Food Science and Technology

International http://fst.sagepub.com/

Comparative Survival Rates of Lactic Acid Bacteria Isolated from Blood, Following Spray-drying and
Freeze-drying
L. M. Zamora, C. Carretero and D. Parés
Food Science and Technology International 2006 12: 77
DOI: 10.1177/1082013206062443

The online version of this article can be found at:

http://fst.sagepub.com/content/12/1/77

Published by:

http://www.sagepublications.com

On behalf of:

Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research)

Additional services and information for Food Science and Technology International can be found at:

Email Alerts: http://fst.sagepub.com/cgi/alerts

Subscriptions: http://fst.sagepub.com/subscriptions

Reprints: http://www.sagepub.com/journalsReprints.nav

Permissions: http://www.sagepub.com/journalsPermissions.nav

Citations: http://fst.sagepub.com/content/12/1/77.refs.html

>> Version of Record - Jan 24, 2006

What is This?

Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014

 Comparative Survival Rates of Lactic Acid Bacteria Isolated
from Blood, Following Spray-drying and Freeze-drying

L.M. Zamora, C. Carretero and D. Parés*

Institut de Tecnologia Agroalimentària – CeRTA – Escola Politècnica Superior, Universitat de Girona 17071
Spain

The effect of two dehydration technologies, spray-drying and freeze-drying, on the viability of 12 lactic
acid bacteria (LAB) were compared. All LAB cultures had been previously isolated from porcine blood
and were candidates to be used as biopreservatives in order to maintain the quality of porcine blood
until further processing to obtain added-value blood derivatives is carried out. The residual viability and
the reductions in microbial counts in dried LAB samples at 20 °C and 5 °C during 60-day storage were
determined. Cellular damage due to freeze-drying was observed immediately after drying whereas cellu-
lar damage due to spray-drying did not become evident until the subsequent phase of storage. For most
of the strains, the faster decrease in viability of spray-dried as compared to freeze-dried cultures was
compensated by the higher percentage of viable cells obtained after dehydration, leading to comparable
survival rates at the end of the storage period. Dehydration resulted in a good alternative to freezing at
80 °C for preservation purposes. Spray-drying has been shown to be as suitable as freeze-drying for
preserving LAB strains during a 2-month storage period. Results suggest the possibility of achieving a
good formulation system for the LAB strains with a high number of viable cells to be used for the indus-
trial development of bioprotective cultures.

Key Words: spray-drying, freeze-drying, bio-preservation, porcine blood, Lactococcus garviae, Entero-
coccus raffinosus, Lactobacillus murinus, Lactobacillus reuteri

INTRODUCTION The use of lactic acid bacteria (LAB) cultures as

Most by-products generated from the commercial
slaughter of meat-producing animals have potential
industrial uses and, at the same time, they can be
highly contaminant if considered and treated as waste
products and thrown out. Upgrading these by-products
by taking advantage of potential industrial applications
is the best way to guarantee good practices in waste
management, thus minimising the environmental
impact of such industrial activities. The use of blood
and blood derivatives as food ingredients seems to be a
useful alternative to the simple discarding of this by-
product (Parés et al., 1998, 2000; Carretero and Parés,
2000; Parés and Ledward, 2001; Saguer et al., 2003;
Toldrà et al., 2004).
*To whom correspondence should be sent
(e-mail: dolors.pares@udg.es).
Received 28 January 2005; revised 6 June 2005.
Food Sci Tech Int 2006; 12(1):77–84
© 2006 SAGE Publications
ISSN: 1082-0132
DOI: 10.1177/1082013206062443
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
bio-preservatives is a fairly widespread technique and
the results of their application in different areas of the
food industry whether alone or in combination with
other food preservation techniques, are quite encour-
aging.
Bio-preservation with LAB has been considered
and investigated by our group as a possible alternative
aimed at improving the microbiological quality of
slaughterhouse blood and maintaining its functional
and nutritional characteristics until further processing
is carried out (Zamora et al., 2001, 2003). From that
work, 12 LAB strains, isolated from blood collected in
industrial slaughterhouses, were selected with respect
to their antagonistic potential, at mesophilic (30 °C) or
psychrotrophic conditions (15 °C), against the typical
contaminant microbiota of this by-product (Zamora,
2003; Zamora et al., 2003; Parés et al., 2004).
Freezing is widely used for long-term preservation
of both the viability and the technological properties of
LAB starters used by the food industry. However,
freezing causes a loss of viability and acidification
activity of LAB (Fonseca et al., 2000). Moreover,
subzero storage temperatures and bulkiness increase
the final cost of the frozen cultures (Johnson and Etzel,
78 L.M. ZAMORA ET AL.
1995; To and Etzel, 1997a). So, bearing in mind not
only the stability of the cultures during storage but also
the final cost of processing, dehydration is often seen
as a good alternative to preserve industrial strains (To
and Etzel, 1997a, 1997b; Mauriello et al., 1999; Gar-
diner et al., 2000).
Dried cultures are commonly produced by freeze-
drying. However, commercial scale freeze dryers are
expensive and production output is low. An alternative
method for microorganism preservation is by spray-
drying, whose production rates are higher and the
operative costs lower; according to Desmond et al.
(2001) spray-drying costs up to six times less per kg of
water removed than freeze-drying.
There is considerable information on the practical
application of dehydration techniques of lactic acid
bacteria (Espina and Packard, 1979; Johnson and
Etzel, 1995; Teixeira et al., 1995b; To and Etzel, 1997a,
1997b; Desmond et al., 2001; Lian et al., 2002;) but,
even nowadays, there is not much available literature
on the direct comparison of various techniques of
dehydration for the same strain. Most of the literature
reports only the application of a specific method for a
single microorganism. Even when the results belong to
the same microorganism dehydrated by different
methods, this is performed by different workers and it
is still difficult to carry out the comparison, since
culture strains, growth conditions, protective agents
and testing methods vary significantly (Carvalho et al.,
2004). Some researchers have reported that freeze-
drying is more suitable than spray-drying for some cul-
tures (Johnson and Etzel, 1995; Costa et al., 2002).
Nevertheless, others have determined that there is no
difference in terms of survival between these methods
(Teixeira et al., 1995a).
So, the aim of this work was to compare the effect of
two dehydration technologies, spray-drying and freeze-
drying, on the viability of the 12 selected LAB cultures
isolated from porcine blood by determining the per-
centage of microorganisms surviving the processes and
the percentage of cells remaining viable after a 60-day
storage period at 20 °C and 5 °C.
MATERIAL AND METHODS
LAB Strains
Twelve strains, out of 144 psychrotrophic lactic acid
bacteria isolated from porcine blood collected in indus-
trial slaughterhouses, were used. They had been
selected as showing the broadest antagonistic spectrum
against four indicator microorganisms (Escherichia
coli, Staphylococcus aureus, Bacillus spp. and
Pseudomonas fluorescens) in agar plates as well as
when growing in blood (Parés et al., 2004). They had
been identified by partially sequencing their 16S rDNA
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
gene and by using the variable regions V1–V3 as the
identification key. From the 12 selected strains, six
were identified as Lactococcus garviae (PS12, PS22,
PS23, PS48, PS95 and PS60), two as Enterococcus raffi-
nosus (PS7 and PS99), three as Lactobacillus murinus
(PS85, PS86 and PS87) and one as Lactobacillus reuteri
(PS77), those four species representing more than 90%
of total LAB species isolated from blood samples
(unpublished data). All LAB strains were preserved
under freezing at 80 °C with 20% glycerol in 2 mL-
vials.
Methods
Freeze-drying
Pure LAB cultures were grown to stationary phase
in MRS broth (OXOID, CM359) for 24 h at 30 °C.
After centrifugation at 2430  g for 20 min at room
temperature (Sorvall RC 5C Plus, DuPont Co.,
Newtown, CT) and decanting, cells were re-suspended
in a sterile cryoprotective solution. According to Car-
valho et al. (2004) in the absence of other relevant
information on the specific LAB cultures, skimmed
milk powder should be selected as drying medium. So
the LAB cultures were freeze-dried using 20% non-fat
skimmed milk (NFSM) (Scharlau Chemie S.A.,
Barcelona, Spain) at neutral pH. Solutions of 10%
D()-Glucose PRS-CODEX (Panreac Química,
Barcelona, Spain) and 12% lactose (Oxoid Ltd, Bas-
ingstoke, UK), selected on the basis of previous studies
on other microorganisms (Costa et al., 2000), were also
tested as protective agents against freeze-drying injury
on Lact. murinus-PS85 and Ent. raffinosus-PS7.
Protective solutions were prepared in water and
sterilised at 121 ºC for 15 min before mixing with a
volume of washed cells of LAB cultures to obtain a
concentration of 108 cfu/mL, and distributed into 2 mL-
vials. Samples were frozen at 80 °C in a freezer for
24 h and dried in a Virtis Unitop SQ freeze-dryer (The
Virtis Co., New York, USA) at 15 °C and 15 °C for
the primary (sublimation) and the secondary (desorp-
tion) drying stages, respectively.
Experiments were carried out in triplicate. The
moisture content and residual viability of freeze-dried
cultures was determined and samples were kept for 60
days at 20 °C and 5 °C for subsequent microbial counts.
The moisture content of the final powder was based on
the weight loss after the process by drying samples
(0.5 g) in a convection oven at 105 °C for 24 h.
Spray-drying
Pure LAB cultures were grown in MRS broth
(OXOID, CM359) for 24 h at 30 °C. After centrifuga-
tion at 2430  g for 20 min at room temperature
(Sorvall RC 5C Plus, DuPont Co., Newtown, CT), cells
 Comparative Survival Rates of Lactic Acid Bacteria Isolated from Blood
were harvested and concentrated ten times by re-sus-
pending in tryptone water (tryptone 10 g/L, NaCl
5 g/L).
Aliquots of 500 mL of 20% NFSM supplemented
with 0.5% yeast extract were pasteurised (90 °C for
30 min) and inoculated with 1% (v/v) cell suspensions.
Cultures were incubated at 37 °C and were stopped
after 4–5 h in order to prevent the milk coagulating
(Gardiner et al., 2000). After incubation, liquid cul-
tures had a final concentration up to 108 cfu/mL of
exponential phase cells and a pH between 5.5 and 5.8.
pH was neutralised (pH 6.7–7) before spray-drying
(Abd El-Gawad et al., 1989; Metwally et al., 1989; Gar-
diner et al., 2000).
Samples were spray-dried by using a Büchi Mini
Spray Dryer B191 (Büchi Labortechnik AG, Flawil,
Switzerland), the process parameters being as follows:
inlet temperature 170 °C; outlet temperature 80–85 °C;
product input flow 670 mL/h.
Experiments were carried out in triplicate. The
moisture content of the final powder and the residual
viability of spray-dried cultures were determined and
dehydrated samples were stored at 20 °C and 5 °C for
60 days.
Microbiological Measurements
The viability of the LAB strains after 60 days at
80 °C, the reductions in microbial counts caused by
the drying processes and the viability during 60-days’
storage, at 20 °C and 5 °C, of the dried cultures were
determined.
After dehydration, freeze-dried and spray-dried
samples were rehydrated using sterile tryptone water.
All samples were homogenised for 1 min with a Vortex
mixer and incubated at 25 °C for 1 h for complete rehy-
dration.
Serial dilutions of rehydrated samples were pour-
plated in Petri plates with MRS Agar (OXOID
CM361) as a culture medium. The plates were incu-
bated anaerobically for 48 h at 30 °C. Sealed jars con-
taining gas packs (AneroGen OXOID, AN25) were
used to set anaerobic conditions. The viability was
determined by enumerating the colony-forming units
after incubation.
Survival levels were expressed as the quotient of
colony-forming units per gram of dry matter powder
on MRS medium before (N0) and immediately
after (Nf) each dehydration process.
Viability  (Nf/N0)  100.
Residual viability of freeze-dried and spray-dried
powders was measured following the same procedure
after storage for 5, 15, 30 and 60 days at 20 °C and 5 °C.
The stability of dehydrated cultures was established
from the percentage of cells remaining viable during
the storage. The viability at a specific time (t),
expressed as a percentage, was calculated as the quo-
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
79
tient of the number of bacteria per gram after a fixed
storage period (Xt) to the initial number (Xi). Xt was
estimated from the linear adjustment of survival data
by using the expression: log10Xt  log10Xi  Kt, where t
is the time elapsed in storage, and K is the linear
regression slope of the relation of viable cells over
time.
Cultures kept at freezing conditions (at 80 °C in
MRS broth containing 20% glycerol) were thawed at
room temperature after 60 days of storage and the
appropriate dilutions were also pour-plated on MRS
Agar plates, and incubated as described above.
Statistical Analysis
The data were analysed with the statistical software
SPSS version 11.5. Analyses of variance (ANOVA)
using the general univariant model procedure were
applied to the results. The significance level for all tests
was   0.05. The Kolmogorov-Smirnov (KS) and the
Levene tests were used to assess the normal distribu-
tion of residuals and the homocedasticity of the results
respectively, and the square root transformation of
percentages was used when necessary.
The strain and the storage temperature were evalu-
ated as potential variability factors for both the per-
centages of survivors after the dehydration and the
percentage of viability after the storage period (60
days). For freeze-dried cultures, the effect of cryopro-
tectant was considered. All possible interactions
(strain-by-temperature, cryoprotectant-by-strain and
cryoprotectant-by-temperature) were also included in
the model. The Bonferroni test with least significant
differences at p  0.05 was used to compare sample
means when the interactions were not significant.
RESULTS AND DISCUSSION
Freeze-drying
NFSM and lactose provided a freeze-dried material
with a moisture content between 6.5% and 7% and
with a light and porous structure that made rehydra-
tion easy, but this was not the case for glucose-contain-
ing samples.
The viability of cells of the LAB cultures (Ent. raffi-
nosus-PS7 and Lact. murinus-PS85) immediately after
freeze-drying was determined. The Lact. murinus
strain revealed the lowest percentage of cell recupera-
tion when glucose was used as a protective agent
(27.80%). Costa et al. (2000) also reported that mono-
saccharides provided viabilities of between 30% and
50% for a freeze-dried strain of Pantoea agglomerans.
Contrasting to this, they also reported that only around
15% of cells remained viable when NFSM was used;
while results from this study showed the highest viabil-
80
ity (81.41%) for the Ent. raffinosus strain dehydrated
in non-fat skimmed milk. No significant differences
(p > 0.05) were obtained among the number of sur-
vivors depending on the cryoprotectant, probably due
to the great variability between replicates. In any case,
the survival was always higher than 10%, whatever the
cryoprotectant used, resulting in viable counts at the
same log level to the initial suspension.
Regarding the stability during a 2-month storage
period at 5 °C and 20 °C (Figure 1), differences in the
viability values at the end of storage between samples
with different cryoprotectants or stored at different
temperatures proved to be significant (p < 0.05). As it
can be observed in Figure 1, samples with the cryopro-
tectants glucose or lactose followed a similar pattern.
The viable counts of both strains decreased gradually
during storage, this effect being much more noticeable
at 20 °C. The viability decreased at room temperature,
ultimately reaching reductions between 1-log and 4-
log, whatever the strain or the cryoprotectant being
analysed. However, in chill conditions, a maximum
reduction of viability of tenfold (1-log) was obtained in
L.M. ZAMORA ET AL.
samples containing glucose. NFSM was the most effect-
ive in maintaining the viability during storage since
maximum counts of viable cells of both strains at the
end of the storage period were found; practically 100%
of viability after 60 days under chill conditions. In
samples containing NFSM, the higher reduction was
observed for the Ent. raffinosus-PS7 stored at room
temperature, although it still showed viability values
higher than 70%.
The rest of the LAB strains were freeze-dried using
only NFSM as cryoprotectant and 100% viability was
obtained in six of the ten dehydrated strains (four
strains of L. garviae (PS14, PS23, PS60, PS95), Ent. raf-
finosus-PS99 and Lact. murinus-PS86), two strains gave
viabilities between 60% and 70% (Lact. reuteri-PS77
and Lact. murinus-PS87) and two of them (L. garviae-
PS22 and PS48) between 3% and 4% only (Table 1).
The stability of the freeze-dried cultures during 60-day
storage followed the same pattern that has already
been described for Ent. raffinosus-PS7 and Lact.
murinus-PS85. In general, final counts in samples kept
at 5 °C were from 67% to 100% of the viable counts at

120 Lact. murinus-PS85 + Glucose 120 Ent. raffinosus-PS7 + Glucose

100 100
Viability (%)

Viability (%)

80 80
60 60
40 40
20 20
0 0
0 15 30 45 60 0 15 30 45 60
Days Days
120 120
Lact. murinus-PS85 + Lactose Ent. raffinosus-PS7 + Lactose
100 100
Viability (%)

Viability (%)

80 80
60 60
40 40
20 20
0 0
0 15 30 45 60 0 15 30 45 60
Days Days
120 Lact. murinus-PS85 +NFSM 120
Ent. raffinosus-PS7 + NFSM
100 100
Viability (%)

Viability (%)

80 80
60 60
40 40
20 20
0 0
0 15 30 45 60 0 15 30 45 60
Days Days

Figure 1. Survival rates of freeze-dried cultures of Lactobacillus murinus-PS85 and Enterococcus raffinosus-PS7
with 10% glucose, 12% lactose and 20% non-fat skimmed milk, during storage at 5 °C (–◆–) and 20 °C (--■--) for
60 days (mean  SD, n  3 standard deviations are indicated by vertical bars).
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
 Comparative Survival Rates of Lactic Acid Bacteria Isolated from Blood 81
Table 1. Survival rates (%) of freeze-dried and spray-dried lactic acid bacteria strains. (a) Immediately after
dehydration, (b) after 60-day storage at 5 and 20 °C; and after 60-day storage at freezing conditions (80 °C).
Freeze-drying Spray-drying

(b) (b)

LAB Strain (a) 5 °C 20 °C (a) 5 °C 20 °C Freezing

L. garviae-PS14 100 79.54 80.62 100 92.54 74.73 55.77

L. garviae-PS22 3.3 107.14 73.14 100 62.42 35.85 42.48
L. garviae-PS23 100 89.43 58.23 100 62.53 44.24 59.77
L. garviae-PS48 3.9 81.65 54.82 100 75.24 44.16 67.97
L. garviae-PS60 100 67.01 61.46 100 58.44 40.87 37
L. garviae-PS95 100 34.40 22.19 100 70.57 45.20 72.8
Lact. murinus-PS85 48.12 109.77 98.56 100 83.40 42.72 33.84
Lact. murinus-PS86 100 67.61 32.74 64.21 67.82 58.73 64.80
Lact. murinus-PS87 61.63 124.11 101.52 100 74.49 33.97 73.05
Ent. raffinosus-PS7 81.41 128.6 72.75 100 86.36 18.88 88.3
Ent. raffinosus-PS99 100 82.55 41.46 55.56 31.31 20.29 25.43
Lact. reuteri-PS77 66.67 85.91 66.60 100 33.05 10.66 95.36

the beginning of the storage. The stability of the same
samples maintained at room temperature was generally
reduced when compared to those at 5 °C. So, the viabil-
ity percentage at the end of storage at 20 °C
(63.67  24.12%) was around 30% lower than that in
chill conditions (88.14  26.41%).
Spray-drying
At the processing conditions used in this work (con-
stant inlet and outlet air temperature of 170 °C and
80–85 °C, respectively), spray-drying provided a dried
material with a moisture content between 5.75% and
6.7%.
Although other workers (Teixeira et al., 1995b;
Boumahdi et al., 1999; Corcoran et al., 2004) suggested
that exponential-phase cells were more susceptible to
spray-drying than cells in the stationary phase of
growth, 100% of viable cells were recovered after
dehydration in ten of the twelve spray-dried LAB
strains. Only the Ent. raffinosus-PS99 and Lact.
murinus-PS86 strains gave lower survival values, up to
55% and 64%, respectively (Table 1). The authors
2-month storage period at 5 °C and 20 °C for the Lact.
murinus-PS85 and Ent. raffinosus-PS7 strains.
Although, lower numbers of viable cells were always
obtained in samples stored at 20 °C with regard to the
samples at 5 °C, differences in the viability values at the
end of storage were shown to be significant (p < 0.05)
only for the Enterococcus strain. The counts of viable
cells at the end of storage in samples maintained at
20 °C were 22% and 58% of those in the spray-dried
cultures at the beginning of storage, while they
remained higher than 80% in the same cultures at 5 °C.
Concerning the behaviour of the rest of the spray-
dried LAB strains (Table 1), it was observed that all of
them were more stable at chill conditions (viabilities of
64.49  17.47% at the end of storage) than at room
temperature (viabilities of 39.19  17.50%).
On the basis of studies from other researchers
(Brennan et al., 1986; Teixeira et al., 1995a; To and
Etzel, 1997a; Conrad et al., 2000; Costa et al., 2002)
lethal thermal injury is the main cause of loss of via-
Lact. murinus-PS85 Ent. raffinosus-PS7
120 120

results on survival after spray-drying seem to be better 100 100

than those from Espina and Packard (1979), Metwally 80 80
Viability (%)

Viability (%)

et al. (1989) and To and Etzel (1997b). Espina and 60 60

Packard (1979) obtained between 1- and 2-log reduc- 40 40
tions in the bacterial counts of Lact. acidophilus, using
20 20
similar process conditions but more concentrated
0
NFSM (25–40%). They reported that major differences 0 15 30 45 60
0
0 15 30 45 60
in total numbers of survivors were related mainly to Days Days

the concentration of NFSM used in drying and that

survival was higher in the lower solids milk concen- Figure 2. Survival rates of spray-dried cultures of
trate. From these results it can be suggested that a 20% Lactobacillus murinus-PS85 and Enterococcus raffi-
NFSM concentration would be even better than the nosus-PS7, during storage at 5 °C (–◆–) and 20 °C
lowest concentration used by them. (--■--) for 60 days (mean  SD, n  3 standard devia-
As an example, Figure 2 shows the stability during a tions are indicated by vertical bars).
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
82 L.M. ZAMORA ET AL.
bility during spray-drying whereas cellular injury
during freeze-drying may result from the additive
effect of freezing and dehydration (Speckman et al.,
1973; Johnson and Etzel, 1995; Teixeira et al., 1995b;
Castro et al., 1997). Freezing may destabilise cells
causing them to become more susceptible to dehydra-
tion. Most of the studies also report that in both
cases, the loss of viability appears to be related to
damage to the cell components, cell membrane, cell
wall and DNA.
In this work, cellular damage due to freeze-drying
was observed immediately after drying whereas cellu-
lar damage due to spray-drying did not become evident
until the subsequent phase of storage. Immediately
following the dehydration process, spray-drying
seemed to be the less aggressive technique for the
LAB strains tested, since it permitted the maximum
recovery of viable cells in 83% of the strains whereas,
in the best case freeze-drying only permitted the total
recovery of 50% of them. This result did not agree with
the results reported by To and Etzel (1997b), who
showed that spray-drying caused considerable reduc-
tion in survival of three different LAB compared to
freezing and freeze-drying. It can be seen that the
effect of treatments resulted hardly depended on the
strain under evaluation, i.e. four out of six strains of L.
garviae gave viability values up to 100% while two
strains of the same species allowed recovery of less
than 4% of viable cells. This confirms results from Lian
et al. (2002) and Carvalho et al. (2002, 2004), who
reported that under comparable conditions, distinct
strains of the same species can differ in their behaviour
during drying.
Maintaining the powdered strains at room tempera-
ture is not recommended, whatever the dehydration
technology (i.e. spray-drying or freeze-drying) since all
dehydrated LAB cultures proved to be more stable at
5 °C than at 20 °C. The same behaviour has already
been described by other researchers for other lactic
bacteria (Abd El-Gawad et al., 1989; Teixeira et al.,
1995b; Gardiner et al., 2000, 2002). Nevertheless, it is
important to notice that the survival rate after 60-day
storage at 5 °C or 20 °C for almost all the dehydrated
cultures was higher than 10%, which supposes less than
1-log reduction with respect to the cell concentration in
the liquid cultures before drying.
Although spray-dried powder showed a faster loss
of viability than freeze-dried cultures during the sub-
sequent storage stage, in 50% of the spray-dried strains
higher percentages were obtained at the end of the
storage at 5 °C, as compared to freeze dried samples
(Table 1). Spray-drying was shown to be a good preser-
vation method for most of the strains of L. garviae and
Lact. murinus, but worse than freeze-drying or freezing
for Ent. raffinosus and Lact. reuteri strains. The results
would lead to the conclusion that the loss of viability
during a 2-month storage of spray-dried cultures would
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
often be compensated by the higher percentage of
viable cells obtained after processing.
From these results, it has been shown that both
dehydration methods resulted in at least equal or
better than freezing at 80 °C, which had been the
method used for maintaining our LAB collection
before this study was performed. Most of dehydrated
LAB showed close to higher percentages of survivors
than the frozen ones at the end of the 60 days (Table
1). The only strain that maintained survival rates at the
end of storage significantly higher in frozen cultures
than the dried ones was Lact. reuteri. Considering that
dried cultures would facilitate their transportation, this
step would facilitate the bio-preservative culture appli-
cation at pilot or industrial scale.
Related to the authors previous studies on the
antagonistic potential in agar plates or in blood, no
changes of the inhibitory properties of the LAB when
strains were maintained under freezing were detected
(20 °C and 80 °C). The evaluation of cultures
following dehydration confirmed that their antagonistic
capacity was not affected by the drying processes since
all the spray-dried or freeze-dried LAB cultures
showed the same antagonistic spectra (Parés et al.,
2004) as the strains kept frozen. The LAB strains also
conserved their inhibitory properties after the 60-day
storage at 5 °C. These results agree with the ones
reported by different authors (Mauriello et al., 1999;
Gardiner et al., 2000, 2002), who also confirmed that
the development of inhibitory systems and probiotic
properties of LAB were not affected by an adequate
dehydration technique.
Results suggest the possibility of achieving a good
formulation system for the LAB strains with a high
number of viable cells to be used for the industrial
development of bioprotective cultures for porcine
blood. The authors think that it is possible and advan-
tageous, at least for the lactic acid bacteria investi-
gated, to concentrate the cultures by spray-drying.
ACKNOWLEDGEMENTS
This work was financially supported by the Spanish
Government (MCYT-AGL 2001–0888). The authors
wish to thank the collaboration of Yolanda Bogado.
They also gratefully acknowledge Anna M. Aymerich,
Rebeca Jiménez and Olga Montojo for helpful techni-
cal assistance.
REFERENCES
Abd El-Gawad I.A., Metwally M.M., El-Nockrashy S.A.
and Ahmed K.E. (1989). Spray-drying of lactic acid cul-
tures: the effect of culture conditions and storage on
 Comparative Survival Rates of Lactic Acid Bacteria Isolated from Blood
microorganisms survival. Egyptian Journal of Dairy
Science 17: 273–281.
Boumahdi M., Mary P. and Hornez J.-P. (1999). Influence
of growth phases and desiccation on the degrees of
unsaturation of fatty acids and the survival rates of rhi-
zobia. Journal of Applied Microbiology 87: 611–619.
Brennan M., Wanismail B., Johnson M.C. and Ray B.
(1986). Cellular damage in dried Lactobacillus aci-
dophilus. Journal of Food Protection 49: 47–53.
Carretero C. and Parés D. (2000). Improvement of the
microbiological quality of blood plasma for human con-
sumption purposes. In: Pandalai S.G. (ed.), Recent
Research Developments in Agricultural & Food Chem-
istry, Vol. 4. Trivandrum, India: Research Singpost, pp.
203–216.
Carvalho A.S., Silva J., Ho P., Teixeira P., Malcata F.X.
and Gibbs P. (2002). Effect of additives on survival of
freeze-dried Lactobacillus plantarum and Lactobacillus
rhamnosus during storage. Biotechnology Letters 24
(19): 1587–1591.
Carvalho A.S., Silva J., Ho P., Teixeira P., Malcata F.X.
and Gibbs P. (2004). Relevant factors for the prepara-
tion of freeze-dried lactic acid bacteria. International
Dairy Journal 14 (10): 835–847.
Castro H.P., Teixeira P.M. and Kirby R. (1997). Evidence
of membrane damage in Lactobacillus bulgaricus
following freeze-drying. Journal of Applied Microbiol-
ogy 82: 87–94.
Conrad P.B., Millar D.P., Cielenski P.R. and Pablo J.J.
(2000). Stabilization and preservation of Lactobacillus
acidophilus in saccharide matrices. Cryobiology 41:
17–24.
Corcoran B.M., Ross R.P., Fitzgerald G.F., Stanton C.
(2004). Comparative survival of probiotic lactobacilli
spray-dried in the presence of prebiotic substances.
Journal of Applied Microbiology 96 (5): 1024–1039(16)
Costa E., Teixidó N., Usall J., Fons E., Gimeno V.,
Delgado J. and Viñas I. (2002). Survival of Pantoea
agglomerans strain CPA-2 in a spray-drying process.
Journal of Food Protection 65 (1): 185–191.
Costa E., Usall J., Teixidó N., Garcia N. and Viñas I.
(2000). Effect of protective agents, rehydration media
and initial cell concentration on viability of Pantoea
agglomerans strain CPA-2 subjected to freeze-drying.
Journal of Applied Microbiology 89: 793–800.
Desmond C., Stanton C., Fitzgerald G.F., Collins K. and
Ross R.P. (2001). Environmental adaptations of probi-
otic lactobacilli towards improvement of performance
during spray drying. International Dairy Journal 11:
801–808.
Espina F. and Packard V.S. (1979). Survival of Lactobacil-
lus acidophilus in spray-drying process. Journal of
Food Protection 42 (2): 149–152.
Fonseca F., Beal C. and Corrieu G. (2000). Method of
quantifying the loss of acidification activity of lactic
acid starters during freezing and frozen storage.
Journal of Dairy Research 67: 83–90.
Gardiner G.E., Bouchier P., O’Sullivan E., Kelly J.,
Collins J.K., Fitzgerald G.F., Ross R.P. and Stanton C.
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
83
(2002). A spray-dried culture for probiotic cheddar
cheese manufacture. International Dairy Journal 12:
749–756.
Gardiner G.E., O’Sullivan E., Kelly J., Auty M.A.E.,
Fitzgerald G.F., Collins J.K., Ross R.P. and Stanton C.
(2000). Comparative survival rates of human-derived
probiotic Lactobacillus paracasei and L. salivarius
strains during heat treatment and spray-drying. Applied
and Environmental Microbiology 66 (6): 2605–2612.
Johnson J.A.C. and Etzel M.R. (1995). Properties of Lac-
tobacillus helveticus CNRZ-32 attenuated by spray-
drying, freeze-drying, or freezing. Journal of Dairy
Science 78 (4): 761–768.
Lian W., Hsiao H. and Chou C. (2002). Survival of bifi-
dobacteria after spray-drying. International Journal of
Food Microbiology 74 (1–2): 79–86.
Mauriello G., Aponte M., Andolfi R., Moschetti G. and
Villani F. (1999). Spray-drying of bacteriocin-produc-
ing lactic acid bacteria. Journal of Food Protection 62:
773–777.
Metwally M.M., Abd El-Gawad I.A., El-Nockrashy S.A..
and Ahmed K.E. (1989). Spray-drying of lactic acid
culture: the effect of spray drying conditions on the sur-
vival of microorganisms. Egyptian Journal of Dairy
Science 17: 35–43.
Parés D. and Ledward D.A. (2001). Emulsifying and
gelling properties of porcine blood plasma as influ-
enced by high pressure processing. Food Chemistry 74
(2): 139–145.
Parés D., Saguer E., Saurina J., Suñol J.J. and Carretero
C. (1998). Functional properties of heat induced gels
from liquid and spray dried porcine blood plasma as
influenced by pH. Journal of Food Science 65 (6):
958–961
Parés D., Saguer E., Toldrà M. and Carretero C. (2000).
Effect of high pressure processing at different tempera-
tures on protein functionality of porcine blood plasma.
Journal of Food Science 65 (3): 486–490.
Parés D., Zamora L., Saguer E. and Carretero C. (2004).
Acid lactic bacteria as biopreservative cultures in
porcine blood for human consumption. Food Info
Online Features 2 April 2004 http://www.foodscience-
central.com/library.html#ifis/13082.
Saguer E., Altarriba S., Lorca C., Parés D., Toldrà M. and
Carretero C. (2003). Color stabilization of spray-dried
porcine red blood cells using nicotinic acid and nicoti-
namide. Food Science and Technology International 9
(4): 301–307.
Speckman C.A., Sandine W.E. and Elliker P.R. (1973).
Lyophilized lactic acid starter culture concentrates:
preparation and use in inoculation of vat milk for
cheddar and cottage cheese. Journal of Dairy Science
57 (2): 165–173.
Teixeira P., Castro H. and Kirby R. (1995a). Spray-drying
as a method for preparing concentrated cultures of
Lactobacillus bulgaricus. Journal of Applied Bacteriol-
ogy 78 (4): 456–462.
Teixeira P.C., Castro M.H., Malcata F.X. and Kirby R.M.
(1995b). Survival of Lactobacillus delbrueckii ssp. bul-
84 L.M. ZAMORA ET AL.
garicus following spray-drying. Journal of Dairy
Science 78 (5): 1025–1031.
To B.C.S. and Etzel M.R. (1997a). Survival of Brevibac-
terium linens (ATCC 9174) after spray-drying, freeze-
drying, or freezing. Journal of Food Science 62 (1):
167–170.
To B.C.S. and Etzel M.R. (1997b). Spray drying, freeze
drying, or freezing of three different lactic acid bacteria
species. Journal of Food Science 62 (3): 576–578.
Toldrà M., Elias A., Parés D., Saguer E. and Carretero C.
(2004). Functional properties of spray dried porcine
red blood cells fraction treated by high hydrostatic
pressure. Food Chemistry 88 (3): 461–468.
Zamora L. (2003). Aislamiento, identificación y conser-
vación de cultivos de bacterias lácticas antagonistas de
Downloaded from fst.sagepub.com at Mount Royal University on August 24, 2014
microbiota contaminante de sangre de cerdo de
matadero. PhD thesis. Institut de Tecnologia Agroali-
mentària (INTEA). University of Girona, Spain.
Zamora L., Carretero C. and Parés D. (2003). Aplicación
de bacterias ácido lácticas como cultivo bioprotector en
sangre de cerdo procedente de matadero industrial.
Alimentación, Equipos y Tecnología 175: 45–50.
Zamora L.M., Guajardo M., Saguer E., Bonaterra A.,
Carretero C. and Parés, D. (2001). Application of lactic
acid bacteria as biopreservative culture of blood from
slaughtered animals. In: Proceedings of III Congreso
Iberoamericano de Ingeniería de Alimentos and I Con-
greso español de Ingeniería de alimentos. Valencia 2001,
7C-14.III.