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Biocontrol Science and Technology

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Survival of the postharvest biocontrol

yeast Candida sake CPA-1 after
dehydration by spray-drying
a a a a a
M Abadias , N Teixid , J Usall , C Solsona & I Vias
a
CeRTA, Centre UdL-IRTA , Postharvest Area , Lleida, Catalonia,
Spain
Published online: 18 Jan 2007.

To cite this article: M Abadias , N Teixid , J Usall , C Solsona & I Vias (2005) Survival of the
postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying, Biocontrol
Science and Technology, 15:8, 835-846, DOI: 10.1080/09583150500187041

To link to this article: http://dx.doi.org/10.1080/09583150500187041

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 Biocontrol Science and Technology, December 2005; 15(8): 835
/846

Survival of the postharvest biocontrol yeast Candida

sake CPA-1 after dehydration by spray-drying

, J. USALL, C. SOLSONA, & I. VIN

M. ABADIAS, N. TEIXIDO AS

Postharvest Area, CeRTA, Centre UdL-IRTA, Lleida, Catalonia, Spain

(Received 7 October 2004; returned 21 January 2005; accepted 30 March 2005)
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Abstract
Spray drying was evaluated as a dehydrating method to preserve the postharvest biocontrol
agent Candida sake CPA-1. The effect of drying temperature, carrier, growth and rehydrating
medium on the survival of the yeast was studied. Outlet temperature had more influence on the
death of the cells than inlet temperature, and survival decreased with increasing temperature.
Spray drying at an inlet temperature of 1508C was optimum in terms of viability, powder
recovery and moisture content of the product. Use of 10% (v/v) skimmed milk as a carrier gave
the highest survival and percentage of powder recovery (34
/47%). Rich rehydration media were
found to be better than water or phosphate buffer, with slight differences on survival. Spray-
dried cells were less effective than fresh ones in controlling Penicillium expansum rot on apples.
Spray drying of C. sake was not a good dehydration method as it gave low cell survival, poor
recovery of product, and low efficacy.

Keywords: Candida sake, spray-drying, dehydration, antagonist, formulation, blue mold,

postharvest diseases, biocontrol

Introduction
Postharvest diseases have the potential to cause considerable losses to fruit and
vegetables (Janisiewicz & Korsten 2002). Losses have mainly been reduced by
applying postharvest fungicides (Eckert & Ogawa 1988) and, to a lesser degree, by
postharvest management practices. Over the past 15 years, biological control has
emerged as an effective strategy to combat major postharvest diseases of fruits
(Janisiewicz 1988, 1998; Wilson & Wisniewski 1989; Korsten et al. 1994). Although
biological control of postharvest diseases is in its infancy, the first commercial
products have been registered in the United States, and are sold under the names
BioSave 100 and 110 (based on Pseudomonas syringae ) and Aspire (based on Candida
oleophila ). In South Africa, YieldPlus, a Cryptococcus albidus-based product is also
commercialized (Janisiewicz & Korsten 2002).
In Europe, the strain CPA-1 of Candida sake has been shown to be an effective
biocontrol agent of the major fungal pathogens of pome fruits at a small, semi-
commercial (Teixido et al. 1998c; Vinas et al. 1998; Usall et al. 2000) and
commercial (Usall et al. 2001) scale. The major obstacle to the commercialization

Correspondence: M. Abadias, Centre UdL-IRTA, Rovira Roure, 191, 25198 Lleida, Catalonia, Spain. Tel:
34 973 003 430. Fax: 34 973 238 301. E-mail: isabel.abadias@irta.es

ISSN 0958-3157 print/ISSN 1360-0478 online # 2005 Taylor & Francis

DOI: 10.1080/09583150500187041
 836 M. Abadias et al.

of biocontrol products is the development of a stable formulated product that

retains biocontrol activity similar to that of the fresh cells (Janisiewicz & Jeffers
1997). Drying the product and maintenance in a dry environment or suspension in oil
are common approaches that allow microbial agents to be handled commercially
during distribution and storage (Rhodes 1993). The feasibility of preserving the
biocontrol agent C. sake as a solid formulation has been recently studied using
freeze drying as a dehydration method (Abadias et al. 2001a,b). Viability of
C. sake cells after freeze-drying was 85%, but the efficacy of the dried product
against Penicillium expansum infection of apples was lower than that obtained with
fresh cells. Moreover, its viability decreased by up to 10% after two months storage at
48C (Abadias et al. 2001b). Other studies have evaluated the possibility of
commercializing the yeast as a liquid formulation. It was found that cells grown
in a molasses medium with reduced water activity (aw) by the addition of sorbitol,
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and preservation in a solution of trehalose at 48C for 7 months, maintained their

viability and efficacy against P. expansum infection of apples (Abadias et al. 2003).
However, to be of practical use, it would be better to maintain the product as a dry
formulation at room temperature, which would lower costs for transport and storage.
Spray drying may be an alternative method to freeze drying because it allows large
quantities of cultures to be dried at low cost (Foster 1962). The main disadvantage
of spray drying is the extent of the destruction of the microbial cells during the
drying process. Survival of the biocontrol agent Pantoea agglomerans strain CPA-2
in a spray drying process has recently been studied and found to be very sensitive
with B/8% of cells surviving after the process using MgSO4 as a carrier (Costa
et al. 2002a).
Growth and rehydration media are important factors affecting the survival of dried
microorganisms (Font de Valdez et al. 1985; Teixeira et al. 1995; Costa et al.
2002a,b). Recent studies have demonstrated that rehydration media had a high
influence on cell recovery after freeze-drying of C. sake (Abadias et al. 2001b).
Therefore, it is possible that the rehydration process could also be a critical step in the
recovery of spray-dried cells. Growth medium had a strong influence on survival of
C. sake cells preserved as a liquid formulation. Cells that were grown in a cane
molasses medium with its aw reduced to 0.98 by the addition of sorbitol and preserved
in a trehalose solution, survived better than those grown in unamended molasses
medium (Abadias et al. 2003). Some studies have demonstrated that growth medium
can influence the pattern of intracellular solutes accumulated in yeasts (Spencer &
Spencer 1978; Teixido et al. 1998a,b; Abadias et al. 2000, 2001c). In some cases, such
modifications resulted in improved tolerance to water stress with retained biocontrol
activity (Teixido et al. 1998a,b).
The aim of this work was to evaluate spray drying as a dehydration method for
preserving C. sake cells. The effect of temperature and different carriers on survival of
C. sake on the thermal inactivation kinetics, and on the recovery and residual moisture
content of the dried powder was studied. The impact of growth and rehydration
medium was also examined. Finally, the efficacy of spray dried cells was tested against
P. expansum on apple fruits (cv. Golden Delicious).
 Spray drying of Candida sake 837

Materials and methods

Yeast strain
Candida sake (strain CPA-1) was obtained from the Postharvest Unit, Centre UdL-
IRTA, Lleida, Catalonia, Spain. It was originally isolated from the surface of apple
fruits, and it is deposited in the Coleccion Espanola de Cultivos Tipo, CECT-10817
(Universidad de Valencia, Campus de Burjasot, Burjasot, Valencia, Spain). Stock
cultures were stored at 48C and subcultured on nutrient yeast dextrose agar
(NYDA), which contained nutrient broth (8 g L1), yeast extract (5 g L1), dextrose
(10 g L1) and agar (15 g L1).

Cell production
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Several single colonies from a 48-h culture of C. sake were transferred from NYDA-
plates to 5-mL of potassium phosphate buffer (PB, 0.05 mol L1, pH 6.5), and this
inoculum was used to inoculate a BIOSTAT-A Modular fermenter (B. Braun Biotech
International, Melsungen, Germany) containing 5 l of molasses-based medium (cane
molasses (40 g L 1), urea (1.2 g L1), and antifoam, 0.5 mL) and set at 258C, 400
rpm and 150 L air h1. After 36
/38 h of growth, cells were harvested by centrifugation
(6981 / g for 10 min at 158C), resuspended in PB and enumerated using dilution
plating on NYDA. Plates were counted after incubation at 258C for 48 h.

Effect of protectants and temperature on the survival kinetics of C. sake

To test the effect of temperature and protectants, which also acted as carriers, on the
survival kinetics of C. sake, a volume of resuspended cells was dispersed into 50 mL of
each protectant to obtain an initial concentration of about 2 / 109 colony forming
units (CFU) mL 1. Based on previous studies (Abadias et al. 2001a; Costa et al.
2002a), the protectants tested were 10% MgSO4, 10% skimmed milk (SM), 10%
lactose (L) and a combination of 10% SM plus 10% lactose (SML). Suspensions of
protectants and yeast cells were incubated at 150 rpm for 30 min at room
temperature. The initial concentration of C. sake in the suspensions was determined
using dilution plating as described before. Thereafter, yeast suspensions were spray-
dried in a laboratory-scale spray dryer (SD-05, Lab Plant, UK). Moisture in the spray
droplets produced by a jet nozzle (0.5 mm in diameter) was evaporated into the drying
chamber (215 mm in diameter and 500 mm long). The powder passed through
a single cyclone separator and was collected in a collector bottle. Feed suspensions
were delivered by a peristaltic pump at 500 mL h 1, and the inlet air was heated to
140, 150, 160 and 1708C by an electrical heater. The percentage of recovered powder
was calculated for each temperature and carrier by the difference between the final
weight of the powder in the collector bottle, and the weight of solids in the initial
suspension.
Approximately 0.04 g of the powder was rehydrated with 5 mL of PB. The dried
cells were shaken vigorously for 1 min and then allowed to rehydrate for 9 min.
An equation was used to correct for small differences in solids after rehydration
(To & Etzel 1997a,b):
NNm Sf =Sm (1)
 838 M. Abadias et al.

where N /corrected CFU mL1 for the rehydrated sample, Nm /measured

CFU mL1 of the rehydrated sample, Sf /solid contents of the feed suspension
(g L1), and Sm /solid contents of the rehydrated sample (g L 1). Viable cells were
enumerated on NYDA by the dilution plate technique. The plates were counted after
incubation at 258C for 48 h.
The moisture content of the final powder was measured for each temperature and
carrier by placing spray-dried powder (1 g) in duplicate in an aluminium-weighing
boat and drying it in a convection oven at 1058C for 24 h.
To calculate the kinetic parameters, Kim and Bhowmik (1990) proposed the
equation

ln(ln N0 =N)ln At - Ea =RT (2)

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where N0 /initial cell count (CFU mL1), N /number of survivors (CFU mL1),
A /constant, t / time, Ea /activation energy (cal mol 1 K 1 or J mol 1 K1), R /
universal gas constant (1.98 cal K1 mol 1 or 8.31 J K1 mol 1), and T /absolute
temperature (K). This equation allowed us to calculate Ea of C. sake as follows: ln(ln
N0/N) was plotted against the reciprocal of T (1/T) and then the activation energy was
calculated from the slope of the straight line. The calculation is based on the
assumption that drying takes place in the same exposure time and that each particle is
subjected to the outlet air temperature during spray drying. Thus, it does not give true
activation energy, but an apparent value. The apparent activation energy is a good
indicator for comparing thermoresistance and death rate of microorganisms during
spray drying, even though it is not a true value (Kim & Bhowmik 1990).

Effect of growth medium on the viability of C. sake after spray drying

In order to study the effect of growth medium on the viability of C. sake after spray
drying, cells were grown either in the molasses-based medium, or in the same medium
but with aw modified to 0.98 with the addition of sorbitol (198.2 g L 1). Suspensions
of cells grown in both media were prepared in SM, MgSO4 and SML as described
above. Suspensions were spray-dried at 1508C and 500 mL h1, and thereafter
rehydrated with PB for 10 min. Cell viability was measured by dilution plating as
described previously.

Effect of rehydration medium and recovery time on viability of spray-dried cells

To evaluate the effect of the rehydration medium, a 200-mL suspension of 10% SM
and 2 / 109 CFU mL1 of C. sake was prepared as described above. C. sake
suspension was spray-dried at an inlet temperature of 1508C and a delivery rate of
500 mL h1. The powder obtained was rehydrated with 10% SM, 10% sucrose
(SUC), 1% peptone (PEP), a mixed medium, PTM peptone (15 g L1), meat extract
(5 g L1), tryptone (10 g L1), PB, water or SML (Abadias et al. 2001b; Costa et al.
2002a,b). The dried cells were allowed to rehydrate for 10 or 30 min. Initial and final
viable cells were determined as described previously. Corrected CFU mL1 values for
the rehydrated samples were calculated using equation (1) to correct differences in
solids after rehydration.
 Spray drying of Candida sake 839

Efficacy of spray dried cells against P. expansum on apple fruits (cv. Golden Delicious)
The efficacy of the best spray-dried treatments was evaluated and compared with the
efficacy of fresh cells. C. sake was spray-dried at 1508C using SM as a carrier.
Thereafter, 0.04 g of spray-dried powder was rehydrated with SM, SML, or water for
10 min and the cell concentration (CFU g1) determined by dilution plating.
According to counting, a suspension containing 2 / 107 CFU mL1 was prepared.
Fresh cells were obtained following growth in 250-mL conical flasks containing
50 mL of molasses medium. After 36
/38 h incubation at 258C and 150 rpm, cells
were centrifuged and resuspended in 20 mL of PB. Cell density was adjusted to
2 / 107 cfu mL1. Solutions of SM, SML without C. sake cells were also tested in
order to check that they did not have any effect on efficacy themselves.
Apple fruits (cv. Golden Delicious) were wounded with a punch, making an injury
of 2 mm in diameter and 2 mm deep at the stem (top) and another at the calyx
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(bottom). Cell suspension (15 mL) of spray-dried or fresh cells was applied to the
wounds. The fruits were allowed to dry at room temperature for about 2 h and then
wounds were inoculated with 15 mL of an aqueous suspension of P. expansum
(104 conidia mL 1) prepared from 10-day-old cultures incubated on potato dextrose
agar (PDA) at 268C. Each treatment was replicated four times with five apples per
replicate. The treated apples were incubated at 208C and 85% relative humidity (RH)
for 6 and 10 days, after which the percentage of infected wounds (incidence) was
determined.

Statistical analysis of results

For C. sake survival kinetics, statistical software (Excel 2000, Microsoft, Redmond,
Washington) was used to analyse the data to obtain the least square fit of the lines.
The General Linear Model procedure of SAS system (GLM, Statistical Analysis
Systems Institute, V8, Cary, NC) was performed on percentage of recovered powder
and residual moisture content for different carriers and temperatures, and on viability
at different growth, rehydration time and rehydration medium. Statistical significance
was judged at the level P B/0.05. Blue mould incidence data was transformed with the
arcsine of the square root of the proportion of infected fruits to improve homogeneity
of variances. When the analysis was statistically significant, the Duncan test was used
to separate the means.

Results
Survival kinetics of C. sake
No powder was recovered in the collecting bottle after spray drying C. sake using 10%
lactose as a carrier. Thus, this carrier was not used in further experiments. The
logarithmic survival ratios of C. sake in all carrier substances decreased when the inlet
air temperature increased (Figure 1). The highest survival was found when cells were
protected with SM, and the lowest when MgSO4 was used. The addition of lactose to
SM did not improve C. sake viability during spray drying and, at each temperature
tested, survival achieved with the combination was lower than that obtained with SM
alone. When extrapolated to lower inlet air temperatures, fitted regression lines
intersected 100% survival at 116.68C, 105.88C and 108.78C for SM, SML and
 840 M. Abadias et al.
Inlet temperature (C)
130 140 150 160 170 180
0

ySM = 00332x + 38724

1
R2 = 08606
Log10 (N No1)

2 1
ySML = 00491x + 53378
R2 = 09641
3

yMg = 00609x + 64442

4 2
R = 09481
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Figure 1. Relationship between inlet air temperature and logarithmic survival ratio of C. sake spray-dried
using: (^) SM; (k) SML; and (2) MgSO4 as carriers. Values are the mean of at least four replicates and
error bars represent the standard error of the mean.

MgSO4, respectively. However, below an inlet temperature of 1408C, powder

obtained with these carriers using this laboratory spray dryer was wet. When the
logarithmic survival ratio of C. sake was plotted against outlet temperature, higher
correlation coefficients were found (Figure 2). Again, an increase in outlet
temperature caused a decrease in survival with all protectants tested. At the same
inlet temperature, the use of SML as a carrier gave higher outlet temperatures. The
use of SML blocked the jet nozzle often, and it was necessary to clean it regularly.
To calculate the activation energy of C. sake , ln(ln(N0/N)) was plotted against the
reciprocal of absolute outlet temperature (Figure 3). The activation energy of C. sake
in SM or SML was higher (12.87 and 10.29 kcal mol 1K 1 or 53.88 and 43.08 kJ

Outlet temperature (C)

70 75 80 85 90 95 100
0
ySM = 00697x + 43318
R2 = 09517
1
log10 (N N01)

2
ySML = 01063x + 65566
R2 = 09784
3
yMg = 01209x + 67733
R2 = 09967
4

Figure 2. Relationship between outlet air temperature and logarithmic survival ratio of C. sake spray-dried
using: (^) SM; (k) SML; and (2) MgSO4 as carriers. Values are the mean of at least four replicates and
error bars represent the standard error of the mean.
 Spray drying of Candida sake 841
3
ySML = 51966x + 16183
R2 = 08383
2.5 Ea=1029 kcal K/mol yMg = 40258x + 13262
R2 = 07998
2 Ea=797 kcal K/mol
ln (ln(N0 / N))
1.5

1
ySM = 65022x + 19312
0.5 R2 = 08917
Ea=1287 kcal K/mol
0
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2.7 2.75 2.8 2.85 2.9

1/T (K)*1000

Figure 3. Relationship between ln(ln(N0/N)) and reciprocal absolute outlet temperature for C. sake spray-
dried using: (^) SM; (k) SML; and (2) MgSO4 as carriers. Values are the mean of at least four replicates
and error bars represent the standard error of the mean.

mol 1K1 respectively) than that of MgSO4 (7.97 kcal mol 1K1 or 33.67 kJ
mol 1K1). This means that more energy was required to kill C. sake in SM or the
combination SML than in MgSO4.
The inlet temperature and protectant significantly influenced the powder recovery
and moisture content of the product (Table I). The recovery was independent of inlet
temperature with SM, whereas with SML and MgSO4, the percentage recovery at
1408C was significantly lower than at higher temperatures. In general, the recovery
was low ( B/48%). The percentage moisture in the final powder was lower at higher
temperatures. Moisture content using MgSO4 protectant was too high for practical
use as it is recommended to dry the microorganism to a final moisture content
of B/8%.

Table I. Percentage of powder recovery (PR,%) and final relative humidity (RH,%) of the powder obtained
after spray drying C. sake at different inlet temperatures for each carrier. Mean of outlet temperature is
shown. Means followed by different letters are significantly different according to Duncan Test (P B/0.05).

Protectant Inlet Temperature (8C) Outlet Temperature (8C) % PR % RH

10% skimmed milk 140 73.8 34.3a 8.4a

150 77.8 43.8a 7.9a
160 81.5 47.3a 7.5ab
170 88.3 45.1a 6.3b

10% skimmed milk/10% Lactose 140 79.3 27.7 b 7.3a

150 81.0 37.5a 5.9b
160 84.0 40.5a 5.8b
170 95.9 37.4a 5.5b

10% MgSO4 140 74.3 16.3b 19.6a

150 77.8 24.7a 18.8ab
160 83.8 22.6a 18.0bc
170 88.8 24.1a 17.2c
 842 M. Abadias et al.

SM and an inlet temperature of 1508C were chosen for subsequent experiments,

as powder recovery was relatively high (43.8%), moisture content was acceptable
(7.9%) and cell survival was one of the best among the treatments tested (about
1-log reduction).

Effect of growth medium on viability of spray-dried cells

To determine the effect of growth medium on yeast population reduction, statistical
analysis was performed for each protectant because the effect of growth medium,
protectant and their two-way interaction were statistically significant (Table II). There
were no significant differences between culture medium when the carrier used was SM
or SML. However, the use of sorbitol-modified molasses medium reduced survival of
C. sake cells when MgSO4 was used as a carrier.
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Effect of rehydration medium on viability of spray-dried cells

Recovery of spray-dried cells at optimum conditions (1508C, SM 10% as carrier) was
investigated as a function of rehydration medium and time used in the rehydration
process. There were no significant differences (P B/0.05) between cells rehydrated for
10 or 30 min. This allowed the data from both times to be pooled (Figure 4). The use
of some enriched medium for rehydrating cells slightly improved the recovery of
C. sake cells compared to those rehydrated with water or PB. The best results were
obtained when SM was used as a rehydrating medium, but without significant
differences from SML, sucrose, PTM or PEP.

Efficacy of spray-dried cells against P. expansum on apple fruits (cv. Golden Delicious)
After 6 days incubation, 50% of control fruits decayed (Figure 5), but apples treated
with fresh cells of C. sake did not show any rot symptoms. There was some reduction
in infection when apples were inoculated with spray-dried cells rehydrated with water,
SM or SML and compared with their respective controls (47, 74, and 41% of rot
reduction, respectively). However, their efficacy was significantly lower than that
obtained with fresh cells. SM and SML favoured rot development, as incidence of
decay was higher than in control fruits (about 90%). After 10 days incubation, there
was excellent control of infection when wounds were inoculated with fresh cells, but
there were no significant differences between untreated apples and those treated with
spray-dried cells.
Table II. Effect of growth medium and protectant on the logarithmic survival rate of spray-dried C. sake . Spray
drying was done at an inlet temperature of 1508C and flow rate of 500 mL min 1. For each protectant,
different letters indicate significant differences in growth medium according to a Duncan Test (P B/ 0.05).

Protectant Culture medium Log (N/N0)

10% skimmed milk Unamended molasses /1.09a

Sorbitol modified molasses /1.56a
10% skimmed milk/10% Lactose Unamended molasses /2.06a
Sorbitol modified molasses /2.00a
10% MgSO4 Unamended molasses /2.43a
Sorbitol modified molasses /3.44b
 Spray drying of Candida sake 843
10
a a a
ab ab
8
b b

Viability (%) 6

0
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SM SML Suc PTM PEP Water PB

Rehydration medium

Figure 4. Effect of rehydration medium on the viability of C. sake cells spray-dried at 1508C using 10% SM
as carrier. Separation of means was conducted according to Duncans Test. Columns with different letters
indicate significant differences (P B/0.05). SM, 10% skimmed milk; SML, 10% skimmed milk plus 10%
lactose; suc, 10% sucrose; PTM, peptone-tryptone-meat extract medium; PEP, peptone 1% and PB,
phosphate buffer.

Discussion
Survival of C. sake after spray drying is much lower than after freeze-drying (Abadias
et al. 2001a,b). It is possible that in the former process, there is more cell inactivation
because cells experienced both thermal and dehydration inactivation simultaneously.

120

x x
100
a a xy
x
xy
Infected fruits (%)

80 y

60 b b

40
c
c
20
z
d
0
CK SM SML CS-Fresh CS-water CS-SM CS-SML
Treatment

Figure 5. Efficacy of spray-dried and fresh cells of C. sake (2/107 CFU mL 1) against P. expansum
infection of apple fruits (cv. Golden Delicious). Fruits were wounded and first treated with: CK, water;
SM, skimmed milk; SML, skimmed milk/lactose; CS-Fresh, fresh cells of C. sake ; CS-water, spray-dried
cells rehydrated with water; CS-SM, spray-dried cells rehydrated with SM; CS-SML, spray-dried cells
rehydrated with SML. Afterwards, fruits were inoculated with P. expansum at 104 conidia mL 1. Inoculated
fruits were stored at 208C for 6 and 10 days. Bars represent the percentage of infected fruit after 6 days (I)
or 10 days (j). Different letters indicate significant differences (P B/0.05) according to a Duncans Test.
 844 M. Abadias et al.

High temperature and thermal stress are extremely detrimental to microorganisms

and non-thermostable enzymes (Hutter et al. 1995).
Residual viability of C. sake after spray-drying decreased with an increase in inlet
or outlet temperature. Similar results were observed when the biocontrol agent
P. agglomerans was also spray-dried (Costa et al. 2002a), and in spray-drying of lactic
acid bacterial species (To & Etzel 1997a,b; Mauriello et al. 1999). Lievense and vant
Riet (1993) pointed out that, because of the evaporative cooling in the first part of the
drying process, the survival of bacterial cells during spray-drying is strongly correlated
to the outlet temperature and not directly to the inlet temperature; the highest survival
rate is found at the lowest outlet temperature. Obviously, low outlet temperature will
result in less thermal inactivation, but possibly also in a higher residual water
concentration, which can also influence the survival.
Theoretically, 100% survival of C. sake cells could be achieved at inlet temperatures
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of about 1008C. However, the small spray dryer that we used was incapable of
completely drying the feed suspensions which were spray-dried at inlet temperatures
below 1408C. Nevertheless, if the size of the spray-drying chamber was greater, it
would be possible to obtain dry products at a lower inlet temperature. To and Etzel
(1997b) observed greater survival of Brevibacterium linens using a 12.5-L drying
chamber than a 4.5-L, suggesting that in the larger drying chamber, increased mixing
of suspension droplets and the hot air caused the cells to dehydrate faster, shortening
the critical period during which damage to cells would occur. Using the same inlet
temperature, they also showed that outlet temperature was reduced in the larger
drying chamber.
The best viability results were found when 10% skimmed milk was used as a carrier.
Moreover, the powder recovery was also higher than that obtained with the other
carriers used. In contrast, Costa et al. (2002a) found that 10% SM provided the
lowest and MgSO4 the greatest viability of P. agglomerans after spray-drying.
Activation energy, Ea, of C. sake in SM, SML and MgSO4 were 12.87, 10.29 and
7.97 kcal/mol 8K, respectively. This means that more energy is required to kill C. sake
in SM or SML than in MgSO4 under the same processing conditions. Lower Ea
values were found for cells of P. agglomerans in suspensions of MgSO4 and SM (5.95
and 5.83 kcal/mol 8K, Costa et al. 2002a). Thus, the bacterial biocontrol agent
P. agglomerans is more sensitive to spray drying than the yeast C. sake .
The use of sorbitol-molasses medium in preserving C. sake cells in liquid
formulations had a positive effect in maintaining cell survival (Abadias et al. 2003).
This is in contrast with our work as no significant differences between media were
found.
There were no significant differences between cells rehydrated for 10 or 30 min.
Font de Valdez et al. (1985), found that the time of exposure of freeze-dried lactic acid
bacteria to a rehydration medium was an important factor in the recovery of dried
cells. Recovery also depended on the bacterial species or strain with the highest
survival rate obtained after rehydration for 10 min. Our results showed that there were
no significant differences in survival between cells rehydrated for 10 and 30 min, but
the recovery medium significantly affected survival. Rehydration with water or PB
gave the lowest survival and some rich media such as SM or SML gave the highest
values. Teixeira et al. (1995) found similar results when rehydrating Lactobacillus
bulgaricus cells with milk, MRS broth or water. Ray et al. (1971) found similar results
during rehydration of freeze-dried Salmonella anatum and proposed that SM used as a
 Spray drying of Candida sake 845

carrier may have supplied all the necessary nutrients to the injured cells and thus
masked any effects of various added nutrients. However, in other studies on
rehydrating freeze-dried C. sake cells, cell viability increased twice after rehydration
with SM compared to water or PB (Abadias et al. 2001b). Costa et al. (2002a,b) also
found that SM was the best rehydration medium tested in spray- or freeze-drying
P. agglomerans, due to the ability of this complex medium to repair damaged cells and
improve the final recovery. In our studies, it is likely that cell damage caused by both
high temperature and dehydration was so severe that cells could not be repaired even
with the presence of a complex medium.
Spray-dried cells reduced the incidence of decay after 6 days incubation, but after
10 days there was no difference between apples inoculated with P. expansum alone.
It may be possible that nutrients present in the suspension favoured the growth of
P. expansum, as there was more incidence of decay on apples inoculated with SM or
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SML. The mode of action of C. sake is probably nutrient competition or site exclusion
(Vinas et al. 1998). Thus, it is possible that surviving spray-dried cells do not become
metabolically active rapidly enough to colonize apple wounds and prevent P. expansum
development. Similar results were found when freeze-dried C. sake cells were used to
control P. expansum on apples (Abadias et al. 2001b). In contrast, a liquid formulation
of C. sake cells performed as well as fresh cells in controlling blue mould on apples
(Abadias et al. 2003).
Using spray drying, we obtained a C. sake dried product with low moisture content
which could be easily rehydrated. However, viability of the product was very low
(maximum 10%) and cells obtained were not able to control P. expansum rot on
apples. Thus, we conclude that this process is not suitable for dehydrating C. sake
cells. Further research should be focused on the use of other milder drying techniques,
such as fluidised bed drying.

Acknowledgements
This work was supported by the Spanish Government (CICYT, Comision Inter-
ministerial de Ciencia y Tecnologa, grant ALI99-0652-C02-01) and European
Commission QoL-PL1999-1065.

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