CRYOBIOLOGY 35, 159–164 (1997)

ARTICLE NO. CY972036

Induced Cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus

LBB by Preincubation at Suboptimal Temperatures
with a Fermentable Sugar

Patricio de Urraza and Graciela De Antoni

Centro de Investigación y Desarrollo en CriotecnologıB a de Alimentos (CIDCA),
UNLP, 47 y 116, 1900, La Plata, Argentina

The resistance of Lactobacillus delbrueckii subsp. bulgaricus to freeze–thaw cycles can be increased by
incubating bacterial suspensions in nongrowing conditions before freezing. Incubation at 307C during 60
min in medium with a fermentable sugar induces cryotolerance of strain LBB. No change of viable counts
but a decrease of extracellular pH and an increase of intracellular ATP during preincubation were observed.
Other preincubation conditions did not show any improvement in cryotolerance. q 1997 Academic Press

Lactobacillus delbrueckii subsp. bulgaricus
is widely used in the elaboration of Italian-
type cheeses and fermented milk. In our coun-
try some factories usually employ starter cul-
tures that must be kept active for several days,
so they are stored frozen at 0207C. L. del-
brueckii subsp. bulgaricus can be metaboli-
cally damaged or killed by freeze–thaw cy-
cles. Thus, it is difficult to preserve bacterial
concentrates in the active state during long
periods. Preservation of frozen cultures in liq-
uid nitrogen is the best condition when com-
pared with subculture, freeze drying, spray
drying, and freezing at 020 or 0807C. How-
ever, the high cost of liquid nitrogen prohibits
its widespread use in the storage of frozen
cultures. Resistance to freezing has been im-
proved by using several cryoprotectants such
as milk, glycerol, trehalose, and adonitol (1,
4, 6). Also, the composition of the growth
medium and its influence on the action of spe-
cific cryoprotectants was studied (1, 10, 13).
However, none of these conditions were ade-
quate enough to preserve a high percentage
of L. delbrueckii subsp. bulgaricus cells in
cultures frozen at 0207C (5).
It is known that hypothermia treatment be-
fore a freeze–thaw cycle increases the resis-
Received May 28, 1996; accepted May 20, 1997.
tance of mammalian cells (7) and similar con-
ditions have been tried in order to increase
the resistance of Lactococcus lactis to freeze–
thaw cycles (9).
Preliminary results showed that bacterial
cultures of L. delbrueckii subsp. bulgaricus
LBB, maintained in fresh medium at non-
growing temperatures before freezing, became
more resistant to freezing and thawing. This
finding prompted us to confirm this behavior
under standard conditions.
In this work we describe the effect of prein-
cubation at suboptimal temperatures on the
development of cryotolerance of L. del-
brueckii subsp. bulgaricus LBB and correlate
this behavior with the accumulation of intra-
cellular ATP.
MATERIALS AND METHODS
Strains and Media
L. delbrueckii subsp. bulgaricus LBB was
isolated and identified at CIDCA, Argentina
(1), it was kept frozen at 0807C in sterilized
12% skimmed milk (Difco, Detroit, MI,
U.S.A.) Frozen bacteria were reactivated by
subculturing in 12% skimmed milk at 377C
before use. Cultures were made in modified
APT broth (in g/liter, tryptone 10, yeast ex-
tract 10, lactose 10, K2HCO3 5, sodium citrate
5, Tween 80 0.2, MgCl2r7 H2O 0.8, and
MnCl2 0.14, pH 7.2). Minimal broth contained

159
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Copyright q 1997 by Academic Press
All rights of reproduction in any form reserved.

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160 URRAZA AND DE ANTONI
in grams per liter tryptone 10 and yeast extract
10, pH 6.8. It was supplemented with lactose,
glucose, or sucrose at a concentration of 1%
(w/v). Solid media were prepared with 1.5 g/
100 ml of agar.
Treatment of Bacterial Suspensions before
Freezing
Bacterial cultures were harvested at the
early stationary phase (DO550nm Å 1.0), at pH
6.0–6.2 unless indicated, washed once and re-
suspended in different media (minimal broth,
lactose broth, glucose broth, sucrose broth,
and APT broth) to a final concentration of 1–
3 1 108 cfu/ml. The final pH after resuspen-
sion was 6.6–6.8 for broths and 7.2 for APT
broth, respectively. Bacterial suspensions
(BS) were frozen immediately at 0207C or
preincubated (BSP) and then frozen at 0207C.
Preincubations were carried out at 0, 7, and
307C during periods of 30, 60, and 180 min.
Freezing and Thawing Methodology
BSP and BS were frozen immediately in
the same incubation medium or after centrifu-
gation and resuspension in freezing medium
(lactose broth, sucrose 0.3 M, or skimmed
milk 12%). Aliquots of 0.5 ml containing 1–
3 1 108 cfu/ml were frozen at 0207C in 1.5
ml polypropylene tubes (cooling rate 47C/min)
and stored at 0207C for different periods. Fro-
zen tubes were thawed at 377C and held for 5
min at that temperature. The number of viable
cells and acid production was determined be-
fore freezing and immediately after thawing.
Cell Viability and Acid Production
Colony forming units were counted by plat-
ing duplicate dilutions in tryptone 0.1% on
lactose broth agar. Results were expressed as
a percentage of surviving bacteria. Acid pro-
duction was determined by diluting the
thawed bacterial suspensions 10 times in lac-
tose broth and incubation at 427C. The pH was
determined after different periods of incuba-
tion at 427C. All experiments were done in
duplicate. DpH was calculated as pHtÅ0 0
pHtÅi , where t Å 0 immediately after dilution
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of the thawed samples and i is the time of
incubation at 427C.
Determination of Adenosine Triphosphate
Determination of intracellular ATP in bac-
teria was carried out using an ATP biolumi-
nescence assay kit. A 50-ml sample of bacte-
rial suspension was blended for 30 s with 100
ml of lysis buffer to extract the bacterial ATP.
The ATP content was then determined by the
luciferine–luciferase reaction. All reagents
were from the ATP biomass kit Bio-Orbit
(Torku, Finland). The emitted light signal was
measured with a luminometer Bio-Orbit 1-2-
3-4 (Torku, Finland).
RESULTS AND DISCUSSION
Figure 1 shows that BSP, that is bacteria
preincubated at 307C for 60 min in lactose
broth before freezing, became more resistant
to freezing and thawing than control, nonpre-
incubated bacteria (BS). Other preincubation
temperatures such 0 or 77C and other incuba-
tion periods (30 or 180 min) did not show a
significant increase in the resistance of bacte-
ria to freeze–thaw cycles. The increased cryo-
tolerance of BSP was observed both after stor-
age at 0207C for 10 days (Fig. 1) and after 45
days (data not shown), while the percentage
survival in the last condition was five times
lower. However, the concentration of viable
bacteria in BSP stored at 0207C for 45 days
was only 1.2 times lower than that obtained
after 10 days of storage. In accordance with
these results, Fig. 2 shows that freeze–thawed
BSP have a higher acidification rate than BS.
These results indicate that the high percentage
survival of BSP is not due to the rupture of
lactobacilli chains. The cryoprotective effect
of preincubation in lactose broth was observed
irrespective of the growth phase at which they
were harvested, provided that they were prein-
cubated at 307C for 60 min before freezing.
However, BSP from cells obtained at the early
stationary phase gave the highest concentra-
tion of survival after freezing and thawing
(data not shown). At this point we wondered
whether preincubation added an extra cryo-
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 INDUCED CRYOTOLERANCE OF Lactobacillus 161

FIG. 1. Survival of L. delbrueckii subsp. bulgaricus LBB preincubated at different temperatures before
freezing. Bacterial suspensions preincubated in lactose broth at 0, 7, and 307C for 0, 30, 60, and 180 min
were frozen in the same medium at 0207C. The pH ranged between 5.0 and 5.3 after preincubation at 0
and 77C and 4.4–4.7 after preincubation at 307C. Frozen suspensions were stored at 0207C for 10 days.

FIG. 2. Acid production of freeze-thawed bacterial suspensions. Bacterial suspensions nonpreincubated

(BS, m) and preincubated at 307C for 60 min (BS, n), were frozen in lactose broth at 0207C for 10 days.

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162 URRAZA AND DE ANTONI
FIG. 3. Survival of BSP and BS frozen with cryoprotec-
tants. Bacterial suspensions were preincubated (BSP) in
lactose broth at 307C for 1 h. Before freezing at 0207C,
BSP and BS were centrifuged and resuspended in lactose
broth, sucrose, or skim milk at pH 6.5. Frozen suspensions
were stored for 10 and 20 days at 0207C.
protective effect to bacteria frozen with cryo-
protectants. Considering that sucrose and
skimmed milk are cryoprotectants for L. del-
brueckii subsp. bulgaricus at 0807C (2) they
were tested at 0207C with BS and BSP. Figure
3 shows that bacteria frozen and stored at
0207C for 10 days have a higher percentage
survival than those stored at 0207C for 20
days. Under all conditions, BSP were more
resistant to freeze–thaw cycles than BS. BSP
frozen in skimmed milk showed a signifi-
cantly higher percentage survival, both after
10 days (Ç80%) and after 20 days (Ç30%)
of storage. These results indicate that cryopro-
tective effects of skimmed milk and preincu-
bation at 307C for 60 min are additive. Figure
4 shows that the rate of acid production of
thawed suspensions is in accordance with the
percentage survival (Fig. 3). A high rate of
acid production by the unfrozen control
(100% survival) and by BSP frozen in
skimmed milk (Ç80% survival) was ob-
served.
Table 1 shows that the protective effect of
incubation (307C, 60 min) was obtained only
when it was performed in the presence of a
fermentable sugar, such as glucose or lactose.
Under these conditions, the final pH after in-
cubation dropped to 4.51–4.81 giving an ex-
tremely rapid decrease in pH (DpH 1.62–
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1.83). However, the concentration of viable
bacteria remained constant during preincuba-
tion. Under this condition the amount of ATP
per bacteria increased 85–100 times in com-
parison with cells preincubated with nonfer-
mentable sugar. This high intracellular content
of ATP may be responsible for the higher re-
sistance to freeze–thaw cycles since the per-
centage survival is 3–10 times higher. Low
extracellular pH in the freezing media does
not have any influence on cryotolerance. BSP
frozen in lactose broth at a final pH 4.5 (Fig.
1) and BSP frozen in the same media at pH
6.6 (Fig. 3), gave a similar percentage survival
after freeze–thaw cycles. Accumulation of in-
tracellular ATP is a consequence of glycolysis
and a halt in biosynthesis due to incubation
in media with a fermentable sugar at low tem-
perature. The fact that incubation with a fer-
mentable sugar in nongrowing conditions in-
duces cryotolerance suggests that the large in-
crease in intracellular ATP could allow rapid
repair of injuries upon thawing. Bacterial sus-
pensions preincubated at 307C for 180 min
show lower cryotolerance than those preincu-
bated for 60 min. Probably, after a long incu-
bation time, some increase of bacterial mass
at the expense of ATP could occur and then
less ATP would be available for the repair
process. Another possibility is that prolonged
exposure to low extracellular pH may produce
more injury to the cells. The cryotolerance of
strain LBB is not induced at 0 or 77C because
at these low temperatures no glycolysis is pro-
duced and so less ATP can be accumulated in
the cells during incubation periods of 30–180
min. Panoff et al. (1995) found that preincuba-
tion of L. lactis at 87C led to an enhanced
capacity to survive exposure to freezing tem-
peratures of 0207C. However, no studies re-
lated to the induction of cryotolerance of ther-
mophilic lactobacilli have been reported. On
the other hand, thermal shock stress in L. lactis
was characterized by the expression of several
proteins (3, 8, 12). Both thermotolerance and
cryotolerance were induced by heat and cold
shocking of the bacteria for different periods.
Other treatments, such as UV irradiation and
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 INDUCED CRYOTOLERANCE OF Lactobacillus 163

FIG. 4. Acid production of BSP and BS frozen with cryoprotectants. Bacterial suspensions were diluted
in lactose broth after thawing suspensions stored at 0207C for 10 days. Bacterial suspensions unfrozen
(control, n), frozen with lactose broth (BSP, ,; and BS, .), 12% skim milk (BSP, h; and BS, j), and
0.3 M sucrose (BSP, s; and BS, l). BSP and BS were obtained as indicated in the legend to Fig. 3.

starvation, promoted enhanced thermotoler- know if some particular proteins were ex-
ance in Lactococcus. In the case of the lacto- pressed during the preincubation period at
bacillus strain studied in this paper we do not 307C, but this could be related to the observa-

TABLE 1
Effect of Preincubation on Cryotolerance and Metabolic Activity of Bacterial Suspensions

pH after
Incubation mediaa incubation DpHb ATP c Survial (%)d

Lactose broth 4.81 1.62 23.578 47.5 { 6.4

APT broth 4.51 1.66 19.297 14.2 { 2.1
Glucose broth 4.60 1.83 17.398 31.9 { 8.7
Sucrose broth 6.55 0.00 0.191 4.9 { 1.6
Minimum broth 6.01 0.05 0.167 4.4 { 2.2

Note. cfu/ml were determined before and after preincubation. In all cases cfu/ml remained unchanged during
preincubation.
a
Bacteria were harvested at the early stationary phase and bacterial suspensions were preincubated at 307C for 1
h. Preincubation media were removed and BSP were frozen in lactose broth (pH 6.6–6.7) at 0207C. After thawing
survival (%) was determined.
b
DpH Å pHi 0 pHf . Where pHi is the pH before preincubation and pHf is the pH after incubation.
c
Intracellular ATP accumulated during preincubation at 307C. Concentration of ATP per bacteria Å (log emitted
light/bacteria) 1 108.
d
Survival after a freeze–thaw cycle.

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164 URRAZA AND DE ANTONI
tion of induced cryotolerance. The results pre-
sented in this study demonstrate that preincu-
bation of L. delbrueckii subsp. bulgaricus
LBB at 307C for 60 min induced cryotoler-
ance. The enhanced resistance to freezing and
thawing was achieved only if the thermal ad-
aptation was performed in the presence of a
fermentable sugar and it correlates with the
accumulation of intracellular ATP. The induc-
tion of cryotolerance in combination with the
use of skimmed milk as a cryoprotectant can
be used to preserve cultures of L. delbrueckii
subsp. bulgaricus LBB at 0207C with a rela-
tively high percentage survival.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. L. Brandi for technical
assistance and Dr. E. A. Disalvo for technical comments.
This work was supported by Consejo Nacional de Investi-
gaciones CientıB ficas y Técnicas (CONICET), Comisión
de Investigaciones CientıB ficas de la Provincia de Buenos
Aires (CICPBA), and Universidad Nacional de La Plata
(UNLP).
REFERENCES
1. Abraham, A., De Antoni, G. L., and Añón, M. C.
Effect of calcium on the cryopreservation of Lac-
tobacillus bulgaricus in different freezing media.
Cryobiology 27, 336–342 (1990).
2. Abraham, A. ‘‘Lactobacillus Bulgaricus en Fer-
mentos Para Yogur.’’ Ph.D. thesis, Universidad
Nacional de La Plata, Argentina, 1992.
3. Auffray, Y., Gansel, X., Thammavongs, B., and Bou-
tibonnes, P. Heat shock-induced protein synthesis
in Lactococcus lactis subsp. lactis. Curr. Micro-
biol. 24, 281–284 (1992).
4. De Antoni, G. L., Pérez, P., Abraham, A., and Añón,
AID CRYO 2036 / a90d$$$103 08-15-97 06:57:21
M. C. Trehalose, a cryoprotectant for Lactobacil-
lus bulgaricus. Cryobiology 26, 149–153 (1989).
5. de Urraza, P., Becerra, A., Abraham, A., Pérez, P.,
Kociubinski, G., Añón, M., and De Antoni, G.
Fermentos lácticos congelados: Optimización del
proceso de conservación. In ‘‘III Congreso Latin-
oamericano de MicrobiologıB a e Higiene de Ali-
mentos, Montevideo, Uruguay’’ 1992.
6. Font de Valdéz, G., de Giori, G. S., de Ruiz Holgado,
A. P., and Oliver, G. Protective effect of adonitol
on lactic acid bacteria subjected to freeze-drying.
Appl. Environ. Microbiol. 45, 302–304 (1983).
7. Glofcheski, D. J., and Kruuv, J. Induction of toler-
ance to freeze-thaw (FT) damage in mammalian
cells by pre-FT hypothermia treatment. Cryobiol-
ogy 30, 353–365 (1993).
8. Panoff, J-M., Legrand, S., Thammavongs, B., and
Boutibonnes, P. The cold shock response in Lacto-
coccus lactis subsp. lactis. Curr. Microbiol. 29,
213–216 (1994).
9. Panoff, J-M., Thammavongs, B., Laplace, J. M.,
Hartke, A., Boutibonnes, P., and Auffray, Y. Cryo-
tolerance and cold adaptation in Lactococcus lactis
subsp. lactis IL 1403. Cryobiology 32, 516–520
(1995).
10. Smittle, R. B., Gilliland, S. E., and Speck, M. L.
Death of Lactobacillus bulgaricus resulting from
liquid nitrogen freezing. Appl. Environ. Microbiol.
24, 551–554 (1972).
11. de Valdéz, G. F., and de Giori, G. S. Effect of freez-
ing and thawing on the viability and uptake of
amino acids by L. delbrueckii ssp. bulgaricus.
Cryobiology 30, 329–334 (1993).
12. Whitaker, R. D., and Batt, C. A. Characterization of
the heat shock response in Lactococcus lactis
subsp. lactis. Appl. Environ. Microbiol. 57, 1408–
1412 (1991).
13. Wright, C. T., and Klaenhammer, T. R. Survival of
Lactobacillus bulgaricus during freezing and
freeze-drying after growth in the presence of cal-
cium. J. Food Sci. 48, 773–777 (1983).
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