Note: Descriptions are shown in the official language in which they were submitted.
WO 9513~026 2 1 9 0 7 6 1 PCr~llS95J0~828
NOVEL PLANTS AND PROCESSES FOR OBTAINING THEM
FIELD OF THE ~VENlION
5 The invention relates to a transgenic or mutated plant having b~enomic maoerial which
alters the normal starch synthesis pathway within the plant. More specifically, the
present invention relates to a plant having a genotype which creates new forms of
starch in significant quantity. Particularly, the invention relates to grain having an
embryo with a genotype h~ u~bu.,.. for two or more wild type genes (for example,10 Aa/Bb) and an endosperm having a genotype ll~,t~,.uL~bu.~. for such genes (for
example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced
therefrom.
Such grain are produced by pollinating a plant having a genotype llul~u~ ~bU..S
15 recessive for at least one gene and wild type for another gene (for example, aalBB) with
pollen from another plant having a genotype llullluL~buuD recessive for at least one
other gene and wild type for the other gene (for example, AA~bb).
BACKGROUND OF THE ~IVEN~ON
Most plants produce and store starch. These plants have a starch synthesis
pathway for starch prûduction. The arnount of starch produced varies with the type of
plant. The most commonly known starch producing plants are the cereal grains. These
cereals include rice, maize, sorghum, barley, wheat, rye, and oats. Additionally, the
25 potato family, including the sweet potatoes and certain fruits, like the banana, are
known as starch producing.
Starch is an impor~ant end-product of carbon fixation during ~Jl.vlu~ ' in
leaves and is an important storage product in seeds and fruits. In economic oerrns, the
3 0 starch produced by the edible portions of three grain crops, wheat, rice and maize,
provide ~ , two-thirds of the world's food calculaoed as calories.
I
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2 1 ~076 1
Starch from plants is used in various ways. For examp~e, it can be extracted
and used for cooking and food processing. Starch can be left in the grain or plant and
35 used for animal and human . Starch can also be used in the distillation
process for processing alcohols, for example, starch can be converted into ethanol.
Additional starch can convert to high-fructose syrup and other industrial
Starch is defined in the dictionary as a granular solid which is chemically a
40 complex carbohydrate which is used in adhesives, sizes, foods, cosmetics, medicine,
etc. More generally, starch is comprised of amylose and ~ y., Amylose and
lu~ is synthesized in the plastid ,:, t...~,.-L (the chloroplast in
1' , ' cells or the amyloplast in non-~,l.v~u,y,.~..,Li., cells). Different plants
generate differing proportions of ~".~, and amylose. r. , the different
45 branching pattems of ~..,!u~li,, and different chain lengths of amylose and
~ lu~lill chains gives rise to different starch propefies~ Thus, the fine structure of
amy]ose and o~ lu~ill is different in different plants so that the branching patterns
and ~ vaty UUllSl~ld~ resulting in new and novel properties which are
useful in different 1." Until now there have been four ways of making
50 starches with special properties: (i) using statches extracted ftom different plant
species, (ii) using statches extracted from mutant lines of palticular plants, (iii) using
natutal and mutant starches which had been chemically modified, and (iv) using natural
and mutant statches which had been physically modified. In all cases the new statches
were valuable because of the special properties provided for by the new starch type.
It is known that mutant genes in plants affect the propetties of the statch. A
variety of statch related mutant genes in maize have been identified and some have
been cled. These mutant genes were named accotding to the physical appeatance
(phenotype) of the mai~e ketnel ot the properlies of the statch. These tecessive mutant
60 genes include waxy (wx), sugaty (su) [which includes but is not limited to sugary-l
(sul), sugary-2 (su2), sugaty-3 (su3), sugary4 (su4)] dull (du), amylose extender (ae),
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WO 9~/35026 ~ 1 ~ 0 7 6 1 PCTIUS95107828
homy (h), shrunken (sh) which includes, but is not limited to, shrunken-l (sh-l),
shrunken-2 (sh-2). Some of these recessive gene mutarts produce an isoform of a
known enzyme in the starch synthesis pathway. The recessive mutant alleles of these
65 genes result in a complete or nearly complete reduction in the activity of a specific
isoform of one enzyme (hereinafter defined as complete reduction of enzyme isoform
activity) in the pathway when IIUIIIU~6V..D in a plarlt or when expressed in sufficient
levels in a transgenic plant. This change in the starch synthesis pathway causes the
fommation of starches with different properties,
Several crop varieties are known which produce different types of starch. The
type of quality of starch makes it suitable for certain purposes, including particular
methods of processing or particular end-uses. Naturally-occurring maize mutants
produce starches of differing fine structure suitable for use in various food products and
75 other l A]though known mutants produce altered starch, some of these
lines are not suitable for crop breeding and/or for the farmers' purposes. For example,
they can give relatively poor yields, and/or are difficult to process and/or carl have poor
. .
In order to generate different starches, single and double mutant plarlts have
been bred. A single mutant is a plant that is IIUIIIV~6~JL.D for one recessive mutant
gene. For example, waxy maize, waxy rice, waxy barley, and waxy sorghum have thehu~llv~y~v~D mutant waxy (wx) gene. Whilst starches from waxy genotypes have very
little or no amylose, another mutation known as amylose extender (ae) results in starch
85 with high amylose. A double mutant is a single plant that has t~ v~ .,., (or full
expression) of two recessive mutant genes. For example, the wxfll double mutant is
taught in U.S. patent 4,789,738. Many other novel starches have been provided in other
starch patents in which double or triple mutants are generated (for example, US Patent
Serial Numbers: 4789557, 4790997, 4774328, 4770710, 4798735, 4767849, 4801470,
90 4789738, 4792458 and 5009911 which describe naturally-occurfiing maize mutants
producing starches of differing fine structure suitable for use in various food products).
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The present invention is highly surprising in light of these ~ because it
produces altered starch and does not require double or triple mutants.
Normal starch is defined as starch which is not chemically modified (by
peop1e) or which is produced from a plant that has the expected genes (wild type)
regulating the starch synthesis pathway. For ease of reading, double lower-case letters,
for example aa, shall refer to a liuil~u~6v~o recessive mutant gene, double upper case
letters, for example AA, shall refer to a l1UIIIU~ D nûn-mutant gene (wild type), and
100 one upper-case and one lowercase letter, for example Aa, shall refer to a non-
I~UIIIU~A~ o set of genes, one mutant, one non-mutant. Different letters in the same
size shall mean different genes; "aalbb" would be a double mutant; "aalbB" would be a
single homozygous mutant gene and a l.~,t~,.u~gu.,. mutant gene in the genome of the
plant. For purposes of this application, the order of any three letters on one side of the
105 slash can be , ' , ' and will not define the parent that donated the gene. For
example, AAa~bbB is defined to be equivalent to aAA/bBb, AaA/Bbb, AaAlbBb,
aAA/Bbb, and the like.
Although maize plants and the embryo are diploid, maize endosperm is triploid.
110 The endosperm genotype has two gene doses which are inherited from the fernale
plant portion and one gene dose which is inherited from the pollen or male plantportion. Thus, if a single mutant plant "aa" is used as the female and crossed to a non-
mutant plant "AA" male, then the endospemm in the kemel of this female plant would
be "aaA". If a non-mutant plant "AA" is crossed to a mutant plant "aa" with the non-
115 mutant as the female, the endospetm on the kemel of the female plant will be "Aaa",because two gene doses come from the female and one from the male plant. Classic
teæhing is that the mutant gene is recessive and the non-mutant is dominant; therefore,
the starch produced by a plant having the following gene doses in the endospemm "aaA"
or "AAA" or "AAa" results in the nommal starch in the expected amounts. However,120 the endosperm of a IIUIIIUL~6~..0 mutant plant "aa" æting as the female crossed to a
l~u~u~ o mutant plant "aa" acting as the male plant results in the endospemm having
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WO 95135026 r~
21 90761
the gene dosage "aaa". This endosperm gives starch with different properties from
normal starch. Likewise, the starch from a double mutant having of an endosperm
which is "aaalbbb" shows differences in starch properties from normal starch. These
12 5 starch differences are useful in that they can replace chemically modified starches or be
used with or in foodstuffs or as grain in alcohol production or in general starch
industria] ~
Clearly, it appears that production of grain having starch with different physical
13 0 properties of starch requires the crossing of two mutated plants to generate grain which
;S IIUIIIUL~ recessive for both genes. Mutant plants are less predictable than
standard plants.
There is a recurrent problem with the production of grain and extraction of
135 starch from double mutant hybrids and/or inbreds and some single mutants. The
amount of starch produced is usually less than the amount of starch produced by the
non-mutant plant, there is also a loss in starch granule size and/or starch granule
integrity. This problem with known double mutant lines which produce !
altered starch in which the quantity of starch produced in the crop is relatively low can
140 result in poor g ' ".y of the seed. r~.tl,~.",u.~, the reduced starch yield of the
seed appears to be I ~ui.ld~l~ since the mutations cause the normal starch synthesis
~ of the cells to be disrupted. There remains a need for a way to produce
grain having structurally altered starch structurally altered starch or altered properties
without a significant loss of yield or reduced starch granule size or integrity.145
SUMMARY OF TE~ INVEN~ON
A first object of the present invention is to provide a method of developing
- hybrid plants having altered complex ~uI,vh~.' content of the grain which does not
15 0 require the crossing of double mutant inbreds.
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W095/35026 2 ~ ~Q76 t PCI/US95/07828
An object of the present invention is to provide plants that produce grain having
altered starch properties.
155 Another object of the present invention is to provide transgenic plants that
produce grain having altered starch properties.
Yet another object of the present invention is to provide a m~ize plant that
produces both altered starch and larger quantities of starch than the associated mutant
16 0 plants produce.
Still another object of the present invention is to provide new plants which
contain genes which produce incomplete reduction of the activity of at least twoisoforms of the specific enzymes in the starch synthesis pathway of the plant.
165
A further object of the present invention is to provide maize plants which have
with the genotype "AAalBBb or AAalbbB or aaAlBBb or aaA/bbB".
Yet another object of the prvsent invention is a plant producirlg the following
17 0 endosperm genotype "wxwxWX/AeAeae".
Still an additional object of the present invention is to provide the altered starch
which can be produced by plants having the genotype "AAalBBb or AAalbbB or
aaA/BBb or aaA/bbB".
175
Yet another object of the present invention is to provide novel uses of the starch
obtained from the Maize plants of the present invention.
The present invention broadly covers a method of producing
180 generally which have the endosperm genotype of AAa/bbB and certain
including endosperm which is waxy, waxy, amylose extender (wxwxWx/AEAEae).
SUBSTITUT~ SHEET (RULE 26)
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21 90761
The method of producing grain with altered starch qualities includes the steps of
planting the female acting parent which is capable of flowering. The female parent
having ' ly complete reduction of at least one specific isoform enzyme in the
185 starch synthesis pathway. This can be by a bul~u~,uu~ recessive mutant or by the
partial down regulation of the wild type gene through the use of a cloned gene using
techniques generally known as antisense or co ~ ' or sense-down regulation.
Additionally the female has incomplete reduction of at least one specific isofoTm
enzyme in starch synthesis pathway. This can be by a l.~.~.u~,~.~ recessive mutant
190 gene or partial down regulation. Regardless of how the female is produced it should
only act as the female portion. To assure this a step includes eliminating the first
parent's capability to produce pollen. The method includes the step of pollinating the
female acting parent with the pollen of the male acting parent which is a non-mutant
parent. Harvesting the grain produced by said first parent. Additionally, the method can
195 include the extraction of starch from the grain.
This invention also ~ a plant having genomic material which
includes genes which give incomplete reduction of the activity of at least two specific
isoforms of the enzymes in the starch synthesis pathway of said planL And the starch it
200 produces which has altered structure when compared with the starch formed by a
similar plant as described but which comprises genomic material which does not form
isoforms of the enzymes in the starch synthesis pathway of the plant.
A plant which forms said starch in grain such as cereal grains. Grain produced
205 by a female plant having for example a waxy genotype (wxwx) crossed with a male
plant having for example an amylose extender genotype (aeae) in which the genotype
of the endosperm of the gr~un is wxwxWx/AeAeae.
In other words, the present invention is a starch producmg plant comprising
210 genomic material which includes genes which give incomplete reduction of the activity
of at least two specific isoforms enzymes in the starch synthesis pathway of said plant
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wo ss/3so26 ~ r~ /n~s
whereby said plant produces ! ' ' " 1~/ more starch than said plant would produce if
said genes gave complete reduction of the activity of the same two specific isoforms of
the enzymes within the starch synthesis pathway.
215
Grain having an endosperm that has two genes which contain one gene dose of
recessive mutant gene and two doses of wild type; and, having two gene doses of
recessive mutant and one dose of wild type. Within this description the invention
r~ grain having endosperm genotypes of wxwxWx/AeAeae, or
220 aeaeAe/WxWxwx, or wxwxWx/DuDudu, or duduDu/WxWxwx, or aeæAe/DuDudu,
or duduDu/AeAeae, or wxwxWx/SuSusu, or susuSu/WxWxwx, or æaeAe/SuSusu, or
susuSu/AeAeæ, or duduDu/SuSusu, or susuSu/DuDudu and the like.
The starch from a grain having a genotype of wxwxWx/AeAeae. The starch
22 5 from a grain having a genotype of A= /WxWxwx.
A female plant having a diploid genotype of aa/BB and having a hiploid
genotype of aaA/BBb where a is a recessive mutant gene and A is the wild type gene,
and b is a recessive mut~nt gene and B is the wild type gene such that the starch is
2 3 0 altered from the normal starch where a and b can be selected from ae, wx, sh, bt, h, su,
fl, op and B and A can be selected from Ae, Wx, Sh, Bt, H, Su, rl, Op.
The starch obtained in accordance with the preænt invention produces a strong
resilient gel which clears from the mouth uniquely fast. The starch of the preænt
235 invention produces a gel with a unique and distinctive texture compared to
.,u,l~. ' starches. The urlique and distinctive texture makes the starch of the
preænt invention suitable as a "' for cu.~ iU~ ge]ling gums such as
natural gums and gelatin, in whole or in part in food r,.. ~ The starch of the
present invention has also been found to produce a more resilient gel than common
2~0 starch. r. ;h~.l,.u.~, it has been found that comstarch produced from maize produces a
gel which has improved clarity compared to a gel made from a common starch. Such
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improved clarity is visible to the human eye and lends itself to a more appetizing
foodstuff.
2 4 5 BRIEF DESCRlPI~lON OF THE DRAWINGS
The present invention will now be described, by way of illustration, by the
following description and examples with reference to the . , g drawings of
which:
250
FIG I is a graph of enzyme activity for different gene dosages of a single
mutant;
FIG 2a is a graph of the DSC scan of waxy, amylose extender and common
255 maize;
FIG 2b is a graph of the DSC scan of a double mutant (~a~t~iAWA WA);
FIG 2c is a graph of the DSC scan of starch from an
2 6 0 (~La ~,AeJ W A W A W A ) .
FlG2d is a graph of the DSC scan of starch from another
(wxwxWx/AeAeae).
2 6 5 FIG 3a is a graph of Brabender data of common starch in various pH.
FIG 3b is a graph of Brabender data of waxy starch in various pH.
- FIG 3c is a graph of Brabender data of 70% amylose starch in various pH.
270
FIG 3d is a graph of Brabender data of double mutant starch in various pH.
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21 9a76~ -
FIG 3e is a graph of Brabender data of a first inter nutant starch in various pH.
2 7 5 FIG 3f is a graph of Brabender data of a second intermutant starch in various
pH.
FIG 4a is a schematic showing the design and KStriCtion enzyrne sites of plant
' vectors used to alter gene expression levels of branching
2 8 0 enzyme 1.
FIG 4b is a schematic showing the design and restriction enzyrne sites of plant
1 "" ,_ r .., ~ ;~ ... vectors used to alter gene expression levels of branchingenzyme 11.
285
FIG 4c is a schematic showing the design and KStriCtion enzyrne sites of plant
vectors used to alta gene eXpKsSion levels of bound
starch synthase (waxy),
2 9 0 FIG 4d is a schematic showing the design and restriction enzyme sites of plant
-- vectors used to alter gene expression levels soluble
starch synthase.
FIG 5 is a plot of the elastic modulus (G') over time comparing A gel made
2 9 5 from the starch of the present invention to a gel made from an aewx
starch and a gel made from a waxy (~) starch; and
FIG 6 is a plot of the elastic modulus (G') plotted against strain for both the
pKsent invention and an aewx starch.
300
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21 90761
DETAILED DESCRlPI ION OF THE INVENTION
Broadly then the present inYention is an improved crop line which has
, ' I e~pression of at least two starch-~y..~ ;"~ enzymes which alter the
3 0 5 amount and type of starch, and, , 1!" alters the grain produced by the plant.
It has been discovered that plants which contain at least two genes which
partial1y down regulated or reduce the ætivity of specific isofomms of enzymes in the
starch synthesis pathway will ~JI;~;II~,ly produce significant amounts of starch in the
310 grain and will produce altered starch types.
Specialty maize or mutant plants differ from "nommal" maize because of its
altered endospemn. The changed endospemm gives rise to a high degree of starch
branching, or changed sugar content, or different kemel structure. The endospemm of15 course is forlned by the spemn and ovule, and the selection of both parents effects the
makeup.
The present invention can be formed by two principle methods. The invention
can be fommed within a selected crop species by the use of mutant breeding. And the
320 invention can be formed in various plants by the use of ~ of the plants
with genes which partially down regulate two or more enzymes in the starch synthesis
pathway. More particularly, down regulation of one of the isoforms of the enzyme in
the starch synthesis pathway to ,.~ , 1/3 of the nommal ætivity and 2/3 of the
normal ætivity in the other isoform enzyme or down regulation of both isoform
325 enzymes in the starch synthesis pathway ~", '~, 2/3 of the nommal activity.
Eæh of these methods has its own ædvantages.
First, the use of mutants to develop unique grain and starches in cereal crops is
widely known. However, the present invention is highly unique and surpnsing because
Il
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21 90761
330 it was expected to prr~duce grain having nortnal starch ~,llola~i~,l;~li-,~. The following
table explains how unexpected the present invention iS.
~ .
3 3 5 Genotyloe of Parents Genotyp Type of Sta~h
(f~male Ist) of endosplm Starch Ybld
Wild-Type MYM AM Normal Nomlal
3 4 0 Gene dosage AAi'aa AAa Nomul Nomlal
aa#AA aaA Normal Normal
Singlemutant aai'aa aaa Altered Lowered
345 Doublemutant a~Vbb*a~Vbb aaa/bbb Altered Lowered
PXESI~NT INV~NTION - EXPECTED RI~SULTS
Intemmut~nt aa/BB~M/bb aaAlBBb Normal Nonnal
AAlBBi'LILVbb AALVBBb Nomlal Nomlal
3 5 0 aLvbb~AAlBB aaA/bbB Nomlal Nomlal
aalbb~aalBB ar~vbbB Nomlal Normal
aalBB~aLvbb aa~vBBb Normal Nomlal
PRESENrl.. vr,~ ACrUALRESULrS
355 Int~nnruant aolBB~AAlbb aaA/BAb Altcrc4 Mc41umlo
AAalBBb AlJc~cd h;~h - 70%
odbb~AA/BB aaAlbbB A~r~4 of Irornu
aolbb~oalBR an~VbbB Alttrc4
3 6 0 aolBO~o~/bb aaalBBb Alt~rc4
na = mut~nt gene " , O
AA = wild tyloe (or non-mutant homoygous gene)
i~ = signifies a ~ ; '" ' between two varieties
3 65 arvbb = two mutant genes (both homoygous)
12
SUBSTITUTE SHEET (RULE 26
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Clearly, since the genotype of the endosperm of the present invention shown in
the table did not have completely recessive genes, the starch yield and structure was
expected to be normal. In fact, according to the present invention, the grain does not
370 evidence normal starch structure altered starch. Historically, when altered starch is
produced only small quantities are usually produced. The altered starch of tbe present
invention was . ~, produced in larger than expe~ted quantities. Additionally,
production of this starch is much simpler than the production of double mutant crops.
Previously only single mutant hybrids have been used extensively for large-scale starch
375 production. Previously, to develop the double mutants, both parents had to carry both
mutations which requires significant research and d~ efforts and results in poorstarch yield and poor seed g ~ y. Heretofore, only small-scale production of thedouble mutants has been possible.
3 80 The present invention: . a method of producing gr~un v~ith altered
starch qualities which includes the steps of planting a parent which is capable of
flowering, this parent having ' "~ complete reduction of at least one specific
isoform of an enzyme (A) in the starch synthesis pathway and having no reduction of at
least one other specific isoform of an enzyme (13) in the starch synthesis pathway. The
3 8 5 other parent has no reduction of one isofolm of an enzyme (A) and ' "~ complete
reduction of at least one other specific iwform of an enzyme (B) in the starch synthesis
pathway. It is then necessary to eliminate said first parents capability to produce pollen
and allow pollination to proceed from said second mutant parent, and finally harvesting
the grain produced by said first parent. Additionally, the method can include the
3 9 0 extracdon of starch from the grain and using said starch as a specialty starch for a variety
of uses for which it is shown to be valuable.
In order to prepare a sol in accordance with the present invention, a slurry is
prepared which comprises water and an effective amount of starch of the present invention
3 9 5 and the sol subject to a cooking step to form a paste. Generally, cooking entails raising
13
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the i , ~ of the slurry to above about the ~ i . of the starch
and subjecting the starch to enough shear such that the granules rupture and a paste is
formed. It is not necessary that all the granules rupture. Preferably, the sol contains the
sta~ich of the present invention in the amount of about 1 to about 20% by weight total sol.00 The sluniy is cooked at a i . of about 90 C and above to provide thickening
prior to adding to the foodstuff. Cooking time is about lO minutes. The
sol in accordance with the present invention need not be cooked if the starch has already
been subjected to a process which makes it cold water swellable. Cooking generally
comprises raising the r ' _ of an aqueous slurry of the starch of the present
4 05 invention to the ~ ~ of the starch and subjecting the starch to shear
such that the starch granules rupture and form a paste.
A sol or a thickener ~ ;- of the starch of the present invention is added to
a foodstuff in a ~,u..v. ' manner in order to provide the benefits of the starch of the
410 present invention to the foodstuff.
In order to prepare the thickened foodstuff, a sol made in accordance with the
present invention is combined with a foodstuff and the . is cooked to tbe
necessary degree to provide a thickened foodstuff. Conventional mixing is employed to
415 combine the sol with the foodstuff. Cooking of the sol and foodstuff ~ . is also
carried out in a ,u.l ~ ' manner,
Altematively, starch of the present invention is mixed ~vith the foodstuff or a
slurry comprising the starch of the preænt invention and water is mixed with a foodstuff
42 0 and the resulting mixture is cooked to the desired degree to obtain a thickened foodstuff,
When the starch itself or a slurry containing the starch itself is mixed with a foodstuff, the
resulting mixture must be cooked in order to provide a thickened foodstuff. The mixing
as well as the cooking is r , " ' ' in a ~u~ t;u~ l manner. Cooking is carried out
at a: , of about 90 C and above. Cooking time is about lO minutes but may
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WO 95~35026 2 1 9 0 7 6 1 PCTIUS9510~828
42 5 vary depending on the amount of foodstuffpresent and the amount of shear that the mix is
subject to during cooking.
Such a thickencr c . ~ ;- can provide ' ' ' economic advantage to the
user. Those familiar with the art have long uscd a variety of gelling gums for their clean
43 0 breaking texture. Application of the present invention have included but are not limited to
gum candies, gellcd desserts, glazes and spreads and can be used to replace ~.u..~. '
gelling gums such as kappa ~ O agar, pectin, or gelatin. These ~.u...~...;~...d
gelling gums can be quite expensive however, and have other ~' " . ,, including the
presence of off-flavors, lack of hcat or acid stability, limited availability, or lack of Kosher
435 approval. It has been found that the starch of the present invention can replace dl or a
portion of these cu~ ' gelling gums.
In order to replace a gdling gum in food r~ . a weight ratio of about 1:1,
starch of the present i..~, ' ,. gum, can be employed. Larger or smdler amounts44 O of the starch of the present invention may be used to replace a gelling Oum. Such gelling
gums include gelatin, pectin" " gum arabic, tragacanth, guar, locust bean,
zanthan, agar, algin and ~I,u~ ' jl cellulose.
Naturally, the starch of the present invention can be qsed in any food '
44 5 where there is a need to provide gel ~ ` ;`1 ;' ` and a clean brcak from the mouth. For
example, the starch of the present invention can be used in a food r ~ '- which had
heretofore employed a common starch, thereby providing the food with improved
properties, i.e. clean break when compared to the same food ' ' using a common
starch.
450
The clean break of a gel made with the starch of the present invention is useful in a
- variety of food nl~ The clean break of the starch gel has vdue in a variety of
bakery ~ ;,. " for example cream or fruit fillings for pies such as lemon, banana
cream or Bavarian cream; and in low or reduced fat high solids fruit centers for cookies,
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455 for example, in fig bars. The starch of the present invention dso creates an improved
Kxture in mousses, egg custards, flans and aspics.
The Krm starch as used in the ~ r; A~ and claims means not only the
,uI,A~,lidl~ pure starch granules as extracted from a starch bearing plant but also grain
4 6 0 products of the starch granule such as flour, grit, hominy and meal.
The following examples of the present invention are given for illustrative purposes
only. These examples are not inKnded to limit the type or the uses of the present
invention. The present invention or the gr~un or starch or sugar thereof can oe useful in,
465 but may not be limited to, the preparation of foodstuff, paper, plastics, adhesives, patnts,
production of ethanol and corn syrup products.
Example I
4 7 0 The physical properties of various e.. ~ " of the present
invention (similar genotypes are from different maize
crosses). These tables display data well known to people
skilled in the arts of evaluating new and novel starches. Data
on moistures, and percent oil, protein, solubles and starch are
475 useful in evaluating yield and milling poKntial. Starch DSC
(Differentid Scanning (~ ) data are vduable for
evaluating starch cooking and g. 1 ~; A' ;~ . properties. Starch
particle size data are vduable for making decisions on starch
milling and separation properties. Brabender and starch paste
4 8 0 data are most important for evduating a new starch's potentiaA
for improved food ~ r ~ where particular starch
thickening and pasting and i' properLies are
mostly desired. Such data when interpreted as a whole
collection of ~ enable one skilled in the art to
16
SUBSTITUT~ SHEET (RULE 26
W095135026 2 19 0 7 6 1 r~l~u~ 6
485 decide whether to conduct further more detailed tests of the
starches properties and potent;al.
Genotype %Moisture %Starch %Protein %Oil %Solubles
aeae/wx 8.01 72.12 11.30 3.63 6.51
aeæ/wx 8.00 7134 11.74 3.79 6AI
aeæ/wx 8.03 67.52 12.6] 3.67 7.01
aeae/æ 8.13 60.11 14.59 6.60 10.14
sulsul/wx 8.18 69.66 12.09 4.03 6.91
dudu/wx 7.98 71.10 11.57 3.92 6.61
dudu/sul 7.90 70.53 11.67 3.93 6.81
flfl/o 7.78 70.23 1 1.60 4.23 6.59
sulsul/du 7.81 69.00 12.20 4.M 7.98
sulsul/æ 7.88 69.03 12.27 4.88 8.02
sulsul/su2 795 69.67 12.28 4.55 7.49
.
.
17
SUBSTITUTE SHEET (RULE 26)
WO 95/3s026 . 2 1 9 0 7 6 ~ PCT/US95/07828
~9 0 Table 2
- Corn Data
S93 % % % % %
EDIr,~ l~n~ Row Moisture ~ta~h l~ill ~lil ~QIIlk~
] aeaewx 4660 9.15 64.80 14.58 4.47 7.64
2 aeaesu 4636 9.27 66.25 14.77 4.54 7.40
3 aeaedu 4612 9.24 66.01 IA65 4.73 7.80
4 wxwxae 4600 9.07 65.72 ]3.91 A38 8.]4
5 wxwxsu 4648 9.06 66.03 ]4.79 4.08 7.83
6 wxwxdu 4624 8.77 65.32 ]5.62 427 8.23
7 susuae 4594 8.85 64.83 13.11 4.71 8.49
8 susudu 4672 8.73 65.39 ]3.20 5.01 8.35
9 susudu 4618 8.77 65.]5 14.52 435 8.26
]0 sing]eae 4582 9.]4 6052 ]5.]9 5.95 9.29
] ]single wx 4654 8.83 65.44 ]4.04 4.77 9.43
12 single su 4630 7.75 54.98 14.25 6.50 9.78
13 duduae 4588 8.97 6926 1230 A89 6.85
]4 duduwx 4666 8.88 70A3 ] 1.25 4.40 6.96
15 dudusu 4642 9A8 70.43 13.48 4.67 6.92
16 single du 4606 850 65.54 12.02 6.03 933
- Starch Data
S93 % % % % %
Entr.~ ~2~1YQ ~ ~Q~ ~ ~ ~ ~ ~8
aeaewx 4660 3.82 86.44 1.68 0.18 31.60 597.1
2 aeaesu 4636 6.51 86.95 1.56 0.20 31.74 603.9
3 aeaedu 4612 5.22 86.44 1.70 0.11 3338 598.8
4 wxwxae 4600 733 87.89 0.39 0.09 21.10 601.1
s wxwxsu 4648 7.27 85.87 1.32 0.09 2A.68 591.7
6 wxwxdu 462A 7.31 86.07 1.12 Q10 24.57 595.2
7 susuae 4594 8.19 86.32 Q93 0.07 29.C0 604.5
8 susudu 4672 8.09 87.16 052 0.08 27.86 599.6
9 susudu 4618 7.67 84.20 0.60 0.12 3Q82 ~501.1
10 sing]eae 4582 7.16 8331 1.37 0.15 6555 6Q~I
Il single wx 4654 8.30 85.16 0.51 0.14 0.43 xx
12 single su 4630 6.82 80.92 5.08 xx 29.32 598A
13 duduae 4588 6.58 84.31 0.62 0.17 29.86 xx
14 duduwx 4666 11.08 84.77 0.48 0.16 28.74 ~x
15 dudusu 4642 8.48 87.34 Q62 Q16 26.07 xx
16 singledu 4606 8.36 8Q70 0.46 0.08 37.88 608.9
18
SUBSrlTUT~ SHEET (RULE 26)
WO 95/35026 2 1 9 0 7 6 1 PCTIUS95/07828
4 9 5 Table 2
- (Starch Data)
%qO % %
aeae/wx 9.56 83.84 0.63 QQS 29.44 593.1
2 ae ae / wx 7.96 85.94 0.70 0.05 27.89 596.6
3 ae ae / wx 11.71 82 54 0.82 Q05 42.54 595.1
4 aeae/ae 6.Q9 84.17 12A 0.14 63.60 602A
5 sul sul /wx 4.37 90.00 Q56 0.Q9 28.36 600.0
6 dudu/wx 5.95 8850 0.61 0.Q9 27.98 596.9
7 dudu/sul 6.93 82~66 Q69 0.10 29.98 600.8
8 flfl/0 9.24 8272 093 0.14 2929 596.8
9 sul sul /du 9.01 84.82 Q71 Q02 2957 600.8
10sul sul /ae 7.78 84.81 0.63 0.03 29.90 6C0.8
Ilsul sul /su2 7.61 85.02 0.49 QQ2 29.68 6023
12White waxy 1834 9A94db 0.77 Q09 2.74 526.9
- Starch DSC Data
Pellk Delta H Peek n Onset Endset
sL cc l¢g C C C
aeae/wx 67.7 11.67 IQ0.0 62.6 76.6
2 aeae/wx 665 10.33 96.8 61.9 74.8
3 aeae/wx 7A6 IQ00 100.8 68.8 83A
4 aeae/ae 81.0 11.83 xx 66.0 107A
5 sul sul /wx 69.7 1200 98.3 64.2 77.7
6 dudu/wx 72.2 1150 99.5 66.6 82.3
7 dudu/sul 702 9.67 98A 65.7 78.3
8 fl fl/0 71.4 12.33 100.9 67.1 8Q7
9 sul sul /du 68.8 7.83 995 63.2 79.1
10sul sul /ae 68.1 IQ33 99.8 61.9 78.3
Ilsulsul/su2 67A 12.00 96.1 61.3 79.5
12White waxy 72.7 1533 xx 66.1 82.6
- Starch DSC Data
S93 Pe~k Delta H Peek n Onset E.ndset
~lY~a~k~ ~ oc J/~ C C ~
Deaewx 4660 73.8 11.17 99A 67.7 82.8
2 aeaewx 4636 73.0 11.50 101.0 66.6 83A
3 aeaewx 4612 73.3 10.83 98.6 66.7 83.0
4 wxwsae 46~0 72.9 12.83 99.9 67A 81.7
5 wxwssu 4648 73.7 12.17 98.2 682 81.6
6 wxwsdu 4624 73.6 1133 99.6 69.3 80.5
7 susllae 4594 72.0 IQ33 97.7 67.2 79.7
8 susudu 4672 71.9 9.83 98.9 67.0 79.1
9 susudu 4618 725 1033 97.0 68A 79.8
10single ae 4582 84.6 15.67 xx 68.7 106.9
I lsingle wx 4654 72.5 16.17 ~ 68.1 81.4
12single su 4630 709 15.50 100.8 63.6 772
13duduae 4588 71.8 1350 1005 67.1 81.1
14duduwx 4666 71.9 10.17 98.8 67.2 79.5
15dudusu 4642 71.7 IQ17 97.2 66.8 79.0
16singledu 4606 71.6 9.50 100.1 65.8 79.8
.
SUBSTITUTE SHEET (RUL~ 26~
W0 95/35026 ~ &
2 ~ 9376 1
Table 2
5 0 0 - Brabender Data
S93 lR HP HF CP CF
ntrY73ackw~und ]35~ ~BI~ C L3U . 1~_ }3U ~
aeaewx 4660 460g/5.5% 65.0 315 285 940 940
2 aeaewx 4636 460g/5.5% 81.5 225 225 470 430
3 aeaewx 4612 460g/5.5% 84.5 180 180 410 380
4 wxwsae 4600 460g/5.5% 74.0 360 355 590 460
5 wxwssu 4648 460g/55% 74D 325 325 540 470
6 wxwsdu 4624 460g/5.5% 80.0 315 270 480 425
7 susuæ 4594 460g/5.5% 77.0 270 270 610 585
8 susudu 4672 460g/5.5% 77.0 295 295 785 610
9 susudu 4618 460g/5.5% 77.0 295 295 620 620
10 single ae 4582 460g/12% 90.5 170 170 240 240
I l single wx 4654 460g/5.5% 68.0 750 360 415 405
12 single su 4630 90g/55% 93.5 35 35 40 40
13 duduae 4588 460g/55% 83.0 225 225 545 520
14 duduwx 4666 460gl5.5% 86.0 245 245 600 550
15 dudusu 4642 460g/55% 84.5 270 270 620 585
16 single du 4606 460gl5.5% 89.0 70 70 160 160
IR. = n~it~ isc
CP = cooling pe~k
CF = cooling final
505
- Brabender Data
IRHP HF CP CF
C~ ~ ~ ~
ae æ / wx 460g/5.5% 83.0 220 220 520 460
2 .aeae/wx 460g/5.5% 89.0 220 220 540 510
3 aeae/wx 460g/5.5% 845 270 270 510 450
4 aeae/ae 460g/129~o 90.5 490 490 1220 845
5 sul sul /wx 460g/5.5% 80.0 250 240 595 535
6 du du / wx 460glS5% 83.0 280 250 560 470
7 dudu/sul 460g/5.5% 50.0/83.0 230 230 575 535
8 flfl/0 460g/5.5% 81.5 280 255 630 560
9 sul sul /du 460g/5.5% 84.5 200 200 500 460
10sul sul /ae 460g/5.5% 86.0 205 205 455 415
Ilsul sul /su2 460g/5.5% 84.5 180 180 420 385
12White waxy 90g/5.5% 68.0 830 220 310 270
SUBSTITUTE SHEET (RULE 26)
woss/3so26 2 ~ ~ 0 7 6 i 1~l,., ",~
Table 2
- Starch Particle Size Data
(Volume Distribution)
S93 % Starch
~Y Backeround Row Mode ~n Mean ~un ~di3nJL_ Re~over,v
aeaewx 4660 16.90 12.57 16.04 45.9
2 aeaewx 4636 16.63 11.82 15.23 57.9
3 aeaewx 4612 16.36 11.48 15.20 52A
4 wxwsae 4600 17.43 11.62 16.53 41.5
5 wxwssu 4648 17.72 12A3 16.59 58.8
6 wxwsdu 462A 17.48 12.22 16.26 54.1
7 susuae 4594 16.36 11.96 15.66 55.5
8 susudu 4672 16.65 9.83 15.29 58.7
9 susudu 4618 16.36 11.82 15.61 55.9
10 single ae 4582 13.09 9.07 12.25 64.0
Il singlewx 4654 18.03 11.99 16.85 55.7
12 single su 4630 7.43 5.16 7.95 5.6
13 duduae 4588 16.90 11.01 15.37 SQ9
14 duduwx 4666 17.19 11.85 16.21 52.5
15 dudusu 4642 17.17 12.22 16.01 44.9
16 singledu 4606 14.41 9.77 13.19 58.9
510
- Starch Particle Size Data
(Volume 1~ ' )
9~o Starch
E~~Ll;L ModeILm ~LU_ ~ Recoverv
aeae/wx 16.63 10.90 15.70 51A
2aeae/wx 16.12 10.44 14.92 52.8
3ae ae / wx 15.60 10.55 14.60 59A
4aeae/ae 11.53 8.40 1130 63.7
5sul sul / wx 14.88 IQ2A 14.21 52.5
6du du / wx 16.38 IQ73 15.12 52.0
7dudu/sul 15.87 11.03 14.98 53.2
8flfl/0 16.12 10.73 1535 69.3
9sul sul /du 15.87 10.73 14.92 53.7
10sul sul /ae 15.11 7.85 14.35 48.5
I lsul sul / su2 15.85 IQ33 14.20 58A
y17.1 845 1~81 71.5
SUBSTITUTE: SHEET (RULE 26~
Wo 95135026 2 1 9 0 7 6 1 PCT~S9S/07828
515 Table 2
- Starch Paste Data
BrQokfield Freeze
Viscosity Gel-2Ahr Thaw
EDg:Y Genot~e CPS. 20 rvm ~m~ cles
~eæ/wx 6,200 166.6 0
2 ae ae / wx 6,700 2272 0
3 æ æ / wx 8,000 256.3 0
4 ææ/ae 14,250 111.1 0
5 sulsul/wx 6,800 173A Q
6 dudu/wx 6,000 141.4 0
7 dudu/sul 7,100 215.4 0
8 fl fl / 0 7,500 209A 0
9 8ul sul /du 6,300 219.7 0
10sul sul /æ 6,000 172.2 0
Ilsul sul /su2 5,500 158.8 0
12White waxy 1.900 15.3 3
- Starch Paste Data
Brookfield Freeze
S93 Viscosity Thaw
~EYGenQtvee Row = ~P,C 2n Q~
aeaelwx 4660 25,000 151.8 0
2 aeæ/wx 4636 6,500 240.1 0
3 ææ/wx 4612 3,150 216.6 0
4 ae æ / ae 4600 5,600 159.5 0
5 sul sul /wx 4648 6,100 126A 0
6 du du / wx 4624 5,700 114.4 0
7 du du / sul 4594 8,700 254.8 0
8 fl fl /0 46?2 12,000 272.8 0
9 sul sul /du 4618 10,250 233.6 0
10 sul sul /æ 4582 2,500 53.7* 0
I Isul sul / su2 4654 3,100 13.3 0
12 White WIXy 4630 275 29.0 3
13 duduæ 4588 7,200 226.5 0
14 duduwx 4666 7,300 233A 0
15 dudusu 4642 5,100 3329 0
16 single du 4606 1,650 99.3 0
520
$ came out as a plug
22
SUBSI ~TUTE SHEET (RULE 26
w095/35026 2 1 9076 1 ~."~ ",~"
DEFlNlTIONS:
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
IR denotes initial rise
525 HP denotes heat peak
HF denotes heat final
CP denotes cooling peak
CF denotes cooling final.
530 BROOKFIELDVISCOMETER
The Brookfield Viscometer measures shear-strength (in centipoise, cP) and
stability of starch pastes.
BRABENDER VISCO-AMYLOGRAPH DATA
53 5 Pasting i , denotes the i , of paste formation.
Peak Viscosity denotes the . . _ needed to provide a useable paste.
Viscosity at 95C denotes the ease of cooking of the starch.
Viscosity at 50C denotes the setback in paste viscosity during cooling of a hot
paste.
540 Viscosity after I hour at 50C denotes the stability of the cooked paste.
ORN PERCENT PROTEIN, STARCH, OIL AND MOISTURE
of oil starch and protein m com give a measure of starch yield how
,~, . ~,.~1~ the starch is.
545
STARCH PERCENT PROTE~, STARCH, OL AND MOISTURE
re. ~ of oil starch and protein in starch give a measure of how well purified
the starch is and indicates millability.
5 5 0 PERCENT AMYLOSE AND L-MAX
These data provide a measure of apparent amylose levels in starch.
23
SUBSTITUTE SHEET (RULE ~6
woss/35026 21 ~76 1
STARCH PARTICLE SlZE DATA
Starch particle size gives an indication of starch yield and ~u._.~;lily through5 5 5 the milling process.
SHORT HAND
aeaewx in table 2 refers to aeaeAE/wxWxWx, likewise
1' ' A_' ' r)U/WXWXWX Throughout this table the wild type is not listed.
Figure 1 is a graph of enzyme activities for individual gene-dosages (e.g., MMM,560 ~MM, mmM, mmm) of mutant alleles of the single mutants of amylose extender
and dull. These data show the enzyme activities of sucrose synthase (SS), UDP-
glucose ~ yl~x, (UDM-PP), O' ' (GK), rl u~luki,.~., (FK),
(PGM), ~ , O isomerase (MI), ATP-dependent
1' , ' ' u~lvl~ill~ (PFK), PPi dependent I ' , ' ', ' (PFP), ADP-
565 glucose ,u,,' yl~_ (ADM-PP), soluble starch synthase (SSS), branching
enzyme (BE) and bound starch synthase (BSS). Enzyme activities are presented as
relative to the wild-type control (MMM). In the case of the full mutants
(mmm) there is a dramatic effect on expression levels of various enzymes in the
pathway of starch synthesis. In the case of the partial mutants (mMM and mmM)
5 7 0 there is almost no detectable change in expression levels. These data mdicated that
the alteration in starch quality observed with the single mutants is a . of
the u . _.~ A,U.~;VII of æveral enzymes as well as elimination of the enzyme coded for
by the mutated allele. By combining two mutant doses (e.g., wxwxWx) with other
doses of another mutation (e.g., AeAeae) there would be partial reduction in two575 enzymes without the U._~AIJ' seen in the rest of the pathway.
hgure 2 is a graph of the DSC scan of starches extracted from grain taken frorn
waxy, amylose extender and common (wild type) com. Such DSC scans enable one
skilled in the art to provide numerical data (see tables in text for data on Pea~A
Tc r ', Delta H, Peak II T~ . , Onset Temperature and Endset
5 8 0 Temperature). It is particularly noteworthy that the profile of the high amylose starch
is different from the common starch and waxy starch.
24
SUBSTITUTE S~IEET (RULE 26
WO 95~026 2 1 9 0 7 6 1 PCTIUS95107828
Figure 2b is a graph of the DSC scan of starches extracted from grain taken fromthe double mutant (aeaeae/wxwxwx) com. Such DSC scans enable one skilled in the
art to provide numerical data (see tables in text for data on Peak T; . Delta
585 H, Peak n T~ . , Onset Temperature and Endset Temperature). It is
particularly noteworthy that the profile of the double mutants is different from the
data provided in Figure 2a on common starch and the single mutants, waxy and high
amylose.
59 0 Figure 2c is a graph of the DSC scan of starches extracted from grain taken from
(aeaeAe/WxWxwx) com. Such DSC scans enable one skilled in the art
to provide numerical data (see tables in text for data on Peak T , Delta H,
Peak ~ T~ r ' , Onset Temperature and Endset Temperature). It is particularly
noteworthy that the profile of the intermutant starch is different from the starch of the
595 double mutant and appears to be similar to that of waxy starch.
hgure 2d is a graph of the DSC scan of starches extracted from grain taken from
(wxwxWx/AeAeae) corn. Such DSC scans enable one skilled in the art
to provide numerical data (see tables in text for data on Peak T~ , ` . Delta H,600 Peak 11 Temperature, Onset Temperature and Endset T: , ). It is particularly
noteworthy that the profile of the intermutant starch is different from the starch of the
double mutant and appears to be similar to that of waxy starch.
Figure 3a is a graph of Brabender data taken from common starch in either neutral
605 or acid conditions. Common corn starch shows substantial breakdown in viscosity
using acid conditions.
F~gure 3b is a graph of Brabender data taken from waxy starch in either neutral or
acid conditions. The waxy mutation most particularly affects viscosity of the starch
610 in neutral conditions.
SUBSTITUTE SHEET (PULE 26
WO 95/35026 . 2 ~ 9 ~ 7 ~ 1 PCT/US95/07828
Figure 3c is a graph of Brabender data taken from amylose extender (70%
amylose) starch in either neutral or acid conditions. High amylose starches increase
in viscosity in either acid or neutral conditions,
615 - - -
Figure 3d is a graph of Brabender data taken from double mutant
WAWA) s~arch in neutral conditions. Double mutant starches maintain
Yiscosity despite being IIVIIIV~J~.JU;~ for the waxy mutation.
62 0 Figure 3e is a graph of Brabender data taken from intemmutant (aeaeAe/WxWxwx)
starch in neutral conditions. It is particularly noteworthy from these data that the new
intermutant starches provide an increasing strength of viscosity similar to tbat seen
with high amylose mutants, despite containing no increase in apparent amylose
content.
625
Flgure 3f is a graph of Brabender data taken from intermutant (wxwxWx/AeAeae)
starch in neutral conditions. It is particularly noteworthy frvm these data that the new
intermutant starches providc an increasing strength of viscosity similar to that seen
with high amylose mutants, despite containing no increase in apparent amylose
630 content.
Example 2
This example illustr~tes the production of maize grain
635 possessing starch of the present invention. Maize plants of
various 1~.. ' can be converted to mutant genotypes
using either traditional breeding and/or v~h.lua~
techniques or else using ,, such as chemical
treatments of pollen. ~ the altemative, waxy mbreds and
6~ 0 hybrids can also be purchased from a number of suppliers and
foundation seed companies. Any maize line with good
26
SUBST~TUTE ~HEET (RULE 26)
WO 95/35026 PCrlUS95107828
2191~
agronomic traits and relatively high yield can be employed. In
the present invention, normal inbred lines are converted to
mutant inbred lines using chemical ,, followed by
6 4 5 careful selection of the mutant grain type from the segregating
offspring. This method is well known to those skilled m the
art (see for example, Neuffer, M.G. and Chang, M.T. 1989.
Induced mutations in biological and agronomic research.
Vortr. I`f' .g 16, 165-178). Any '~,
650 valuable inbred line may be used for this process. The lines
were confirmed to carry the mutation of interest by an allelism
test in which the line may be crossed with a known mutant
line, a process well known to those skilled in the art.
r. i ~, the kernels from the line will have the
655 appearance and iodine-staining ~ typical of the
mutation selected, a method well known to those skilled in the
art. In order to obtain the highest yields from the plants it is
best next to produce a hybrid cross between two inbreds, both
inbreds carrying the same mutation (e.g., both inbreds being
660 waxy or amylose extender types). It is preferred to produce
two hybrids, one being the male and being l.~ L~b~.,D for
one mutation and the other being the female and being
IIU~ IL~6~JUD for the other mutation. The male and female
hybrids can be made up of the same or different genetic
665 1 ~ ' it is merely import3nt for the two lines to have
similar maturities in the field (i.e., require similar heat units
from, to silking and pollen shed). In order to
make the intermutant aoss in the field it is necessary to
eliminate pollen production from the female. This can h
670 done by a variety of methods including, but not limited to,
hand pollination, hand and mechanical ~'~
27
SUBSTITUTE SHEET (RULE 26)
WO 95/35026 2 ~ 9 0 7 6 ~ PCT/US95107828
v6.~ genetic or ~: r~ male sterility into the
female plants, introducing male sterility through genetic
r ~ ti.~ll and use of chemical detasseling agents. The
675 grain of this crvss contains the present invention with a
genotype in the endosperm of aaAlBBb, with the starch froln
this genotype called intemmutant starch. It is well known to
those skilled in the art that the genetic v~k6.. ' can be
optimized for best starch qualities.
680
Example 3
Starch may be extracted from gr~un by a number of different
methods. The most commonly used method involves a "wet
685 milling" procedure known and used throughout the world
The basic principle involves steeping and starch separation.
The key step in this process involves softening the grain in a
steep tank a process which has been optimized to permit
optimal separation of the com grain ----r This method
690 was employed to extract the starch, wxwxWx/AeAeae from
the grain of an intermutant developed in accordance with
Example 2. The gemm was easily liberated intact and freed
from adhering endosperm and hull. The endospemm is
macerated under water, the starch was easi]y separated as a
695 white floc and gluten proteins are obtained as a yellow floc.
The grain was steeped for 30-40 hrs at 48-52C in tanks
usually holding 50-90 metric tons of grain. The steep water
contains 0.2% sulphur dioxide (SO2 gas is bubbled-in) and so
is mildly acidic (pH 4.0). The sulphur dioxide helps break-up
700 the protein matrix permitting the endosperm matrix to break-
up into granules. After steeping, the grain were coarsely
28
SUbSTlTUTE SHEET (RULE 26
w0 9~/35026 2 1 9 0 7 6 ~ 8
ground or pulped. The oil-rich embryo floats Io the surface
and dense starchy endosperm sinks, Separation is achieved
through hydreclones (continuous separation). The starch was
705 purified further after being milled through "an impact mill
known as Entoleter mill" which smashes the slurry at high
speed through counter-rotating grooYed plates made of
hardened steel alloy, followed by impact with an outer impact
ring. The defibered starch was separated from gluten by
710 ~ .~;r.. ~,. ;.. to give two fractions: protein (70% protein) arld
starch (2% protein). Processing i , ~ was maintained
above 45C to prevent microbial growth. The starch was dried
by flash-drying by injection into an air stream heated to 200
260F.
715
Varjous intermutant grains can have tbe starch purified and
prepared in this manner is suitable for a variety of food, feed
and industrial uses. It may be used directly as unmodified com
starch. It may be modified by chemical or physical treatments
72 0 that preserve granule structure and granules may be washed to
remove residual reactants. Bleaching is sometimes used lo
create , ..' starches. The starch ean be gelatinized
using a high l . treatment and sold directly as
gel$inized starch. Such starch may be ehemieally modified
7 2 5 and dried. The polymer itself may be hydrolysed partially or
completely to produce ' ' or glucose. Such
products ean be further modified by F~ to produce
ethanol for the gasoline industry, or the glueose ean be
converted to high-fructose corn syrup for the sweetener
7 3 0 industry.
29
SUBSTITUTE SHEET (RULE 26
w0 95135026 r~l,u~
2 1 9~76 ~ --
Example 4
The mutations called shrunken-2 (sh2), brittle-2 (bt2), dull
735 (du), sugary (su), waxy (wx) and amylose extender (ae)
encode isoforms of ADP glucose ~,~ U ~JIIUI ~l~,
enzyme, soluble starch synthase, bound starch
synthase and branching enzymes:
740 Shrunken-2 encodes one subunit of ADP glucose
Brittle-2 encodes one subunit of ADP glucose
~v~ h~
Waxy encodes granule bound starch synthase,
745 Amylose Extender encodes an isoform of branching enzyme,
Dull alters expression of an isoforms of soluble starch
synthase and branching enzyme,
Sugary alters expression and activities of soluble starch
synthases and ' ' ' ~ enzyme.
750
Using known mutants and the gen~dosage crossing
regimes we have examined the effects of altered gene
expression on starch deposition in griun (See figures). With
the bt2 mutan~ we see a progressive loss in measurable ADP
755 Glc l~J.. ' ,' ~' activity which correlates well v~ith a
loss in starch synthesis in the grain. The control strength
exerted by this enzyme over flux to starch cannot be
quantified from these data. In fæt our studies indicate that this
enzyme is one of the major 1' of the duration of
7 60 starch synthesis and may have little cont~ol over rate of starch
synthesis. This mutation does not appreciably alter starch
SUBSTITUTE SHEET ~RULE 26
w095135026 2190761 r~ s
structure. When the mutations are with sugary. dull, waxy and
amylose extender we now do detect changes in starch fine
structure (branched chain length changes as well as changes in
- 765 ~.. ,~ i.. ratios). In these cases there is more
minor control of flux to starch (except with the sugary mutant
which is used to make sweet-com genotypes). In all of these
cases it is the changes in ratios of the starch synthases and
branching enzymes which have resulted in alterations in starch
770 fine structure. A dramatic new finding in these studies was the
discovery that not only does the mutation reduce expression of
key enzymes, but also it induces an u. .~ - - of other
enzymes in the pathway. r. IS, it is only in the full
mutant (mmm) genotypes where we see changes in starch fine
775 structure .~ that the structural changes occur only
when there is an enzyme isoform loss in u ' - with an
enzyme isoform u. , Whilst not wishing to be
bound by this proposal, these data illustrate the means by
which starch structure may be influenced by not only reducing
7 8 0 expression (eg using antisense constructs) an enzyme but also
be ' '~ increasing expression (eg using sense
construct).
Plant ~ r, vectors for use in the method of the invention may be
785 constructed using standard techniques. Since these enzymes are localized in the
amyloplast . , of the cell, the gene construct requires the presence of an
amyloplast transit peptide to ensure its correct localization in the amyloplast. The
construct may carry the gene either in the partial ænse orientation or
in the antisense orientation. Expression of said gene in the plant results in a reduction
790 in expression of the enzyme by effects well known in the art as "sense . .,or antisense". When only a reduction in expression is needed the transit peptide is not
31
SUBSTITUT~ SHEET ~ULE 26)
wo 9~J3~026 9 ~ 7 6 1 ~ liD
required. However, when enzyme U.vlvhlJlCDD;vll iS rvequired then a correct piastid
targeting se~iuence is needed in the construct. Key enzymes required for this
invention include branching enzyme and soluble and bound starch synthase.
795 Branching enzyme [1,4-a-D-glucan: 1,4~-D-glucan v~ -D-(1,4-a-D-glucano )
transferase] convertD arnylose to .u.-.t' r ", (a segment of a 1,4-a-D-glucan chain
is transferred to a primary hydroxyl group in a similar glucan chain) sometimes cailed
Q-enzyme. Soluble starch synthase [ADPglucose: I ,4-a-D-glucan 4-a-D-
vUD~ ;lal~D~v~aDC] extends the ~ of alll~5v~vlill and perhaps also
8 0 0 amylose. Bound starch synthase LAi~Pgiucose: I ,4-v~-D-glucan 4-a-D-
yltl~lDfvlaDv] extends the chain length of amylose and perhaps aiso
alll,r~utfvvlill~
For any antisenæ or senæ- . . construct only a partiai cDNA clone is
805 re~iuired to be expresæd in a transgenic plant. Where enzyme U~v~CALllvDD;UII iS
rc~iuired, then a full length cDNA clone is needed. The se~iuence of maize branching
enzyme-I waD ~ , ' by Baba, T., Nishihara, M., Mizuno, K., Kawasai~i, T.,
Shimada, H., KobayaDhi, E., Ohnishi, S., TanalAa, K, and Arai, Y. (1~1. . ;r;.~
cDNA Cloning, and Gene ~xpression of Soluble Starch Synthaæ in Rice (Oryza-
810 sativaL) Immature Seeds. Plant Physiology. 103:565-573, 1993) . Starch branching
enzyme-ll from maize endosperrn was ~ ' by Fisher, D.K., Boyer, CD., and
Hannah, L.C. (Starch Branching Bnzyme-ll from Maize Fn~1n~i~rm Plant
Physiology. 1021045-lvn46~ 1993). The article by Mu, C., Harn, C., Ko, Y.,
Singletary, G. W., Keeling, P. i,. and Wasserman, B. P. shows an association of a
815 76i~Da l,ul.~ livie with soluble starch synthase I ætivity in maize (cv B73)
endosperm. Plant Joumai 6, 151-159 (1994). The maize waxy locus for
UDP-glucose starch glycosyl transferaæ was cloned in 1986 by Kioesgen, R.B.,
Gierl, A., Schwarz-Sommer, Z. and Saedler, H (Molecuiar anaiysis of the waxy locus
of Zea. Mol. Gen. Genet. 203, 237-244). Recently, the sequence for the maize sugary
820 locus was ûbserved by James, M. and Wright, A. (The Plant Journai) using
transposon ~, to locate the gene. The gene for any such i~rotein is thought
32
SUBSTITUTE SHEET (~ULE 26
-
WO 95/35026 2 l 9 0 7 6 1 PCTIUS95107828
to be a 1' ' ' ~ enzyme and may be used in constructs according to this
invention.
82 5 It is believed that the chloroplast transit peptides have similar sequences (Heijne et
al describe a database of chloroplast transit peptides in 1991, Plant Mol Biol
Reporter, 9(2)~ 126). Other potential transit peptides are those of ADPG
(1991, Plant Mol Biol Reporter, 9:1û4 126), small subunit
RUBISCO, ' synthase, ~1~ '' ' /Je-3P-d~.b~ub~ c and nitrite
830 reductase. For examp~e, the consensus sequence of the transit peptide of small
subunit RUBISCO from many genotypes has the sequenoe:
MASSMLSSAAVATRTNPAQAS~ VAPFTGLKSAAFPVSRKQNLDlTSlAS
NGGRVOC
The com small subunit transit peptide of RUBISCO has the sequence:
8 3 5 MAPI`VMMASSATATRTNPAQAS AVAPFQGLKSTASLPVARR.~R.~T r.
VASNGGRIRC.
The transit peptide of leaf starch synthase from com has the sequence:
MAALATSQLVATRAGLGVPDAS TFRRGAAQGLRGARASAAADTL
SMRTASARAAPRHQQQARRGGR FPSLWC.
840
Example ~
Production of fertile transgenic maize plants has been done
since 1990. Although a number of DNA deliwry systems are
845 known, the selection is a particle ~ ' .' As noted
above, constructs of the various maize mutant genes are
available from ~ in the U.S. and Europe. Attached
are a few examples of some of these constructs as shown in
Figures 4a - d. Figure 4c shows a promoter, which is CaMv
850 (cauliflower mosaic virus), an Adhl, the waxy gene, the
and the pat gene which is useful as a selectable
33
SUBSTITUTE SHEET (RULE 26
wo g~ 026 2 1 9 0 7 6 I PCT/[IS95/078Z8
marker and amp. Figure 4d is similar but shows the soluble
starch syntheses first isoform gene in the construct. Figure 4a
again has the same construct but shows the branching enzyme
855 first isoform. Figure 4b shows the second branching enzyme
second isoform. Of course, other constructs associated with
the gene mutants used in maize breeding are also available.
For purposes of this example reference is made to Figure 4c, the waxy construct.8 6 0 The purpose of this experiment is to form an inbred that has partial down regulation
of the waxy gene. If the inbred selected is already a mutant for ae, then the gr~un
produced by crossing with a non-mutant inbred will be the grain of an
Depending on the strength of the down regulation, the female inbreds grain will
resemble the mm*tmmY' or the mm*tm~ type of starch and grain. Clearly, the
865 ~.~, r~ " allows a more precise way of down regulation of the starch synthesis
activity such that the alteration of tbe starch can be finely tuned.
To assure reasonable levels of down regulation of the waxy gene, the
target tissue is immature zygotic embryos, through cll~blylO
870 callus can also be employed. Immature zygotic embryos from A188 plants 12 days
after pollinated with the B73 ae inbred can be selected. The medium for the callus
was 6 mM L,proline, 2% (w/v) sucrose, 2 mg/l 2,4- d;~ lu~ acid (2,4-
D) and 0.3% (w/v) Gelrite (Caroline Biological Supply) (pH 6.0). Callus is grownand suspension cultures were initiated.
875
A MS-based liquid medium containing 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2
mg/l l-l .' ' ' ~ acid (NAA), 6 mM proline, 200 mg/l casein hydrolysate
(Difco T ' ), 3% (w/v) sucrose, and 5% (v/v) coconut water (Difco
T ~ ) (pH 6.0). Cell , were maintained in this medium in 125 ml
880 r ~ , . flasks at 28C in the dark on a gyrating shaker at 125 rpm.
34
SUBSTITUT~ SHEET (RULE 2~)
wo ssl3so26 2 1 9 0 7 6 ~
The . ' Il.dli~". vector in Figure 4c is selected. This plasmid contains a 355-
ladh-pat nos 3' selectable gene expression cassette.
8 85 The cell r ' are sieved and then suspended in 5 ml of suspension medium
and placed on filter paper through vacuum. The construct was coated into particles
as is know in the art. The plates were then ~ ' ' ' The cells are then transferred
to a N-6 medium and after 14 days II_~- F~ cells are selected by I mg/l
bialaphos. The cells are then suspended in a medium containing .6% (w/v) (Sea-
8 9 o Plaque; FMC) and held at 37C.
Two to five weeks later, growing calli are removed and transferred to the surface
of fresh selection medium. Plants were ~ O ' in a MS based medium having
6% sucrose Ig/l myo-inositol, I mg/l NAA (34), and 0.3% (w/v) Gelrite (pH 6.0).
895 Next the embryo " cccurred in a MS media of 0.25 mgll NAA and 3%
(w/v) sucrose and light. Plants are grown and transferred to the O ' The
expression levels in the plant can then be evaluated.
The plant is bred and developed to an inbred having the mutant and the down
900 reO~ulated pathway. Alternatively, the selected inbred can have the mutant crossed
onto a transgenic after 1.. r ....- ~;.... to form the desired starch in the grain when the
trdnsgenic plant is employed as the female.
Exampk 6
905
This example illustrates the i ' I . . of the
starch of present invention compared to other starches. The
O ' i . are listed in Table 3 below.
SUBSTITUTE SHEET (RULE 26)
WO 95/35026 PCT/I~S9~/07828
2~ qQ~
910 Tsble3
h Samvles % Amylose~ T~ ture C
NatiYe common maize 2S 71
2 AMY V native 57 80
3 AMY Vll native 73 90
4 Present Invention 21 73
5 Native aewx 25 gO
6 Native wxwxwx 3 72
~Values rounded to a whole number
Sample I was a commercial product sold by American Maize-Products Company
of Hammond, Indiana. The percent amylose and the ~ A~ for
915 Sample l above are mear~ values determined by random sampling of product. The
9g% confidence level for percent amylose and ~;, IA~ A~ I j r ' ~ are 25.9 to
29.3 and 68.7 to 72.9, ~
AMY V and AMY VII are commercial high amylose com starches sold by
920 American 1'~ -Pr. ' Company of Hammond, Indiana. The percent amylose
and the t, l''; ~';'' , . in Table 3 above are mean values determined
from a random sampling of product. The 99% confidence interval for the percent
amylose in AMY V and AMY Vll was 53.4 to 62.5 and 65.5 to 73.8"~li~
The 99% confidence interval for the E,~ A~ -- i r ' for AMY V and
925 AMY vn was 72.8 to 84.4 and 83.1 to 90.8, .~li~ Both AMY V and AMY
Vll were grown in native maize.
Starch Sa~nple 4 w~ u~ to the Pr~sent Invention, while Sample 5
WII~IUIIJ~ to the average values from ~xample I of U. S. Patent No. 5,009,911.
930 Sample 6 cu.-. r to a commercial waxy starch sold by American Maize-
Products Company.
36
SUBSTITUTE SHEET ~RULE 26)
WO 95135026 2 1 9 3 7 6 1 r~l,o~)s. I~ ~
The method for ~ ~ botb the percent amylose and the g.
_ was:
935 The percent amylose ~vas determined using standard iodine
procedures wherein the starch is first gelatinized with sodium hydroxide and then
reacted with an iodine solution and the resulting sample measured using a
;"~11" ' in a I cm cell at 600 nm against a blank of 2% iodine solution.
940 The DSC ,, ' i , was measured using a scanning calorimeter
~ by Mettler Moddle No. 300 using 30% starch solids following the
procedure outlined in the owner's manual for that model.
It is readily apparent from Table 3 above that the L ' : . _ of the
9 4 5 srarch of the present invention is comparable to common com starch.
Example 7
T.his example illustrates the gel strength of a sol made from
950 the com starch of the present invention compared to a sol
made from aewx com starch, a sol made from common com
starch, and a sol made from wxwxwx starch. The results of
the test are reported in Table 4 below.
955 ~ Table 4
SamDle str~ne!h (grams)
Present ~vention 159.5
Common Com Starch 225.0
aewx Com Starch 55.0
WaxyComStarch 16.0
37
SUBST~TU~E SHEET (RULE 2
WO 95/35026 PCT/US95/07828
2l 90761
In order to perform the gel strength test rcported in Table 4 above, sols were
prepared by mixing water with starch and subjecting the slurry to a rapid heat mode
in the Brabender Visco-Amylograph to heat the sample to 50 C. Once 50 C was
960 reached, the instrument was set at a controlled rate of heating, ~.5C1minute, until a
of 95 C was reached. The sample was then held at 95 C for 30
minutes. Next, the sample was cooled at 1.5 C to a i . c of 50 C for 30
minutes. Portions of these sols were added separately to 4 ounce jars into which a
plumger was placed. The sols were then allowed to stand at ambient conditions for
9 65 24 hours. Gcl strength was measured by ,:" ,, the force needed to remove the
plunger from the sol.
This example illustrates that the gel strength of a sol made in accordance with the
present invention is comparable to common com starch sols.
970
E:xample 8
This example illustrates the difference between aewx
starch, a waxy starch wherein the plant had a triple dose of the
975 waxy gene, and the starch of the present invention. All
starches were obtained from maize.
All starches were tested for their rheological prope~ties. Each starch was subjected
to the same test procedure using the same method. The starch granules were pasting
9 8 0 using a Brabender Visco-Amylograph with the cooling probe down and with the 750
g cm cartridge. The starch slurry, 5.5% initial solids, was rapidly heated to 60 C in
the Brabender cup, and then pasted while increasing the I . to 95 C at 1.5
C per minute. The starch paste was held at this t~ . for 20 minutes, and then
' 1~ loaded onto the measuring geometry of the rheometer which had been
9 8 5 preheated to 70 C. A four-patt rheological ~ -, was performed. The gel
38
SUBSTITUTE SHEET ~RULE 26
woss~3so26 2 1 90 7 6 1 ~ C,07828
cure segment which monitored the formation of structure at .2 hertz and .2% strain
(well within the linear viscoelastic region) was measured as the sample was cooled
from 70 C to 25 C and held for 4 hours. Also messuled was the strain sweep,
which measures the rheological response of the paste or gel to increasing levels of
9 9 0 strain, also at 02 hertz, at 25 C.
Using this technique, distinct differences between aewx starch and the starch ofthe present invention were found. Figure 5 shows the results of the gel cure analysis.
The starch of the present invention, while having an initial Modulus (G') lower than
9 9 5 aewx starch, more quickly formed structure or gelled as evidenced by the rapid rise in
G'. Thus the starch of the Present Invention was distinct from aewx starch in the rate
of gel formation despite similar apparent iodine binding contents. Figure 6
shows the results of the strain sweep analysis only for the starch of the present
invention and the aew% starch. Here the starch of the present invention showed
10 0 0 dilatant behavior as evidenced by the increase in G' as the strain level was increased.
Under the applied levels of strain, the structure was not destroyed. In contrast, the
structure of the ~ç~ starch was rapidly destroyed when the applied strain becamegreater than 4%. Thus, when compared to aewx starch, the starch of the present
invention formed a gel which didn't break under the applied strain of this test. In
1005 contract, aewx starch showed much less time-dependent structure formation and did
"break" or was destroyed by the strains applied in this technique.
Example g
1010 This example illustrates preparing a thickena ~ in
accordance with the present invention.
The starch of the present invention is mixed with water in an amount to produce a
slurry having 10% by weight starch. The sol has a short texture and a bland taste.
1015 The sol when cooked at about 90 C for lO minutes produces a thickener
39
SUBSTlTUT~ SHEET (RULE 26
w095/3s026 21 q~7~ &
." which had better clarity than a similar thickener ~ ;.- . made from
common com starch and a shorter texture.
Example 10
1020 ~
This example compares the mouth feel of a gel made from the
starch of the present invention to a gel made with a common
starch.
1025 The common starch and starch of the present invention were pasted using a
Brabender visco-a~ . The starch was slurried at 5.5% solids, and then heated
using the rapid heat mode to 50 C. Using controlled heat of 1.5 C per minute the
slurries were heated to 95 C, and then held at this i , ~i for 30 minutes. The
final solids was 5.9%. The sample starch pastes were then poured into small jelly
1030 jars, covered with cellophane, and allowed to age 24 hours before analysis. A taste
panel was then asked to rank the samples for the following attributes.
First they ranked the two for relative firmness to the touch.
~Qmm~ Present ~vl~n~inn
5.1 5.6
1035
Next, they ranked the relative break, firmness, and dearing of this sample whilebeing masticated.
Present
Common ~j~
Degree of clean break 3.2 8.5
Firmness 4.4 4.5
Rate of clearing 2.5 4.6
SUBSTITUTE SHEET (RULE 26)
wo gs/35026 2 1 9 0 7 6 1 PCTIUS95JO'1828
These results show that the firmness of these samples is similar. However the
10 4 0 starch of the present invention has a much cleaner break while being masticated.
r. I~ u~e~ it tends to clear from the mouth faster than a common based starch
gel. The panel all agreed that the starch of the present invention produced a gel
which had a "clean" mouth feel similar to that of a gelatin or a pectin.
10~5
Exsmple ll
This example illustrates making a gum candy using the starch
of the present invention.
10 5 0 The following ingredients and procedure are used:
Table 5
% by Weight
In redients Present~v~.n~inn
44/62 Corn Syrup Unmixed 56.34
Sugar, fine granular 23.98
Water 7.73
P~sent Invention Starch l 1.80
Citric Acid Qo7
Sodium Citrate 0.08
100.00
Procedure
1055 All ingredients are mixed and then cooked to 340 F using
,UIl~,l..iUllal equipment such as a jet cooker. The cooked
slurry is then poured into candy molds and allowed to solidify.
41
SUESTITUTE SHEET (RULE 26)
woss/3so26 2.~ q~761 r~l,u .
Example 12060
This example illustrates making a Bavarian cream pie using
starch of the present invention.
The following ingredients and procedure are used:
1065
Table 6
% by Weight
In~redients Present ~vention
Whole milk, fresh 3.5% 72.794
Sugar, fine grain ] 7.586
Salt, Flour QlOl
Present Invention SAI0
Banana Flavoring 0.300
Egg Yolk, fresh 3.809
100.000
Procedure
1070 All of the pie filling ingredients except for egg yolks are
combined and cooked at 195 F for 3 to 5 minutes. Then the
ingredients are cooled to 120F with constant stirring. Next,
egg yolks are added and the admixture well blended. This
mixture is then added to a w~ iullal pie crust and allowed
1075 to cool to room i r ' before ser~ing.
42
SUBSTITUTE SHEET (RUL~ 2~)
W09S/35026 2 1 9 0 7 6 1 PCTIUS9Sl07828
Example 13
This example illustrates preparing a lemon pie filling with the
- 10 8 0 starch of the present invention.
The following ingredients and procedure are used:
Table 7
% By Weight
In~redie~t~ p~cPnt InV~ntj~n
Water 62.94
Sugar 19.30
~'' ' 6.67
Present Invention 4.50
Corn Syrup Solids 2.50
Lemon Juice 2.50
Vegetable Shortening 1.03
Salt 0.23
Citric Acid 0.20
Emulsifier o.lo
Lemon Oil (2x) 0.03
100.00
1085
P~cedure
Half of the water is combined with the sugar and brought to
a boil. All of the remainmg ingredients are slurried together
and then added to the boiling sugar and water. The
1090 ; , of this mixture is then adjusted to 200 F. and
held there for two minutes. The mixture is then poured mto
prepared pie crusts and allowed to cool and solidify.
43
SUBST~TUTE SHEET (RUL~
wo ss/3so26 r~m~
Example 14
10 9 5 This example illustrates making a chocolate mousse using the
starch of the present invention. The formulation in Table g is
employed to prepare a mousse mix.
Tllble X
1100
%
Frodex 24-924 39.20
Sugar (Baker's) 30.75
Whiptreme 3554 (Kerry b~gredients) 12.88
Starch of Present Invention 9.80
Cocoa, Dutch Red (Gill .5c Duffus Products) 7.17
Leceitreme 40 (Kerry Ingredients) 0.20
Flavor as desired
100.00
Procedure
Combine ingredients to form a uniform blend.
1105 Use
Combine 200 gms. of mousse mix with l cup (250 grs.)
milk. Using an electric mixer, combine on low speed for l
minute. Scrape bowl. Mix on high speed for 3 minutes, until
light and fluffy. Spoon into serving dishes and refrigerate for
1110 l hourbeforeserving.
To prepare the mousse mix, all the ingredients are mixed.
To prepare the mousse itself, 200 grams of mousse mix are
44
SUBSTITUTE SHEET ~RULE 26)
Wo 9513s026 1 ~ ~
~ 2 1 ~076 1
combined with 250 grams of milk and combined at a low
1115 speed. Then the mixture is sti~ed at a high speed to make it
light and fluffy and the mixture is ,, ' ,, ' for one hour.
In this way, a light, fluffy mousse is prepared.
Accordingly, the present invention has been described with some degree of
112 0 I,~u Li~u L ;~y directed to the preferred ~ ' " of the p~sent invention. It should
be Tr 1, though, that the present invention is defined by the following claimsconstrued in light of the prior art so that ,~ or changes may be made to the
preferred e ' ' of the present invention without departing from the inventive
concepts contained herein.
112 5
SU~STITUTE SHEET (RUL~ 2~)
