The present invention provides refrigeratable yeast-leavened dough compositions and methods of making such doughs. The dough composition and the strain of yeast used therein are chosen to limit the total leavening action of the yeast by controlling the amount of substrate in the dough fermentable by the yeast. Dough compositions made in accordance with the invention are capable of being leavened at elevated temperatures, yet stored in a sealed container at refrigeration temperatures for extended periods of time. In one embodiment, a maltose negative yeast is used and sucrose or the like is added to the dough to serve as a fermentable substrate for the dough; this dough is suitable for storage times of up to 30 days or so. In a particularly useful embodiment which is suitable for even longer storage at refrigeration temperatures, the yeast used in the dough is substantially incapable of fermenting carbohydrates native to the dough and a predetermined quantity of a non-native carbohydrate fermentable by the yeast (e.g. galactose) is added to the dough to provide the desired amount of proofing.

FIELD OF THE INVENTION 
The present invention relates to refrigeratable dough products for use in 
making edible baked goods. In particular, the invention provides a 
yeast-leavened dough which can be stored for extended periods of time at 
refrigeration temperatures. 
BACKGROUND OF THE INVENTION 
A wide range of refrigeratable dough products are currently available to 
consumers for producing numerous different baked products. These 
refrigerated doughs range from doughs for biscuits and breads to sweet 
rolls to cornbread products. These dough products are rather popular with 
consumers because they are very convenient and easy to use, Most of these 
products are sold in a pre-proofed state so that they can be opened to 
remove the dough and the dough can be baked immediately. Packaging and 
selling doughs in a pre-proofed state omits any necessity on the part of 
the consumers to carefully proof the dough for an extended period of time 
before baking it. 
In producing refrigeratable dough products, suitably sized portions of 
unproofed dough are placed in individual containers. The dough is then 
proofed within the container, such as by holding the dough at an elevated 
temperature, causing the dough to expand. The dough will continue to proof 
until a positive internal pressure of about 15-20 psi is attained; most 
such containers will rupture or explode if the internal pressure of the 
container substantially exceeds about 40 psi. Such products are desirably 
capable of storage at refrigeration temperatures for at least a couple of 
weeks, and desirably as long as a few months, without any significant 
degradation of the quality of the dough or any substantial likelihood of 
having the containers rupture. 
One disadvantage of refrigeratable dough products on the market today is 
that these doughs generally cannot be leavened with yeast. When yeast is 
used in a dough, the yeast cells will tend to continue to grow, or at 
least continue metabolization, even at refrigeration temperatures. The 
yeast therefore continues to produce carbon dioxide over the entire 
storage time, unless the dough is stored in a frozen state. Although 
allowing yeast to ferment for the entire shelf life of the dough may work 
if the dough is intended to be used immediately, extended storage (e.g. 
about two weeks or more) in a sealed container generally will not work 
because the pressure in the container will quickly build and rupture the 
container. If a conventional yeast-leavened dough were placed in a 
standard dough product container, the container may be expected to fail in 
no more than about two days. Additionally, continued activity of the yeast 
beyond the desired degree of proofing can deleteriously affect the 
organoleptic and rheological properties of the dough, producing 
unacceptable final baked products. 
To date, manufacturers of refrigeratable doughs have had to replace yeast 
with chemical leavening agents, such as baking soda or the like. Such 
chemical leavening agents generally comprise a combination of a leavening 
acid and a leavening base, with the acid and base portions reacting to 
generate carbon dioxide, causing the dough to rise. One of the primary 
advantages of such leavening agents is that their behavior is based upon a 
predictable chemical reaction, permitting one to readily control the 
volume of carbon dioxide produced to leaven the dough. Once the chemical 
reaction of the leavening agents has proceeded to completion, carbon 
dioxide production ceases. 
Although a chemically leavened dough product can be stored for extended 
periods of time at refrigeration temperatures, the final baked product 
obtained by baking such a dough is noticeably inferior to a product made 
with a yeast-leavened dough. Products made from yeast-leavened doughs are 
widely acknowledged to have superior taste, aroma and texture than those 
made with chemical leavening agents. Commercial dough manufacturers 
frequently add ingredients for the sole purpose of simulating 
yeast-leavened doughs. For instance, these manufacturers frequently add 
yeast flavoring, such as inactive pasteurized yeast cultures, to the 
chemically leavened dough. Even with such additives, baked products made 
from chemically leavened doughs lack the characteristic flavor and aroma 
of yeast-leavened dough and continue to exhibit relatively poor texture. 
Others have attempted to solve the problems associated with storage of 
yeast-leavened doughs by storing the doughs at freezing temperatures 
rather than refrigeration temperatures. Frozen yeast-leavened doughs can 
yield baked goods which are noticeably better than chemically leavened 
refrigerated doughs. Yeast becomes inactive when frozen, thereby avoiding 
the problems associated with continued carbon dioxide evolution at 
refrigeration temperatures. 
In a published European patent application (Published European Patent 0 442 
575, published Aug. 21, 1991), Gist-Brocades describes a dough composition 
which uses a substrate limitation concept. In accordance with this 
disclosure, a dough is leavened with a maltose negative yeast (a yeast 
which cannot ferment maltose) and the dough is frozen. Gist-Brocades 
states that the dough may be thawed, proofed and baked anytime the same 
day without having to carefully monitor the proofing time. However, this 
dough is not designed by Gist-Brocades to be stored at refrigeration 
temperatures for extended periods of time, e.g. two weeks or more. 
However, frozen doughs simply are not as convenient as pre-proofed 
refrigerated dough products. Whereas such refrigerated doughs can be baked 
immediately after removal from the container, frozen doughs must be 
allowed to thaw prior to baking. Also, since proofed dough does not 
survive freezing very well, frozen doughs generally must be proofed after 
thawing and prior to baking. This can further delay the baking of the 
dough. The consumer must spend more time monitoring the proofing process 
to avoid over-proofing the dough, making sure to place the dough in the 
oven for baking at the right time. Not only do such frozen doughs require 
more attention than do refrigerated dough products, it also requires the 
consumer to plan well in advance so the dough can be thawed and proofed to 
provide the baked goods at the desired time. 
Hence, there has been a long-felt need in the industry for a yeast-leavened 
dough that can be stored at refrigeration temperatures for extended 
periods of time. To date, though, commercial producers have been unable to 
make and sell refrigeratable yeast-leavened doughs suitable for 
large-scale commercial production and extended shelf life, despite the 
obvious economic potential of such a product. It appears that the problems 
associated with the continued generation of carbon dioxide by the yeast 
have precluded any such product. 
SUMMARY OF THE INVENTION 
The present invention provides a method of making refrigeratable 
yeast-containing doughs and dough products made therewith. In another 
aspect, the invention provides a yeast-leavened refrigeratable dough 
composition, a dough product comprising refrigeratable dough in a 
container, and a baked product made from such refrigeratable dough. In 
accordance with the invention, a preselected strain of yeast is mixed with 
flour and water and, perhaps, other ingredients to form a dough. The yeast 
and the dough composition are chosen so that the total amount of 
carbohydrate or carbohydrates fermentable by the yeast in the dough is 
limited. 
In one preferred embodiment, the yeast is substantially incapable of 
fermenting carbohydrates native to the flour used in the dough and a 
non-native carbohydrate, such as galactose, is added to the dough in an 
amount selected to provide the desired volume of carbon dioxide. By so 
doing, one may limit the maximum volume of carbon dioxide which the yeast 
can generate. This, in turn, prevents generation of sufficient carbon 
dioxide to rupture a sealed container of dough, even if the temperature of 
the dough is inadvertently elevated. 
In another preferred embodiment, the yeast is capable of fermenting 
selected sugars native to the dough system which are naturally present in 
only limited amounts. Such sugars should be naturally present in amounts 
no more than, and desirably less than, that necessary to generate the 
volume of CO.sub.2 necessary to proof the dough, with any additional sugar 
required to proof the dough being supplied by adding quantities of that 
sugar to the dough composition. 
In one such embodiment, the yeast is substantially incapable of fermenting 
any carbohydrate native to the dough except fructose. Fructose 
concentration in wheats is initially on the order of less than 0.1 weight 
percent (wt. %). Through the action of various enzymes that can break down 
the disaccharide sucrose in the wheat into glucose and fructose 
monosaccharides, fructose concentration in the yeast may increase over 
time. Nonetheless, the concentration of fructose in most wheat-based dough 
systems is less than that necessary to generate the 100-200 ml CO.sub.2 
per 100 g of dough required to adequately proof the dough. Additional 
fructose is added to the dough to generate the desired degree of proofing. 
The method may also include the additional steps of placing the resultant 
dough in a pressurizable container and heating the dough within the 
container to an elevated temperature for proofing. Once the dough in the 
container has been proofed, the temperature of the dough within the 
container is reduced to refrigeration temperatures and the dough is stored 
at refrigeration temperatures for an extended period of time. A method of 
this embodiment may further comprise the step of removing the dough from 
the container and baking it to produce a baked good.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the present invention, a dough product is prepared 
wherein the dough composition and the yeast used therein are chosen in a 
manner that effectively and controllably limits the leavening action of 
the yeast by controlling the amount of substrate fermentable by the yeast 
in the dough. Strains of yeast which do not ferment certain carbohydrates 
are known in the art; often, two different strains of the same species of 
yeast are unable to ferment the same sugars. Therefore, a strain of yeast 
may be utilized in a dough composition which is capable of fermenting only 
selected sugars. By controlling the total amount of those sugars in the 
dough composition; the amount of fermentation can be controlled. 
As explained above, even at refrigeration temperatures, most yeast will 
generate carbon dioxide. If the sugar substrate fermentable by the yeast 
is limited, carbon dioxide generation will substantially cease when the 
sugar is exhausted. Hence, by either allowing the yeast to-metabolize the 
fermentable sugars in the dough for a given period of time prior to 
canning or controlling the sugar content of the dough, carbon dioxide 
generation by the yeast can be substantially terminated once a certain 
predetermined volume has been reached, regardless of the temperature of 
the dough. Accordingly, the total volume of carbon dioxide generated in 
the container can be prevented from reaching a level sufficient to 
increase internal pressure and rupture the container. 
Wheat flours used in most commercial dough manufacturing operations contain 
about 5 weight percent (wt. %) of damaged starch. Alpha- and beta-amylases 
(inherent in wheat flour) convert such starch into maltose, among other 
sugars. Maltose and some of the other sugars produced by the action of the 
amylase are metabolizable by many strains of yeast. 
In an earlier embodiment of the invention, a strain of yeast which did not 
ferment maltose, referred to as "maltose negative," or just "MAL-," was 
chosen. Such yeast can usually ferment other types of sugars, such as 
sucrose or dextrose. A number of yeasts which ferment sucrose but not 
maltose ("SUC+/MAL-") are commercially available, including the following 
strains of Saccharomvces Cerevisiae: DZ (CBS 109.90), DS 10638 (CBS 
110.90), DS 16887 (CBS 111.90) V 79 (CBS 7045), and V 372 (CBS 7437) 
Approximately 100-200 ml of CO.sub.2 per 100 grams of dough at 32.degree. 
C. is usually sufficient for proofing. The total amount of fermentable 
sugar in the dough was adjusted in an attempt to limit the volume of 
carbon dioxide gas produced by fermentation of the entire fermentable 
sugar supply. 
EXAMPLE 1 
In order to test a dough product leavened with a MAL- yeast as a means of 
providing a refrigeratable yeast-leavened dough composition, water and a 
MAL- yeast were slurried together to produce a total combined weight of 
approximately 194 grams. The slurry contained 189 grams of water and 4.8 
grams of the yeast. The yeast used in making the slurry was a MAL- strain 
of yeast which was obtained in a paste form. The paste was mixed with 
water at room temperature (approximately 23.degree. C.) and allowed to sit 
at room temperature for about 10-15 minutes. 
To this slurry was added 261.74 grams of flour, 18.77 grams of wheat gluten 
pre-blend, 3.60 grams of salt and 1.20 grams of dextrose. The wheat gluten 
pre-blend was 75 wt. % vital wheat gluten, 21.9 wt. % hard, high gluten, 
enriched ingredient flour, 2.50 wt. % xanthan gum, and 0.616 wt. % 
azodicarbonamide premix. The resulting dough composition therefore 
contained 54.53 wt. % flour, 3.91 wt. % gluten pre-blend, 0.75 wt. % salt, 
and 0.25 wt. % dextrose, with a final concentration of 1.00 wt. % MAL 
yeast. 
The dough composition was mixed in a Farinograph.TM. mixing bowl at 60 rpm 
for 4.5 minutes. Immediately after mixing, a 50-gram sample of the dough 
composition was placed into a Risograph.TM. testing machine. The Risograph 
is commercially available from Sheldon Manufacturing, Inc. for detecting 
the volume of gas, e.g. carbon dioxide, generated by a sample and the rate 
at which the gas is generated. 
FIGS. 1 and 2 show the data collected in the Risograph for the sample. Of 
particular interest, the dough appeared to effectively cease producing 
carbon dioxide after about 1500 minutes at 32.degree. C. Dough products 
made with this dough by placing the dough in standard spirally wound 
refrigeratable dough containers were found to maintain acceptable internal 
pressures, e.g., below about 20 psi, for about 25 days. However, carbon 
dioxide once again began to be generated by the dough after about 25 days. 
This renewed activity of the yeast in the dough was projected to be 
sufficient to generate enough carbon dioxide to cause all of the 
containers of Example 1 to rupture after about 50-55 days. 
It has not been conclusively determined why the yeast became active after 
apparently substantially ceasing fermentation. However, one factor which 
is believed to have contributed to the generation of additional carbon 
dioxide, and subsequent failure of the containers, is a change in the 
carbohydrates present in the dough. As noted above, alpha- and 
beta-amylases, which are inherent in wheat flours, act on carbohydrates 
present in the dough, and particularly in the flour. Over time, these 
amylases break down oligosaccharides which are not fermentable by the 
yeast, such as maltose and maltotriose, into sugars which can be fermented 
by the yeast. Accordingly, it is anticipated that, even if the yeast used 
in such a dough composition were truly maltose negative, the changing 
carbohydrate profile of the dough may present sugars which are fermentable 
by the yeast. Accordingly, the dough could continue to generate carbon 
dioxide and cause containers to rupture. 
Thus, a dough product made with a MAL- yeast and a limited amount of 
initial maltose in the composition can be useful for storage at 
refrigeration temperatures for shorter periods of time, with a storage 
period on the order of about 30 days or less. If such dough products were 
stored for significantly longer periods of time, it is likely that the 
containers would begin to fail. Although a shelf life of 30 days may be 
suitable for some applications, current refrigerated dough products are 
expected to have an anticipated shelf life at refrigeration temperatures 
of 90 days or more. Accordingly, this MAL- embodiment of the invention may 
have only limited commercial application, with commercial use being 
limited to institutional markets, such as in-store bakeries and the like, 
where an anticipated shelf life of 30 days may nonetheless be considered 
acceptable. 
In accordance with a preferred embodiment of the present invention suitable 
for significantly longer storage at refrigeration temperatures, the strain 
(or strains) of yeast used in the dough are substantially incapable of 
fermenting carbohydrates which are native to the flour. In the case of 
doughs using wheat flour, these native carbohydrates include sugars such 
as maltose, sucrose, glucose, fructose and various oligosaccharides made 
up of these sugars. If other flours were to be used, of course, there may 
be some variation in the sugars native to such a flour. 
Use of such a yeast has been found to effectively prevent the yeast from 
fermenting any carbohydrates in the dough which are either initially 
present in the dough composition or result from the action of alpha- and 
beta-amylases on the carbohydrates initially present in the dough. A 
predetermined quantity of a non-native carbohydrate which is fermentable 
by the yeast may be added to the dough to provide the desired amount of 
proofing. Once that substrate is consumed, the fermentation activity of 
the yeast appears to substantially cease, preventing further carbon 
dioxide generation and avoiding over fermentation of the dough. It has 
been found that dough compositions in accordance with this embodiment of 
the invention can be used to make dough products which can be stored for 
periods of time in excess of 90 days without rupturing or exploding. 
The non-native carbohydrate which can be fermented by the yeast strain in 
the present dough can be virtually any carbohydrate which does not 
naturally occur in the flour. This carbohydrate is preferably a sugar or 
an oligosaccharide, though. For instance, the fermentable, non-native 
sugar may be galactose or lactose, a disaccharide of glucose and 
galactose. 
In one particularly preferred embodiment, the yeast is capable of 
fermenting galactose, which is not native to wheat flours, but is 
substantially unable to ferment any sugars which are native to wheat 
flour; this yeast is referred to below as a "galactose positive" or "GAL+" 
yeast. This GAL+ yeast is mixed with flour, water and galactose to form a 
dough. The amount of galactose in the dough is selected to limit the 
activity of the yeast so that the dough is proofed no more than the 
desired degree. As noted above, in most circumstances about 100-200 ml of 
carbon dioxide per 100 grams of dough at 32.degree. C. is sufficient to 
proof the dough. Accordingly, the weight percentage of galactose in the 
dough composition should be chosen to generate no more than approximately 
200 ml of carbon dioxide per 100 grams of dough at 32.degree. C. The 
amount of galactose necessary to generate this volume of carbon dioxide 
will have to be determined on a case-by-case basis as the amount may vary 
for different strains of yeast. 
Given the present disclosure, it will be well within the ability of those 
skilled in the art to make yeasts which are substantially incapable of 
fermenting carbohydrates native to flour but capable of fermenting other 
carbohydrates. Such yeasts can be made through standard methods of 
crossing yeast strains, isolating suitable strains having the desired 
properties and the like. These types of common techniques are described, 
for example, by Sherman et al. in Methods in Yeast Genetics, the teachings 
of which are incorporated herein by reference. Of particular interest in 
the Sherman et al. publication is Section III, entitled "Making Mutants", 
which appears on pages 273-369 of this reference. 
Lobo and Maitra teach a method of rendering a hexokinase negative strain of 
S. Cerevisiae glucokinase negative (i.e., a method for making a GAL+ yeast 
strain) using standard techniques in "Physiological Role of 
Glucose-Phosphorylating Enzymes in Saccharomyces Cerevisiae," Archives of 
Biochemistry and Biophysics 182, 639-645 (1977), the teachings of which 
are incorporated herein by reference. In accordance with that method, the 
hexokinase negative strain was mutagenized with 
N-methyl-N'-nitro-N-nitrosoguanidine in yeast extract-peptone medium (YEP) 
containing 50 mM glucose-free galactose, and a glucokinase-negative mutant 
was isolated by replica plating from a YEP galactose plate to a YEP 
glucose plate as a glucose-negative colony. The genotype of the mumant, 
determined by independent genetic analysis, was hxk1 hxk2 glk1, where hxk1 
and hxk2 stand for genes coding P1 and P2 hexokinases respectively, and 
glk1 for the genetic determinant for glucokinase synthesis. 
Although Lobo and Maitra teach one suitable method of making a yeast for 
use in accordance with the present invention, others methods will be 
apparent to those skilled in the art. Those in the art will also realize 
that other strains of yeast which are substantially incapable of 
fermenting carbohydrates native to a particular flour but capable of 
fermenting non-native carbohydrates other than galactose can be made by 
known methods. 
EXAMPLE 2 
In order to test the ability of a GAL+ yeast to ferment carbohydrates which 
are native to a common dough system, a dough composition containing GAL+ 
yeast was prepared. This dough formula included 870075 g (58.05 wt. %) 
wheat flour, 529.80 g (35.32 wt. %), water, 58.20 g (3.88 wt. %) of the 
wheat gluten preblend used in Example 1, 11.25 g (0.75 wt. %) salt and 
28.50 (2.00 wt. %) yeast. The yeast used in this experiment was a GAL+ 
strain of Saccharomyces Cerevisiae designated as D308.3; this yeast was of 
the genotype .alpha.hxk1 hxk2 glk1 ade1 trp1 his2 met4. This yeast is 
available to the public from the Yeast Genetic Stock Center at the Donner 
Laboratory in the Department of Molecular and Cell Biology of the 
University of California, Berkeley (YGSC); in the Seventh Edition of the 
catalog of the YGSC dated Mar. 15, 1991, this strain of yeast was listed 
under stock no. D 308.3. This yeast strain was also deposited with the 
American Type Culture Collection of 12301 Parklawn Drive, Rockville, Md. 
20852, USA (ATCC), on Mar. 5, 1993, under number ATCC 74211. 
Isolated colonies of the D308.3 yeast from solid galactose agar plates were 
used to inoculate six 50 ml culture flasks containing liquid yeast 
extract-peptone ("YEP") and galactose. The samples were incubated for 
approximately 20 hours at about 30.degree. C. and then used to inoculate 
six one-liter flask samples, which also contained YEP and galactose. These 
1 L flasks were incubated for about 24 hours at 30.degree. C., followed by 
incubation at about 24.degree. C. for approximately 20 hours. 
This yeast was then harvested using a GSA rotor, which is commercially 
available from Sorval Instruments. Sample containers for use with the GSA 
rotor were filled so that the total weight of the sample, lid and 
container was about 300 g. The sample container was spun at 2500 rpm for 
20 minutes, and the supernatant fluid was immediately decanted. Enough 
distilled water to raise the total weight of the sample, lid and container 
to 300 g was added to the sample container, and the container was swirled 
to bring the yeast pellet back into suspension. This sample container was 
then spun at 2500 rpm for 20 minutes again, and the supernatant fluid was 
again decanted. 
The washed yeast paste and water were combined to form a slurry. This 
slurry was mixed with the other ingredients in a table-top Hobart mixer. 
The dough was mixed at speed 1 for 30 seconds, followed by mixing at speed 
2 for between about 4 and about 5 minutes. Two 100 g samples (A1 and A2 in 
FIGS. 3 and 4) and two 50 g samples (A3 and A4 in FIGS. 3 and 4) were 
placed in the Risograph testing machine used in connection with Example 1 
above. The samples were incubated at about 30.degree. C. for about 17 
hours (1,000 minutes). The results of this Risograph testing are shown in 
FIGS. 3 and 4. 
As can be seen in FIG. 3, carbon dioxide was generated fairly rapidly in 
all of these samples for the first 40-50 minutes, after which the rate of 
evolution tapered off to about zero. Although the rate of carbon dioxide 
generation appears to have fluctuated between slight positive and negative 
rams, it appears as though the samples generated very little or no carbon 
dioxide between about 120 minutes after incubation began and the end of 
the experiment. 
Furthermore, although the rate of carbon dioxide generation was noticeable 
at the beginning of the experiment, it should be noted that the total 
volume of carbon dioxide generated in this sample was no more than about 7 
ml; this result is best seen in FIG. 4. As noted above, in order to 
adequately proof dough, between about 100 and about 200 ml of carbon 
dioxide/100 g of dough is generally considered to be necessary. The volume 
of carbon dioxide generated in these galactose-free samples, though, fell 
well below those limits. The indication that about 7 ml of gas was 
generated in these samples may actually be attributable primarily, if not 
entirely, to an expansion of the headspace in the Risograph sample 
containers when the containers were heated for incubation. In other words, 
it appears likely that no appreciable carbon dioxide was generated by the 
dough samples in this experiment. 
Accordingly, the D308.3 yeast used in this Example can be said to be 
substantially incapable of fermenting, or otherwise metabolizing, the 
carbohydrates native to this dough system. Hence, it is believed that the 
D308.3 strain of yeast can be accurately referred to as GAL+, as that term 
is used herein, and this yeast provides one example of a yeast suitable 
for use in the present invention. As noted above, though, one of ordinary 
skill in the art could make other GAL+ yeasts, as well as other yeasts 
which are capable of fermenting only carbohydrates not native to the flour 
in the dough, in light of the present disclosure. 
EXAMPLE 3 
In order to test the responsiveness of the GAL+ yeast used in Example 2, 
four different dough compositions, with varying non-native carbohydrates, 
were prepared. Each of the four doughs included 290.25 g of flour, 176.60 
g of water, 3.50 g of salt and 12.00 g of the D308.3 GAL+ yeast used in 
Example 1. The formulas of the four different doughs varied in the nature 
of the other ingredients which were added. In a control sample, no other 
ingredients were added; in a second sample, 5.00 g of galactose was 
included; in a third sample, 10.00 g of lactose was provided; and the 
final sample included 20.00 g of non-fat dry milk (NFDM), which is used as 
a flavoring ingredient in some doughs and typically contains some lactose 
and may contain slight amounts of galactose. 
Yeast paste was grown and harvested in substantially the same manner as set 
forth in connection with Example 2. For each of the samples, the washed 
yeast was slurried with the water, and this slurry was added to the other 
ingredients in a table-top Hobart mixer. Each sample was then mixed at 
speed 1 for about 30 seconds, followed by mixing at speed 2 for about 4 
minutes. Two 100 g samples of each of the dough compositions were placed 
into Risograph sample jars immediately after mixing and held in the 
Risograph at about 28.degree. C. for approximately 20 hours. FIGS. 5 and 6 
show the total volume of carbon dioxide evolved and the rate of carbon 
dioxide evolution, respectively, for each of the samples. 
As can be seen from FIGS. 5 and 6, only the dough composition which 
included galactose generated appreciable volumes of carbon dioxide. The 
control sample, the lactose-containing sample and the sample with the NFDM 
all generated less than about 10 ml of carbon dioxide over a period of 
about 20 hours. Furthermore, essentially all of the carbon dioxide 
generation measured for the non-galactose doughs was generated in the 
first one to two hours of incubation. This slight change in gas volume in 
the Risograph sample jars may be wholly attributable due to thermal 
expansion of the headspace in the sample jars, as explained above. 
Accordingly, the samples which did not contain non-native galactose quite 
likely did not generate any significant amount of carbon dioxide during 
the course of this test. 
The results of this experiment show that the D308.3 yeast can metabolize 
galactose but it is substantially incapable of fermenting any 
carbohydrates which are native to flour of the dough composition. It also 
appears that this yeast is substantially incapable of fermenting either 
"straight" lactose or lactose in non-fat dry milk. During the course of 
this experiment, the galactose-containing dough appears to continue to 
generate carbon dioxide, indicating that not all of the galactose was 
used. Furthermore, at the end of the 20-hour incubation, the galactose 
dough had generated slightly more than 100 ml of carbon dioxide, with 
carbon dioxide generation appearing to continue beyond the end of the 
experiment. 
The dough containing galactose was about 1.0 wt. % galactose (5.00 g 
galactose/487.35 g total dough).degree. Based on the results of this 
experiment, it appears that about 1 wt. % galactose is more than adequate 
to generate the desired 100-200 ml of carbon dioxide per 100 g of dough. 
Additional experimentation using standard, spirally wound composite 
containers of about 250 cc capacity, such as are commonly used in 
packaging commercial refrigerated doughs, has established that about 0.5 
wt. % to about 1.0 wt. % galactose is sufficient to generate enough carbon 
dioxide to reach an internal pressure of about 10-20 psi. Accordingly, in 
making a refrigeratable dough product of the invention, the dough placed 
in the container optimally includes between about 0.5 wt. % and about 1.0 
wt. % galactose. 
EXAMPLE 4 
The D308.3 yeast was added to a chemically-leavened dough product in order 
to see if the presence of the GAL+ yeast affected the integrity of the 
container if no galactose was added to the dough. Two batches of a dough 
containing the D308.3 yeast and two separate batches of chemically 
leavened dough were prepared. The chemically leavened doughs had the 
following formula: about 1590 g (56 wt. %) flour, 947 g (33.43 wt. %) 
water, 110 g (3.9 wt. %) of the wheat gluten pre-blend of Example 1, 89.2 
g (3.15 wt. %) of yeast flavorings, 42.5 g (1.5 wt. %) glucono delta 
lactone (GDL), 32.0 g (1.13 wt. %) baking soda, and 21.3 g (0.75 wt. %) 
salt. The two batches of dough containing yeast had a very similar 
formula, with the approximately 947 g (33.4 wt. %) of water being replaced 
with about 890 g (31.4 wt. %) of water and about 56.7 g (2.00 wt. %) 
D308.3 yeast. 
The water in each of these batches was first mixed with the flavoring 
ingredients before being charged with the flour and gluten pre-blend into 
a McDuffy mixing bowl. In the batches containing yeast, the yeast was 
slurried with the water before the flavoring ingredients were added to 
this slurry. The ingredients were mixed at speed 1 for about 30 seconds, 
followed by mixing at speed 2 for about 5 minutes. The salt and the 
leavening agents (GDL and soda) were then added to this dough and the 
mixture was mixed at speed 1 for approximately 30 seconds and at speed 2 
for about 2.5 minutes. 
Each batch of dough was sheeted to a thickness of about 1/4 inch (about 
0.64 cm) and rolled into a long "log" of dough. Each log of dough was 
divided into a series of samples weighing about 210 g and each sample was 
sealed into a standard, spirally wound composite can having a 250 cc 
capacity. These dough products were then proofed at about 
32.degree.-35.degree. C. until an internal pressure of about 10-15 psi in 
the containers was reached. After this proofing, the dough products were 
transferred to refrigerated storage at about 4.degree. C. 
FIG. 7 plots the measured can pressure, i.e., the internal pressure of the 
container, as a function of time. As can be seen in FIG. 7, there does not 
appear to be any significant difference between the pressure in the dough 
product containing the standard chemically leavened dough and the dough 
product containing the chemically leavened dough with the GAL+ yeast. 
A variety of other physical measurements were made on the different samples 
to compare the standard chemically leavened dough with the yeast-doped 
dough. Among the physical measurements compared were water retention, pH, 
and sugar content. Samples of the doughs were also baked at approximately 
375.degree. F. (163.degree. C.) for about 20 minutes. The specific volume, 
as well as the appearance, aroma and other sensory properties, of the 
resulting baked goods were compared. Aside from a slightly lower specific 
volume for the sample containing the GAL+ yeast, there did not appear to 
be any significant differences between these two dough compositions. 
EXAMPLE 5 
The relationship between galactose content of the dough and the resultant 
internal pressures of dough products containing dough in accordance with 
the invention was tested. Four different batches were prepared, with the 
batches differing only in the amount of galactose added. Each dough 
composition contained about 870.75 g (58.05 wt. %) wheat flour, 529.80 g 
(35.32 wt. %), water, 58.20 g (3.88 wt. %) of the wheat gluten preblend 
used in Example 1, 11.25 g (0.075 wt. %) salt and 28.50 g (2.00 wt. %) 
D308.3 yeast. Additionally, one batch contained about 5.92 g (0.5 wt. %) 
galactose, another contained about 7.40 g (0.63 wt. %) galactose, a third 
contained. about 8.87 g (0.75 wt. %) galactose, and the final batch 
contained about 11.83 g (1.00 wt. %) galactose. 
The D308.3 yeast was grown and harvested in substantially the same manner 
as that detailed above in Example 2. In forming batches of dough 
containing the 0.5 wt. % and 1.0 wt. % galactose, the yeast paste was then 
mixed with the water and the galactose in a 1 L culture flask and 
incubated in the flask for about 1 hour at about 30.degree. C. while the 
flask was agitated. This slurry was then added to a McDuffy mixing bowl 
and mixed with the other ingredients at speed 1 for about 30 seconds, 
followed by mixing at speed 2 for about 7 minutes. The 0.63 wt. % and 0.75 
wt. % galactose batches were prepared slightly differently in that the 
yeast, water and galactose were not incubated prior to being mixed with 
the other ingredients. Instead, these three ingredients were slurried in a 
table-top Hobart and were mixed at speed 2 for only about 4 minutes with a 
dough hook. 
After the doughs were mixed, two 50-gram samples from each batch of dough 
were placed in Risograph sample jars and incubated in the Risograph at 
about 28.degree.-30.degree. C. The dough was then rolled, divided into 
210-gram samples, and packaged in a standard refrigeratable dough 
container, as outlined above in Example 4. The resultant dough product was 
incubated at about 35.degree. C. for about three hours and subsequently 
stored at about 4.degree. C. 
FIGS. 8 and 9 illustrate the can pressures of the samples as a function of 
time, with the can pressures for samples from each batch being averaged 
together to generate these plots. It can be seen that the ultimate can 
pressure of the sample is generally proportional to the amount of 
galactose in the dough. Whereas the sample containing 0.63 wt. % galactose 
had a can pressure of about 5-6.5 psi, the 0.75 wt. % dough had can 
pressures of about 9-10.5 and the pressure in the dough with 3 wt. % yeast 
and 1 wt. % galactose generated a maximum pressure of just under 16 psi. 
Accordingly, it appears as though the desired pressure in a container of 
the invention can be fairly readily controlled as a simple function of the 
amount of galactose added to the dough - once the galactose is exhausted, 
the dough will substantially cease producing carbon dioxide. 
EXAMPLE 6 
The D308.3 yeast perhaps adversely affected the sensory appeal of baked 
doughs containing such yeast in that the final baked product exhibited a 
slightly off-white color. Although all of the other organoleptic qualities 
of the dough were exemplary, doughs which would not exhibit this slight 
discoloration would probably be more appealing to consumers. It was 
determined that the discoloration of the dough was most likely due to 
inability of the D308.3 yeast to make adenine, causing the yeast to 
develop a pinkish or reddish hue when it is grown in a medium without 
adenine supplementation. This discoloration of the yeast is presumed 
attributable to a build up of metabolites which are toxic to the yeast 
(but not to humans). 
Spontaneous revertant strains of the D308.3 yeast which do not require 
adenine for metabolization, referred to herein as RD308.3 yeast, were 
isolated. First, a concentrated paste of the D308.3 yeast was formed by 
spinning down the yeast in a rotor, as outlined in Example 2. This yeast 
paste was then diluted with a potassium phosphate monobasic buffer (about 
43 mg KH.sub.2 PO.sub.4 added to a liter of distilled water, with the Ph 
adjusted to about 7.2 with NaOH) and spread on an "adenine drop out" (ADO) 
medium, i.e. a medium which does not contain any supplemental adenine, at 
a concentration of about 1.times.10.sup.7 colony forming units (CFU)/ml. 
The ADO medium contained, for each liter of distilled water, about: 6.7 g 
of bacto-yeast nitrogen base without amino acids, 20 g galactose, and 20 g 
of bacto-agar, 2 g of a "drop out mix" which contained alanine, argenine, 
asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, 
histidine, inositol, isoleucine, leucine, lysine, methionine, 
para-aminobenzoic acid, phenylalanine, proline, serine, threonine, 
tryptophan, tyrosine, uracil, and valine. (Substantially the same formula 
is taught by Rose et al. in Appendix A of Methods in Yeast Genetics, A 
Laboratory Course Manual (1990), which is incorporated herein by 
reference, at pages 179-180, but that formula used glucose rather than 
galactose.) 
These ADO plates were incubated at about 25.degree. C. for approximately 4 
days and colonies of the yeast which did not require adenine were 
isolated. Identifying these colonies was greatly simplified by the fact 
that the non-revertant strains tended to be pinkish or reddish in hue 
while the revertant colonies were whitish. The isolated yeast was then 
once again plated onto a fresh ADO medium and incubated under 
substantially the same conditions. Colonies of revertant strains of the 
yeast were once again isolated from any strains inadvertently carried over 
in the first isolation and the platting and incubation were repeated one 
final time. Although it is believed that one skilled in the art could 
readily make such a yeast in light of the present disclosure, this 
resulting strain of RD308.3 yeast has been deposited with the ATCC on Mar. 
5, 1993, under number ATCC 74212 and this strain is available to the 
public from the ATCC. 
Two samples were prepared, with one sample containing the original D308.3 
yeast and the other containing the RD308.3 yeast. These samples were 
prepared by mixing an isolated colony (about one loop) of the desired 
yeast with about 5 ml of YEP/galactose (which contained about 10 g of 
bacto-yeast extract, 20 g of bacto-peptone, and about 20 g of galactose 
per 1 liter of distilled water) and incubating for about 12-15 hours at 
about 30.degree. C. (The formula for the YEP/galactose medium is 
substantially the same as the YEP/glucose formula taught on page 177 of 
Appendix A of Methods in Yeast Genetics, noted above, except that the 
glucose in that formula was replaced with galactose in the present 
medium.) Titer results indicated a population of approximately 
48.+-.2.times.10.sup.5 CFU/ml for each strain. For each of the resulting 
samples, about 100 .mu.l of the sample was added to three separate 5 ml 
potions of media, with one medium comprising just YEP, another comprising 
YEP and glucose and the third comprising YEP and galactose. 
The absorbance of each resulting sample was measured over time and is 
graphically illustrated in FIG. 10. The growth behavior of the D308.3 and 
RD308.3 yeasts appeared to be essentially the same for all three of these 
growth media. Furthermore, both of these yeasts appear able to readily 
metabolize galactose, but can only grow slightly on YEP or YEP/glucose. It 
is also interesting to note that both the D308.3 strain and the RD308.3 
strain grew slightly less on the YEP/glucose than on YEP alone. This 
further demonstrates the substantial inability of these yeasts to 
metabolize glucose. 
The auxotrophic markers adenine, histidine, methionine and tryptophan as 
growth supplements for the D308.3 and RD 308.3 strains were compared by 
standard techniques. The D308.3 yeast was not able to grow on galactose 
minimal media unless all four of these growth supplements were present, 
but the RD 308.3 yeast was able to grow if only the histidine, methionine 
and tryptophan were added. 
Thus, the only significant difference noted between the auxotrophic markers 
of these two strains was that the D308.3 yeast requires adenine 
supplementation while the RD308.3 yeast does not. Accordingly, it is 
believed that the RD308.3 yeast will behave substantially as described 
above in connection with the D308.3 yeast when added to dough, but the 
slight discoloration of baked goods associated with doughs containing the 
D308.3 yeast should be substantially eliminated. 
FIG. 11 is a schematic representation of the process of glycolysis. As is 
well known in the art, various sugars are broken down by glycolysis into 
pyruvic acid via glycolysis and the resulting pyruvic acid can be utilized 
by yeast to generate carbon dioxide through fermentation in an anaerobic 
environment. 
As schematically illustrated in FIG. 11, glucose can be converted into 
glucose 6-phosphate by either hexokinase (HXK) or glucokinase (GLK). This 
glucose 6-phosphate can then be converted into fructose 6-phosphate by one 
of two pathways. In the normal glycolytic pathway, glucose 6-phosphate is 
converted into fructose 6-phosphate by the action of 
phosphoglucoisomerase. 
In an alternative pathway for converting glucose 6-phosphate to fructose 
6-phosphate, the glucose 6-phosphate is first converted into 
6-phosphogluconolactone by the action of glucose 6-phosphate dehydrogenase 
(G6PDH), also known as zwischenferment (ZWF). Through the pentose 
phosphate pathway, also called the pentose phosphate shunt, this 
6-phosphogluconolactone can be converted into fructose 6-phosphate. 
In the embodiment described above wherein the yeast is substantially 
incapable of fermenting any sugars native to the dough systems, the yeast 
mutation was substantially incapable of generating either hexokinase (HXK) 
or glucokinase (GLK). As can be seen from the schematic illustration of 
FIG. 11A, this prevents glucose or fructose from being converted into 
fructose 6-phosphate. Since the yeast is substantially incapable of 
converting either glucose or fructose into fructose 6-phosphate, glucose 
and fructose generally cannot be converted into pyruvic acid and therefore 
cannot be utilized effectively by the yeast to produce CO.sub.2. 
As can be seen in the schematic representation of FIG. 11A, galactose can 
be converted into glucose 1-phosphate by the action of galactokinase. This 
glucose 1-phosphate can then be converted into glucose 6-phosphate through 
the action of phosphoglucomutase. This glucose 6-phosphate can then be 
converted into fructose 6-phosphate, and thence to pyruvic acid as 
outlined above, by either phosphoglucoisomerase (PGD or glucose 
6-phosphate dehydrogenase (G6PDH). Accordingly, the yeast in the 
embodiment set forth above can utilize galactose through the action of 
galactokinase, but the pathways for the glycolysis of glucose and fructose 
are disabled due to a lack of hexokinase and glucokinase. 
As noted above, in an alternative embodiment, the present invention does 
not utilize a non-native sugar, but instead ferments a sugar which is 
native to the dough system, but is present only in a limited quantity. In 
one particularly preferred embodiment of this invention, the native sugar 
is fructose. Fructose is commonly present in wheat flours, but in a 
concentration of less than 0.1 wt. %, with concentrations on the order of 
0.02-0.08 wt. % being common for most wheat flours. Once a wheat flour is 
combined with water and yeast, this relatively low concentration of 
fructose will be diluted even further. As doughs leavened with haploid 
yeasts commonly require at least about 1.0 wt. % of a fermentable 
substrate to generate the 100-200 ml of CO.sub.2 necessary to proof about 
100 g of dough, the quantity of native fructose generally will be less 
than that necessary to adequately proof a dough. 
In accordance with the present invention, a yeast which is capable of 
phosphorylating only selected sugars to produce fructose 6-phosphate is 
mixed with flour and water to form a dough composition. In one preferred 
embodiment, the yeast is capable of fermenting fructose but is 
substantially incapable of fermenting any sugar naturally occurring in the 
flour in significant concentrations. An additional quantity of fructose is 
added to the dough composition to provide additional fermentable substrate 
for the yeast. The quantity of fructose added to the dough should be 
sufficient to allow the yeast to generate about 100-200 ml CO.sub.2 /100 g 
of dough as measured at about 30.degree. C. The amount of fructose 
necessary to generate these quantities of carbon dioxide should be 
determined on a case-by-case basis for different strains of yeast to yield 
the desired degree of proofing. 
As described above and illustrated in FIG. 11, fructose requires hexokinase 
in order to be converted into fructose 6-phosphate through 
phosphorylation. Accordingly, the yeast used in accordance with the 
present invention must be capable of generating hexokinase. However, 
hexokinase also breaks glucose down into glucose 6-phosphate, which can 
then be converted into fructose by either PGI or G6PDH. In order to allow 
the yeast of the present invention to utilize fructose but not glucose, 
the two pathways for the conversion of glucose 6-phosphate into fructose 
6-phosphate are blocked. 
As schematically depicted in FIG. 11B, this is accomplished in accordance 
with the instant invention by using a yeast which is both 
phosphoglucoisomerase negative ("PGI-") and glucose 6-phosphate 
dehydrogenase negative ("G6PDH-"), i.e. the yeast lacks PGI and G6PDH. As 
such a yeast cannot generate either PGI or G6PDH, it is substantially 
incapable of utilizing glucose 6-phosphate by converting it into fructose 
6-phosphate by phosphorylation. Accordingly, even if glucose is converted 
to glucose 6-phosphate by the action of hexokinase or glucokinase, the 
process of glycolysis is substantially blocked at that point and the 
glucose cannot be converted to pyruvic acid. 
As illustrated schematically in FIG. 11B, this leaves only one pathway 
through the glycolysis process, that being the conversion of fructose to 
fructose 6-phosphate by hexokinaseo Thus, the amount of fructose in a 
dough of the present invention will determine the volume of carbon dioxide 
which is generated. 
In accordance with one embodiment of the present invention, a yeast which 
is both PGI- and G6PDH- is mixed with flour, water and an amount of 
fructose sufficient to provide the desired degree of proofing of the 
dough. The amount of fructose added to the dough is optimally selected to 
produce a volume of 100-200 ml CO.sub.2 /100 g of dough as this is usually 
considered necessary to proof the dough, as noted above. The precise 
amount of fructose added to the dough will depend on a number of factors, 
including the specific strain of yeast being used and the amount of native 
fructose in the flour. Accordingly, the amount of fructose added to a 
given dough composition should be determined on a case-by-case basis for 
different dough formulations. However, once a suitable formula has been 
determined for a given composition, the volume of carbon dioxide produced 
can be predictably varied as a function of the amount of fructose added to 
the dough. 
Although there do not appear to be any PGI-/G6PDH- yeasts currently 
commercially available, a skilled artisan in the field will be able to 
make such a yeast using known techniques, such as those outlined by 
Sherman et al., supra. In making such a yeast, one can cross a PGI- yeast 
with a G6PDH- yeast through known techniques. 
Once the two yeasts have been crossed and sporulated to yield haploid 
strains, the strains should be tested to ensure that they are indeed 
PGI-/G6PDH-, i.e. they behave as though they have substantially no 
phosphoglucose isomerase and substantially no glucose 6-phosphate 
dehydrogenase activity. Once the PGI-/G6PDH- nature of the yeast is 
confirmed (e.g. by confirming substantially zero growth on glucose media), 
the yeast can be use to make a dough of the invention. For example, 
PGI-/G6PDH- yeast of the invention was made as follows. 
EXAMPLE 7 
A number of putatively PGI- yeast strains and a number of putatively G6PDH- 
yeast strains were obtained from publicly available sources. In this 
experiment, the following strains of Saccharomyces cerevisiae yeast were 
used: 
______________________________________ 
Yeast Strain 
Genotype 
______________________________________ 
N543-9D .alpha. pgi1 leu2 can.sup.r cyh.sup.r SUC2 mal mel gal2 CUP1 
N548-8A a pgk1 leu2 can.sup.r cyh.sup.r SUC2 mal mel gal2 CUP1 
9520T4C .alpha. pgi1 ade1 trp1 ura3 his2 metl4 
YM3269 a zwfl URA3 ura3-52 his3-200 ade2-101 
lys2-801 try1-501 met- 
______________________________________ 
The YM3269 yeast'sgenotype includes the designation zwf1, indicating that 
the yeast is deficient in ZWF, i.e. glucose 6-phosphate dehydrogenase 
(G6PDH). The first three strains of yeast were all obtained from the YGSC 
at UC Berkeley, noted above; the last strain (YM3269) was obtained from 
Dr. M. Johnston, Department of Genetics, Washington University School of 
Medicine, St. Louis, Mo., USA. 
Although these particular yeast strains were selected for the present 
experiment, it is well within the ability of those skilled in the art to 
select or develop other suitable PGI- and G6PDH- strains of yeast. For 
instance, in "Identification of the Structural Gene for 
Glucose-6-phosphate Dehydrogenase in Yeast. Inactivation Leads to a 
Nutritional Requirement for Organic Sulfur", The EMBO Journal, vol. 10 no. 
3 pp. 547-553 (1991), the teachings of which are incorporated herein by 
reference, Thomas et al. teach that a defect in the MET19 gene of pu S. 
Cerevisiae pk will cause such a yeast to be G6PDH- because the MET19 gene 
encodes glucose-6-phosphate dehydrogenase from yeast. Thomas et al. also 
describe methods by which such a defect can be cloned into other yeast 
strains. 
The ability of each of these yeasts to utilize different substrates was 
tested by inoculating a sample of each of a number of different media with 
a sample of each yeast and measuring the absorbance of the samples over 
time at 25.degree. C. The different media were all employed liquid YEP as 
a base and varied in the addition of sugars to the media, with one medium 
having no additional substrate, a second including glucose, a third 
including fructose, a fourth including sucrose, and a fifth including 
maltose. This process of testing the ability of yeast to grow on different 
media is well known in the art and need not be discussed in great detail 
here. 
By using such known testing protocols, it was determined that the N543-9D 
yeast grew readily on fructose, sucrose and glucose but not on maltose. 
The ability of this yeast to grow on glucose indicates that it is not 
actually PGI-, as reported. 
The 9520T4C yeast strain also grew readily on fructose and sucrose, but 
grew poorly on maltose and initially could not grow well on glucose. After 
about 6 days of incubation in liquid glucose media, this strain did adapt 
to growth on glucose, though. It has been surmised that this strain is 
most likely at least partially PGI-, but that the yeast may be able to 
process glucose by shunting the glucose through the pentose phosphate 
pathway (noted above in connection with FIGS. 11 ) or the PGI- mutation 
may be "leaky", i.e. PGI is not fully disabled. 
Strain YM3269 was able to grow readily on glucose, fructose and sucrose, 
although the yeast seemed to have difficulty growing on maltose. As can be 
seen in FIGS. 11, one would expect the yeast to be able to use glucose, 
fructose and sucrose despite a lack of G6PDH because the glucose 
6-phosphate can proceed through the normal glycolysis pathway without 
having to go through the pentose phosphate pathway disabled by the lack of 
G6PDH. 
The N548-8A yeast grew slowly on glucose, fructose and sucrose and was 
generally unable to grow on maltose. This is consistent with an 
understanding from the literature that this yeast has a "leaky" 
phosphoglucokinase (PGK) mutation. 
Thus, the YM3269 yeast was apparently G6PDH negative and the 9520T4C yeast 
appeared to be PGI negative. Accordingly, these two yeast strains were 
selected for crossing to yield PGI-/G6PDH- haploids. In mating these two 
strains to make the desired haploids, a protocol was derived from Methods 
in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor 
Laboratory Press, pp. 53-59 (1990), the teachings of which are 
incorporated herein by reference. 
In accordance with this method, YM3269 yeast and 9520T4C yeast were plated 
onto separate YEP+ fructose plates using a sterile loop to apply the 
strains on their respective plates in a series of parallel lines about 7 
mm apart. These plates were allowed to incubate at approximately 
30.degree. C. for about one day. 
An impression of the G6PDH- YM3269 strain was made on a replicate plate 
pad. This impression was imprinted onto a fresh plate including a YEP+ 
fructose medium (about 1 wt. % bacto-yeast extract, about 2 wt. % 
bacto-peptone, about 2 wt. % bacto agar, and about 2 wt. % fructose, with 
the balance being distilled water) supplemented with adenine, histidine, 
methionine and uracil. Using a fresh replicate plate pad, an impression of 
the 9520T4C PGI- strain was made. The second replicate pad was imprinted 
on the same YEP+ fructose plate used for the previous imprinting, but at 
an orientation generally perpendicular to the first imprint, resulting in 
a pattern of yeast strains resembling a checkerboard. This doubly 
imprinted YEP+ fructose plate was incubated at approximately 30.degree. C. 
overnight (i.e about 12-15 hours). 
The YEP+ fructose plate thus prepared was imprinted on a fructose minimal 
media plate containing adenine, histidine, methionine and uracil. The 
fructose minimal media included about 6.7 g of bacto-yeast nitrogen base 
without amino acids, about 20 g of bacto-agar and about 20 g of fructose 
in about 1 liter of distilled water; the adenine, histidine, methionine 
and uracil were added to this formula in aqueous form. Such a minimal 
medium, utilizing dextrose instead of fructose, as well as the formulas 
for the supplements are taught in Methods in Yeast Genetics, A Laboratory 
Course Manual, Cold Spring Harbor Laboratory Press, pp. 178-179 (1990), 
the teachings of which are incorporated herein by reference. 
These fructose minimal media plates were incubated for about two days at 
about 30.degree. C. Growth at the intersections of the "checkerboard" 
pattern was scored and plated onto a fresh fructose minimal media plate to 
isolate the diploid (crossed) colonies from the haploid colonies. The 
diploid colonies isolated on the fructose minimal media plate were 
streaked onto a plate of sporulation media and incubated for about 4-5 
days at about 25.degree. C. The sporulation media contained about 10 g (1 
wt. %) potassium acetate, about 1.0 g (0.1 wt. % ) bacto-yeast extract, 
about 0.5 g (0.05 wt. %) fructose, about 20 g (2.0 wt. %) bacto-agar, with 
the balance being about 1000 ml distilled water. 
About one loopful of yeast cells was taken from the sporulation plate and 
combined with about 300 microliters distilled water and approximately 15 
microliters glusulase in an Eppendorf.TM. microfuge tube. This solution 
was mixed by vortex and incubated at around 30.degree. C. for 
approximately 30 minutes. The incubated sample was briefly sonicated to 
separate spore clusters. Within about 20 minutes of being made, serial 
dilutions of about 10.sup.-4, 10.sup.-5 and 10.sup.-6 of the sonicated 
sample were plated onto YEP+ fructose plates. 
These serial dilutions were then exposed to ethyl ether fumes in a manner 
adapted from "Guide to Yeast Genetics and Molecular Biology", Guthrie and 
Fink editors, in Methods of Enzymology, vol. 194, pp. 146-147 (1991), the 
teachings of which are incorporated herein by reference. In this process, 
a 4 mm .times.4 mm piece of filter paper was placed into the inverted lid 
of each petri dish containing one of the serial dilutions. In a ventilated 
hood, 0.75 ml of ethyl ether was added to each filter paper and the lids 
and dilutions were placed in a glass chamber along with a beaker 
containing 10 ml of ethyl ether to maintain the vapor pressure in the 
chamber elevated. 
The chamber was sealed and the samples were incubated for about 15 minutes 
at room temperature, at which time an additional 0.75 ml portion of ethyl 
ether was added to each filter paper square. These samples were again 
incubated in the glass chamber at room temperature for about 15 minutes, 
following which the samples were removed from the chamber and allowed to 
sit in the open atmosphere with the lid of each sample ajar for about 30 
minutes. 
Isolated yeast colonies from each of the 10.sup.-4, 10.sup.-5 and 10.sup.-6 
dilution plates and a sample of each of the YM3269 and 9520T4C parent 
strains were grid plated onto YEP+ fructose plates and incubated at about 
25.degree. C. for approximately 24 hours. Each of these samples was then 
replicate plated onto one plate of YEP+ fructose and one plate of YEP+ 
glucose. These plates were then incubated at about 30.degree. C. for about 
1-2 days to determine which of the isolated putative YM3269.times.9520T4C 
strains were able to grow on the fructose-enriched medium but not the 
glucose-containing medium. 
The growth rate of each isolated strain was then evaluated by inoculating a 
5ml volume of YEP+ fructose with one loop of the yeast. Control samples 
for each of the YM3269 and 9520T4C parent strains were also prepared by 
inoculating similar media with a loop of the parent yeast. These samples 
were incubated at about 30.degree. C. for about 24 hours and 100 
microliter samples of each resultant yeast were used to inoculate separate 
5 ml samples of YEP, YEP+ fructose and YEP+ glucose, with one such set of 
three samples being prepared from each isolated strain and each of the 
parent strains. The absorbency of each sample was measured at 600 nm and 
the samples were incubated at about 25.degree. C. for about 2 weeks, with 
absorbency measurements being taken about twice a week for each sample. 
Approximately 200 colonies of putative YM3269.times.9520T4C haploid strains 
were initially obtained from the "checkerboard" plating. Of these 200 
colonies 47 were found to grow on the fructose medium but substantially 
unable to grow on the YEP or YEP+ glucose media in the initial stages of 
the incubation. As noted above, though, the 9520T4C parent strain was 
found to be able to adapt to the glucose media after about 6 days in 
incubation. Of the 47 strains which did not initially grow in the glucose 
medium, all but 15 demonstrated an ability to adapt to the glucose like 
the parent yeast. Accordingly, only 15 of the 200 initially isolated 
colonies were evaluated as being truly PGI-/G6PDH- in that they were able 
to grow on fructose but not on glucose. These 15 colonies are referred to 
herein as PGI-/G6PDH- 1 through PGI-/G6PDH- 15. 
This experimental example therefore readily provided some 15 colonies of 
yeast which appear to be useful in the present invention. Although this 
experimental example illustrates one straightforward method of making 
yeast of the present invention, it is to be understood that other 
variations of this method or other procedures for making such yeasts will 
be apparent to those skilled in this field. 
The PGI-/G6PDH- 1 yeast was deposited with the ATCC on Jul. 2, 1993 under 
designation number ATCC 74230. This strain has been deposited simply as an 
example of a suitable yeast strain in accordance with the invention; it is 
to be understood that any one or more of the 15 isolated PGI-/G6PDH- 
strains are believed likely to work as well and that other strains of 
yeast in accordance can be readily produced by those skilled in the art in 
accordance with the present disclosure. 
In accordance with a further embodiment of the present invention, such a 
yeast of the invention is incorporated into a dough composition. In 
accordance with this embodiment, a yeast of the invention is mixed with 
flour, water and a substrate fermentable by the yeast. The substrate 
fermentable by the yeast may occur naturally in the dough system, but if 
so the quantity of such substrate in the dough should be no more than that 
necessary to generate 200 ml of CO.sub.2 per 100 g of dough, and is 
desirably substantially less than that amount. As used herein, a substrate 
is said to be naturally occurring in the dough if it is either native to 
the flour or is generated over time by the action of enzymes in the flour 
on other carbohydrates initially present in the flour. 
For instance, fructose occurs naturally in wheat and will generally 
comprise between about 0.02 wt. % and about 0.08 wt. % of wheat flour. 
Such fructose can be said to be native to the flour as it is present in 
the flour in its natural state. Wheat flours also include sucrose, which 
is a disaccharide of glucose and fructose, and sucrose can be broken down 
by the action of commonly occurring enzymes into its constituent 
monosaccharides, giving rise to additional fructose in the dough over 
time. The concentration of sucrose in most wheats is generally on the 
order of about 0.2 wt. %, which if completely broken down would yield 
approximately 0.1 wt. % additional fructose to the initial 0.02-0.08 wt. % 
fructose in the flour. Also, various wheat flour oligosaccharides (e.g. 
glucofructose) contain varying amounts of fructose which could possibly 
also contribute the total amount of fructose in a wheat flour dough system 
over time. Both the native fructose and that produced by the degradation 
of native sucrose and other native oligosaccharides is considered to be 
"naturally occurring" as that term is used herein. 
Continuing with the example of fructose, it should be noted that the total 
naturally occurring fructose in most wheat flours will be no more than 
about 0.2 wt. %, even if the sucrose is completely degraded into glucose 
and fructose. By the time the flour is mixed with water and the other 
ingredients of the dough, the weight percentage of naturally occurring 
fructose in the dough composition will be even less, frequently on the 
order of about 0.12 wt. % of the dough. However, a concentration more on 
the order of 1 wt. % of a fermentable substrate is usually necessary to 
generate the 100-200 ml CO.sub.2 /100 g of dough necessary to adequately 
proof the dough. Accordingly, the naturally occurring fructose is 
substantially less than that which one would expect to be required to 
properly proof a dough made with the flour. 
Fructose would therefore serve as a suitable substrate fermentable by yeast 
of the present invention, particularly where the yeast is to be used with 
wheat flours. Naturally occurring glucose, on the other hand, while 
initially present at relatively low concentrations in a refrigerated wheat 
flour dough (e.g. native concentrations on the order of about 0.2 wt. %) 
will, over time, increase in concentration to as much as 1 wt. % or more 
by the end of about 90 days of refrigerated storage. Thus, the "naturally 
occurring" glucose in wheat flour doughs can be as much or more than that 
necessary to suitably proof the dough. 
Without further treatment of the wheat to limit the amount of naturally 
occurring glucose, the additional amount of glucose generated in the dough 
system over the anticipated 90-day shelf life of the product could very 
well generate more than the 100-200 ml of CO.sub.2 /100 g of dough desired 
for proper proofing of the dough. Furthermore, when dough is proofed in a 
standard container, the expansion of the dough during proofing is used to 
flush the container of air initially present in the container and to 
substantially seal the container. The amount of native glucose in wheat 
flours generally will not be sufficient to adequately flush and seal 
standard containers in commercial use today. 
Inadequate flushing and sealing would permit some quantities of air to 
remain in the container and, perhaps, enter the container before it is 
sealed. Significant oxygen concentrations in the container can have a 
number of adverse effects on the dough, such as graying of the dough and 
promoting growth of deleterious bacteria. Thus, the native glucose may be 
insufficient to generate the 15-20 psi internal pressure desired in dough 
packages, but yet the total volume of CO.sub.2 generated in the dough from 
the total naturally occurring glucose may well exceed the desired volumes. 
Accordingly, glucose would not be a good candidate for a fermentable 
substrate for a yeast-leavened dough of the invention. 
By utilizing a yeast which is capable of fermenting only a substrate (most 
commonly a sugar) which is present in the dough in naturally occurring 
amounts no more than that necessary to proof a dough, the total volume of 
carbon dioxide which the yeast can generate will also be limited. Hence, 
by selecting the concentration of fermentable substrate in the dough and 
using a yeast of the present invention, the proofing of a dough with yeast 
can be controlled on a commercial basis. This is critical, as outlined 
above, in that it can permit the dough to be stored for extended periods 
of time, e.g. on the order of 90 days or more, without rupturing the 
container in which it is placed. 
EXAMPLE 8 
In order to test the ability to use the yeast produced in Example 7 to 
proof a dough composition and to survive extended refrigerated storage, 
such a yeast was mixed with flour and water to form a dough. In 
particular, yeast strain PGI-/G6PDH- 1 (ATCC designation number ATCC 
74230) was used in four different dough compositions, with each dough 
composition including about: 428.3 g (57.7 wt. %) wheat flour; 259.7 g 
(35.0 wt. %) distilled water; 26.5 g (3.56 wt. %) wheat gluten preblend of 
a formula substantially the same as outlined in Example 1; 5.63 g (0.76 
wt. %) salt; and 15.0 g (2.02 wt. %) PGI-/G6PDH- 1 yeast. The four dough 
compositions differed in the amount and type of substrate added to the 
dough, with the first composition having no added sugars, the second 
having about 7.5 g (1.0 wt %) fructose, the third dough having about 7.5 g 
(1.0 wt %) glucose, and the fourth dough having about 7.5 g (1.0 wt %) 
sucrose. 
The doughs were made by slurrying the PGI-/G6PDH- 1 yeast with the water 
and the sugar, if any, and mixing this slurry with the remaining 
ingredients in a table top Hobart mixer. The dough was mixed at speed I 
for about 30 seconds, followed by mixing at speed 2 for approximately 4 
minutes. 
After the doughs were mixed, two 100 g samples of each dough composition 
were placed in separate Risograph sample jars, These sample jars were then 
held in the Risograph at about 30.degree. C. for about 24 hours and the 
gas evolution of these samples was monitored by the Risograph. FIG. 12 is 
a graph of the total volume of CO.sub.2 generated over time for each 
sample while FIG. 13 is a graph of the rate of CO.sub.2 generation for the 
same samples. (The data collected from the two samples from each batch of 
dough was averaged together to yield the data for that sample shown in 
these drawings.) 
Additionally, two 210 g samples of each of the fructose-containing and 
sucrose-containing doughs were placed in standard, commercially available 
spirally wound cans (about 2.25" diameter by about 4" in length). The 
canned dough samples were then incubated at about 97.degree. F. 
(36.degree. C.) for about 3.5 hours and stored at about 4.degree. C. for 
about 90 days. FIG. 14 is a graph of the pressure in the sealed container 
for each of these doughs over time. (The pressure measurements were begun 
after the initial proofing incubation at 97.degree. F. and the data for 
the two samples of each dough were averaged together to yield the 
illustrated data for that dough.) 
The data of FIGS. 12 and 13 indicate that the dough containing fructose 
generated significantly more CO.sub.2 than the other three samples. This 
is much as one would expect for a dough leavened with a yeast which is 
both phosphoglucose isomerase (PGI) negative and glucose 6-phosphate 
dehydrogenase (G6PDH) negative. As illustrated in FIGS. 11 and 11B, such a 
yeast should be able to readily utilize fructose in the dough, but not 
other sugars. 
The dough which did not include any additional sugar did generate some 
CO.sub.2, although it was substantially less than that generated by the 
fructose-supplemented dough. The yeast may have been able to utilize the 
naturally occurring fructose in the dough as a substrate in generating 
carbon dioxide. It is interesting to note that the 100 g control sample 
containing no non-naturally occurring fructose (i.e. no added fructose in 
the composition) generated somewhat more than 100 ml CO.sub.2. 
The reliability of this result is not certain, though. In some 
circumstances, various bacteria in a dough will generate CO.sub.2 or other 
gases if the dough is proofed for an extended period of time, such as more 
than 10 hours. As the doughs of FIGS. 12 and 13 were held at about 
30.degree. C. for much longer than 10 hours, it is believed that at least 
some of the volume of gas generated by the control sample may be 
attributable to the action of bacteria, i.e. to a source other than the 
yeast. It is well known that the growth of bacteria in yeast can not only 
yield undesirable byproducts which can adversely affect the dough. 
Accordingly, commercially proofed doughs generally are proofed for as 
short a period of time as possible and one should not rely on the 
generation of CO.sub.2 from sources other than the yeast in these doughs 
for leavening purposes. 
Accordingly, it is believed that the volume of CO.sub.2 generated by the 
yeast is less than 100 ml for this 100 g sample of dough. Since this falls 
below the level believed to adequately proof the dough, it appears as 
though the dough lacks sufficient naturally occurring fructose to proof 
the dough to the desired degree. 
The dough containing 1 wt. % sucrose actually generated slightly less 
CO.sub.2 than the control sample, which did not contain any additional 
sugars. At first, this may seem anomalous in that the additional sucrose 
could be broken down into glucose and fructose, adding to the fermentable 
substrate in the dough. This is not completely understood, but it is 
believed that this may be attributable either to an inability of the 
PGI-/G6PDH- 1 yeast to cleave sucrose into its constituent monosaccharides 
or to metabolic suppression of yeast due to the abundance of unconsumed 
glucose in the dough. The fact that the glucose-supplemented formulation 
produced even less gas than the sucrose dough would seem to bear out that 
the presence of glucose, which cannot by fermented by the yeast, is 
suppressing the metabolism of the yeast. 
As noted above, FIG. 14 shows the pressure in the canned dough stability 
test. The fructose-supplemented dough reached an internal pressure of 
about 18 psi in the first 10 days of storage, with the pressure gradually 
creeping up to about 24 psi by the end of the 68 days. As explained above, 
it is desirable to have an internal can pressure of at least about 15-20 
psi, but that the internal pressure of the can should not exceed about 40 
psi or the container may rupture. Accordingly, the fructose-supplemented 
dough of FIG. 14 appears to readily meet the desired operational 
parameters of standard canned doughs and can be stored for extended 
periods of time without rupturing the container. In this case, the dough 
was stored for 90 days at refrigeration temperatures without any adverse 
effects on can pressure, which meets the requirements for most current 
commercially produce refrigeratable dough products, as noted above. 
The sucrose-supplemented dough did not generate as much internal pressure 
as the fructose-supplemented dough. As seen in FIG. 14, the sucrose dough 
reached a pressure of less than about 10 psi in the first 10 days of 
refrigerated storage and gradually crept up to an internal pressure of 
about 14 psi by the end of the 68 days shown in this graph. It is 
interesting to note that this product did not reach the desired 15-20 psi 
of internal pressure even after more than two months of storage. 
Accordingly, by varying the concentration of non-fermentable sugar (e.g. 
glucose) in the dough product system, one can effectively limit or control 
the gas production of the dough, and hence internal pressure of a package 
containing the dough, during the dough's shelf life. 
Another embodiment of the present invention provides a method of forming a 
dough which can be stored at refrigeration temperatures for extended 
periods of time without generating significant volumes of carbon dioxide. 
This method may further include the steps of packaging the dough, proofing 
the dough in the package, and storing the dough for an extended period of 
time at refrigeration temperatures. 
In making a dough of the invention, flour, water, a yeast substantially 
incapable of fermenting carbohydrates native to the flour, and a quantity 
of a carbohydrate fermentable by the yeast are mixed together, as outlined 
above. The amount of the fermentable carbohydrate added to the dough is 
desirably sufficient to provide only the necessary degree of proofing of 
the dough; adding too much fermentable substrate could cause adverse 
changes in dough rheology due to overfermentation. This amount is 
optimally determined on a case-by-case basis for a given strain of yeast 
as different strains of yeast may utilize the fermentable substrate more 
efficiently than others. 
In a particularly preferred embodiment of the method of the invention, the 
yeast used in making the dough is a GAL+ yeast and a predetermined 
quantity of galactose is added to the dough to provide the desired degree 
of proofing. This GAL+ may be the D308.3 yeast or the RD308.3 yeast 
described above, but it is to be understood that other GAL+ yeasts can be 
made in accordance with the present disclosure which will also work in 
accordance with the invention. 
As noted above, the method may further include the steps of packaging the 
dough, proofing the dough in the container, and storing the dough at 
refrigeration temperatures for an extended period of time. Virtually any 
known refrigeratable dough package known in the art may be used in this 
method. For instance, spirally wound dough containers such as those 
currently used in commercially manufactured refrigeratable dough products 
should suffice. A quantity of dough somewhat less than that necessary to 
fill the container is placed in the container, leaving a headspace in the 
container when it is sealed. 
The dough may then be proofed in the container, expanding to fill the 
container and flush out any air in the headspace. The proofing is 
continued until substantially all of the fermentable carbohydrate is 
consumed by the yeast, at which point an internal pressure of about 15 to 
about 20 psi is attained in the container. This proofing may be 
advantageously carried out at an elevated temperature, e.g. about 
30.degree. C. to about 40.degree. C., to allow the yeast to ferment, and 
thus proof the dough, more rapidly. 
This proofed dough may then be placed in refrigerated storage for extended 
periods of time, desirably up to at least about two weeks. The dough of 
the invention is optimally capable of storage at refrigeration 
temperatures for at least about 90 days, the anticipated shelf life of 
current doughs, as explained above. By "refrigerated storage", storage at 
temperatures between about 12.degree. C. and about 0.degree. C., and 
preferably between about 4.degree. and bout 7.2.degree. C., is intended. 
Such temperatures are referred to in the present specification as 
"refrigeration temperatures 
While preferred embodiments of the present invention have been described, 
it should be understood that various changes, adaptations and 
modifications may be made therein without departing from the spirit of the 
invention and the scope of the appended claims.