Food Bioprocess Technol (2014) 7:29622968

DOI 10.1007/s11947-014-1303-0

ORIGINAL PAPER

Postharvest Control of Gray Mold in Apples with Lyophilized

Formulations of Cryptococcus laurentii: the Effect of Cold Stress
in the Survival and Effectiveness of the Yeast
Leonardo G. Navarta & Juan Calvo & Paola Posetto &
Soledad Cerutti & Julio Raba & Delia Benuzzi &
Mara I. Sanz

Received: 20 October 2013 / Accepted: 16 March 2014 / Published online: 4 April 2014
# Springer Science+Business Media New York 2014

Abstract Cryptococcus laurentii BNM 0525 adapted to cold

was used for developing a lyophilized formulation for controlling Botrytis cinerea (gray mold) in apples. For cold stress,
the yeast was grown at 1 C for 48 h. The trehalose content of
stressed cells reached 109 mg g1 after 24 h, but it did not
show significant modification in unstressed cells which were
grown at 28 C. The skimmed nonfat milk 10 %, yeast extract
0.5 %, and glucose 1 % (SMYG) medium was chosen as
freeze-drying protectant for stressed and unstressed cells.
After the freeze-dried process, stability of stressed yeast cells
was significantly higher along 90 days of storage at 4 C than
that of unstressed cells. The effectiveness in protection against
B. cinerea was also improved. When apple protection was
performed with freeze-dried cells, the maximum protection
was obtained with stressed cells. In this case, decay reduction
percentage was 79.30 %, and there were no significant differences when a lyophilized formulation stored for 90 days was
used. Unstressed cells were less effective immediately after
the freeze-dried process (69.12 %) and less resistant to storage. The percentage of decay reduction was less to 60 % when
applied with unstressed freeze-dried cells stored during
90 days. Cold stress increased the trehalose content in
C. laurentii cells and improved the behavior of the yeast in
front of preservation operations and also its effectiveness for
controlling B. cinerea.
L. G. Navarta : J. Calvo : P. Posetto : D. Benuzzi : M. I. Sanz
Laboratorio de Microbiologa Industrial, rea de Tecnologa
Qumica y Biotecnologa, Departamento de Qumica, Facultad de
Qumica, Bioqumica y Farmacia, Universidad Nacional de San Luis,
Ejrcito de los Andes 950, 5700 San Luis, Argentina
S. Cerutti : J. Raba : M. I. Sanz (*)
Instituto de Qumica San Luis, INQUISAL, CONICET, Universidad
Nacional de San Luis, Ejrcito de los Andes 950, 5700 San Luis,
Argentina
e-mail: msanz@unsl.edu.ar

Keywords Cryptococcus laurentii . Cold stress . Botrytis

cinerea . Biocontrol . Freeze-drying . Gray mold . Postharvest
diseases

Introduction
Postharvest losses of fruit and vegetables can reach very high
values, representing more than 25 % of the total production in
industrialized countries and more than 50 % in developing
countries (Nunes 2012). Postharvest diseases limit the storage
period and marketing life of fruits and vegetables. The woundinvading fungus Botrytis cinerea is one of the most important
postharvest pathogens affecting pome fruits. This fungus is
the causal of gray mold in apples. The control of postharvest
mold rots relies on the use of synthetic fungicides, but the
demanding requirements in sustainable agriculture, integrated
crop management, and organic production have resulted in the
need of developing other methods to control postharvest decays (He et al. 2003; Sansone et al. 2005; Nunes 2012).
Several antagonistic microorganisms have been shown as
biological controllers because they reduce postharvest fungal
decay on pome fruits (Janisiewicz and Korsten 2002; Calvo
et al. 2010); however, nowadays, there are only few biological
products available in the market. The major obstacle in the
commercialization of biocontrol products is the development
of a shelf-stable formulated product (Coulibaly et al. 2010;
Droby et al. 2008). Freeze-drying under vacuum is the most
convenient and successful method of preserving bacteria,
yeast, and fungi (Ming et al. 2009). However, not all strains
are able to survive in quantitative rate and or retain biocontrol
activity after a freeze-drying process (Bonaterra et al. 2005;
Janisiewicz and Korsten 2002). This is critical since a high cell
concentration is necessary in order to obtain a good formulated product for commercial application that, moreover, can be

Food Bioprocess Technol (2014) 7:29622968

handled using the normal channels of distribution and storage

(Sharma et al. 2009). The microbial cell viability and efficacy
resulting after the freeze-drying process are dependent on
many factors, including the initial microorganism condition
(Morgan et al. 2006), the protective medium, the freezing
temperature, and the rehydration conditions (Hublek 2003).
Physiological manipulation of biocontrol agents can be used
to improve the behavior in front of preservation operations,
i.e., freeze-drying process, or to allow a best adaptation to
environmental conditions when they are applied. Moreover,
physiological manipulation may also enhance mechanisms of
biocontrol. Osmotic shocks using modified growth media
with NaCl (Texeid et al. 2005) exposition to high temperatures (Hernndez-Oropeza and Aranda-Barrada 2011) or limitations of nutrients (Aranda et al. 2004) are the different ways
used for improving the survival and effectiveness of the biocontrol agents.
Another study (Kandror et al. 2004) showed that below
10 C, yeasts have an adaptive response that protects viability
to subsequent exposure to low or freezing temperatures. More
recently, it was shown that cells of industrial strains growing at
15 C displayed enhanced freeze and frozen storage resistance
than those grown at 30 C. The adaptation of yeast cells to low
temperatures implies a change in gene expression with consequences at the level of metabolism, membrane physicochemical properties, and expectedly the production and accumulation of trehalose (Kandror et al. 2004; Tulha et al. 2010).
One potential microorganism that could be developed for
commercial applications is the naturally occurring yeast
Cryptococcus laurentii (Kuffer) strain BNM 0525, which
was isolated from the surface of apple fruits in a commercial
orchard in Cuyo region (Argentina) (Calvo et al. 2003). The
principal goal of this study was to develop a formulation with
C. laurentii BNM 0525 adapted to cold, with the aim of using
it against gray mold decay in apples. For reaching this objective, the proposal was to (1) investigate the effect of cold stress
on intracellular trehalose levels in the yeast, (2) select the
adequate cryoprotectant for freeze-dried process, and (3) assess the viability and efficacy of lyophilized formulations in
controlling gray mold in apples.

Material and Methods

Microorganisms
C. laurentii BNM 0525 was isolated and identified in our
laboratory from microbial consortiums obtained by picking
the surface of apple fruit throughout the growing season
(Calvo et al. 2007). The strain was deposited in the National
Bank of Microorganisms (WDCM938) of the Facultad de
Agronoma, Universidad de Buenos Aires (FAUBA),
Argentina. Stock cultures of C. laurentii were stored at 4 C

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on yeast extract 5 g L1, glucose 10 g L1, and agar-agar

20 g L1 (YGA).
B. cinerea was a kind gift to us from National Institute of
Agricultural Technology (INTA, Lujan de Cuyo, Argentina).
It was maintained on potato dextrose agar (PDA) medium
(200 mL of extract of boiled potatoes, 20 g of dextrose, 20 g
of agar, and 800 mL of water). To maintain virulence, it was
periodically grown on apples and reisolated. For conidial
production, B. cinerea was grown on PDA at 2025 C.
When the mycelium appeared, cultures were kept at 15 C
for inducing sporulation. After a week, spores were harvested
and suspended in 10 mL of sterile distilled water containing
0.05 % (v/v) Tween 80. The concentration of spore suspension
was determined with a Neubauer chamber and adjusted with
sterile distilled water to 1105 CFU mL1.
Cold Stress
C. laurentii cells were grown using yeast extract 5 g L1 and
glucose 10 g L1 (YG) broth. Cultures were made in 1-L
Erlenmeyer flasks with baffles containing 250 mL of medium
at 28 C on rotary shaker (2.5 eccentricity140 rpm). Cells
were harvested at the beginning of the stationary phase by
centrifugation 10,000 rpm for 10 min in a Sorvall SS-3
(DuPont Instruments). Then, yeast cells at 1108 CFU mL1
in YG broth were exposed to cold stress. The cultures in
Erlenmeyer flask on rotary shaker (120 rpm) were placed in
cold rooms at 1 C and were sampled at 12, 24, and 48 h.
Trehalose and colony-forming unit per milliliter were determined at these times. A culture of the yeast at 28 C was used
as control.
Extraction and Determination of Trehalose
Yeast cells exposed to cold stress (stressed cells (SCs)) were
harvested by centrifugation at 5,000 rpm for 5 min and
washed three times with cold distilled water in order to remove residual medium. Cells cultured at 28 C (unstressed
cells (UCs)) were used as control. The yeast paste was
dried in stove at 40 C overnight, and then, 10 mg was
taken for trehalose determination. Trehalose was extracted
from the dry yeast with water and determined using
high-pressure liquid chromatography. An Acquity Ultra
High-Performance LC system (Waters, Milford) equipped
with autosampler injection was used. The mobile phase was
formic acid 0.1 % at 0.5 mL min1. The determinations were
repeated twice.
Sample Preparation for Lyophilization
SCs and UCs were harvested by centrifugation at 10,000 rpm
for 10 min in a Sorvall SS-3 (DuPont Instruments). The
growth medium was decanted and the cell paste suspended

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in 15 mL of 0.05 M potassium phosphate buffer (PB), pH 6.5,

and centrifuged again. The resulting paste was suspended in
the protecting medium, and the initial cell concentration of
each suspension was adjusted to 109 CFU mL1 by the standard count method on YGA plates.
Selection of Protecting Medium
A basic protecting medium (SM10) was formulated with 10 %
(w/v) of powdered skimmed nonfat milk (SM) in deionized
water. SM10 was used as protective agent, alone (SM10) or with
additives. The additives were sugars such as trehalose, fructose,
and sucrose. These were tested in concentration of 10 %. Also, it
was assayed by other protecting mixture that consisted of SM
10 %, yeast extract 0.5 %, and glucose 1 % (SMYG).
For each protecting medium to assay, three autoclaved vials
were filled with 5 mL of sample (SC or UC) and placed at
70 C overnight. Then, the vials were desiccated in chamber
of a freeze-drier (Labconco7740030; Labconco Corp., Kansas
City, MO, USA), operating at 45 C and 0.05 mbar for 12 h.
The freeze-dried samples were rehydrated until reaching
the original volume (5 mL) with the medium to test and were
left at room temperature for 20 min. Then, serial dilutions
were performed employing the rehydration solution. The
number of CFU per milliliter was determined by the plating
as described previously. Plates were incubated at 28 C for
24 h. Then, the number of viable cells (survival level) was
calculated by comparing them before (N0) and after (Nf)
freeze-drying and rehydration processes. The determination
of Nf was carried out 24 h after freeze-drying treatments and
was repeated twice.
Stability of Freeze-dried Cells
Stability of freeze-dried cells (UC and SC) was assessed by
determining their viability after 90 days of storage (at 4 C).
The viability was determined as described previously.

Food Bioprocess Technol (2014) 7:29622968

were wounded (222 mm) in two places (midway between the

calyx and the stem end) with a punch. A 20-L suspension
(106 CFU mL1) of freeze-dried or fresh C. laurentii cells was
put in each wound. After 3 h at room temperature, the treated
wounds were inoculated with 20 L of B. cinerea spores (1
105 CFU mL1). Immediately, the apples were stored at 4 C and
905 % RH. The lesion diameters (severity) were determined
after 40 days of cold storage. Then, decay reduction percentage
was calculated. Fifty fruits constituted a single replicate, and each
treatment was replicated three times.
Population Dynamics of C. laurentii
Growth curves were done in fruit (Red Delicious apples). The
apples were wounded in the equatorial zone (222 mm) with a
punch. The wounds were inoculated with 20 L of stressed or
unstressed yeast cells (24 h after freeze-dried process) in suspension of known concentration (106 CFU mL1) and incubated for
35 days at 4 C and 905 % RH. For monitoring the population,
the wounded tissue was extracted by using a cork borer (6-mm
internal diameter), was suspended in 50 mL of sterile distilled
water, and was shaken on a rotatory shaker for 20 min at
240 rpm. Serial dilutions of the washings were made and plated
on YGA plates. The colonies were counted after 48 h of incubation at 28 C. There were three replicates of three fruit per
treatment, and the experiment was repeated twice.
Data Analysis
Survival percentages of C. laurentii were estimated on the
basis of CFU per milliliter counted before (fresh cells) and
after freeze-dried treatments:
% Viability N f =N 0  100:
The percentage of decay reduction was calculated on the
basis of lesion diameters () as follows:
% Reduction decay controltreatment=control  100:

Determination of Antagonistic Activity of Freeze-dried

C. laurentii Cells Against B. cinerea on Apples in Cold
Storage
Antagonistic activity of freeze-dried C. laurentii cells (SC and
UC) against B. cinerea was tested on apples cv. Red Delicious.
The treatments applied consisted in SCs and UCs before and 24 h
after the freeze-dried processes and 90 days after this operation.
The fruit came from a commercial orchard in San Luis,
Argentina, and was selected free of wounds and rots and, as
much as possible, homogeneous in maturity and size. Before the
assay, apple surfaces were disinfected by immersion for 1 min in
a dilute solution of sodium hypochlorite (1 % active chlorine),
washed two times by immersion in distilled water, and left in a
dry place to remove excess water off the surface. Then, the apples

Differences in percentages were analyzed by one-way

analysis of variance (ANOVA) followed by multiple comparison test of Tukey (P<0.05).
Data from trehalose content were compared in a Students t
test. Significance was judged at level P<0.05.

Results
Intracellular Trehalose Content of C. laurentii After Cold
Stress
The content of trehalose in SC and UC cells of C. laurentii was
measured, and the results are shown in Fig. 1. Trehalose

Food Bioprocess Technol (2014) 7:29622968

2965

protectant and the internal content of trehalose. The viability

of C laurentii cells in SM10 was, for UC and SC, 11.40 and
16.10 %, respectively, while in SM10 plus trehalose, the
viability reached 36 and 43 % for UC and SC, respectively.
The best protection was given by the SMYG (81.91 % for UC
and 96 % for SC). In all cases, the viability of cells with high
trehalose content (SC) was higher, independent of the cryoprotectants used.
Taking into account that SMYG medium gave the best
results, all following assays were carried out using SMYG
as freeze-drying protectant.
Stability of Freeze-dried Cells
Fig. 1 Effect of cold stress on intracellular trehalose content of
C. laurentii. Data from trehalose content were compared in a Students t
test. Significance was judged at level P<0.05

content of SC was significantly increased from 60 to

109 mg g1 after 24 h of cold stress, while the contents of this
disaccharide in UC did not show significant modifications
along the time of incubation. Taking into account that the
accumulation of trehalose was observed at 24 h, all studies
were carried out with cells harvested at this time.
Effect of Preservation Media on Viability of C. laurentii BNM
0525 After Freeze-drying
The viability of cells (UC and SC) of C laurentii after freezedrying using different preservation media is shown in Fig. 2.
As can be seen in Fig. 2, significant differences in the viability
of cells after freeze-drying were observed depending on the
Fig. 2 Effect of preservation
media on viability of C. laurentii
BNM 0525(SC and UC) after
freeze-drying. Differences in
percentages were analyzed by
one-way analysis of variance
(ANOVA) followed by multiple
comparison test of Tukey
(P<0.05)

Viability of cells stored during 90 days was compared with the

cells recently processed (24 h after freeze-dried process)
(Fig. 3). Results showed that the SCs were more resistant
and more stable than the unstressed ones. After 90 days of
storage, there was no significant difference between the SCs,
while the UCs showed a statistically significant decrease in
their viability.
Antagonistic Activity of Freeze-dried C. laurentii Cells
Against B. cinerea on Apples in Cold Storage
After 40 days of cold storage, apples inoculated with
B. cinerea and protected with freeze-dried C. laurentii cells
(UC and SC) were examined, and the lesion diameter was
registered to calculate percentage of decay reduction. Fresh
cells (UC and SC) were used as control. Results are shown in
Fig. 4. Decay reduction percentage was of 91.62 % when
apples were protected with fresh cells, and there was no

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Food Bioprocess Technol (2014) 7:29622968

significant difference in number of cells between UCs and

cold SCs. The lag period for UC was 12 days while for SC, the
lag period was only 7 days (Fig. 5).

Discussion

Assay of population dynamics in apple wounded at 4 C

showed an evident difference in the lag period and a

The cold stress of C. laurentii BNM 0525 improved its survival

and effectiveness against B. cinerea perhaps because to the
increment in intracellular trehalose. Different authors have
reported accumulation of trehalose in various microorganisms
in response to heat shock and osmotic shock (Kandror et al.
2004) or induced by a culture medium formulated with citric
acid (Li and Tian 2006) or trehalose ( Kandror et al. 2002; Li
et al. 2008). Also, trehalose is considered as a suitable protectant
for liquid formulation of C. laurentii. Moreover, combining
L-ascorbic acid with this sugar improved the protective
efficiency (Liu et al. 2009). Adaptation to cold of different
microorganisms, among them yeasts, is manifested with the
accumulation to intracellular trehalose (Kandror et al. 2004)
demonstrated an adaptive response in yeasts that was activated
below 10 C and increased tolerance to low temperatures and
freezing. This response involved induction of trehalosesynthesizing enzymes. Aguilera et al. (2007) revised the
mechanisms of response to cold stress in Saccharomyces
cereviseae, among them, the induction of enzymes related
with trehalose synthesis. However, so far, there were no
reported works with induction of trehalose as a response to
cold stress in C. laurentii as we report here. In the present
study, the maximum accumulation of trehalose in cold SCs
was reached at 24 h, and the level was similar to one reported
by Li and Tian (2006). An interesting fact was the decrease of
this sugar after 24 h which coincided with the beginning of

Fig. 4 Antagonistic activity of freeze-dried C. laurentii cells (SC and

UC) against Botrytis cinerea on apples in cold storage (40 days at 4 C).
Differences in percentages were analyzed by one-way analysis of variance (ANOVA) followed by multiple comparison test of Tukey (P<0.05)

Fig. 5 Population dynamics of C. laurentii BNM 0525 (SC and UC; 24 h

after freeze-dried process) on the surface of wounded apples incubated at
4 C for 35 days

Fig. 3 Stability of freeze-dried C. laurentii cells (SC and UC). Differences in percentages were analyzed by one-way analysis of variance
(ANOVA) followed by multiple comparison test of Tukey (P<0.05)

significant difference between SCs and UCs. When protection

was performed with freeze-dried cells, the maximum protection was obtained with SC. In this case, decay reduction
percentage was 79.30 %, and a similar result was obtained
with freeze-dried SC (no significant differences) stored for
90 days. UCs were less effective immediately after the freezedried process (69.12 %) and less resistant to storage. As can be
seen in Fig. 4, decay reduction percentage was less to 60 %
when applied with freeze-dried UC with storage of 90 days.
Population Dynamics of C. laurentii

Food Bioprocess Technol (2014) 7:29622968

growth in cold conditions while at this same time, at 28 C, the

yeast reached the stationary phase (data not shown). Studies
carried out with the yeast S. cerevisiae determined that the
biological function of trehalose is protecting certain cell
structures such as membranes and proteins in the cell cytosol
when culture conditions are not optimal. Furthermore, the
sugar is hydrolyzed during the budding which would mean
that it also has a function as reserve carbohydrate for the
reproduction of the yeast (Hernndez-Oropeza and ArandaBarrada 2011). Stability of SC was significantly higher along
90 days of storage at 4 C than that of UCs. In fact, cold stress
significantly improved the survival of the yeast. Also,
effectiveness against B. cinerea was improved. The best results
were obtained with SC in SMGY as protecting medium. These
results suggested two important points to consider: The first
point is related to the internal accumulation of trehalose
induced by adaptation to cold which significantly improved the
survival and the ability of cells for growth in the apple wounds,
decreasing the lag period and increasing the number of cells
reached after 35 days of incubation at 4 C. Consequently, the
effectiveness against B. cinerea was improved since the main
mode of action of yeasts as biological control agents is
competition for space and nutrients (Sharma et al. 2009).
The second point is the nature of the cryoprotectant used.
SMYG is a mixture of cryoprotectants, which showed to
be a good vehicle for using in lyophilized formulations
of biological control agents since similar results were
obtained previously when SMYG was used for freezedried process of Rahnella aquatilis (Navarta et al. 2011).

Conclusions
The results reported here showed a promising way to preserve
antagonist yeast as C. laurentii for commercial application.
The cold stress combined with SMYG as cryoprotectant
seems to be a good tool for reaching an adequate stability of
lyophilized formulations of the yeast for application to biological control of B. cinerea. Moreover, adaptation to cold
showed to be a good strategy for improving the effectiveness
of C. laurentii as a controller of this fungus.
Acknowledgments The support by the Universidad Nacional de San
Luis, the Agencia Nacional de Promocin Cientfica y Tecnolgica, and
the Consejo Nacional de Investigaciones Cientficas y Tcnicas
(CONICET) is gratefully acknowledged.

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