Yeast strains, method of production and use in baking

Novel biologically pure quick acting bakers yeast strains NRRL Y-15338 and NRRL Y-15339 are provided which show good performance in sweet, regular, and lean doughs, and superior performance in sweet and lean doughs, particularly when used in the active dry yeast form. A method of obtaining these and other novel bakers yeast strains by hybridization via protoplast fusion of petite mutants is also provided. Improved methods of baking using these novel bakers yeasts especially in the active dry yeast form are also provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to improved biologically pure, novel strains of 
quick acting general purpose bakers yeast which show good performance in 
sweet, regular and lean dough systems and especially superior performance 
in sweet and lean doughs, and methods of obtaining the same from existing 
strains of quick acting bakers yeast by hybridization via protoplast 
fusion of petite mutants of such quick acting yeast, to quick acting 
active dry yeast made from these strains yeast and to improved baking 
methods using said novel yeast strains in active dry yeast form. 
2. Description of the Prior Art 
So-called quick acting strains of the yeast Saccharomyces cerevisiae are 
known and are commercially available in active dry yeast form. These 
yeasts are generally characterized by the rapid production of a relatively 
large amount of carbon dioxide in a given time frame when mixed with 
doughs which basically contain flour and water, and which usually fall 
into one of the general categories of sweet, lean, or regular doughs. The 
value of these so called quick bakers yeasts are especially significant in 
the commercial baking field particularly when used in the active dry yeast 
form containing low amounts, i.e. 4 to 8 percent, of moisture. Some of the 
quick acting yeasts which are available are characterized by a tolerance 
to sweet doughs which contain substantial amounts of sugar, i.e., up to 
about 20 to 25% sugar. Generally speaking, these fast acting, or so called 
quick yeasts of commerce have only moderate activity with a regular or 
straight dough which contains 4 to 5% sugar and relatively poor activity 
in lean doughs which contain no added sugar. Likewise quick lean dough 
yeast of commerce are not useful for sweet doughs. Actually, it is 
generally acknowledged in the baking industry that improvements in 
performance of yeast in sweet dough formulations is at the expense of lean 
dough performance and vice versa. 
This recognition has led to commercial yeast producers manufacturing 
separate yeast products for commercial use in either sweet doughs or lean 
doughs. Insofar as it is presently known, there is no single yeast strain 
which demonstrates quick and superior leavening activity in both lean 
doughs and sweet doughs. 
SUMMARY OF THE INVENTION 
This invention relates to novel strains of the bakers yeast Saccharomyces 
cerevisiae which are characterized by quick leavening action and 
particularly by suitability for use in leavening of bakery products made 
from both lean doughs which contain no sugar and high sugar containing or 
so called sweet doughs. 
This invention also relates to methods of forming novel hybrids of yeast 
strains via protoplast fusion of petite mutants of certain yeast strains, 
the use of these novel yeast strains in the production of active dry yeast 
for use in the bakery industry, and to improved baking methods using these 
novel yeast strains in the active dry yeast form. 
Broadly, the procedure and method of the present invention involves 
hybridizing, by protoplast fusion of petite mutants of dissimilar, 
sexually incompatible, heterozygotic strains of the yeast Saccharomyces 
cerevisiae. More particularly, the process of the present invention 
involves the steps of 
(a) growing two dissimilar yeasts on a fermentable sugar-containing culture 
medium, the said yeast strains being characterized by a relatively high 
tolerance to sugar and a capacity for proliferative growth on nutrient 
substrates in which glycerol is the sole carbon source nutrient, 
(b) isolating spontaneous petite mutant yeast colonies which are 
characterized by their inability to metabolize glycerol, 
(c) enzymatically removing the cell wall material to produce yeast 
protoplasts, 
(d) hybridizing the yeast by fusing the yeast protoplasts then in the 
presence of polyethylene glycol, 
(e) recovering the fused hybridized yeast cells from the fusion step, and 
(f) growing the protoplast fusion yeast cells on a nutrient substrate with 
glycerol as the sole carbon nutrient source, and 
(g) recovering the yeast cells produced by said procedure. 
Broadly, the yeast strains used to generate the petite mutants are 
Saccharomyces cerevisiae strains which demonstrate relatively high 
osmotolerance and relatively quick acting characteristics when placed in 
dough formulations containing flour, water and sugar. Such yeasts are well 
known commercially and have also been referred to in the literature--see 
for example U.S. Pat. No. 3,993,783 which refers to strains Ng 2031 and Ng 
2103 and U.S. Pat. No. 3,394,008 which refers to strains Ng 740 and Ng 
1777, said accession numbers having been assigned to yeasts deposited in 
the Central Bureau Voor Schimmelcultures, Delft, Netherlands. 
Preferably the strains used in the hybridization and protoplast fusion 
procedure of this invention are also characterized by the fact that the 
strains cannot mate in nature nor can the petite mutants arising from the 
culture of said yeast be mated. The yeasts are also characterized by their 
capacity to grow on substrates in which glycerol is the sole carbon source 
nutrient. 
The two seletced yeast strains chosen as petite mutant sources, are first 
cultivated on nutrient substrates in which a fermentable sugar such as 
glucose, or in some caases sucrose, is the sole carbon nutrient source. 
Such growth procedure gives rise to spontaneous petite mutant colonies 
which are distinguished from the grande colonies by size and color. These 
colonies are treated with the dye 2,3,5-triphenyl tetrazolium chloride 
(TTC) which stains normal colonies pink while petite colonies remain 
white. 
In a typical procedure, the petite colonies are isolated and sub-cloned on 
yeast extract, peptone and dextrose media (YPD) plates. The sub-clones are 
then replicated on yeast extract, peptone, glycerol (YPGLY) plates to 
confirm and select the desired petite mutants which do not grow on 
substrates having glycerol as the carbon nutrient source. The selected 
sub-clone isolates are then separately grown on a YPD liquid broth culture 
for 48 hours and harvested by separation (centrifugation) from the 
nutrient media which is washed from the cells. The sub-clone cultures of 
the petite mutant yeast cells are then treated with enzymes (i.e., 
zymolyase or .beta.-glucuronidase) to remove the cell walls thereby 
creating yeast protoplasts which are thoroughly washed. 
The separate yeast protoplasts thus produced from the source yeast petite 
colonies following either of the two protocols, are then combined and 
suspended in a fusion buffer of polyethylene glycol (mol. wt. 4000-6000), 
sorbitol and a calcium salt, and incubated for a period of from 15 to 30 
minutes, preferably about 30 minutes, and the fusion buffer is then 
removed. 
The recovered cells are incubated overnight in a recovery broth comprising 
hypertonic glucose which contains 1% yeast extract, 0.8 molar sorbitol and 
0.2% glucose. 
Following incubation in recovery broth, the cells were plated on hypertonic 
glycerol agar which consisted of 1% yeast extract, 2% peptone, 3% 
glycerol, 0.8M sorbitol, and 3% agar,. Only fusion products successfully 
proliferate on glycerol substrates. 
The hybrid yeast cells arising on hypertonic glycerol were streaked to YPD, 
and selected clones were replicated to sucrose, glycerol and sporulation 
media (potassium acetate) plates. Selected clones which grew on all media 
were cultured for about 48 hours in a YPD broth. Clones yielding 0.3 grams 
of yeast solids/100 mL of broth were selected for further evaluation. 
The selected protoplast fusion yeast hybrids are further selected by gas 
production (carbon dioxide) tests in three test dough systems for lean and 
sweet dough activity. Fifty (50) mg (dry weight) of yeast were added to 
the model mixes shown below. The yeast was suspended in the 15 mL of added 
water. 
A. 
Flour 20 g (4X, Pillsbury) 
Water 15 mL 
B. 
Flour 20 g 
Water 15 mL 
NaCl 0.4 g 
C. 
Flour 20 g 
Water 15 mL 
NaCl 0.4 g 
Sucrose 4 g 
The dough was mixed for 45 to 60 seconds and the mix incubated at 
30.degree. C. for four hours, during which the gas volume was measured at 
half hour (30 min) intervals. Strains which produce more than 300 mL of 
carbon dioxide per 100 mg yeast solids for test A and at least 200 mL of 
gas for tests B and C over a four hour test period are selected. 
An active dry yeast is readily prepared from these hybrids by standard 
procedures, i.e. growing the yeast strains in typical multi-stage batch 
fermentation stages using a molasses as the nutrient carbon source. The 
yeast harvested from the last or trade fermenter is concentrated and dried 
under temperature and humidity conditions that maintain its viability, to 
final moisture contents of between about 4 and 8%, preferably about 6%. 
Two novel strains produced by the procedure of this invention were 
deposited with the Northern Regional Research Laboratory, Peoria, Ill., 
accession numbers NRRL Y-15338 and NRRL Y-15339. 
The yeast strain Saccharomyces cerevisiae NRRL Y-15338 has the following 
physical description: 
Cells grown three days at room temperature in 10.degree. Brix malt extract 
are ovoid to ovate-deltoid, to ellipsoidal. Cell sizes (lenth.times.width) 
of unmatured daughter cells are in the 5.8-7.0.times.4.7-5.8 micron range. 
Mature cells range from 9.3-11.7.times.5.8-8.2 microns. Most cells are in 
the 9.times.7 micron range. On 1% potassium acetate, 0.1% glucose, 0.25% 
yeast extract sporulation medium, sporulation efficiency is 77.9.+-.9.4%. 
The frequency of asci with four spores was 5.59.+-.1.64%. Four spored asci 
are rhomboid or tetrahedral. Ascospores are spherical to 
prolate-ellipsoidal in shape. 
The culture ferments .alpha.-methylglucoside and melezitose slowly. Gas 
first appears in .alpha.-methylglucoside broth durham tubes after 3 to 5 
days with a standardized 200,000 cell/mL inoculum. With melezitose, first 
gas appeared in 3 days. Trehalose was not fermented in 21 days of 
observation. Protocols used were those of Lodder, North Holland Publishing 
Co., Amsterdam (1970), pp. 66-73. 
The colony morphology on uncrowded (15-30 colonies/plate) Wallerstein's 
Laboratory (WL) medium after 5 days is dark green in color, 
peaked-pulvinate in shape with an entire edge. After 10 days on WL medium 
some colonies demonstrate papillae at the edge of the colony. Color 
sectoring is occasionally seen. On less crowded plates (less than 15 
colonies/plate) colony shape is flatter and almost umbonate in 
configuration. 
On glycerol medium (uncrowded plates) morphology after 5 days is smooth, 
convex, with entire edges. After 10 days many colonies show papillae at 
the edges. On crowded plates (50 per plate) after 5 days colonies are 
flatter and concentrically rugose leaving the colony center smooth. 
A second strain of bakers yeast NRRL Y-15339 was prepared by the procedure 
described above, but using the .beta.-glucuronidase cell wall digestion 
protocol. This yeast had the same balanced baking characteristics as NRRL 
Y-15338. 
This strain differs in having a 64.2.+-.16.0 percent asci and 5.95.+-.1.91 
percent four spored asci. After 48 hours on 10.degree. Brix malt extract 
the cells are ovoid to ellipsoidal in shape. Large cells 7.2.times.10.4 
microns are seen but most of the population is in the 5.7.times.8.3 micron 
range. Unmatured, but separated from the mother cell, daughters average 
4.0.times.5.5 microns. 
On uncrowded WL medium after 6 days colonies are raised pulvinate to 
papillate, concentrically colored dark green and light green. The colony 
edge is entire and dark green, the colony center is dark green with the 
raised tip lighter in color. On crowded plates the concentric coloring is 
less pronounced. After 10 days on WL color sectoring and peripheral light 
papillae are seen. 
On uncrowded glycerol medium after 6 days, colonies are white, slightly 
pulvinate with a flattened rugose or wrinkled edge. This becomes more 
apparent at 14 days, with some papillae seen at the colony edges. On 
crowded plates colonies appear the same but the peripheral wrinkling is 
more pronounced. 
The strain ferments melezitose and .alpha.-methyl glucoside slowly. With 
.alpha.-methylglucoside gas first appears in the durham tube in 3 days; 
with melezitose, first gas also accumulates after 3 days. Trehalose is not 
fermented in 21 days observation. 
The following examples illustrate the preparation of the novel yeasts of 
the present invention.

EXAMPLE 
Two separate strains of so-called quick acting sweet dough yeasts are 
selected. These strains are characterized by ability to grow on glycerol 
and by being unable to mate in nature. 
The two strains of the yeast Saccharomyces cerevisiae were cultivated on a 
culture media containing 0.3% glucose, 1% potassium acetate, 1% yeast 
extract, 2% peptone, and 1.5% agar for a period of 4 to 5 days. This plate 
was overlaid with agar containing 0.067 molar concentration of phosphate 
buffer, pH 7.0, containing 0.1% of TTC and 1.5% agar. After an incubation 
of 4 to 5 hours as indicated above, two types of colonies were produced, 
namely grande colonies of pink to red coloration and petite colonies of a 
white color. The petite colonies are considerably smaller than the grande 
colonies. This follows the protocol as outlined in Science 125, 198 
(1957). The petite colonies were removed and streaked for isolation on 
yeast extract, peptone and dextrose (YPD), a non-selective media, from 
this, a master plate was formed which was replicated on a YPGLY plate. The 
pure sub-clone isolates of the original petite cultures which did not 
proliferate on the glycerol substrate were cultivated in a flask using a 
YPD broth media for 48 hours and harvested by centrifugation. The yeast 
cells were washed free of media with water and recovered. 
PROTOPLAST FORMATION 
Zymolyase Protocol 
The two separate yeast cell cultures from the above procedures were 
suspended in a zymolyase enzyme cell wall digestion media to form the 
protoplasts. The protocol involved the inoculation of approximately 
5.times.10.sup.8 cells into the following medium: 
5.0 .mu.L -mercapto-ethanol 
100 .mu.g zymolyase 
Total volume 2.0 mL buffered 1M sorbitol 
The cell concentrations in the suspension were about 5.times.10.sup.8 
cells in 2 mL of total volume of solution. The digestion of the yeast cell 
walls was microscopically followed and when concluded (about 45 minutes), 
the protoplasts were washed 5 times with spheroplasting buffer until free 
of enzymes. The procedure generally follows that of J. Bacteriol. 130, 
946-948 (1977). The yeast arising from this protoplast formation protocol 
following the procedure set out below, was later given the accession 
number NRRL Y-15338. 
.beta.-Glucuronidase Protocol 
Another procedure for protoplast formation involves the use of 
.beta.-glucuronidase enzyme in place of zymolyse in protoplast formation. 
This .beta.-glucuronidase protocol is as follows: 
5.times.10.sup.8 yeast cells were incubated 30 minutes in 0.5M sodium 
thioglycollate in a 0.1M TRIS pH 8.8 buffer. The yeast cells were 
harvested by centrifugation, washed once, resuspended in 2 mL buffered 1M 
sorbitol and 0.1 mL of a 1:10 dilution of .beta.-glucuronidase (Sigma 
Chemical Co.) was added. 
Digestion was followed microscopically. After completion (i.e., about 4 
hours), the cells were washed 5 times with spheroplasting buffer. The 
procedure generally follows J. Molec. Biol. 52, 323-335 (1970). 
The yeast which was ultimately obtained following the balance of this 
procedure set out below, but using the .beta.-glucuronidase protocol, was 
also deposited at the Northern Regional Research Laboratory and given the 
accession number NRRL Y-15339. 
PROTOPLAST FUSION 
The protoplasts to be fused were recovered from the cell wall digestion 
procedures set out above. These protoplasts were mixed in a fusion buffer 
containing sorbitol (1M), calcium chloride (0.01M), and 40% polyethylene 
glycol having a molecular weight range of 4000 to 6000. The mixture of 
protoplasts was incubated for 30 minutes, centrifuged and the fusion 
buffer poured off. The procedure used generally follows J. Bacteriol. 
ibid. Recovery broth was added to the recovered fused yeast protoplasts 
comprising a 1% yeast extract, 0.2% glucose and 0.8M sorbital which 
mixture was incubated overnight (12-18 hours) at a temperature of 
30.degree. C. 
RECOVERY OF HYBRIDS 
Reconstituted fused protoplast yeast cells were plated by pour plate 
techniques using hypertonic glycerol plating media. Cells were 
incorporated into a molten agar mixture. The hypertonic glycerol plating 
media contained 1% yeast extract, 2% peptone, 3% glycerol, 0.8M sorbitol 
and 3% agar. The colonies showing growth on the recovery plate represented 
only the successful fusion products. These were streaked on YPD plates and 
selected clones replicated to yeast extract, peptone, sucrose media 
(YPSUC); a sporulation media containing acetate as the carbon source and a 
YPGLY media. Selected fused protoplast yeast hybrids were inoculated into 
YPD broth medium and incubated for 48 hours to determine yield. Those 
yeasts were selected which had a yield of at least 0.3 g (dry weight) of 
yeast per 100 mL of broth. 
RISE TIME SCREENING 
After the first screening for yield described above, the yeast hybrid was 
harvested and subjected to various further gas output measurement 
screening tests. These tests involved the addition of 50 mg of yeast 
solids in 15 mL H.sub.2 O to various model dough systems. These systems 
were 
(A) 20 g of Pillsbury (4X) flour, 15 mL of water with yeast. 
(B) 20 g of Pillsbury (4X) flour, 15 mL of water with yeast, 0.4 g of NaCl 
had been added. 
(C) 20 g of Pillsbury (4X) flour, 15 mL of water with yeast, 4 g of 
sucrose, 0.4 g of NaCl. In all cases the yeast flour and other ingredients 
were mixed for 45-60 seconds in each test batch. 
Gas evolution was measured 1/2 hour for a 4 hour period. Only those strains 
were selected which produced greater than 300 mL of carbon dioxide per 100 
mg of yeast solids in rise time test A and 200 mL in rise time tests B and 
C over the 4 hour test period. The procedure and model dough systems are 
after Harrison and Burrows, J. Inst. Brew. 65, 35-45 (1959). 
Novel strains of bakers yeast prepared according to the procedures 
described above, which passed the gas tests in the model baking dough 
systems A, B, and C above, and which yielded 0.3 g yeast/100 mL of broth 
in growth tests, were propagated in a series of fermenters with a 14 liter 
last stage fermenter. Molasses yields were 80-85% and considered 
acceptable. That propagation procedure is set forth in the following 
example. 
MANUFACTURE OF PRESS CAKE YEAST AND ACTIVE DRY YEAST EXAMPLE 
The pure culture of the yeast strain Saccharomyces cerevisiae NRRL Y-15339 
was propagated in a series of laboratory fermenters, the yeast recovered, 
and reduced in moisture to the cream stage (18-21% solids). Emulsifier 
(sorbitan monostearate, 1% on yeast solids) or sodium chloride (1-1.5% on 
yeast solids) was added to the yeast cream which was then reduced to a 
moisture content of as much as 30 to 90 percent moisture and preferably a 
moisture content of from about 60% to 70%, particularly 66%, (to form a 
fresh bakers yeast in a cream, compressed or press cake form) following 
generally the procedures set forth in Reed and Peppler, Yeast Technology, 
AVI Pub.Co., Westport, Conn. (1973) pp. 83-88. 
An active dry yeast is prepared following the procedure generally set forth 
in Reed and Peppler, ibid. pp. 90-97. 
The fresh bakers yeast press cake (66% moisture) containing surface active 
agent is extruded as a noodle through a perforated plate (0.02-inch 
orifices) and dried in a commercially available drier, such as a fluidized 
bed drier made by Aeromatic Co., Mutenz, Switzerland, under controlled 
humidity conditions (25 to 60 minutes; 220.degree. F. to 100.degree. F.) 
to a moisture content of about 4.0 to about 6.5% to produce a high 
activity active dry yeast (HADY). Usually the yeast used to form the 
noodle has a moisture content of preferably between about 60-70% or 65 to 
70% moisture. 
This type of yeast, HADY, can be directly added to the dough without 
reconstitution in water. 
An active dry yeast (ADY) is also prepared by a similar procedure using 
orifice sizes of about 0.065 inch dried to a moisture content of about 
7.4-8.2% based on dry yeast solids. This type of yeast (ADY) should be 
reconstituted in water before mixing with the dough. The results of the 
fermentation and drying sequence are set forth in the table below for the 
two strains of bakers yeast NRRL Y-15,338 and NRRL Y-15,339, run by the 
same procedure as set out below. 
TABLE I 
______________________________________ 
Molasses 
DRY YEAST DATA 
Strain Yields % H.sub.2 O 
% N % P.sub.2 O.sub.5 
______________________________________ 
NRRL Y-15338 
89.0 5.34 7.12 1.99 
93.1 6.14 6.48 1.87 
NRRL Y-15339 
84.8 5.12 6.58 2.03 
91.5 5.85 6.63 1.85 
______________________________________ 
Bake tests showed the following results. 
BAKE TESTS 
Bake tests were run on the different yeasts in three different dough 
systems (i.e., sweet, lean, and regular) using S.J.A. gas measuring 
equipment. The formulations are as follows: 
______________________________________ 
REGULAR DOUGH 
______________________________________ 
Premix 
500 g flour 
20 g sugar 
20 g non-fat dry milk 
10 g salt 
15 g shortening 
Regular Dough Test 
500 g of premix (above) 
5.5 g of dry yeast 
290 mL of 50.degree. F. water 
______________________________________ 
The test ingredients are mixed in a Hobart Model A-120 mixer for 6 minutes. 
A 250 g aliquot of dough is placed in the S.J.A. gas measuring equipment 
maintained at 100.4.degree. F. and the gas evolved is measured at 60 and 
90 minute intervals. First rise is considered gas production at 60 
minutes. The gas production in the period from 60 to 90 minutes (by 
difference) is considered proof time for purposes of this test, 
correlatable with commercial operations. The gas volume units are 
translated into minutes for first rise and proof time using standard 
curves derived from actual baking trials. 
The same procedure is followed in sweet and lean dough systems. The dough 
formulations and procedure are: 
______________________________________ 
Sweet Dough 
______________________________________ 
500 g of premix (above) 
72.0 g sugar 
11 g dry yeast 
250 mL water at 70.degree. F. 
______________________________________ 
Procedure: Mix for 8 minutes (Hobart), 155 g of dough placed in a S.J.A. 
apparatus. Measure gas produced at 60 and 120 minute intervals as 
exemplary of first rise and proof time as described above. 
______________________________________ 
Lean Dough 
______________________________________ 
442 g Pillsbury 4 .times. flour 
13 g shortening 
8.0 g salt 
5.5 g dry yeast 
300 mL water, 70.degree. F. 
______________________________________ 
Procedure: Mix in Hobart 6 minutes. Place a 200 g aliquot of dough in 
S.J.A. apparatus. Readings of gas evolved are taken at 60 and 90 minute 
intervals as above and converted to minutes for first rise and proof 
times. 
The yeast strains produced in table I were used in bake tests for regular, 
sweet, and lean dough systems. The results are shown in table II below. 
TABLE II 
______________________________________ 
Rise Times (Min.) 
YEAST STRAIN Reg. Lean Sweet 
______________________________________ 
NRRL Y-15338 122 117 124 
121 116 124 
NRRL Y-15339 124 112 121 
116 115 101 
______________________________________ 
Minimum standards for performance based on commercial averages are lean 
dough-122 min; regular-140 min; and sweet-155 min. 
A similar yeast propagation fermentation procedure was run in a 200 liter 
fermentor (Fermatron, New Brunswick Scientific Co.). The results obtained, 
including the bake tests, are shown in Table III: 
TABLE III 
______________________________________ 
Dry Yeast 
Data Rise Times (min.) 
Strain Molasses % N % P.sub.2 O.sub.5 
Reg. Lean Sweet 
______________________________________ 
NRRL Y-15338 
87.9 6.62 1.91 119 116 114 
NRRL Y-15339 
91.6 6.74 2.01 123 104 129 
______________________________________ 
The foregoing procedures provide methods for producing novel man made yeast 
hybrids which are useful in providing to the baking industry an improved 
baking procedure, especically when the yeast is marketed in the active dry 
yeast form. They provide an instant active dry yeast of good activity over 
the entire dough spectrum, but show especially good and superior results 
in both sweet and lean dough systems. Insofar as it is known, there are no 
instant active dry yeast on the commercial market which show superior 
performance in both sweet and lean dough systems. Commercial yeasts are 
usually selective only for a particular dough system and are so identified 
by the manufacturer. This versatility permits the commercial baker greater 
flexibility in his work allowing him to use a single yeast for both these 
sugar containing systems. This not only minimizes work effort, but also 
minimizes possible error by the production personnel which could occur by 
inadvertently using the wrong yeast.