Improvement of the cryopreservation of the fungal starter Geotrichum
candidum by artificial nucleation and temperature downshift controlq
G. Missousa,b, B. Thammavongsa, V. Dieuleveuxb, M. Gue ´guena, J.M. Panoffa,*
aLaboratoire de Microbiologie Alimentaire (EA 3213), IBFA—ISBIO, Universite ´ de Caen Basse-Normandie,
Esplanade de la Paix, 14032 Caen cedex, France
bLaboratoire De ´partemental F. Duncombe, 1 rte Rosel, 14053 Saint Contest, France
Received 8 February 2007; accepted 30 May 2007
Available online 8 June 2007
Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter pro-
duction. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some
specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out
using a Peltier cooler–heater incubator, which permits to control the temperature downshift from +20 to ?10 ?C in time period ranges
from 20 to 40 min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from
Pseudomonas syringae (SNOMAX?) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of
this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucle-
ation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from ?8.6 to ?2.6 ?C, (iii) a
significant decrease of the lethality of G. candidum cells subjected to a freezing–thawing cycles challenge.
? 2007 Elsevier Inc. All rights reserved.
Keywords: Geotrichum candidum; Starter; Freezing; Nucleation; Stress; Cryopreservation
Any modification of environmental parameters that
leads to a response by living organisms may be considered
as a stress [1,33,35]. Currently, two types of stress are pro-
posed: abiotic stresses including physical and chemical ones
and biotic stresses. As a global phenomenon, it can be actu-
ally extended to anthropogenic pressure such as pollution
or genetic engineering .
From prokaryotes [25,31] to humans , including
plants , animals  and fungi  cold temperature
is one of the major stresses that most of the living organ-
isms have to confront . At subzero temperatures, condi-
tions considered to equate to ‘moderate’ or ‘severe stress’
according to Yousef and Courtney , the response of
most microorganisms is passive, leading to a slow lethality
of cells. Freezing stress is considered to be the combination
of both osmotic and mechanical stresses, leading to cryo-
injury of cellular structures and macromolecular damage.
Freezing is a transition from liquid to solid state by a
mechanism called nucleation. Two types of nucleation
can be observed: the first one, called homogeneous nucle-
ation, occurs in pure liquid whereas the second one, called
heterogeneous nucleation, occurs in a liquid which contains
foreign substances around which the ice crystal develops.
Those foreign substances act as nucleation sites at a rela-
tively high sub-zero temperature [15,36,3,16] and are so
called ice nucleators. Numerous ice nucleators have been
described in literature such as silver iodide, river sand
[37,4] and specific microbial proteins. Indeed, some micro-
organisms, particularly phytopathogenic ones such as
Pseudomonas syringae, Erwinia herbicola, Xanthomonas
campestris and Fusarium moniliforme, synthesize proteins
which initiate nucleation and consequently induce frost
0011-2240/$ - see front matter ? 2007 Elsevier Inc. All rights reserved.
qThis work was supported by the ‘‘Ministe `re de l’Education Nationale,
de l’Enseignement Supe ´rieur et de la Recherche’’, the ‘‘Conseil Re ´gional
de Basse-Normandie’’ and the ‘‘Conseil Ge ´ne ´ral du Calvados’’.
*Corresponding author. Fax: +33 (0) 2 31 56 61 79.
E-mail address: email@example.com (J.M. Panoff).
Cryobiology 55 (2007) 66–71
damages to their hosts, in order to access to nutrients
Geotrichum candidum is a filamentous yeast-like fungus.
This ubiquitous microeucaryote, considered as the ana-
morph of Galactomyces candidus , is widely used as food
starter to ripen soft and semi soft cheeses and in fermented
milks [19,39,2]. More recently, G. candidum has been inte-
grated to the malting process as a bioprotective agent
. Its optimal growth temperature is between 22 and
25 ?C  and the average highest growth temperature
around 35–36 ?C even if some strains can tolerate higher
temperatures and develop until 39 ?C . According to
some authors, the lowest growth temperature is around
4 ?C [8,27].
Most of the few studies related to the cryopreservation
of G. candidum are focused on the physiological adaptation
of this microorganism to freezing stress by mild stress pre-
treatment. This phenomenon, which is reversible and not
heritable, leads to a transient increased resistance to the
freezing challenge. Two types of physiological adaptation
to negative temperatures have been observed with G. candi-
dum depending on the type of pre-treatment: (i) Homolo-
gous adaptation when cells were pre-incubated at low
positive temperatures . (ii) Heterologous adaptation
(cross protection) when cells were pre-exposed to specific
chemicals . Recently, cryopreservation experiments com-
pleted on an artificial microbial community (consortium)
of dairy interest, including G. candidum and two prokary-
otes, highlighted an interspecies cryoprotective phenome-
Our main goal is to improve the cryopreservation of G.
candidum. The work presented in this manuscript has been
carried out as a necessary step to increase our understand-
ing of the fundamental mechanism related to the cold stress
responses. We have developed a new freezing process,
which brings together the control of the temperature down-
shift (Peltier cooler–heater) with the addition of an indus-
Pseudomonas syringae and used in different cryobiological
Materials and methods
Strain and culture conditions
The study was carried out with Geotrichum candidum
ATCC204307, obtained from the laboratory collection
(UCMA91). This strain initially isolated from a cheese
(Pont l’Eve ˆque, Protected Designation of Origin) made
with raw milk, is cryopreserved (?80 ?C) as a cell suspen-
sion in glycerol 15% (v/v).
Cells were spread on MEA (Malt Extract Agar) medium
, incubated 48 h at 25 ?C, and suspended in 3 mL of
0.9% (w/v) NaCl in order to prepare a preinoculum for fur-
One milliliter of the preinoculum was added to 100 mL
of MEB (Malt Extract Broth) medium  in a 1 L Erlen-
meyer flask, and incubated 48 h at 25 ?C with orbital shak-
ing at 150 rpm (AS850, LSL Biolafitte SA, St. Germain en
Laye, France). Cells (stationary growth phase) were pel-
leted at 2400g for 10 min (Eppendorf centrifuge 5810 R,
Hamburg, Germany), washed and centrifuged twice
(2400g, 10 min) in 0.9% (w/v) NaCl. Cells were then resus-
OD620 nm= 1 ± 0.05 (3.5 · 106TFU/mL) using a spectron-
ic 301 spectrophotometer (Bioblock Scientific, Illkirch,
France) and divided in 1 mL aliquots into 1.5 mL micro-
Freezing was performed using a Peltier cooler–heater
(PCH-2, Grant-bio, Cambridgeshire, England) which
enables to control the temperature downshift from +20
to ?10 ?C in time period ranges from 20 to 40 min accord-
ing to the experiments. This apparatus has a stand of 20
wells which contain 1.5 mL microtubes. The temperature
of the samples was measured and recorded every 10 s using
a thermic probe (Testo175-T3, Germany) inserted into one
Freezing–thawing challenges on G. candidum were per-
formed as follow: 1 mL aliquots were incubated 5 min at
20 ?C then 40 min at ?10 ?C. This challenge was repeated
five times per experiment. At different cycles, cells suspen-
sions were sampled, diluted in NaCl 0.9% (w/v) and spre-
aded on MEA medium. The plates were incubated at
25 ?C for 48 h before TFU counting.
Depending on the experiments, different concentrations
(from 10 fg/mL to 1 mg/mL) of ice nucleant [SNOMAX?
(York International, York, PA, USA)] were added to ster-
ile distilled water or G. candidum suspensions. The effective
freezing of the samples was evaluated by visual assessment.
The results are the means of at least three experiments.
Results and discussion
Asynchronous freezing of G. candidum
In order to optimize the freezing–thawing process of
G. candidum, cells suspensions were subjected to a con-
trolled challenge using a Peltier cooler–heater as described
above. Dynamic of freezing of G. candidum was followed
concomitantly in 20 microtubes. Cooling and warming rate
were continuously measured using a thermic probe inserted
in a sample as control. The temperature reached 0 ?C
10 min after the beginning of the controlled temperature
downshift. Interestingly, as described in the Fig. 1(A), the
20 suspensions did not freeze simultaneously: some samples
were frozen 20 min after the beginning of the experiment
whereas others remained liquid 50 min after.
Fig. 2 shows, through the example of two samples, that
the temperature profiles were clearly similar with a nucleat-
ing temperature of ?8.6 ?C. Nevertheless, time to reach the
nucleation point was different between the two samples
G. Missous et al. / Cryobiology 55 (2007) 66–71
[29 min (S1?) and 38 min (S2?)]: In our conditions, the
spontaneous and asynchronous freezing process main-
tained G. candidum suspensions in a supercooled state dur-
ing various periods.
This unexpected chaotic behavior of the nucleation pro-
cess of the cell suspensions has led to a new method being
developed in order to avoid the asynchronous freezing of
the samples. According to the literature  a spontaneous
nucleation can be shifted to an artificial one by addition of
ice nucleating agents. This procedure was tested, first, to
sterile distilled water and, second, to G. candidum
Water as control
Dynamic of freezing of sterile distilled water was fol-
lowed concomitantly in 20 microtubes as described above
for G. candidum suspensions. Depending on the experi-
ments, different concentrations of ice nucleator were
added to the samples. According to Fig. 3, the freezing
kinetic of the water is a function of the concentration of
SNOMAX?. Without ice nucleator, freezing of the sam-
ples was highly asynchronous, distributed from 15 min
to more than 17 h. Even after 48 h, not all the samples
0 600120018002400 30003600
Fig. 2. Cooling rate of G. candidum suspensions with [(h), 100 lg/mL] and without ice nucleator [(d), (·)]. The cooling temperature values were obtained
and recorded with a thermic probe each 10 s. The nucleation points S+ (?2.6 ?C) and S1?/S2? (?8.6 ?C) correspond to experiments with and without ice
Number of frozen samples
Fig. 1. Ice nucleator effect on the dynamic of freezing of G. candidum. Twenty samples were prepared as described in the text and the evolution of the
number of frozen samples was assessed visually each 5 min. The graphs correspond to six independent experiments: (A) without ice nucleator [(h); (s);
(D)] and (B) with 100 lg/mL of ice nucleator [(+); (·); (})]. Results corresponding to the experiments with SNOMAX?are superimposed.
G. Missous et al. / Cryobiology 55 (2007) 66–71
were frozen (from 2 to 19 microtubes depending on the
experiments; data not shown). After addition of SNO-
MAX?(100 lg/mL), all the samples were frozen 15 min
after the beginning of the temperature downshift, which
corresponds to five min exposure at subzero temperature.
As an intermediate condition, addition of 100 fg/mL of
SNOMAX?lead to a relative asynchronous freezing of
all the samples, distributed from 15 to 40 min.
Interestingly, the standard deviations were signifi-
cantly lower in this intermediate condition than in the
control one (without ice nucleator). SNOMAX?not
only reduced the time to reach the freezing state but also
the chaotic distribution of the nucleation among the
In parallel, addition of the ice nucleator lead to a signif-
icant increase of the nucleation temperature as shown in
Fig. 4. Below 105fg/mL, SNOMAX?effect was not
observed and the nucleation temperature of water, in our
conditions, was comprised between ?7.3 and ?7.6 ?C.
Above 108fg/mL, SNOMAX?effect was optimal and the
nucleation temperature was comprised between ?1.9 and
Number of frozen samples
Fig. 3. Ice nucleator effect on the dynamic of freezing of water samples. Twenty samples were prepared as described in the text and the evolution of the
number of frozen samples was assessed visually. Bars correspond to three different conditions: without (white bars) and with ice nucleator [100 fg/mL (grey
bars) and 100 lg/mL (black bars)]. Error bars indicate the standard deviations of the mean (white bars n = 4, black and grey bars n = 3).
Nucleating agent (Log10[x(fg/ml)+1])
Nucleation temperature (˚C)
Fig. 4. Effect of SNOMAX?concentrations on the nucleation temperature of water samples. The concentrations were varied from 10 fg/mL to 1 mg/mL
in steps, each subsequent step being a factor of 10 higher than the previous one. Error bars indicate the standard deviations of the mean of three
G. Missous et al. / Cryobiology 55 (2007) 66–71
The nucleating agent SNOMAX?(100 lg/mL) was then
added to G. candidum suspensions and, as shown in
Fig. 1(B), all the samples froze simultaneously 15 min after
the beginning of the temperature downshift, with a freezing
temperature value of ?2.6 ?C as shown in the example (S+)
in Fig. 2. Indeed, ice nucleator addition initiated the nucle-
ation at a relatively high subzero temperature leading to an
(Fig. 2S1?/S2?), the suspensions remained supercooled
until the spontaneous nucleation occurred. Finally, the
addition of ice nucleator to cells suspensions led to the syn-
chronous freezing of all the samples, improving the exper-
The effect of the ice nucleator on the survival of G. candi-
dum submitted to freezing–thawing stress was ultimately
evaluated. Three concentrations of SNOMAX?
chosen as leading to an optimal synchronous freezing
according to Fig. 4. As shown in Fig. 5, after the first freez-
ing–thawing cycle, the percent of survival was 60 ± 4% with-
out nucleating agent and, interestingly, 71 ± 4%, 74 ± 5%
and 79 ± 6% with 0.1, 1 and 10 lg/mL of SNOMAX?,
respectively. This decrease of lethality, related to the addi-
tion of the ice nucleator, was confirmed after three and five
freezing–thawing cycles, and seems concentration depen-
dent. However, the freezing–thawing stress generated by
the first cycle lead to a lethality level which stabilized during
the third and fifth cycles. The presence of both vegetative
(Hyphae) and differentiate (arthrospores; ?60%; unpub-
lished results) cells in G. candidum cultures might be the
cause of this non-linear death rate of the whole population.
Cold stress is, in parallel to its interesting fundamental
aspect, associated to many biotechnological applications,
particularly the freezing processes . Two types of appli-
cation are related to freezing stress: (i) quality optimization
of the frozen starters used in food technology and (ii) ex
situ preservation of biodiversity by frozen storage of
Microbial ice nucleators release the nucleation at a rela-
tively high sub-zero temperature, reducing the temperature
of supercooling and, concomitantly, the cell lethality .
This phenomenon gives them potential biotechnological
applications in food freezing processing, like cryopreserva-
tion and freeze-concentration [13,38].
In the present work, we have developed a two-step
methodology in order to optimize the cold storage of
G. candidum. Freezing experiments were carried out using
a Peltier cooler–heater (PCH TD) incubator concomitantly
to the addition of an industrial ice nucleator (SNOMAX?)
to the microbial suspension. To summarize, three comple-
mentary effects arise from this new methodology: the
increase of the nucleating temperature, the synchronization
of the nucleation and the decrease of the lethality to freez-
ing–thawing stress of G. candidum. These results should
permit to control the impact of freezing stress on biological
material such as G. candidum, using an improved process of
Number of freezing / thawing cycles
Fig. 5. Survival of G. candidum as a function of the number of freezing–thawing cycles (?10 ?C/+20 ?C). Cells were subjected to a freeze–thaw challenge
as described in the text. Bars correspond to four different conditions: without (white bars) and with ice nucleator [0.1 lg/mL (light grey bars); 1 lg/mL
(dark grey bars); 10 lg/mL (black bars)]. Error bars indicate the standard deviations of the mean of four independent experiments.
G. Missous et al. / Cryobiology 55 (2007) 66–71
Acknowledgments Download full-text
This work was supported by the ‘‘Ministe `re de l’Educa-
tion Nationale, de l’Enseignement Supe ´rieur et de la
Recherche’’, the ‘‘Conseil Re ´gional de Basse-Normandie’’
and the ’’Conseil Ge ´ne ´ral du Calvados’’. The authors
would like to thank ‘‘York Neige’’, Dardilly, France for
providing a sample of SNOMAX?.
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