten of a high quality is
obtained.
The two main components of flour, such as wheat flour, are
gluten and starch. After separation the vitality of gluten is
is preserved due to controlled drying. When hydrated, the dried
gluten must possess the same vitality as the fresh gluten. The
vitality of the separated gluten may be determined by the means
of gluten vitality tests, such as the farinograph test, or the
SDS (sodium dodecyl sulphate) sedimentation test used to deter-
2o mine the degree of denaturation of the gluten. The denaturation
of the gluten may occur during the drying step, and great care
must therefore be taken to ensure a gentle drying procedure.
Yet, a further parameter correlating to the vitality of
gluten is the protein dry matter ratio, describing the purity
25 Of the gluten. The higher the amount of protein of dry matter,
the better the quality of gluten. In the present context the
term "protein" is meant to equal the term "gluten". The purity
of the gluten may be determined by applying the Kjeldahl analy-
sis, and a dry matter determination analysis.
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Gluten may be used in the food industry, such as in bakery
products, pet foods, meat products and as mentioned above in
flour fortification. In the case of the latter application it
is important that the gluten creates a visco-elastical dough
s ball, having good elasticity and extensibility properties, and
that it is capable of cohering to a wide variety of products,
thereby improving texture, strength and nutritional content of
the food item in question.
In the baking industry, a high vitality of gluten is corre
to fated with a high baking quality, i.e., a high bread volume.
The higher the denaturation degree, measured by the methods de
scribed above, the lower the vitality of the gluten, and the
lower the baking quality. Further, the higher the amount of
protein of dry matter, measured as mentioned above, the better
is the baking quality.
By applying the method of the present invention it is pos-
sible to obtain a gluten fraction consisting essentially of
gluten, i.e., higher purity of gluten fraction. In the present
context the term "consists essentially of" is meant to define a
zo fraction wherein the content of gluten is at least 80 % protein
of dry matter. In a preferred embodiment the gluten content is
at least 90%, more preferred 95%, even more preferred 97% pro-
tein of dry matter, even more preferred 99 protein of dry mat-
ter, and in an even most preferred embodiment the gluten frac-
z5 tion is consisting of protein only (i.e., gluten only - 100%).
The method of the invention additionally provides for an in-
creased yield of gluten from, e.g., wheat independent of the
crop variety.
The method may be applied to any kind of crop. However, espe-
so cially contemplated are wheat, but also crops such as corn,
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rice, sorghum bean, barley, rye, or fruit hulls are contem-
plated.
Wheat
s Modern wheat varieties are classified as winter wheat and
spring wheat. Wheat varieties may be hard grained or soft
grained. Hard grained varieties normally have a large content
of gluten. The flour of the latter varieties is preferably used
in the baking industry for making products, such as bread. In
to case of the soft grained wheat varieties the flour thereof may
be used for the production of cakes, biscuits, and fodder. Du-
rum wheat is hard grained and the flour is used for pasta prod-
ucts. Wheat in general may also be used for the manufacture of
beer and whiskey. Independent of the wheat variety the present
is method has improved the gluten yield obtainable from said soft
varieties. Besides gluten another main wheat component is
starch.
Gluten Quality
2o Wheat starches are classified according to their degree of
purity. The grades are denominated A and B, respectively.
Grade-A starch has a particle size of 20-35 microns and
grade-B starch has a particle size of 2-10 microns. Grade-A
starch is a versatile product providing strength and a pleasing
z5 texture to a variety of foods. Low levels of protein and fibre
insure that grade-A starch gelatinise at low temperatures pro
ducing a smooth paste. The paste may be applied to foods such
as, baby food, soups, sauces, gravies, sour cream, and dips.
Grade-B starch has a higher protein content and a lower den
3o sity.
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Application of starches
There are a vast number of areas in which starches may be
applied, such as in glucose syrup production and in sweetening
s production in general.
According to this invention at least one other fraction is
obtained, said fraction comprising starch and optionally other
wheat constituents, such as fibres. Preferably, the at least
one other fraction consists essentially of no gluten.
to Accordingly, the other fractions) of the present invention
consist of essentially no gluten. By the term "essentially" is
meant that the gluten content in the other fractions are less
than 20%, preferably less than 10%. In a more preferred embodi-
ment at least two fractions are obtained, one of said other
is fractions being a starch fraction. The starch fraction is sub-
stantially free of gluten and also substantially free of wheat
constituents, being an essentially pure starch fraction.
It is an object of the invention to obtain a substantially
pure starch fraction.
zo The method is conducted by mixing flour, in particular
wheat flour, and a liquid, said liquid being acceptable in
products intended for animal and human consumption, and an oxi-
doreductase enzyme.
The mixing may be carried out using any suitable method
z5 known in the art, which may be by the use of an electrically
operated mixer. In a preferred embodiment of the invention wa
ter is preferred as a liquid.
The mixing may be conducted by mixing the flour and the en-
zyme in a first step, followed by the addition of the liquid.
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Furthermore, the enzymes) of the invention may be in a dry
form or in a liquid form, and may be applied to the flour as
such, dependent on the requirement of the timing of the separa-
tion process as described below.
s In one embodiment of the invention the flour is mixed with
the enzymes) in a dry form. This method allows for the prepa-
ration of f lour and enzyme ( s ) mixture that may not be further
processed immediately after mixing, but may in fact be kept for
use at a later point in time. As the process is desired to pro-
to ceed, in a second step the liquid may be added to the flour and
enzymes) mixture.
In another embodiment the flour may be mixed with the en-
zymes) in a liquid form. By doing so the enzyme reaction (s)
may begin immediately after the mixing, and may thus be fully
is complete by the time the flour separation process is meant to
continue by, in a second step, adding the liquid. When adding
the enzymes in a dry form the holding time for the flour and
enzyme mixture may be up to 3 months, such as up to six months,
or longer dependent on the type of enzymes applied and physical
zo parameters, such as storage conditions, humidity and tempera
ture. By using this procedure the resting time may be mini
mised, preferably eliminated once the actual separation process
begins, due to the fact that the enzyme reaction by then may be
partly or fully completed, again dependent on the parameters
as mentioned above.
In a further embodiment of the invention the flour and the
liquid are mixed in a first step obtaining a slurry, and
whereto in a second step the enzymes) are added. This provides
for the initiation of the immediate action of the enzymes) ac
3o cording to the invention, in the wheat separation process.
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Accordingly, the flour mixture (i.e., flour and enzymes)
of the invention may be dry or it may be liquid. In case of the
former it is advantageous that the enzyme preparation is a dry
product, e.g., a non-dusting granulate, whereas in the latter
s case the enzyme preparation may be in liquid form.
For the enzyme reactions) of the invention to occur oxy-
gen must be present. The level of oxygen present must be suffi-
cient to ensure the reaction to take place, i.e., oxygen may not
be the limiting factor of the chemical reaction.
to According to the invention in one embodiment step a)
may comprise mixing the flour and a liquid obtaining a dough,
resting the dough, and adding at least one oxidoreductase en-
zyme to the dough.
The time needed for resting the dough may be any suitable
is time. The resting time may be dependent upon the method chosen
to mechanically process the dough for the purpose of obtaining
individual fractions of gluten and starch, or it may depend upon
the crop (e. g., wheat) variety used.
In yet another embodiment of the present invention step a)
zo may comprise mixing the flour and a liquid and the at least one
oxidoreductase enzyme obtaining a dough, followed by resting
the dough prior to the separating step b) of the invention.
In a further embodiment of the invention the dough is di
luted prior to the separating step b). The dilution of the
z5 dough may be in the order of 1.5:1, preferably 2:1.
The term "dough" in the present context is meant to be
distinguished from the term "batter", the latter containing
more liquid than dough, such as 3 to 4 times more liquid than
dough. Accordingly, the diluted dough of the invention may be
3o referred to as batter.
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In the present context, the term "oxidoreductase" in-
cludes enzymes capable of creating S-S (sulphur-sulphur)
bridges in gluten, or making reduction and exchange between
chains thereby creating a network. The oxidation reaction is as
s follows: SH-OZ -~ S-S and the reduction reaction is as follows:
S-S ~ SH. The enzymatic activity of the enzymes according to
the invention may be determined by standard assays.
According to the invention the enzymes may be selected from
the group consisting of Peroxidase (EC 1.11.1.7), Tyrosinase
to (EC 1.14.18.1), Catechol oxidase (1.10.3.1), Laccase (EC
1.10.3.2), Bilirubin oxidase (EC 1.3.3.5), (Glutathione oxidase
- Sulfhydryl oxidase (EC 1.8.3.3), Glucose oxidase (EC
1.1.3.4), Pyranose oxidase (EC 1.1.3.10), Hexose oxidase (EC
1.1.3.5), L-amino acid oxidase (EC 1.4.3.2), Lysyl oxidase (EC
is 1.4.3.13), xylitol oxidase, galactose oxidase (E. C. 1.1.3.9),
alcohol oxidase (E. C. 1.1.3.13) alone or in combination.
The oxidoreductase(s) in question should be added in an effec-
tive amount.
Dependent on the flour and the purpose of the use of the
zo product, the enzymes may be applied alone or in combination.
The enzymes of the invention may be applied to the flour mix
ture alone or the enzymes may be applied in combination with
another enzyme, such as an enzyme selected from the group con
sisting of hemicellulase, cellulase, xylanase, proteases or de
25 hydrogenases.
The pH value is preferably within a range suitable for the
enzymatic activity. In one embodiment of the invention the
dough has a pH value of between 4.5 and 8.0, preferably between
5.0 and 6.5. It is preferred that the pH is the non-regulated
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pH of the slurry and dough, and that no pH regulators are
added.
Further, according to the invention the temperature of
the dough or slurry is preferably between 10-60°C, more pref
s erably between 20-50°C, and most preferably between 35-45°C.
When the mixing step is accomplished the dough obtained
is subjected to a separating step.
The separating step may be conducted by a variety of
methods suitable for the separation of the dough of the inven
to tion, i.e., the separating method may rely on differences in
particle size between gluten and starch (fibres) and thus rely
on particle weight.
In one widely used embodiment of the invention the dough
is separated by the means of centrifugation. According to this
is method the dough is centrifuged thereby obtaining a heavy phase
containing pure starch and a free flowing light phase contain-
ing the gluten.
In another embodiment of the present invention the sepa
rating process is performed by the means of screening. The
2o screening may be conducted by arranging at least a gluten
screen, for obtaining the gluten fraction. The screen size may
vary dependent on the nature of the material to be screened.
For example the screen for gluten may have the size of
500 ~, or 400 ~, or 200 ~, or 125 ~ .The screening method may
25 furthermore comprise two or more screens, the first for gluten,
and the others) for one or more starch fractions and/or fibre
fractions.
It is of importance that the screens are adapted to the
gluten particle size, which is regulated by the enzymatic
3o treatment during the mixing step.
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The screening method is preferably conducted with diluted
dough, whereby the starch and fibres are washed through the
gluten particle network, leaving the gluten particles on the
first screen.
s In another embodiment of the present invention the sepa-
rating process is performed by the means of decanting. The de-
canting process may begin by homogenising the batter in a ho-
mogeniser. Here shear forces break up the matrix. After this
the mixture is passed through a decanter centrifuge capable of
to separating the dough into distinct phases, such as starch and
gluten phases. The gluten phase may be further processed by ad-
ditional washing and centrifugation or screening.
Yet another method of separating according to the inven
tion may be air classification. In this method the wheat flour
is is separated into fractions, i.e. starch and gluten, by passing
the flour through a spiral air stream. The particles in the
flour will separate according to size, resulting in starch and
gluten fractions. This method may advantageously be applied to
the separation of the stored flour and enzymes) mixture de
zo scribed earlier.
In another aspect of the present invention separating
process is by the means of a hydrocyclone. In the hydrocyclone
apparatus the diluted mixture is applied to the top of a static
cone shaped container. The mixture is rotating inside the con-
z5 tamer and the heavier particles will settle in the lower frac-
tion of the mixture, whereas the lighter particles will be pre-
sent in the top fraction of the mixture.
The methods applied for the separating process may be one
method, or it may be a combination of more processes.
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The separating step is followed by a recovering step,
wherein the gluten fraction is recovered. The gluten fraction
thus obtained may be kept as a suspension of gluten in a liquid
or it.may subsequently be dried. The latter provides for the
s option of processing and storing the gluten for later purposes.
It is an object of the invention that the separated gluten
maintains its characteristic properties, having properties
identical to the original gluten. The drying step is especially
crucial for the conservation of the gluten properties. Too
to forceful a drying process may result in a considerable loss in
gluten quality.
According to the invention the gluten may be dried in a
ring dryer, or it may be dried in a fluid bed dryer. In the
ring dryer process the wet gluten is fed into a ring duct after
is a size reduction in a disintegrator. Upon entering the ring
duct the gluten is mixed with circulating gluten particles that
are already partially dried. Dried gluten particles are removed
from the ring by a manifold. The principle behind the fluid bed
dryer is similar to the ring dryer, except the fluid bed dryer
zo is arranged horizontally and air is entering the bed from be-
low.
In a preferred embodiment at least one starch fraction is
obtained, which fractions) may be further processed as appli-
cable.
as The present invention further relates to a composition
for the separation of wheat flour into one gluten fraction and
at least one other fraction, comprising at least one oxidore-
ductase enzyme.
The composition is preferably suitable for mixing with
3o the flour as described above.
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According to the invention the composition may comprise
any of the oxidoreductase enzymes described above alone or in
combination. Furthermore, the composition may comprise at least
one other enzyme. Said other enzyme may be an enzyme for en-
5 hancing the gluten separation, e.g., an enzyme with affinity to
the non-starch carbohydrate fractions, fibres or soluble arabi-
noxylan fractions.
The more specific combination of components for the com
position according to the invention is dependent upon the type
to of flour used for the separation, and upon the purpose of the
application of gluten obtained by the invention.
According to the invention the enzymes may be from fungal
(including filamentous fungi and yeasts) or bacterial origin.
The enzymes may be derived from the bacterial strains)
15 of strains of the order Actinomycetales, e.g., Streptomyces
spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO
12382) or Streptoverticillum verticillium ssp. verticillium;
strains of Bacillus sp., e.g., Bacillus pumilus (ATCC 12905),
Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas
ao palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC
15958) or Pseudomonas fluorescens (NRRL B-11); or strains of
Myxococcus sp., e.g., M. virescens.
Further the enzymes may be derived from the fungi strains
belonging to the subdivision: Deuteromycotina, class Hypho
z5 mycetes, e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Ver
ticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,
Cladosporium or Dreschlera, in particular Fusarium oxysporum
(DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium
verrucana (IFO 6113), Verticillum alboatrum, Verticillum dahlie,
3o Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocla-
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diem chartarum, Embellisia allior Dreschlera halodes; strains
belonging to the subdivision Basidiomycotina, class Basidiomy-
cetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in
particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus
s macrorhizus, Phanerochaete chrysosporium (e. g. NA-12) or Tram-
etes (previously called Polyporus), e.g., T. versicolor (e. g.,
PR4 28-A); or strains belonging to the subdivision Zygomycotina,
class Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor
hiemalis.
to
Laccases
The lactase may be derived from a fungi such as Collybia, Fomes,
Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora, Phle-
bia, e.g., P. radiata (WO 92/01046), Coriolus sp., e.g. C. hir-
15 situs (JP 2-238885), or Botrytis.
Specifically contemplated laccases are the laccases derived
from a strain of Polyporus sp., in particular a strain of Poly-
porus pinsitus or Polyporus versicolor, or a strain of My-
celiophthora sp., e.g., M. thermophila or a strain of Rhizocto-
2o nia sp., in particular a strain of Rhizoctonia praticola or
Rhizoctonia solani, or a strain of a Rhus sp., in particular
Rhus vernicifera.
In a preferred embodiment of the invention the enzyme is a
microbial lactase derived from a strain of genus Myceliophthora,
2s such as a strain of the species Myceliophthora thermophila,
e.g., the purified lactase described in WO 95/33836 from Novo
Nordisk, which is hereby incorporated by reference.
In another preferred embodiment the enzyme is a lactase
derived from a strain of the genus Polyporus, such as a strain
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of the species P. pinsitus lactase, especially the purified lac-
tase described in WO 96/00290 from Novo Nordisk.
Other laccases include a Scytalidium sp. lactase, such as the
S. thermophilium lactase described in WO 95/33837 (from Novo
s Nordisk Biotech inc.) or a Pyricularia sp. lactase, such as the
Pyricularia oryzae lactase which can be purchased from SIGMA un-
der the trade name SIGMA no. L5510, or a Coprinus sp. lactase,
such as a C. cinereus lactase, especially a C. cinereus IFO
30116 lactase, or a Rhizoctonia sp. lactase, such as a Rh. so-
lo lani lactase, especially the neutral Rh. solani lactase de-
scribed WO 95/07988 (from Novo Nordisk A/S) having a pH optimum
in the range from 6.0 to 8.5.
Lactase may be added in an effective amount. In an embodiment
(as shown in the examples) the lactase may be the above
15 mentioned Polyporus pinsitus lactase. A lactase may preferably
be added in an amount of from 0.1 to 50 LACU/g DS flour, more
preferably 0.2-10 LACU/g DS flour, even more preferably 0.5-5
LACU/g DS flour.
2o Bilirubin Oxidase
Bilirubin oxidases may be derived from a strain of Myrothe-
cium sp., such as M. verrucaria.
Bilirubin oxidase may be added in an effective amount.
z5 L-amino acid oxidase
L-amino acid oxidase may be derived from a starin of Tri-
choderma sp. such as Trichoderma harzianum, such as the L-amino
acid oxidase described in WO 94/25574 (from Novo Nordisk A/S),
or Trichoderma viride.
3o L-amino acid oxidase may be added in an effective amount.
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Glucose Oxidase
A suitable glucose oxidase may originate from Aspergillus
sp., such as a strain of Aspergillus niger, or from a strain of
s Cladosporium sp. in particular Cladosporium oxysporum, espe
cially C1. oxysporum CBS 163 described in WO 95/29996 (from Novo
Nordisk A/S).
Glucose oxidase may be added in an effective amount. As
shown in the examples below the glucose oxidase may be derived
to from Aspergillus niger. Glucose oxidase may preferably be added
in amounts of 0.001-10,000 GODU/g DS flour, more preferably
from 0.005-5,000 GODU/g DS flour, even more preferably from
0.01-2,000 GODU/g DS flour.
is Hexose Oxidase
A hexose oxidases may be derived from the red sea-weed
Chondrus crispus (commonly known as Irish moss)(Sullivan and
Ikawa, (1973), Biochim. Biophys. Acts, 309, p. 11-22; Ikawa,
(1982), Meth. in Enzymol. 89, carbohydrate metabolism part D,
zo 145-149) oxidises a broad spectrum of carbohydrates, such as D-
glucose, D-galactose, maltose, cellobiose, lactose, D-glucose 6-
phasphate, D-mannose, 2-deoxy-D-glucole, 2-deoxy-D-galactose, D-
fucase, D-glucurnic acid, and D-xylose. Also the red sea-weed
Iridophycus flaccidum produces easily extractable hexose oxi-
25 Bases, which oxidise several different mono- and disaccharides
(Bean and Hassid, (1956), J. Biol. Chem, 218, p. 425; Rand et
al. (1972, J. of Food Science 37, p. 698-710).
Hexose oxidase may be added in an effective amount.
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Xylitol Oxidase
Another relevant oxidoreductase is xylitol oxidase (see,
e.g., JP 80892242), which oxidises xylitol, D-sorbitol, D-
galactitol, D-mannitol and D-arabinitol in the presence of oxy-
gen. A xylitol oxidase can be obtained from strains of Strepto-
myces sp. (e. g., Streptomyces IKD472, FERM P-14339).
Xylitol Oxidase may be added in an effective amount.
Sulfhydryl oxidase (SOX)
to Glutathione oxidases or Sulfhydryl oxidases may be derived
from Calodon and Cortinarius sp. (US patent no. 4,610,963); or
a sulfhydryl oxidase from Aspergillus, in particular A. niger
(US patent no. 5,529,926 and EP 321 811-A1), Aspergillus awamori
or Aspergills sojae; or Penicillium, in particular Penicillium
ochrochloron.
Sulfhydryl oxidase may be added in an effective amount. As
shown in the examples below the sulfdryl oxidase may be derived
from Penicillium ochrochloron. Sulfhydryl oxidase may prefera-
bly be added in amounts of 0.001-5 milli SOX/ g DS flour, more
zo preferably from 0.01-3 milli SOX/g DS flour, even more prefera-
bly from 0.1-2 milli SOX/g DS flour.
Pyranose oxidase
Examples of pyranose oxidases as described in JP 61177986
z5 and include pyranose oxidases derived from strains of the genera
Irpex, such as a strain from the species Irpex lacteus; Auricu
lariea, such as a strain of the species Auricularia polytricha,
in particular Auricularia polytricha (FERM-P 7119), Coprinus,
such as a strain of the species Coprinus micaceus, in particular
3o Coprinus micaceus ATCC 20122; and Trametes, such as a strain of
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the species Trametes cinnabarinus, in particular Trametes cinna-
barinus IFO 6139.
Pyronose oxidase may be added in an effective amount.
s Peroxidase
The peroxidase may be derived from plants (e. g., horseradish
peroxidase) or microorganisms including fungi and bacteria such
as a strain of Coprinus sp., such as Coprinus cinereus or Copri
nus macrorhizus, or bacteria such as Bacillus, such as Bacillus
to pumilus. Peroxidase may be added in an effective amount.
The enzymes of the invention may be obtained from the mi-
croorganism in question by the use of any suitable technique.
For instance, the enzyme preparation may be obtained by ferment-
ing a microorganism and subsequently isolating the enzyme con-
15 taming preparation from the fermented broth or microorganism by
methods known in the art. According to the invention a more pre-
ferred embodiment is the use of recombinant DNA techniques as
known in the art. Such methods normally comprise the cultivation
of a host cell transformed with a recombinant DNA vector capable
20 of expressing and carrying a DNA sequence encoding the enzyme in
question. The host cell is grown in a culture medium under con-
ditions permitting the expression of the enzyme, and is followed
by the recovery of the enzyme from the culture.
Additionally the present invention relates to the use of
z5 the composition as previously described.
By the method of the invention at least two different
fractions are obtained. There are various products that may
comprise such fractions.
In one embodiment the obtained gluten may be added to
3o wheat flour of poor quality, i.e., wheat flour having low glu-
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21
ten content. Accordingly, flour normally used for products,
such as fodder may after fortification with gluten be used for
the manufacture of products, such, as bread. Consequently, the
present invention presents a broadening of the types of avail
s able applications for flour having low gluten content.
The gluten fraction obtained by the method of the inven
tion is applicable in any of the applications mentioned above
and in a further aspect the present invention relates to a glu
ten fraction consisting essentially of gluten and to a product
to comprising said fraction.
Further, starches may be used in products of the adhe-
sive, gypsum, paper, corrugating, mining and food industries.
Starch and starch products may also be used as adhesive com-
pounds, such as in the production of bags and adhesive tapes,
is laminates and wound tubes, wallpaper and poster glues, abrasive
paper. Other applications include components of concrete re-
tarders, sizing agents for synthetic, natural and mixed yarns
in the textile industry, and thickeners for the printing of
textiles.
2o In the pharmaceutical industry starch products may be
used as disintegration agents in tablets and surgical glove
powder. Within the ceramic industry field the addition of
starch increases the strength of the ceramic products. Starches
may also be applied to detergents for the purpose of being
as dirt-deposit inhibitors. A completely different application is
in the field of oil well drilling, wherein starch solutions may
be used as agents to help seal drilling cores and to increase
the viscosity of drilling mud and cooling water.
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Yet another application of starch is for the use in water
treatment plants serving the purpose of flocculating various
aqueous suspensions.
A further application is in the plastic industry, wherein
s starches may be used to improve the biological degradation of
plastic products.
Currently one of the applications of starch is for paint
stripping using wheat starch blasting. Wheat starch blasting is
a user-friendly blasting process wherein wheat starch can be
to used in systems designed for plastic media blasting (PMB), as
well as systems specifically designed for wheat starch blast-
ing. The wheat starch abrasive media is a crystallised form of
wheat starch that is non-toxic, biodegradable, and made from
renewable resources. The media is similar in appearance to
15 plastic media, except that it is softer. Wheat starch is a
plentiful natural resource that is biodegradable. Waste gener-
ated from this process may be treated in a bio-reactor using
amylase enzymes. The wheat starch blasting process may be used
for removing coatings from both metallic and composite materi-
zo als. This process is easy to control. It may be used to selec-
tively remove from one and up to all coating layers. Wheat
starch blasting does not cause fatigue to the substrate sur-
face, and it allows for moderate stripping rates, whilst main-
taining a gentle stripping action.
MATERIALS & MATHODS
Enzymes:
Polyporus pinsitus laccase: Disclosed in W096/00290 from Novo
Nordisk (available on request from Novo Nordisk, Denmark)
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Aspergillus niger glucose oxidase (available on request from
Novo Nordisk, Denmark).
Penicillium ochrochloron sulfdryl oxidase (available from Novo
Nordiks, Denmark)
s Glutomatic° System (Perten) (for washing out gluten from
flour) .
SOX units (Sulfhydryl Oxidase Unit):
One sulfhydryl oxidase Unit is the amount of enzyme re
to quired to deplete 1 micromole of O2 per minute from an assay
mixture containing 30 mM L-cystein in 100 mM sodium phosphate
at pH 6.0 and a temperature of 30°C. The oxygen was measured
with an oxygen electrode conneted to an Oxi 3000 Oximeter
( MTW ) .
Determination of Polyporus Laccase Activity (LACU)
Laccase activity is determined from the oxidation of syrin-
galdazin under aerobic conditions. The violet colour produced is
photometered at 530 nm. The analytical conditions are 19 micro M
2o syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30°C, 1 minute
reaction time. 1 laccase unit (LACU) is the amount of enzyme
that catalyses the conversion of 1.0 micro mole syringaldazin
per minute under these conditions.
2s Determination of glucose oxidase activity (GODU)
1 GODU is defined as the amount of enzyme which, under stan-
dard conditions, catalyses the formation of 1 micromole of H202
per minute. The analytic method AF266 is available upon request
from Novo Nordisk A/S).
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Determination of peroxidase activity units (POXU)
Peroxidase activity is measured in POXU/ml. (1 POXU (peroxi-
dase unit) is defined as the amount of enzyme that catalyses the
conversion of 1 micro mole H202 per minute in a system where
s 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonate] is oxidised in
the presence of 1 mM H202, pH 7.0, at a temperature of 40°C.)
A. The enzyme treatment
a. 100 g of wheat flour (type Pelikkan 441/1 from Meneba Meel
to BV, Holland) is mixed, using the Hobart mixer at speed III
(high speed) with 70 mL of tap city water (ca. 20 °dH) in
cluding added enzyme solution. The water temperature is
37°C ~ 2°C. The specific enzyme and dosage hereof is de-
scribed under each example.
15 b. The Hobart Mixer is applied on the pre-dough at speed III
(high speed) for 2 minutes.
c. The 170 g of dough rested for 8 to 40 minutes at 37°C in
the mixing cup, which is placed in a water bath.
Thereby the enzyme reaction time is the resting time plus the
2o mixing time (approximately 3 minutes) for all the tests. Some
further enzyme reaction time is possible during the dough wash
ing (see B. Preparation of gluten) below. However the dough is
washed continuously with water at 37°C, so that the part of the
enzyme that is not attached to the substrate is washed out
as rather quickly.
B. Preparation of gluten.
The Glutomatic~ System (Perten Instruments AB, Sweden)
consists of Glutomatic 2200 mixing and gluten washing device
3o used for preparing a wet sample of gluten. This sample is at
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flourmills used for determining gluten quantity and quality
quantified as the gluten index by sieve-centrifugation using a
Glutomatic Centrifuge 2015. The index value characterizes the
gluten as being weak, normal or strong.
5 The Glutomatic 2200 gluten washer consists of a washing
chamber, and a powerful stirrer. The washing and stirring is
performed by continuous addition of water. The wash water in
cluding suspended starch particles leaves the washing chamber
through an 88-micron filter at the same continuous flow rate at
to which fresh water was added.
3. 20.0 g of dough prepared as described above is placed in
the mixing cup and 4.2 mL water is added.
4. The stirring and continuous flow of water is performed for
minutes using a flow rate of 46.3 mL/minute. Water
15 heated to 37°C + 2°C was used.
5. The washed piece of gluten was weighed and evaluated visu-
ally for elasticity.
6. The piece of gluten was freeze-dried using conventional
freeze-drying technique and weighed. The weight of dried
2o gluten in relation to the weight of the wet gluten sample
is equivalent to the dry matter content.
7. The dried gluten was grinded to a fine powder using a mor-
tar for 15 minutes, and it was assayed by the micro baking
test.
C. Assay for measuring gluten aualitv by micro baking tests.
One to five days before the test begins portions of 12 g
flour + gluten was equilibrated at 30°C. Solutions of salt and
sugar were prepared. Also a solution of the yeast was prepared
(see the table below) and stored in refrigerator.
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The mass and volume of ingredients for the single breads
are the following:
Ingredients Mass or
volume
Flour 12.0 g
Water (from the city) 4.3 mL
Yeast solution (40 g + 60 mL water) 1.2 mL
Solution of 10.8 g sugar + 10.8 g 2.5 mL
salt + 64.2 mL water
Gluten (2 % w/w of the amount of 0.24 g
f lour)
s 2. To a thermo-equilibrated kettle (25-30°C) 12.24 g of
flour including gluten, the yeast solution, the salt plus sugar
solution and water is added (in this order). The kettle is the
mixer device for the Micro Mixer type NSI-33R that is used.
3. The kettle was mounted on the micro-mixer and kneading is
to performed for 3.0 minutes.
4. 18.0 g of the dough is weighed and kneaded by hand. The
temperature in the dough must be 27-29°C. The dough is
flatten out and shaped using a long rolling pin and it
rested for 15 minutes at 28-29?C.
is 5. It is again rolled and shaped using the special long roll-
ing pin and it rested for 10 minutes at 28°C.
6. Finally it is rolled out again and shaped to bread using
the special long rolling pin. The dough is then placed in a
37 mL baking tin and placed in a conditioning cabinet at
20 32.0°C, and 86 % relative humidity (RH) for 45 minutes.
7. It was then baked in the oven at 230°C for 13 minutes.
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8. After cooling the volume of the bread was weighed and meas-
ured using a "mini-PUP size" volume-measuring device. The
baking result was presented as the specific bread volume
(mL/g) . The higher the specific bread volume the better is
s the baking effect of the gluten.
In the following examples the enzyme treatment, the prepa-
ration of gluten and the assay for measuring gluten quality by
micro baking tests were used. The enzymes used and the results
to of the tests are mentioned under each example.
Experimentals
The following are examples of methods of separation of
wheat flour using enzymes. Fig. 1 is an illustration of the
is steps in one of the possible separation processes and Fig. 2 is
an example of a decanter process for the separation of wheat
flour.
Example 1
so 5 kg of wheat flour is mixed with 3.5 L of water having
at temperature of 25°C. The enzyme of Polyphenol oxidase - Lac-
case is added.
The dough is resting for approximately 8 minutes followed
by the addition of 5 L of water. The dough suspension is mixed
2s for approximately 18 minutes. After mixing the dough suspension
is diluted with 4 L of water and is circulated for 20 minutes.
The diluted dough suspension is then separated on screens by
addition of water.
The fractions obtained are gluten, hemicellulose and
3o starch fractions.
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Example 2
200 kg of wheat flour is mixed with 400 L of water. The
enzyme of Polyphenol oxidase-Laccase is added.
s The dough is continuously pumped into a homogeniser at
30-50 bar and is homogenised at a temperature of approximately
35°C. After homogenising, the mixture is passed through a de-
canter centrifuge separating the mixture into a supernatant
fraction and a pellet fraction. The supernatant comprises the
to gluten and the pellet comprises the starch. The supernatant is
kept in a tank at a pH of approximately 7.0, and is then passed
onto a 150 ~ sieve. The gluten of the supernatant is thereby
separated from the effluent. The wet gluten is freeze dried and
milled.
is The A-starch (sediment) is liquefied and subsequently
saccharified and made into syrup.
Example 3
Test of laccase
ao Enzyme treatment, preparation of gluten and baking test
are shown in table 1.
The washing of the gluten was performed at approximately
22°C .
Enzyme Type Dosage Resting Dry mat- Baking
of time, ter of test
enzyme minutes wet glu- Specific
activ- ten, ~ volume
ity. w/w of
bread,
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LACU/g
~/9
Dry mat-
ter of
f lour
Laccase Poly- 1.1 40 33.3 3.29
porus
pinsitus
Lactase Poly- 3.0 40 32.2 3.33
porus
pinsi tus
No 0 40 33.9 3.26
enzyme (average
of 3
tests)
Table 1. Effect of lactase treatment of the flour.
A clear effect of the lactase treatment was seen, as i1-
lustrated by the dosage response trial.
s Example 4
Test of sulfhydryl oxidase:
The specific activity of the Penicillium ochrochloron
sulfhydryl oxidase was 5.12 SOX/A-280. A preparation having an
activity of 6.45 SOX/mL was used.
to Enzyme treatment, preparation of gluten and baking test are
shown in table 2.
The washing of the gluten was performed at approximately
37 . 7-38 . 1°C
Enzyme Type Dosage Resting Dry mat- Baking
of en- time, ter of test
zyme ac- minutes wet glu- Specific
tivity. ten, s volume
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w/w of
Milli bread,
SOX/g
~/g
Dry mat-
ter of
flour
Sulfhy- Penicil- 1.0 10 34.0 3.36
dryl lium
oxidase ochro-
chloron
Sulfhy- Penicil- 1.0 45 33.9 3.38
dryl lium
oxidase ochro-
chloron
No 0 40 33.9 3.26
enzyme (average
of 3
tests)
Table 2. Effect of sulfhydryl oxidase
The wet gluten produced by use of sulfhydryl oxidase was
found extraordinary elastic when evaluated by the visual test
5
Example 5
Test of glucose oxidase:
Enzyme treatment, preparation of gluten and baking test
to are shown in table 3.
The washing of the gluten was performed at 36.1-37.8°C
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The enzyme preparation NovozymT"" 771 (batch no. OGN 00002) (As-
pergillus niger GOX) was analyzed to 1639 GODU/g according to
the internal analyses procedure EAL-SM-0244 (available on re-
quest from Novo Nordisk).
Enzyme Type Dosage Resting Dry Baking
of time, matter test
enzyme minutes of Specific
activ- wet volume
ity. gluten, of
w/w bread,
GODU/g
~/g
Dry mat-
ter of
flour
Glucose Novozym 0.010 10 33.4 3.35
oxidase 771 from (average
Asper- of 2
gillus tests)
ni ger
Glucose Novozym 0.010 45 34.4 3.35
oxidase 771 from (average
Asper- of 2
gillus tests)
ni ger
Glucose Novozym 0.100 10 34.1 3.28
oxidase 771 from (average
Asper- of 2
gillus tests)
niger
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Glucose Novozym 0.100 45 33.9 3.28
oxidase 771 from (average
Asper- of 2
gillus tests)
niger
No 0 40 33.9 3.26
enzyme (average
of 3
tests)
Table 3. Effect of glucose oxidase
An increasing effect of the specific volume of the bread
is clearly seen as a result of the treatment with the glucose
oxidase.
