Food

Microbiology, 1999, 16, 45]51 Article No. fmic.1998.0212

Intracellular concentration of exogenous

glycerol in Saccharomyces cerevisiae
provides for improved leavening of
frozen sweet doughs
D. K. Myers1 and P. V. Attfield 1, 2 *

Addition of glycerol to baker’s yeast coupled with an incubation period under conditions of
refrigeration to allow equilibration of the polyol across the yeast membrane, resulted in
improved frozen sweet (10% sugar) dough leavening and keeping, relative to doughs made
with yeast containing no added glycerol. Glycerol-loaded yeast showed smaller proof time
increases after initial freezing and thawing. Also, glycerol treatment led to slower deteriora-
tion of the frozen sweet doughs during storage at y218C for up to 8 weeks (proof time
increases were significantly lower in stored frozen doughs made with glycerol-added yeast).
Benefits of glycerol loading of yeast for the purpose of production of frozen plain (unsugared)
doughs were less significant. The glycerol treatment effects became more significant as the
yeast was stored refrigerated prior to mixing and freezing of doughs, i.e. shelf-life of yeast for
use in frozen dough manufacture was improved by addition of glycerol. The improvements
were dependent on glycerol concentration. Q 1999 Academic Press

Introduction ¨¨
Rasanen et al. 1995.. Furthermore, proof
times continue to increase with prolonged Received:
Frozen doughs represent great potential for frozen dough storage time and inferior baked 10 February 1998
the baking industry in terms of automation loaf volume, grain and texture is achieved, 1
Burns Philip
and convenience ŽHsu et al. 1979, Jackel relative to unfrozen doughs ŽVarriano- Technology
1991.. Unfortunately, relative to unfrozen Marston et al. 1980, Wolt and D’Appolonia and Research
doughs, proof times Ži.e. time taken for Centre, P.O. Box 219,
¨¨
1984, Autio and Sinda 1992, Rasanen et al. North Ryde,
doughs to rise to a defined height. for frozen 1995, El-Hady et al. 1996.. Deterioration of NSW 2113
doughs increase significantly after initial dough properties due to freezing and then Australia
2
´
freezing and thawing ŽGelinas et al. 1994, School of
prolonged frozen storage are believed to be Biological Sciences,
due, in part, to reduced yeast cell viability Macquarie University,
Sydney,
and poorer gas retention by the dough ŽKline NSW 2109,
* Corresponding author. and Sugihara 1968.. Australia

0740-0020r99r010045q 07 $30.00r0 Q 1999 Academic Press

46 D. K. Myers and P. V. Attfield
Loss of viability of cells upon freezing has
been attributed to intracellular freezing and
increased internal solute concentrations
causing effects such as low pH, dehydration,
ionic toxicity, damage to essential membrane
processes, impairment of cytoskeletal ele-
ments and lowering of activities of glycolytic
enzymes ŽLovelock 1954, Mazur 1961 and
1965, Meryman 1968, Komatsu et al. 1987,
Grout et al. 1990, Hatano et al. 1996.. The
extent to which these effects arise is deter-
mined, in part, by the rate of cooling of the
sample ŽMazur 1966, 1970.. Intracellular
fluid usually freezes after the external
medium and, in the case of yeast, internal
freezing occurs at ; y10 to y158C ŽMazur
1961, 1966.. At freezing rates ŽF108C miny1 .,
as applied in frozen dough manufacture, ice
formation outside the cell leads to a relative
increase in external solute concentration and
hence hyperosmotic conditions that cause ef-
flux of intracellular water and relative dehy-
dration. This is also driven by the relative
increase in vapour pressure of intracellular
supercooled water ŽMazur 1970.. Freezing
rates ŽG 1008C miny1 . can result in forma-
tion of small ice crystals inside cells and,
although these are not necessarily damaging
per se, they can eventually recrystallize into
larger crystals that can rupture membranes
ŽMazur 1970, Grout et al. 1990.. This recrys-
tallization can happen during prolonged
frozen storage and especially during slow
thawing at low temperatures ŽMazur 1961,
1970. such as occurs in frozen dough
technology.
Addition of solutes to various cell types
has been used to reduce loss of viability dur-
ing freezing, but this has usually involved
rapid cooling Že.g. with liquid nitrogen. and
very low storage temperatures ŽMazur 1970.,
Carpenter et al. 1986, Crowe et al. 1987..
Sm all m olecular w eight com patible
solutes can be added, which act as intracellu-
lar components that mediate reduced shrink-
age of the cells ŽMeryman et al. 1968.. These
stop other solutes from reaching toxic or
damaging levels, as well as possibly protect-
ing proteins and membranes ŽMazur 1970,
Crowe et al. 1990, Grout et al, 1990.. Glyc-
erol and trehalose are compatible solutes
synthesized naturally by baker’s yeast ŽAtt-
field et al. 1992, Van Dijck et al. 1995, Myers
et al. 1997. and which protect against dehy-
dration ŽBrown and Edgley 1980, Crowe et
al. 1987. that can arise as a result of slow
cooling. Minimum levels of trehalose in yeast
have been identified for freezerthaw toler-
ance ŽAttfield et al. 1992. and the production
of satisfactory frozen doughs ŽMeric et al.
1995, Van Dijck et al. 1995.. Yeast cultural
methods including unlimited aeration
´
ŽGelinas et al. 1989. and heat shock ŽKomatsu
et al. 1990. have been developed to maximize
trehalose levels.
Compatible solutes can also function as
cryoprotectants which reduce formation of
lethal intracellular ice crystals ŽTan et al.
1995.. Although glycerol has been implicated
in cryotolerance in a number of non-baking
applications, no studies involving glycerol
have been reported for frozen doughs. A
process for addition of 1]20% wrw glycerol
to fresh compressed yeast, to produce a
pastyrliquid yeast with greater shelf-life and
freezerthaw tolerance has been described
ŽSuoranta 1991., but this was for frozen stor-
age of yeast. The need for adjustment of
yeast to hyperosmotic conditions through in-
trinsic production and retention of the osmo-
protectantrcompatible solute glycerol in
order to achieve effective sweet dough fer-
mentation has been reported previously
ŽMyers et al. 1997.. The beneficial effects of
exogenous glycerol addition to, and equilibra-
tion with, baker’s yeast for the purpose of
improving sweet dough fermentation and
shelf-life of compressed yeast has also been
described ŽMyers et al. 1998.. Given the com-
monality of dehydration through hyperos-
motic and freezerthaw stresses in sweet and
frozen doughs respectively, we have investi-
gated the effects of glycerol addition to
baker’s yeast on subsequent leavening and
storage of frozen doughs. The shelf-life of
yeast with or without added glycerol to be
used for frozen dough production was also
studied because currently yeast must be
used as fresh as possible for satisfactory
frozen dough manufacture.

Materials and Methods
Materials
Commercially produced ‘Pinnacle’ com-
pressed baker’s yeast was obtained from
Mauri Yeast, Melbourne, Victoria, Australia.
Trehalose content of yeast was ;10% wrw
of dry yeast and was unchanged by glycerol
addition Ždata not shown.. Food grade glyc-
erol was obtained from Mauri Foods, Regents
Park, New South Wales, Australia. Baker’s
flour Ž;12% protein, ;4% damaged starch.
was purchased from Defiance Mills, Sydney,
New South Wales, Australia, and sugar from
CSR, Sydney, Australia. DPS2 shortening
and Bakedoh bread improver, were obtained
from Mauri Foods, Altona, Victoria, Aus-
tralia. Analar NaCl was purchased from
BDH-Merck, Kilsyth, Victoria, Australia.
Glycerol addition to yeast
Yeast was suspended in 48C distilled water
to ;20% dry weight of yeast and glycerol
added to a final concentration up to 0.4 M on
total water basis, and mixed by magnetic
stirrer for 10 min before storing at 48C as
described in Myers et al. 1998. A yeast sus-
pension, designated control, was prepared
and treated in the same manner, but no
glycerol was added. Yeast " glycerol was
stored for 72 h before the whole sample was
filtered by vacuum through a double layer of
Whatman No. 541, 18.5-cm diameter filter
paper. The resultant yeast filter-cake Žabout
500 g. was wrapped in waxed paper and
stored at 48C for up to 20 days prior to
Table 1. Frozen dough compositions
Component Plain dough Žg.
Flour 2500
Salt 50
Improver 25
Shortening 50
Sugar 0 250
Water 1450
Yeast 125
Enhanced frozen dough leavening 47
preparation of frozen doughs. Filter-cakes
contained 30 Ž"1.5.% yeast dry matter.
Preparation of frozen doughs and
measurement of proof times
Frozen dough components are shown in Table
1. Dry ingredients, yeast and water were
mixed until doughs were fully developed.
Mixing time was determined by farinograph
and water absorption by extensigraph. Final
dough temperatures were 20 " 28C. Seven
520 g dough pieces were produced and
moulded Ž6 in mono-moulder., with one being
tested immediately for proof time Žtime taken
for dough to rise from a standard rolled di-
ameter of 70 mm to 120 mm at 378C. and the
other six blast frozen at y408C until the core
temperature was ; y58C for ;1 h. Frozen
doughs were stored at y218C and duplicate
doughs defrosted at 48C for 16 h and tested
for proof time after 1, 4 and 8 weeks. Proof
times were observed and measured manu-
ally.
Results and Discussion
Reproducibility of results
Experiments were performed over a period of
12 months using three different batches of
industrially grown compressed baker’s yeast
and also different batches of the same brand
of flour. The results presented in this work
are typical of the three separate but repro-
ducible experiments which gave comparable
trends. Within any single experiment stan-
dard errors for proof times of all doughs were
- 5%. Bread volumes were 2200 ml " 5%.
10% sugar dough Žg.
2500
50
25
50
1325
125
48 D. K. Myers and P. V. Attfield
Breads were scored according to external and
internal appearances. External appearances
were 31 " 2% and were obtained as the sum
of subjective grading of crust Žarbitrary scale
of 1]10. and oven spring, which was the
difference in height in mm between the
proofed and baked loaf. Internal appearances
were 44 " 2%, estimated by subjective grad-
ing of crumb Žout of 30., colour Žout of 10.
and softness Žout of 20..
Effect of glycerol addition to yeast on
leavening of frozen doughs
Previous studies showed that addition of 0.2
M glycerol Žon total water basis. to yeast
followed by a period of 72 h to allow for
equilibration, resulted in marked improve-
ment of unfrozen sweet dough leavening and
compressed yeast shelf life ŽMyers et al.
1998.. Initial experiments testing the effect
of 0.2 M glycerol addition on yeast perfor-
mance in frozen doughs showed insignificant
effects on frozen dough leavening and shelf
life Ždata not shown.. The concentration of
glycerol was therefore increased to 0.3 and
0.4 M, with a 72 h period of equilibration at
48C to allow uptake of glycerol into yeast
cells prior to preparation of doughs. Table 2
shows that the addition of glycerol at 0.4 M
resulted in a 10% improvement of plain
dough leavening prior to freezing, but this
was less significant in doughs frozen and
stored for one week. This effect was noted for
doughs made with fresh yeast Žstored for ;4
days at 48C. and yeast that had been stored
at 48C for 20 days. Addition of glycerol at 0.4
M to yeast improved the leavening of sweet
dough by 15% prior to, and 17% post, frozen
storage for one week ŽTable 2.. Improve-
ments were most noticeable with yeast that
had been stored at 48C for 20 days prior to
dough preparation: the aged yeast containing
0.4 M glycerol performed as well in sweet
doughs as the fresh yeast containing no added
glycerol. Addition of glycerol at 0.3 M resulted
in a similar pattern of improvements in
frozen dough leavening, but effects were less
substantial than with 0.4 M Ždata not shown..
Addition of 0.4 M glycerol to yeast resulted
in significant improvement in prolonged
shelf-life of frozen sweet dough ŽTable 2, Fig.
1.. For fresh yeast, addition of glycerol gave
proof times in 8 weeks stored frozen doughs
that were better than 1 week stored doughs
made with yeast containing no added gly-
cerol. Effects on aged yeast were even more
notable. There was only an ;4% increase in
proof times for frozen sweet doughs stored
for 8 weeks relative to 1 week storage, when
made with glycerol-treated yeast that had
been stored refrigerated for 20 days prior to
dough preparation ŽTable 2.. By contrast,
frozen sweet doughs made with identically
aged yeast containing no added glycerol
showed a 35% increase in proof time over the
8 weeks storage. Effects on prolonged storage
of plain doughs were less notable. Indeed,
generally there was a greater increase in
proof times between unfrozen and frozen
doughs stored for 1 week, than be-
tween doughs stored frozen for 1 to 8 weeks
ŽTable 2..
The pattern of proof time change with
frozen sweet dough storage shown in Fig. 1
highlights the point that the major benefit of
glycerol addition is to be gained with yeast
that are stored refrigerated prior to dough
preparation. The proof time of frozen sweet
dough made with yeast containing no added
glycerol increased as frozen storage time pro-
ceeded. This effect became more pronounced
as yeast was stored refrigerated ŽFig. 1..
However, addition of glycerol to the yeast
prior to refrigerated storage resulted in
maintenance of subsequent frozen dough
shelf-life: there was relatively little differ-
ence between fresh yeast plus glycerol and
20 day stored yeast plus glycerol with respect
to rate of proof time increase over 8 weeks
frozen dough storage ŽFig. 1.. This suggests
that glycerol synthesis andror retention
ŽMyers et al. 1997. and presumably yeast
viability is compromised by the freezing pro-
cess which worsens as the yeast used for
frozen dough production is stored, and added
glycerol compensates for this more effectively
with ageing yeast. The benefits of glycerol
addition are dependent upon the equilibra-
tion of the compatible solute into yeast cells.
Its addition to yeast immediately prior to
dough preparation fails to give the effects
Enhanced frozen dough leavening 49
50 D. K. Myers and P. V. Attfield

Figure 1. Effect of glycerol loading of yeast on proof times of stored frozen sweet doughs. Commer-
cially prepared baker’s yeast was mixed or not with glycerol and allowed to equilibrate for 72 h at 48C.
Yeast filter-cakes were then stored refrigerated for Ža., 1; Žb., 10; Žc. 20 days prior to use in preparation
of 10% sugared doughs. Doughs were proofed either prior to blast freezing to a core temperature of
;y58C Žzero time. or after 1 to 8 weeks storage at y218C. Control yeast with no added glycerol Žv.;
yeast with glycerol added to 0.4 M Žtotal water basis. ŽB.. Data points represent averages of duplicate
doughs and errors between duplicates were less than 5%.

observed with equilibrated yeast ŽMyers et References

al. 1998.. In the case of frozen dough produc-
tion, yeast has insufficient time at physio-
logically active temperatures to produce
significant levels of glycerol and hence is
ill-equipped to survive the dehydration and
hyperosmotic effects of freezing and thawing.
In effect, yeast cannot regain turgor and
commence growth, which is linked to leaven-
ing activity unless it can synthesise suffi-
cient glycerol in doughs ŽMyers et al. 1997..
Thus, addition of glycerol to yeast with a
period for uptake into cells, provides the
necessary protection against hyperosmotic
pressure arising in sweet doughs and from
freezing and thawing of doughs.
In conclusion, the addition of glycerol to
baker’s yeast with a period for equilibration
of the compatible solute into cells provides
for better leavening of frozen dough. This
effect is most prevalent in sweet frozen dough
and is increasingly obvious in yeast that has
been stored refrigerated prior to preparation
of dough.
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