Journal of Applied Microbiology 2000, 89, 793ÿ800

Effect of protective agents, rehydration media and initial cell

concentration on viability of Pantoea agglomerans strain CPA-2
subjected to freeze-drying
E. Costa, J. Usall, N. TeixidoÂ, N. Garcia and I. VinÄas
Postharvest Unit, CeRTA, Centre UdL-IRTA, Lleida, Spain

225/3/00: received 10 March 2000, revised 28 June 2000 and accepted 24 July 2000

 , N. GARCIA AND I. VIN

E. COSTA, J. USALL, N. TEIXIDO The effect of initial cell
Ä A S . 2000.

density, protective agents and rehydration media on the viability of biocontrol agent Pantoea
agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were
tested as protective agents against freeze-drying injury. Maximum viability of the bacterial
cells was obtained with disaccharides (survival levels >60%). Freeze-dried samples were
rehydrated with several media; the highest percentage viability was obtained with 10% non-
fat skim milk (100%‡). The effect of initial bacterial load on the ®nal recovery was
dependent on protectant but not on rehydration media. Sucrose was an effective protectant
when a high initial concentration (1010 cfu mlÿ1) was used; the opposite occurred with non-
fat skim milk. The use of 1010 cfu mlÿ1 as an initial concentration, sucrose as a protectant
and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to
be conserved after freeze-drying. Results suggest the possibility of achieving a good
formulation system for the studied biocontrol agent with a high number of viable cells to be
used toward pathogens, which is desirable for the industrial development of the product.

INTRODUCTION and Denis-Arrue 1992; Arras and D'hallewin 1994; Huang

et al. 1995). Some of these are being developed commer-
Fruit and vegetables suffer signi®cant losses after harvest cially.
due to fungal diseases (Eckert 1975; Dennis 1983; Several studies carried out in this laboratory have
Snowdon 1990, 1991). Post-harvest application of synthetic demonstrated that the strain CPA-2 of Pantoea agglomerans
fungicides has been the main method of combating these (Gavini et al. 1989), previously classi®ed as Erwinia herbi-
diseases (Eckert and Ogawa 1988). However, the develop- cola, is an effective antagonist to the main fungal pathogens
ment of resistance to fungicides (Spotts and Cervantes of citrus and pome fruits. Immersion of citrus fruit in solu-
1986; VinÄas et al. 1991, 1993) and public demand for a tions of P. agglomerans at 2  108 cfu mlÿ1 reduces the sub-
reduction in pesticide use have provided the impetus to sequent incidence of post-harvest mould caused by
develop alternative and effective natural methods of con- Penicillium digitatum and P. italicum. In the case of pome
trolling post-harvest diseases. fruit, apples and pears are drenched with 8  107 cfu mlÿ1
Biological control using microbial antagonists has been to reduce incidence of Penicillium expansum, Botrytis cinerea
considered as a desirable alternative to the use of chemicals. and Rhizopus nigricans during storage in packing-houses
In recent years, success has been achieved through (VinÄas et al. 1999).
the development of microbial antagonists effective against In order to use a biological agent as a commercial pro-
fungal pathogens on pome (Janisiewicz and Bors duct it is essential to optimize its formulation. This is
1995; VinÄas et al. 1998), stone (Pusey and Wilson 1984) necessary to provide the product in a suitable form, and to
and citrus fruits (Chalutz and Wilson 1990; Smilanick optimize the ef®cacy, stability, safety and ease of applica-
tion of the product (Rhodes 1993). Dehydrated cells have
the advantage of not requiring cool temperatures during
Correspondence to: Dr Elena Costa, Postharvest Unit, CeRTA, Centre UdL-
IRTA, 177 Rovira Roure Ave., 25198 Lleida, Spain (e-mail: storage and distribution and thus make the product more
[email protected]). economic.
= 2000 The Society for Applied Microbiology
794 E . C O S T A E T A L .

Drying can be accomplished by a number of means This strain was originally isolated from an apple surface
including freeze-drying, drying on silica gel and spray dry- (VinÄas et al. 1999).
ing (Rhodes 1993). Freeze-drying causes little shrinkage
and results in a completely soluble product that is easily
Cell preparation
rehydrated (Powell 1992). Moreover, lyophilization is fre-
quently used to preserve lactic acid bacterial starter cul- Pantoea agglomerans was sub-cultured weekly on Starch
tures involved in dairy and food fermentations (Kearney Agar Medium plates (SAM) which contained (g lÿ1 dis-
et al. 1990). tilled water): 5, peptone; 5, yeast extract; 3, soluble starch
Microbial cell survival during the freeze-drying process and 15, agar (Atlas 1995). After 24 h at 25 ‹ 1  C, plates
is dependent on many factors, including the initial micro- were stored at 4  C.
organism concentration (Bozoglu et al. 1987), the protective The biocontrol agent was grown at 25 ‹ 1  C in a bench
medium (Font de Valdez et al. 1983) and the rehydration top fermentor (Modular Fermenter, Gallenkamp,
conditions (Sinha et al. 1982; Font de Valdez et al. 1985b). Loughborough, UK), containing 3 l of Nutrient Yeast
Protective additives have an important role in the conserva- Dextrose Broth medium (NYDB) which consisted of (g lÿ1
tion of viability. A good protectant should provide cryopro- distilled water): 8, nutrient broth; 5, yeast extract; 10,
tection to the cells during the freezing process, be easily anhydride glucose and 15, agar. Cells were harvested at the
dried, and provide a good matrix to allow stability and ease beginning of the stationary phase (24 h) by centrifugation
of rehydration. Various groups of substances have been (6981 g for 10 min at 15  C). Cell paste was resuspended in
tested for their protective action, including polyols, poly- 0
05 mol lÿ1 phosphate buffer (pH 6
5) and a volume of
saccharides, dissacharides, amino acids and protein hydro- resuspended cells was dispersed into the protective medium
lysates, proteins, minerals, salts of organic acids and in order to obtain the initial concentration. This suspension
vitamins-complex media (Berny and Hennebert 1991; was incubated for 20 min at room temperature and con-
Champagne et al. 1991). However, protection afforded by a stantly shaken to allow cell adaptation.
given additive during these processes will vary with the
species of micro-organism (Font de Valdez et al. 1983).
Freeze-drying
Rehydration is a critical step in the recovery of freeze-
dried micro-organisms. Cells that are subjected to sub- Three vials were ®lled with 5 ml of bacterial suspension
lethal injury may not be able to repair the damage that has produced as described above and placed at ± 20  C for 24
occurred if they are rehydrated under inappropriate condi- h. After overnight storage in the freezer, samples were con-
tions (Champagne et al. 1991). The medium itself, its nected to a Cryodos model freeze-drier (Telstar S.A.,
molarity and the rehydration conditions can signi®cantly Terrassa, Spain) operating at 1 Pa pressure and ± 45  C for
affect the rate of recovery (Ray et al. 1971; Font de Valdez 24 h.
et al. 1983).
In order to obtain a suitable commercial product it is
Rehydration
necessary to achieve a high density of viable dried cells.
Some studies have shown that the initial bacterial load After freeze-drying, samples were immediately brought to
affects the survival rate during treatment. Bozoglu et al. their original volume (5 ml) with each rehydration medium
(1987) demonstrated that there were higher survival levels at 25  C. Then, samples were homogenized for 1 min with
when the highest initial cell densities of lactic acid bacteria a Vortex mixer (SA-5, Stuart Scienti®c, Redhill, UK) and
were freeze-dried. incubated at room temperature for 9 min. Serial dilutions
The aim of the present study was to evaluate the effect were spread-plated onto the surface of 9 cm Petri plates
of protective agents, rehydration media and initial cell con- containing SAM medium. These plates were incubated for
centration on viability of the biocontrol agent Pantoea 24 h at 25 ‹ 1  C and the viability was then determined.
agglomerans CPA-2 when subjected to a freeze-drying pro- Survival levels were expressed as the quotient of colony-
cess. forming units per millilitre (cfu mlÿ1) on SAM medium
before (N0) and after (Nf) freeze-drying. Viability ˆ (Nf/
N0)  100.
MATERIALS AND METHODS
Protectants used in assays
Culture
Suspensions of protectants were prepared in water. The
Pantoea agglomerans (strain CPA-2) was obtained from the additives tested as protective agents against freeze-drying
Postharvest Unit of Centre UdL-IRTA, Lleida, Spain. injury were divided into ®ve groups: (i) sugars: trehalose,
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 793ÿ800
 PANTOEA AGGLOMERANS FREEZE-DRYING 795

glucose and fructose (5%), sucrose (10%) and lactose procedure of the Statistical Analysis System (SAS
(7%); (ii) amino acids: sodium glutamate (1 mol lÿ1) and Institute, version 6
03, Cary, NC, USA). Statistical signi®-
cystine (0
04 mol lÿ1); (iii) polymers: dextran, MW ˆ 17500 cance was judged at the level P < 0
05. When the analysis
g molÿ1 (0
33 mol lÿ1) and Polyethylene glycol (PEG) MW was statistically signi®cant, Duncan's Multiple Range Test
ˆ 200 g molÿ1 (0
05 mol lÿ1); (iv) polyols: glycerol (5 mol was used for separation of means. Variables were analysed
lÿ1); (v) others: reconstituted non-fat skim milk; NFSM as ®x variables, except for the initial concentration that was
(Sveltesse, Nestle, Vevey, Switzerland) (10%), phosphate considered as an aleatory variable and a random test was
buffer (pH 6
5) and water, as controls. These protectants performed.
were selected on the basis of previous studies on other
micro-organisms (Font de Valdez et al. 1983; Champagne
et al. 1991). Protectant solutions were sterilized at 121  C RESULTS
for 15 min before mixing with a volume of washed cells of
antagonist to obtain an initial concentration of 5±8  109 Assay of protectant agents
cfu mlÿ1. The general procedure for cell preparation,
Using a range of protectants, signi®cant differences in the
freeze-drying and rehydration was described above. After
viability of cells of P. agglomerans after freeze-drying were
freeze-drying, all samples were rehydrated with phosphate
observed, depending on the protectant used (Fig. 1). The
buffer to the original volume and the level of survival eval-
best protection was given by sugars. Sugars could be
uated. The experiment was repeated twice.
divided into two groups: on the one hand, disaccharides
which gave viabilities >60% and on the other hand,
Rehydration assay media monosaccharides which gave viabilities of between 30 and
50%. The most effective protectant was trehalose at 5%
In this assay, the initial concentration used was 1  1010
(83% viability), followed by sucrose at 10% concentration
cfu mlÿ1 and sucrose was used as the protectant. Freeze-
(75% viability). The viability of cells of P. agglomerans was
dried samples were rehydrated with the following rehydra-
less than that obtained with the sugars, with all the other
tion media: 10% non-fat skim milk, 10% sucrose, 5%
protectants examined.
sodium glutamate, 10% peptone, water, phosphate buffer,
NFSM or dextran provided a freeze-dried material with
or a combination of 1
5% Peptone, 1% Tryptone, and
a light and porous structure that made rehydration easy,
0
5% Meat extract (PTM medium, Font de Valdez et al.
but only around 15% of the cells remained viable. The rest
1985b). Each rehydration medium was also used for serial
of the assayed substances (amino acids and polyols) showed
dilution plating to assess viability of cells. The general pro-
viabilities <10%, and there was no signi®cant difference
cedure of cell preparation, freeze-drying and rehydration
from the controls (distilled water and phosphate buffer).
was as described above. The experiment was repeated
twice.
Assay for a rehydration medium
Effect of initial concentration of cells on survival of
Percentage viability of the P. agglomerans strain with differ-
freeze-drying
ent rehydration media, and 10% sucrose as a protectant,
Washed cells of the tested micro-organism were resus- are shown in Fig. 2. All samples were rehydrated to the
pended in two suspension media used as protectants, 10% initial volume, because in previous studies it had been con-
sucrose and 10% NFSM, at three concentrations: 108, 109 ®rmed that an increase in the rehydration medium volume
and 1010 cfu mlÿ1. Three different rehydration media, resulted in a decrease in the antagonist viability (data not
10% sucrose, 10% NFSM and phosphate buffer, were shown).
used. Each rehydration medium was also used for serial The results obtained indicated that there were differ-
dilution. The effect of initial bacterial concentration was ences in viability of the bacteria depending on the rehydra-
evaluated for each protective agent in each rehydration tion media used. Recovery of cells was greater in complex
medium. The general methodology of cell preparation, media, such as NFSM or PMT, than in the other media
freeze-drying and rehydration was as previously described. used. The most promising results were obtained where
The experiment was repeated three times. freeze-dried cells were recovered in NFSM or PMT
(100% viability). In contrast, <60% of the cells rehydrated
with water were able to form colonies. Sucrose also showed
Statistical treatment of the results
a high capacity for injury repair, while sodium glutamate
Percentage viability of P. agglomerans cells was estimated in and phosphate buffer gave an intermediate recovery of
all trials and analysed by a general linear model (GLM) cells.
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 793ÿ800
796 E . C O S T A E T A L .

100

90
a
80 b
70 c
Viability (%)

60

50 d

40
e
30

20
f f
10 g
g g g g g
0 †
5%

7%

5%

5%

0%

er

M
ffe
M

M
10

at
1

04

l5
bu
*1

33

05
W
se

se

ro
0·
e

os

os

at
0·
M

0·
lo

co

e
os

ce
§
ct

ct

am

at
FS
ha

n
lu
cr

tin

ly
La

ph

G
ra
G
e

Fr
Su

ut
N

G
ys

PE
t
Tr

os
ex

gl

Ph
D

um
di
So

Protective agents

Fig. 1 Comparative effect of different protective agents on Pantoea agglomerans CPA-2 viability after freeze-drying and rehydrating with
phosphate buffer. The separation of means was conducted according to Duncan's Multiple Range Test. Columns with different letters
indicate signi®cant differences (P < 0
05).*NFSM: Non-fat skim milk; {M: mol lÿ1; xPEG: Polyethylene glycol

120
a
110
ab
100

90
abc
80 bc bc
c
Viability (%)

70

60
c
50

40

30

20

10

0
NFSM* PTM† Sucrose Phosphate Sodium Peptone Water
buffer glutamate
Rehydration media

Fig. 2 Effect of rehydration media on the viability of Pantoea agglomerans CPA-2 cells freeze-dried in 10% sucrose as protective agent.
The separation of means was conducted according Duncan's Multiple Range Test. Columns with different letters indicate signi®cant
differences (P < 0
05).*NFSM: Non-fat skim milk; {PTM: Peptone-Tryptone-meat extract medium

= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 793ÿ800
 PANTOEA AGGLOMERANS FREEZE-DRYING 797

Effect of initial concentration of cells on survival of 100

(a)
freeze-drying 90
80
Statistical analysis of viability results was performed and
four factors considered: experimental replication, initial 70
concentration, protectant type and rehydration medium.

Viability (%)
60
Experimental replication and its two-way interaction were 50
not signi®cant; therefore, the results for the three different a
40
experimental replications were pooled.
b
Rehydration medium and two-way interaction of rehy- 30
a a
dration medium  protectant, and protectant  initial con- 20 a a
centration, were statistically signi®cant. In order to study 10 c
b b
the effect of initial concentration and rehydration media on
0
viability, a statistical analysis was consequently performed NFSM* Sucrose Phosphate buffer
for each protectant used. Rehydration media
In general, viability obtained when sucrose was used as a 120 a
(b)
protective agent was higher than that obtained with NFSM 110
as protectant (Fig. 3). Moreover, results showed that the 100
effect of initial concentration had a marked dependence on 90 ab
b
the type of protectant used. There were no signi®cant dif- 80 a
a
ferences between rehydration media used when NFSM was
Viability (%)

70
used as protectant (Table 1). However, initial concentra- 60 a
b
tion, and two-way interaction between rehydration medium 50
a
and initial concentration, were signi®cant. This indicated 40
that depending on the rehydration media used, the effect 30
b
of initial concentration was different. The highest viabilities 20
in NFSM±protectant medium were obtained at low con- 10
centration (108 cfu mlÿ1) in all rehydration media used. 0
Recovery decreased when the concentration was increased NFSM* Sucrose Phosphate buffer
to 1010 cfu mlÿ1 (Fig. 3a). Rehydration media
When sucrose was used as a protectant, rehydration
medium and concentration were signi®cant (Table 2). With Fig. 3 Percentage of viability of Pantoea agglomerans freeze-dried
this protectant, ®nal viability was higher after rehydration in (a) non-fat skim milk and (b) sucrose as protector and
with NFSM, followed by sucrose and phosphate buffer in rehydrated in non-fat skim milk (NFSM), sucrose and phosphate
the three initial concentrations tested (Fig. 3b). The effect buffer at three initial concentrations 108 ( & ), 109 (+) and 1010
of initial concentration was also noticeable but in this case, (&) cfu mlÿ1. The separation of means for each protector was
the highest recovery was obtained at higher concentrations conducted according to Duncan's Multiple Range Test (P <
of 109 and 1010 cfu mlÿ1. 0
05). Columns with different letters indicate signi®cant
differences between rehydration medium

Table 1 Analysis of variance of effect of rehydration media (reh) and initial concentration (con) two-way interaction on viability of Pantoea
agglomerans after freeze-drying when non-fat skim milk was used as protectant

Source DF MS F P>F

Reh 2 312
251 1
882 0
2651 NS

Con 2 312
251 9
201 0
0317*
Reh  Con 4 90
661 3
456 0
0124*

*Signi®cant P < 0
05; NS Not signi®cant.

MS ˆ Mean square.

= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 793ÿ800
798 E . C O S T A E T A L .

Table 2 Analysis of variance of effect of rehydration media (reh) and initial concentration (con) two-way interaction on viability of Pantoea
agglomerans after freeze-drying when sucrose was used as protectant

Source DF MS F P>F

Reh 2 565
727 30
085 0
0039**

Con 2 565
727 7
910 0
0407*
Reh  Con 4 518
551 1
091 0
3677 NS

*Signi®cant P < 0
05; **Signi®cant P < 0
005; NS Not signi®cant.

MS ˆ Mean square.

For the freeze-dried biocontrol agent, 100% viability industrial use. However, sucrose, which has elicited viabil-
was obtained at 1010 cfu mlÿ1 of initial concentration, ities >75%, is a cheap sugar which could be used econom-
using sucrose (10%) as a protectant and NFSM (10%) as ically.
the rehydration medium. The ®nal cell viability obtained using NFSM and dex-
tran as protectants was only 14 and 12%, respectively.
However, these compounds provide the freeze-dried mate-
DISCUSSION rial with a light and porous structure which makes rehydra-
tion easy. Berny and Hennebert (1991) obtained similar
Freeze-drying has been studied as a dehydration process results with Trichoderma viride, Brettanomyces bruxellensis,
for bacteria in order to achieve a solid formulation. This Saccharomyces cerevisae and Arthrobotrys arthrobotryoides.
study showed the impact of protective additives, rehydra-
The amino acids sodium glutamate and cystine tested in
tion media and initial concentration of micro-organism on
this study were not effective in protecting cells of P.
viability after freeze-drying. It was demonstrated that P.
agglomerans. In contrast, some authors found sodium gluta-
agglomerans is highly resistant to freezing, thawing and
mate to be effective in conserving viability of streptococci
dehydration during the processes of freezing and freeze-
and mesophilic lactobacilli (Font de Valdez et al. 1983) in
drying, which is useful from a commercial viewpoint. This
Gram-negative bacteria (Ashwood-Smith and Warby 1972),
result was expected because many bacteria are known to
and also in protecting yeast cells subjected to a freezing
survive freeze-drying well, including strains of Erwinia
process (Takagi et al. 1997). PEG (polyethylene glycol) and
which have been stored for up to 10 years after freeze-dry-
ing without loss of viability (Rudge 1991). glycerol were found to be completely ineffective in the pro-
The differences exhibited in cell survival in this study tection of the studied biocontrol agent. Polymers such as
indicate that certain additives are more effective than PEG might accelerate drying, and over-drying is detrimen-
others in protecting P. agglomerans. Maximum protection tal to survival during freeze-drying (Clementi and Rossi
of cells of P. agglomerans during freeze-drying was achieved 1984; Font de Valdez et al. 1985a). Glycerol is an effective
with sugars. The use of disaccharides resulted in viabilities cryoprotectant and is widely used in frozen concentrates.
>60% while monosaccharides resulted in cell viability of However, it provided no protection for cells of P. agglomer-
30±60%. Sugars replace structural water in membranes ans subjected to freeze-drying.
after dehydration (Clegg 1986; Crowe and Crowe 1986) When using freeze-dried micro-organisms, rehydration
and prevent unfolding and aggregation of proteins by has been considered as an important step. High recoveries
hydrogen bonding with polar groups of proteins (Hanafusa obtained by complex rehydration media, such as NFSM
1985; Carpenter et al. 1990). Trehalose was the best protec- and PTM, might be related to the rate of hydration of the
tive agent for cells of P. agglomerans when subjected to samples. An environment of high osmotic pressure may
freeze-drying (>80% of viability), followed by sucrose and control the rate of hydration and avoid osmotic shock.
lactose. Differences exhibited by sugars are connected with Moreover, these complex media may have a greater ability
their water-binding capacity and prevention of intracellular to repair damaged cells and to improve the ®nal recovery
and extracellular ice crystal formation (Baumann and obtained (Ray et al. 1971). In fact, the viability afforded by
Reinbold 1964; Burke 1986). Trehalose has less of a ten- complex media was close to 100% and in some samples,
dency to crystallize than sucrose and lactose (Aguilera and the calculated values exceeded 100%. Viabilities greater
Karel 1997). Unfortunately, the cost of trehalose limits its than 100% after freeze-drying were also obtained by To
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 793ÿ800
 PANTOEA AGGLOMERANS FREEZE-DRYING 799

and Etzel (1997) with Streptococcus thermophilus. Sucrose is REFERENCES

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dependent on the protective medium used, the rehydration Champagne, C.P., Gardner, N., Brochu, E. and Beaulieu, Y.
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Clementi, F. and Rossi, J. (1984) Effect of storage and drying
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to the Spanish Government for its ®nancial support (INIA, root/tuber crops. Annual Review of Phytopathology 26, 433±469.
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