International Journal of Food Microbiology 117 (2007) 36 – 42
www.elsevier.com/locate/ijfoodmicro
Osmotic stress induced by salt increases cell yield, autolytic activity, and
survival of lyophilization of Lactobacillus delbrueckii subsp. lactis
Stefanie Koch a , Gaëtan Oberson a , Elisabeth Eugster-Meier b , Leo Meile a , Christophe Lacroix a,⁎
a
b
Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland
Agroscope Liebefeld-Posieux, Swiss Federal Research Station for Animal Production and Dairy Products, Schwarzenburgstrasse 161, 3003 Berne, Switzerland
Received 27 June 2006; received in revised form 21 November 2006; accepted 20 January 2007
Abstract
Growth and stress adaptation of an autolytic strain of Lactobacillus delbrueckii subsp. lactis FAM-10991 was studied during pH-controlled
batch fermentations. After an initial growth to an optical density at 650 nm of 0.8 under controlled optimal growth conditions (pH 5.5, 37 °C, no
salt), exponentially growing cells were exposed to salt at concentrations from 1 to 3.5%, and temperatures between 48 and 53.5 °C, without pH
control or with pH controlled at 5.5 or 4.5. Autolysis was induced by salt concentrations of 2.5 or 3.5% and suppressed at 53.5 °C or pH 4.5. Salt
at concentrations of 2.5 or 3.5% or a temperature of 53.5 °C, without pH control or with pH controlled at 5.5, significantly enhanced (p b 0.05)
survival of lyophilization as compared with the survival of cells in control cultures or cultures with salt at concentration of 1 and 1.5%. The former
conditions increased survival by 125- and 200-fold, respectively. However, no correlation was found between autolytic activity and survival of
lyophilization. Cultures grown with salt at 2.5% gave high yields of viable cells in broths before and after lyophilization, with numbers being 27fold higher than with control cultures, but with autolytic activity that was 2.5-fold higher than in cells from control cultures.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Lactobacillus delbrueckii subsp. lactis; Stress; Cross resistance; Autolytic activity; Survival of lyophilization; Cell yield
1. Introduction
Lactobacilli are widely used as starters in food fermentations
(Giraffa and Mora, 1999). For cheese production, frozen or
freeze-dried starter cultures are often used to inoculate milk
(Monnet et al., 2003). Transportation and storage costs are
lower for dried than for liquid cultures. However, during
lyophilization, considerable greater inactivation of lactobacilli
than of enterococci occurs (Carvalho et al., 2004).
Bacterial resistance to freezing and drying can be improved
by applying moderate stresses during growth of cultures
(Fonseca et al., 2001). Survival mechanisms exhibited by
bacteria exposed to stresses are generally referred to as stress
responses. Lactic acid bacteria react to unfavorable conditions
by producing stress proteins or by modifying their membrane
compositions (Fonseca et al., 2001). These changes allow
⁎ Corresponding author. Tel.: +41 44 632 48 67; fax: +41 44 632 14 03.
E-mail address: [email protected] (C. Lacroix).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.01.016
bacteria to resist subsequent exposure to a higher level of the
same stress, and some other stresses also (Lorca and Font de
Valdez, 2001). The development of cross resistance to stresses
can be used to enhance desirable technological properties,
such as resistance to lyophilization.
Lactobacilli that are highly autolytic are often used as
primary and secondary starters for production of semi-hard
and hard cheeses (Husson-Kao et al., 1999; Fröhlich-Wyder
and Bachmann, 2005; Lortal and Chapot-Chartier, 2005).
Autolysis releases intracellular enzymes into the cheese curd.
These enzymes accelerate peptidolysis during cheese ripening, and so increase the rate of free amino acid production and
the rate at which bitter taste decreases (Husson-Kao et al.,
2000). Autolytic cultures may exhibit low survival of
treatments such as lyophilization since autolysis can be
induced by various environmental stresses (Gaudreau et al.,
2006). A correlation between stress response and autolysis in
lactic acid bacteria, i.e. streptococci, has been investigated by
Husson-Kao et al. (1999). However, in a previous study, no
correlation was found between autolytic activity and survival
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
of freezing or lyophilization of 51 strains of Lactobacillus
delbrueckii subsp. lactis isolated from Swiss semi-hard and
hard cheeses (unpublished results). Stress responses of
autolytic strains and the effects of stresses during growth of
lyophilization have not been studied.
In the present study we investigated the responses of
L. delbrueckii subsp. lactis FAM-10991 to sublethal stress
treatments applied during growth. This strain was selected for
its high degrees of autolysis and sensitivity to lyophilization
(unpublished results). Various osmotic, heat and acid conditions
were tested, with or without pH control, for their effects on
growth, carbohydrate metabolism, autolytic activity and
survival of lyophilization.
2. Materials and methods
2.1. Stock culture and reactivation of L. delbrueckii subsp.
lactis FAM-10991
L. delbrueckii subsp. lactis FAM-10991 was obtained
from the culture collection of Agroscope Liebefeld-Posieux
(Berne, Switzerland). The strain was subcultured three times
in de Man, Rogosa and Sharpe medium (MRS; Biolife
Italiana S.r.l, Milano, Italy) and was kept as frozen stock in
40% glycerol at − 40 °C. For inoculum preparation, 1% of
frozen stock culture was subcultured twice in MRS medium at
37 °C for 12 h.
2.2. Batch fermentations
Control fermentations in MRS medium supplemented with
55 g of glucose-mono-hydrate (Merck, Darmstadt, Germany)
per liter were carried out in a 2-l fermenter (Bioreactor 2.0 l,
Multiple Bioreactors and Sterile Plants AG, Zürich, Switzerland) with addition of a 1% (v/v) inoculum. The fermenter was
flushed with CO2 and stirred with a Rushton impeller at
200 rpm, at 37 °C for 26 h, with pH controlled at 5.5 by addition
of 5 N NaOH. For studying growth conditions, cells were grown
to an optical density of 0.8 at 650 nm (OD650), determined using
a spectrophotometer (Ultraspec II; Biochrom, Berlin, Germany)
under controlled optimum growth conditions. To induce
osmotic stress, autoclaved, dry NaCl was added to the culture
medium to obtain concentrations of 1, 1.5, 2.5 or 3.5% (w/v).
For temperature stress, the temperature was increased to 48, 51
or 53.5 °C. For acid stress, the pH was adjusted to 4.5 by
addition of 5 N lactic acid and subsequently was controlled at
4.5 using 5 N NaOH. A combination of temperature and acid
stresses was also tested, with the temperature at 53.5 °C and
uncontrolled pH after an OD650 of 0.8 was reached. All
fermentations were carried out at least twice with randomization
of experimental units.
Samples were taken at 2 h intervals for optical density
measurements, viable cell enumeration, dry biomass determination, and sugar and organic acid analyses. Samples (60 ml)
were taken for survival of lyophilization after 14 to 15, 22 and
26 h. PepX activities were determined after 22 and 26 h and
autolytic activity was measured after 26 h.
37
2.3. Viable cell, biomass determinations and overall cell yield
calculations
Biomass concentration was determined by measuring the
optical density at 650 nm. Numbers of colony forming units
(cfu/ml) were determined by plating diluted samples on MRS
agar (Biolife, Pully, Switzerland) and incubating for 48 h at
37 °C in anaerobic jars (BBL Microbiology Systems, Becton
Dickinson, Mississauga, Ontario, Canada).
To directly measure dry biomass concentration, a 10 ml
sample was centrifuged at 5000 g for 12 min at 4 °C. The pellet
was washed twice with distilled water and dried to constant
weight at 60 °C under vacuum. Analyses were done in duplicate
and means were reported.
The relative numbers of cells in chains of cells were
estimated from the biomass concentrations at times tM, where
maximum viable cell counts were reached, by reference to the
cell morphology of control cultures. For this, dry biomass
concentration at time tM, was multiplied by the ratio of the
maximum number of cfu to the corresponding dry biomass
concentration for the control cultures.
Overall cell yields were calculated after lyophilization by
multiplying the maximum cfu measured during cultivation by
the appropriate rates of cell survival after lyophilization. If cell
morphology was affected by culture conditions, overall cell
yields were estimated from both the maximum cfu values and
the estimated relative cell numbers, the latter being adjusted for
chain length by reference to the control culture, as described
above.
2.4. Glucose and lactate analyses
Glucose and lactate concentrations in broth samples were
determined by high pressure liquid chromatography (HPLC)
using an Agilent 1100 Series HPLC (Agilent Technologies
Schweiz AG, Basel, Switzerland) and an Online Process
Analyzer (HPLC: Biospectra, Schlieren, Switzerland). Ten
mM sulfuric acid was used as the mobile phase for an Aminex
HPX-87H column (Bio-Rad Laboratories AG, Reinach,
Switzerland) at a flow rate of 0.6 ml/min. Each sample was
analysed once.
2.5. Microscopic observations
Samples were prepared for microscopy by mixing 0.7 ml
portions of broths with 0.3 ml of 1.5% (w/v) agar (Oxoid,
Hampshire, England) placing a drop of each preparation on a
slide, covering the preparation with a cover slip and view it at a
magnification of 1000 using an optical microscope (Olympus
CX 41, Aigle, CH).
2.6. PepX activity
Samples were centrifuged at 14,000 ×g for 10 min at 4 °C,
then the supernatant was analysed for dipeptidylpeptidase
(PepX) activity. PepX was evaluated using L-alanyl-L-prolineP-nitroanilide (Bachem, Bubendorf, Switzerland) as substrate as
38
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
described by Meyer and Spahni (1998). One unit of
aminopeptidase activity (U) was defined as the amount of
enzyme required to release 1 μM/ml p-nitroanilide under the
assay conditions. This test was carried out in duplicate.
2.7. Determination of cell autolytic activity in buffer system
Before analysis, culture samples were centrifuged at 5000 ×g
for 15 min at 4 °C and cells were washed in distilled water. Each
cell pellet was resuspended in 0.2 M NaCl at pH 5.5 to obtain a
cell suspension with an OD650 of 0.8–1.2, which was incubated
for 24 h at 37 °C. The degree of autolysis was expressed as the
percentage decrease of OD650 after 24 h (Kang et al., 1998).
Each analysis was performed in duplicate.
and 2% (w/v) glycerol. Before freezing, viable cells in the
suspension were enumerated. Two 2.5 ml portions of the
suspension were frozen at − 40 °C and stored for 10 to 30 days
until analysis. Lyophilization was performed using freeze
drying equipment (Item No. N 22015; Usifroid Minilyo,
Maurepas, France) for 42 h, at − 40 °C and 100 μbar for
sublimation, and at 20 °C and 70 μbar for secondary drying.
Rehydration for 10 min at room temperature to the original
volume was performed according to Font de Valdez et al. (1985)
and numbers of viable cells were determined. The survival rate
reported was the percent fraction of cells surviving lyophilization. Lyophilization was performed in duplicate for each culture
sample.
2.9. Statistical analyses
2.8. Lyophilization
A 60 ml sample of broth was centrifuged at 4600 ×g for
12 min at 4 °C. The pellet was washed in distilled water and
resuspended in 6 ml of a solution containing 8% (w/v) dextrane
Data sets that did not meet the assumptions of ANOVA for
normally distributed and independent residuals were transformed for statistical analyses; by the arcsin square root
function, after dividing percentage fractions by 100, for survival
Fig. 1. Photomicrographs at 1000 × magnification of L. delbrueckii subsp. lactis FAM-10991 cultured for 26 h in MRS medium under conditions of: A) 37 °C, pH 5.5,
no salt (control culture); B) 37 °C, pH 5.5, 3.5% salt; C) 37 °C, pH 4.5, no salt; D) 53.5 °C, pH 5.5, no salt E) 53.5 °C, uncontrolled pH, no salt.
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
rates; by the inverse of the fourth exponent after adding 0.4 for
PepX activity; by log10-transformation for bacterial counts and
yield; and by square root transformation for autolytic activity.
Univariate ANOVA or repeated measures ANOVA, for survival
rates at three different times, were performed using the general
linear model of SPSS, ver. 13.0 (SPSS, Chicago, IL, USA) at a
significance level of p b 0.05. When differences were statistically significant, treatment means were compared using
Tukey's multiple comparison test. Correlations among PepX
activity, autolytic activity and survival rates after lyophilization
were analysed using Spearman's test at a significance level of
39
0.01. Means of untransformed data are presented in figures and
tables.
3. Results
3.1. Morphology
Microscopic observation of cells grown under control
conditions showed chains 20 to 50 μm long composed of 2 to
5 cells (Fig. 1A). All salt concentrations tested did not affect the
chain length, but empty cells were apparent in cultures grown
Fig. 2. Dry biomass concentration (open symbols) and numbers of colony forming units (cfu; closed symbols) in batch cultures of L. delbrueckii subsp. lactis FAM10991 during cultivation for 26 h under conditions of: A) 37 °C, pH 5.5, no salt (control culture); B) 37 °C, pH 4.5, no salt; C) 37 °C, pH 5.5, 1% salt; D) 37 °C, pH 5.5,
3.5% salt; E) 53.5 °C, pH 5.5, no salt; F) 53.5 °C, uncontrolled pH, no salt. Arrows indicate the times of imposition of stresses. Fermentations were performed twice.
40
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
with 3.5% salt (Fig. 1B). When the pH was 4.5, the arrangement
of cells gradually changed from chains to short, single or paired
cells (Fig. 1C). At 53.5 °C and pH 5.5, cells elongated to form
long hair-like and curled cells (Fig. 1D). In contrast, cells grown
at 53.5 °C without pH control showed the same short rod
morphology as acid-stressed cells (Fig. 1E).
3.2. Cell growth in different conditions
Fig. 2 shows growth profiles of L. delbrueckii subsp. lactis
FAM-10991 during batch cultures, and Table 1 shows
maximum dry biomass concentrations and maximum numbers
of cfu for each condition tested.
For control cultures, maximum numbers of cfu and dry
biomass concentrations were reached after about 14 h or 22 h,
respectively (Fig. 2 A). When the pH was reduced from 5.5 to
4.5, growth was slower, with a lower maximum dry biomass
concentration being reached after 24 h. However, the maximum
numbers of cfu were not significantly different (Fig. 2 B,
Table 1).
For cultures supplemented with 1 and 1.5% salt, the
maximum numbers of cfu were similar to these of the control
cultures, but the maximum dry biomasses for cultures with 1%
salt were significantly higher (p b 0.05) than those for the
control cultures or cultures with 1.5% salt. Addition of 3.5% salt
resulted in an immediate cessation of cell growth, and
significantly lower (p b 0.05) maximum numbers of cfu and
dry biomass concentrations compared with the controls (Fig. 2
C and D, Table 1). For cultures supplemented with 2.5% salt,
maximum numbers of cfu after 26 h were significantly higher
(p b 0.05) than those for cultures with 3.5% salt (Table 1). Dry
biomasses were also higher but not significantly (p N 0.05).
Growth at 48 °C gave maximum numbers of cfu similar to
those obtained with control cultures but maximum dry
biomasses that were significantly greater (p b 0.05), whereas
with temperatures of 51 or 53.5 °C, maximum numbers of cfu
and biomasses were similar to those of the control cultures
(Table 1). The maximum numbers of cfu for heat and acid
stressed cultures were adjusted for cell elongation and cell
separation, respectively, for estimation of the numbers of viable
cells relative to the numbers in control cultures. The adjustments gave values for maximum viable cell numbers for acid or
temperature stressed cultures at pH 5.5 that were, respectively,
smaller or larger than the maximum numbers of cfu. The
adjustments had no effect on the outcomes of statistical tests
(Table 1).
The combination of 53.5 °C and uncontrolled pH gave an
approximately three-fold decrease in maximum dry biomass
(p b 0.05) but maximum numbers of cfu after 11 h or 10 h were
not significantly different (p N 0.05) from those of the control
cultures. Further incubation after 10 h produced rapid
decreases of cfu numbers to values of about 105 cfu/ml after
26 h (Fig. 2 F).
3.3. Metabolic activity
After 26 h culture, glucose and lactate concentrations in the
fermented media with 3.5 or 2.5% salt were significantly higher
and lower (p b 0.05), respectively, than in media with 1% or
1.5% salt, or in control cultures (Table 1). Glucose and lactate
concentrations in pH 4.5 cultures were significantly higher and
lower (p b 0.05), respectively, than those in the control cultures
and not different to those in the cultures with 1 or 1.5% salt. In
contrast, cultures at high temperature with pH controlled at 5.5
showed almost complete glucose utilization and high lactate
production at the end of fermentation. The increased metabolic
activity of cells grown at high temperatures was not correlated
with enhanced cell growth because maximum estimated relative
cell numbers were not significantly different from the numbers
of cfu in control cultures grown at 37 °C. Residual glucose and
lactate concentrations in cultures grown at 53.5 °C with no pH
control were not significantly different (p N 0.05) from the
concentrations in cultures grown with 2.5 or 3.5% salt. Cultures
grown at 53.5 °C and pH 5.5 showed a significant increase
Table 1
Times of the maximum viable counts (tM), biomasses at tM, maximal biomasses, maximum numbers of colony forming units (cfu), maximum estimated numbers of
viable cells and glucose and lactate concentrations after 26-h, in batch cultures of L. delbrueckii subsp. lactis FAM-10991 grown under various conditions
Conditions
Maximum dry Maximum numbers tM
biomass
of cfu
Dry biomass at
tM
Estimated relative number of Glucose b Lactate b Specific lactate
viable cells a
production
Temp. (°C) pH
Salt (%) (g/l)
(×108/ml)
(h)
(g/l)
(×108 cfu /ml)
(g/l)
(g/l)
(glactate/gbiomass)
37
37
37
37
37
48
51
53.5
53.5
37
0
1
1.5
2.5
3.5
0
0
0
0
0
6.19cef
8.32edf
5.75cef
1.08b
0.27a
8.67ef
2.49bc
2.99be
2.62bcd
8.67f
14
23
10
26
9.5
14.3
18.5
8
10
24
0.96
4.01
1.7
0.48
0.39
4.33
2.64
1.94
0.88
2.43
6.19cd
8.32d
5.75cd
1.08b
0.27a
8.91d
5.44cd
3.99c
1.79b
4.98c
20.0b
23.4bc
26.7bc
49.1d
58.2d
1.65a
0.95a
0.41a
55.1d
32.4c
45.0cd
42.5bc
45.0bcd
13.1a
5.64a
59.6e
55.8de
61.5e
14.4a
35.1b
14.5abc
10.4ab
14.5abc
14.5abc
18.7abc
13.3ab
20.2bc
22.8c
14.3abc
14.0ab
a
b
c
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
NC c
4.5
3.09c
4.10d
3.10c
0.71a
0.39a
4.50d
2.77bc
2.72bc
1.01a
2.52b
Calculated using the formula: relative numbers of cells(stress) = dry biomass concentration at tM(stress) × [max CFU(control) / max. dry biomass concentration(control)].
Initial glucose and lactate concentrations were 63.4 ± 4.2 and 0.65 ± 0.52 g/l in supplemented MRS medium.
NC, no pH control.
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
41
Table 2
Rate of survival of lyophilization, PepX and autolytic activities, yields of colony forming units (cfu) and yields of estimated relative numbers of viable cells after
lyophilization of L. delbrueckii subsp. lactis FAM-10991 grown for 26 h in batch cultures under various growth conditions
Conditions
Rate of
survival a
PepX activity at
22 h
PepX activity at
26 h
Autolytic
activity
Yield of
cfu b
Yield of relative numbers of viable
cells c
Temp. (°C)
pH
Salt (%)
(%)
(U)
(U)
(%)
(×107 /ml)
(× 107 cfu/ml)
37
37
37
37
37
48
51
53.5
53.5
37
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
NC d
4.5
0
1
1.5
2.5
3.5
0
0
0
0
0
0.20a
1.06ab
0.72ab
25.0ef
39.7f
2.15ab
3.95abc
7.48bd
15.0cde
6.22ad
0.06c
0.03bc
0.23d
1.53d
0.50d
0.02bc
0.00ab
0.00ab
0.00ab
0.04c
0.07d
0.04cd
0.33e
1.73e
0.70e
0.03bc
0.00ab
0.00ab
0.00ab
0.05cd
14.1d
11.9cd
9.9cd
38.6e
48.6e
9.69cd
7.79bcd
3.36ab
1.89a
6.25cb
0.1a
0.9bc
0.4ab
2.7cd
1.1bc
1.9cd
1.0bc
2.3cd
12.4e
5.4de
0.1a
0.9c
0.4b
2.7d
1.1c
1.9d
2.1d
3.0d
2.7d
3.1d
Means in the same column with the same letter are not significantly different (p b 0.05).
a
Means for three sampling times, with for each two replicates.
b
Maximum CFU × rate of survival of lyophilization.
c
Maximum estimated numbers of viable cells × rate of survival of lyophilization.
d
NC, no pH control.
(p b 0.05) in specific lactate production compared to those for
cultures with 1% salt, or grown at 48 °C or pH 4.5.
3.4. PepX and autolytic activities
PepX activities of cultures were significantly higher (p b 0.05)
after cultivation for 26 h than after 22 h (Table 2). At both
sampling times, significantly higher (p b 0.05) PepX activities
were observed for cultures grown with 1.5% or higher salt
concentrations compared with the activities of cultures subjected
to other treatments. The PepX activities of the control cultures and
cultures stressed by temperature, 1% salt or acid pH were very
low.
Cells grown with 2.5 or 3.5% salt showed significantly
higher autolytic activities than those grown under other
conditions (Table 2). Autolytic activities of cells grown at
53.5 °C, with or without pH control, or at 51 °C or pH 4.5 were
significantly lower (p b 0.05) than those for cells grown under
other conditions.
A highly significant correlation (r = 0.8, p b 0.001) was
calculated between PepX and autolytic activities.
3.5. Survival of lyophilization
Repeated measure ANOVA showed no significant differences (p N 0.05) between the three sampling times but indicated
significant effects (p b 0.05) for stress treatments. Cells grown
under the control conditions were very sensitive to lyophilization, with a survival rate of only 0.2%. In contrast, cells grown
with 3.5% or 2.5% salt had survival rates of 39.7% and 25.0%,
respectively, that were high in comparison with the survival
rates of cells grown with 1.5 or 1% salt at pH 4.5, or at 48 or
51 °C, or control cells (Table 2). Survival rates of cells grown at
53.5 °C and pH 5.5, or with uncontrolled pH were significantly
higher (p b 0.05) than those for cells grown under control
conditions but were significantly lower (p b 0.05) than those for
cells grown with 2.5% or 3.5% salt.
There was no significant correlation between survival rates
after lyophilization and PepX (r = 0.045, p = 0.85) or autolytic
activities (r = 0.032, p = 0.9).
4. Discussion
In this study addition of 55 g/l glucose to MRS medium was
used to avoid carbon limitation which can induce cross
resistances to high temperature, salt concentration or acid
stresses, as previously reported for Lactococcus lactis (Sanders
et al., 1999).
Our finding on the effect of pH on cell morphology and the
lengths of cell chains are consistent with those of Norton et al.
(1993) for Lactobacillus helveticus. Those workers suggested
that there might be a relationship between the low rate of
synthesis of an autolytic enzyme and the long hair-like form of
L. helveticus at pH values above 6.5. However, in our study,
autolytic enzyme activity was not higher at low pH, because cell
lysis during fermentation, as measured by PepX activity, was
similar for pH 4.5 and control cultures, and autolytic activity
was even less at lower pH values.
Since cell morphology was greatly affected by culture
conditions when treatments produced long chains the numbers
of viable cells were underestimated by plate counts. Therefore,
biomass measurements may be more indicative for estimating
maximum cell production. However this parameter cannot
distinguish between live and dead cells, as was clearly shown
during the death phase of cultures supplemented with 3.5%
NaCl and incubated at 53.5 °C with a pH of 5.5. With such
cultures, rapid cell death was observed while dry biomass
concentration remained constant. For applications of autolytic
starter cultures in cheese production, it is important to control
both acidification during cheese making and enzymatic
42
S. Koch et al. / International Journal of Food Microbiology 117 (2007) 36–42
activities during ripening, properties that are correlated with the
numbers of viable cells added to the milk.
Little is known about the effects of high temperatures on the
metabolic activities of lactobacilli. For L. delbrueckii, the
enzymes responsible for glucose metabolism seemed to be more
active at high temperatures, as lactate production by cells grown
at 51 and 53.5 °C and pH 5.5 was relatively high. In contrast, the
lytic enzymes seemed to be less active at those higher
temperatures than at 37 °C, as both PepX and autolytic
activities were reduced at the higher temperatures.
NaCl is a well known triggering factor for autolysis (Kang
et al., 1998; Husson-Kao et al., 1999). In our study, autolysis
was induced by 2.5% or 3.5% salt, but not by high temperature
or low pH. Husson-Kao et al. (1999) showed that pH values
below 5 reduced autolysis of Streptococcus thermophilus
whereas a temperature increase from 42 to 50 °C resulted in
greatly increased autolysis. This suggests that triggering factors
for autolysis in lactic acid bacteria are species-dependent.
During drying of cultures, the ionic strength of the medium
increases and bacteria are subjected to osmotic stress (Kets and
de Bont, 1994). Lactobacilli react to osmotic stress by
accumulating compatible solutes like betain or carnitin, which
acted to balance the difference in osmotic pressure between the
intracellular and extracellular environments and give increased
protection against drying (Kets et al., 1996). In the present
study, the very large increased survival of lyophilization by
osmotically stressed cells might be partly due to the
accumulation of compatible solutes during growth.
It has been suggested that autolytic strains of lactobacilli
poorly survive the stresses imposed during processes such as
lyophilization (Gaudreau et al., 2006). In this study we did not
observe a correlation between autolytic activity and survival of
lyophilization for L. delbrueckii subsp. lactis FAM-10991 cells
grown under different conditions. That finding is in agreement
with data from a previous study with 51 strains of the same
subspecies isolated from Swiss cheeses (unpublished data).
For economic production of autolytic cultures and applications in the cheese industry, high cell yields during both
fermentation and in downstream processes must be obtained.
Yields of cfu and relative numbers of viable cells for most
growth conditions were significantly higher than for the control
cultures. The highest cell yield of cfu was obtained with cultures
grown at 53.5 °C without pH control, in which short rods were
observed, instead of chains of longer cells as in the control
cultures. The best condition for production of L. delbrueckii
subsp. lactis FAM-10991 that would be used to produce
lyophilized cultures was the addition of 2.5% salt to the growth
medium, which gave high cell yields and although the cells had
greater autolytic activity than control cells.
Acknowledgements
This work was supported by Agroscope Liebefeld-Posieux
(Berne, Switzerland). The authors are grateful to C. Birrer (ETH
Zurich), R. Liesch (ETH Zurich) and U. Bütikofer (ALP) for
advice in statistics.
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