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O R I G I N A L A R T I C L E
Effects of copper supplement on growth and viability ofstrains used as starters and adjunct cultures for Emmentalcheese manufacture
L. Mato Rodrguez and T. Alatossava
Department of Food Technology, University of Helsinki, Helsinki, Finland
Introduction
In Switzerland, Emmental cheese is traditionally made in
copper vats, and accordingly, copper levels typically lie
between 76 and 165 ppm (Sieber et al. 2006). Copper
ions leached from the copper cheese vat are mainly
bound by casein proteins and are therefore transferred
into the cheese; many cheese makers believe that copper
has a beneficial effect on Swiss cheese quality (Sieber et al.
2006). However, Emmental cheese-processing plants in
many countries currently use stainless-steel vats and thus
no copper is leached into the cheese. In Finland, where
stainless-steel vats are used for Emmental manufacture,
extra copper is added as CuSO4 salt solution into the
cheese milk to increase the copper concentration of the
milk from less than 01 t o 13 ppm. This supplement
brings the copper level in Finnish Emmental cheese close
to the traditional Swiss-make Emmental cheese, but is still
less than 15 ppm, which is the highest acceptable copper
level.
The consumption of organic foods is increasing popu-
larity in developed countries. In the manufacture of
organic Emmental cheese, the addition of copper salt sup-
plement is not allowed. As same type of vats (stainless
steel) are used, the effect of the nonaddition of copper, as
a step in the technology, should be investigated.
Because of the essential and the toxic nature of
copper, microbial organisms have mechanisms to regulate
Keywords
adjunct culture, bacterial growth, cell viability,
copper, Emmental cheese, lactic acid bacteria,
Lactobacillus, propionibacteria, starter.
Correspondence
Tapani Alatossava, Department of Food
Technology, Viikki campus, PO Box 66,
FIN-00014 University of Helsinki, Helsinki,
Finland. E-mail: [email protected]
20071717: received 25 October 2007,
revised 31 January 2008 and accepted 5
March 2008
doi:10.1111/j.1365-2672.2008.03849.x
Abstract
Aims: To determine the effects of supplemented copper (Cu2+) on growth and
viability of strains used as starters and adjunct cultures for Emmental cheese
manufacture.
Methods and Results: Thirteen strains belonging to Lactobacillus delbrueckii,
Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus orPropionibacterium freudenreichii species were exposed to various copper con-
centrations in the proper growth medium at relevant growth temperatures, and
the effects of supplemented copper on bacterial growth and cell viability were
determined by optical density and pH measurements, also by platings. Among
the species considered, L. delbrueckii was the most copper resistant and S. ther-
mophilus the most sensitive to copper. Anaerobic conditions increased this
sensitivity significantly. There was also a considerable amount of variation in
copper resistance at strain level.
Conclusions: Copper resistance is both a species- and strain-dependent
property and may reflect variability in copper-binding capacities by cell wall
components among species and strains. In addition, the chemical state of
copper may be involved.
Significance and Impact of the Study: This study revealed that copper resis-
tance is a highly variable property among starter and adjunct strains, and this
variability should be considered when strains are selected for Emmental cheese
manufacture.
Journal of Applied Microbiology ISSN 1364-5072
1098 Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 109811062008 The Authors
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intracellular copper concentration (OHalloran 1993). In
Escherichia coli, at least three systems are involved in
copper tolerance (Franke et al. 2003). In Saccharomyces
cerevisiae yeast, the two main homeostatic systems for
copper metabolism have been characterized (Dancis et al.
1994). There are only a few isolated reports on the effects
of copper on physiological and biochemical activities oflactic acid bacteria (LAB) and propionibacteria starters
(Kiermeier et al. 1961; Maurer et al. 1975; Lee et al.
2005a,b).
The principal starters used in the production of Swiss
hard cheese varieties are thermophilic LAB, often as
mixed cultures of lactobacilli and streptococci belonging
to Lactobacillus helveticus andor Lactobacillus delbrueckii
ssp. lactis, and Streptococcus thermophilus species. These
LAB guarantee the homofermentative catabolism of lac-
tose to more than 90% lactate, and the proteinases and
peptidases of lactobacilli play a major role in the break-
down of casein during cheese ripening. Some decades
ago, L. helveticus species was still the major lactobacilli in
starter cultures used in the manufacture of Swiss Emmen-
tal. However, because of its intensive proteolytic activity,
which promotes undesired late fermentation, L. helveticus
has been replaced by L. delbrueckii ssp. lactis more
recently. Facultative heterofermentative lactobacilli are
often used as adjunct cultures in the manufacture of
Emmental cheese to slow down the propionic acid fer-
mentation. Lactobacillus casei and Lactobacillus rhamnosus
are among the most-utilized species. A particular species
of propionibacteria, Propionibacterium freudenreichii, is
employed as a secondary starter in the manufacture of
Emmental cheese in order to achieve the characteristiceyes and nutty flavour (Frohlich-Wyder and Bachmann
2004).
The final quality of the Emmental cheese could be
influenced by different amounts of copper present in milk
and cheese matrix through the modification of bacterial
metabolic activities andor bacterial enzyme activities,
such as peptidases that are important in cheese flavour
formation. In the present study, strains used as starters
and adjunct cultures for the production of Emmental
cheese in Finland were exposed to different concentra-
tions of copper in proper growth medium in order to
investigate the effects of copper both on growth and via-
bility of these strains and to elucidate possible variability
in copper resistance at strain and species levels.
Materials and methods
Bacterial strains
Lactobacillus delbrueckii ssp. lactis ATCC 15808 was
obtained from American Type Culture Collection
(ATCC). Probiotic L. rhamnosus strain GG (also known
ATCC 53103, this strain is of human intestinal origin)
was isolated from a commercial Gefilus product made
by Valio Ltd. The rest of the 11 strains S. thermophilus
strain T101; P. freudenreichii ssp. freudenreichii strain
P131; L. helveticus strains 1129, 1175 and 1518; L. del-
brueckiissp. bulgaricus strain LB270 and L. delbrueckii ssp.lactis strains LKT, LL23 and LL78; and L. rhamnosus
adjunct strains Lc705 and 13 were of industrial origin
and obtained from the dairy company Valio Ltd
(Helsinki, Finland). The stability and purity of each strain
was confirmed by a specific carbohydrate profile using
API 50 CHL identification system according to the direc-
tions of the manufacture (BioMerieux, France).
The experiments with S. thermophilus strain T101 were
carried out in M17 broth (Oxoid) supplemented with 2%
(wv) lactose (M17L). For P. freudenreichii ssp. freud-
enreichii strain P131, Na-lactate broth (Tuomola et al.
1999) and de Man Rogosa Sharpe (MRS) broth (Merck)
were used, and for all lactobacilli strains, MRS broth was
used as growth medium.
Culture conditions and stock preparation
The culture of each strain at exponential growth phase
was stored together with 20% glycerol at )80C for long-
time storage. Each culture was refreshed in 5 ml broth
twice before each experiment. Strains ofL. rhamnosus and
L. delbruecki ssp. lactis were incubated at 37C and strains
ofL. helveticus and L. delbruecki ssp. bulgaricus were incu-
bated at 395C for 24 h under anaerobic conditions.
Propionibacterium freudenreichii ssp. freudenreichii P131was incubated for 24 h at 30C under anaerobic condi-
tions. Anaerobic atmosphere was created using the BBL
(Baltimore Biological Laboratory) gas pack anaerobic sys-
tem (Becton Dickinson and Co., Cokeysville, MI, USA).
The strain S. thermophilus T101 was incubated at 37C
for 24 h under aerobic or anaerobic conditions depending
on the character of the following experiment with this
strain.
Effects of copper supplements in the medium on
bacterial growth and viability
In all experiments, a 5-ml final volume of the corre-
sponding broth medium (M17L, Na-lactate or MRS) and
01% (vv) bacterial inoculum of each strain in exponen-
tial growth phase were used. Copper supplements (final
Cu concentration 75, 15 or 30 ppm Cu) in sterile broth
were made by using freshly made and sterile filtered
(pore size 045 lm; Schleicher & Schuell, Dassel,
Germany) 064% (wv) CuSO45H2O stock solution
(copper content of the stock solution was 2550 ppm Cu).
L. Mato Rodrguez and T. Alatossava Effects of copper on starters for Emmental cheese
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 10981106 1099
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For S. thermophilus strain T101, copper supplements in
M17L medium were adjusted to lower levels based on
preliminary tests. The final concentrations used were 2 5,
5, 75, 10 or 15 ppm Cu in sterile M17L. Each medium
itself (without Cu supplement) was considered to contain
insignificant amount of copper (based on data from the
medium producers), and accordingly, the medium wasused as the 0-ppm Cu control medium for experiments.
All experiments were carried out in duplicate. Following
incubation periods of 24, 48 and 72 h at temperatures
indicated earlier for each group of strains, under anaero-
bic (plus aerobic for strain T101) conditions, bacterial
growth and viability of each culture were measured by
pH and OD600 measurements and by platings as
described later.
Measurements of optical density, pH and total colony
counts
Optical density of the bacterial culture at 600 nm
(OD600) was measured with a spectrophotometer
(Novaspec II, Amersham Pharmacia, Sweden) using a
particular blank. The blank was prepared using identical
incubation conditions to the test culture tubes but not
inoculated with bacteria for each copper supplement con-
dition to correct any possible colour change in the growth
medium considered caused by the presence of supple-
mented copper. In addition, the pH values were measured
both for each bacterial test culture and each blank after
incubation periods considered. Platings of S. thermophilus
T101 were carried out using M17 agar (Merck) supple-
mented with 2% (wv) lactose (M17L agar). A 10-folddilution series in 085% NaCl solution was used, and
1 ml of an appropriate dilution was mixed with 20 ml of
melted M17L agar, and further plated using the poured
agar method. The solidified M17L plates were incubated
at 37C for 48 h in aerobic conditions, and the colony-
forming units (CFU) in each M17L plate were counted.
Platings of P. freudenreichii ssp. freudenreichii P131 were
carried out using Na-lactate plates, which were prepared
by supplementing Na-lactate broth with the 16% (wv)
agar (Merck). A 10-fold dilution series in 085% NaCl
solution was used, and 50 ll of a suitable dilution was
spread on the solid surface of a Na-lactate plate. The Na-
lactate plates were incubated at 30C for 5 days in anaer-
obic conditions using the BBL system described earlier,
and the CFU in each plate were counted. Platings of all
Lactobacillus strains used in this study were carried out
using MRS agar (Merck) plates. A 10-fold dilution series
in 085% NaCl solution was used and 50 ll of a proper
dilution was spread on the solid surface of an MRS plate.
The MRS plates were incubated at 37C for 4872 h in
anaerobic conditions using the BBL system described ear-
lier, and the CFU in each plate were counted. All platings
were carried out in duplicate and the mean values were
used for CFU ml)1 calculations.
Statistical analysis
The correlations between the studied variables (pH,OD600 and log10 CFU ml
)1) were evaluated, and the
results of some experiments were submitted to statistical
analysis using one-way analysis of variance (anova). The
analyses were performed with spss 150 for Windows and
the data was analysed using Fishers protected least signifi-
cant difference (LSD) test with 95% confidence level.
Results
Effects of copper supplements in MRS broth on growth
and viability ofLactobacillus delbrueckii, Lactobacillus
helveticusand Lactobacillus rhamnosus
As all important Lactobacillus species used in Emmental
cheese manufacture are able to grow in MRS broth, it is
possible to study the effects of copper supplements for
growth and viability of various strains of starter and
adjunct Lactobacillus separately in this medium. In this
study, the dairy strains of L. delbrueckii ssp. lactis, Lacto-
bacillus helveticus and Lactobacillus rhamnosus species
have been considered.
The three variables studied (pH, OD600 and log10CFU ml)1) showed a significant (P< 001) correlation
between them; for pH vs OD600 r= )0974, pH vs log10
CFU ml)1
r=)
0613 and log10 CFU ml
)1
vs OD600r= 0568 (primary data not shown). Based on these cor-
relation (r) values, the plating variable log10 CFU ml)1
was chosen for the presentation of the results concerning
the three Lactobacillus species (Tables 13).
Among the three Lactobacillus species, L. delbrueckii
was most resistant to copper showing growth inhibition
only in the presence of 30 ppm Cu in a strain-dependent
way (Tables 13). Among the four ssp. lactis strains
included in this study, the strain LKT was the most resis-
tant and the strain LL78 was most sensitive as it was the
slowest one to recover in the presence of 30 ppm. Still,
the strain LL78 was more resistant than the ssp. bulgaricus
strain LB270, which was used as a noncheese starter refer-
ence strain in this study (Table 1).
The three L. helveticus Emmental cheese starter strains
included in this study indicated strain-dependent copper
resistance (Table 2). The most copper-resistant strain
1175 was about as resistant as the L. delbrueckii ssp.
bulgaricus strain LB270, and it was capable of recovering
slowly in the presence of 30 ppm Cu. The two other
L. helveticus strains, 1518 and 1129, lost cell viability after
Effects of copper on starters for Emmental cheese L. Mato Rodrguez and T. Alatossava
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24- and 48-h incubation, respectively, in the presence of
30 ppm Cu, but could recover in the presence of 7 5 ppm
Cu. Strain 1175 still could retain cell viability in the pres-
ence of 15 ppm Cu after 72-h incubation period.
The three L. rhamnosus adjunct strains included in this
study again indicated strain-dependent copper resistance
(Table 3). All three strains were more sensitive to growth
inhibition by copper supplement in MRS broth than
thoseL. helveticus and L. delbrueckii strains considered, as
no growth was observed during the first 24-h incubation
in the presence of 75 ppm Cu. Among the three
L. rhamnosus strains, the strain Lc705 was the most sensi-
tive and the strain GG (ATCC 53103) was the most resis-
tant to the growth-inhibiting effect of copper, although
Table 1 Counts of viable cells expressed in
log10 CFU ml)1 SD of Lactobacillus
delbrueckiistrain cultures in MRS broth
supplemented with different copper
concentrations (030 ppm) and incubated at
37C for ssp. lactis and at 395C for ssp.
bulgaricus in anaerobic atmosphere up to
72 h. Samples were taken at times indicated,and counts of viable cells were determined
by plating in MRS agar as described in
Materials and methods
Strain T(h)
log10 CFU ml)1 SD
Copper supplemented in medium (ppm)
0 75 15 30
ssp. lactis:
ATCC 15808 0 562 041 562 041 562 041 562 041
24 878 0
28 8
76 0
24 8
50 0
22 8
43 0
16
48 646 019 641 009 709 021 721 018
72 440 014 509 012 549 002 579 002
LKT* 0 596 008 596 008 596 008 596 008
24 896 005 894 003 896 008 876 016
48 873 012 870 014 877 022 853 001
72 770 010 804 016 843 017 819 002
LL23 0 484 012 484 012 4,84 012 484 012
24 871 001 877 002 876 016 649 006
48 732 021 818 023 834 008 836 030
72 461 004 577 012 720 050 807 014
LL78 0 500 018 500 018 500 018 500 018
24 876 007 872 004 865 005 586 019
48 767 015 812 017 834 008 827 013
72 6
48 0
65 6
88 0
33 7
65 0
64 8
23 0
17ssp. bulgaricus:
LB270 0 595 012 595 012 595 012 595 012
24 886 008 915 004 818 016 329 017
48 807 011 862 011 845 061 775 034
72 781 006 836 004 848 009 842 034
CFU, colony-forming units; MRS, de man Rogosa Sharpe.
*Significant at P 005.
Table 2 Counts of viable cells expressed in
log10 CFU ml)1 SD of Lactobacillus helveti-
cus strain cultures in MRS broth supple-
mented with different copper concentrations
(030 ppm) and incubated at 39
5
C inanaerobic atmosphere up to 72 h. Samples
were taken at times indicated, and counts of
viable cells were determined by plating in
MRS agar as described in Materials and
methods
Strain T(h)
log10 CFU ml)1 SD
Copper supplemented in medium (ppm)
0 75 15 30
1518 0 610 012 610 012 610 012 610 012
24 891 002 791 015 443 057 285 008
48 552 017 750 031 817 017 000 000
72 239 009 389 026 660 032 000 000
1175* 0 575 003 575 003 575 003 575 003
24 893 005 826 034 775 026 600 007
48 607 001 676 032 782 014 760 042
72 467 085 585 022 750 014 790 017
1129 0 580 023 580 023 580 023 580 023
24 871 017 695 005 438 013 406 009
48 683 004 817 024 758 033 439 009
72 493 009 681 019 712 019 000 000
CFU, colony-forming units; MRS, de man Rogosa Sharpe.
*Significant at P 005.
L. Mato Rodrguez and T. Alatossava Effects of copper on starters for Emmental cheese
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 10981106 1101
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no statistically significant differences could be obtained
between the strains. On the other hand, both these strains
could retain their cell viabilities even in the presence of
30 ppm Cu in MRS broth, contrary to the L. rhamnosus
strain 13, which shows similar behaviour with L. helveti-
cus strains 1518 and 1129 in this respect (Tables 2 and 3).
Effects of copper supplements in Na-lactate broth and
MRS broth on growth and viability ofPropionibacterium
freudenreichii
Propionibacteria are essential secondary starter added in
Emmental cheese manufacture together with the primary
LAB starters if raw milk for cheese is heat treated by ther-mization or low pasteurization. The most widely
employed propionibacteria in this case is P. freudenreichii
ssp. freudenreichii or ssp. shermanii. In this study, the
P. freudenrecihii ssp. feudenreichii cheese starter strain
P131 was used to study the effects of copper supplements
on the growth and viability of P131 in Na-lactate broth
(Fig. 1a,b) and MRS broth (Fig. 1c,d) under anaerobic
conditions at 30C. Na-lactate broth was shown to be a
more favourable medium (as indicated by higher log10CFU ml)1 and OD600 values obtained) for the growth of
this strain (Fig. 1). As shown in Fig. 1a,b, the growth of
the strain P131 was strongly inhibited in the presence of
75 ppm Cu in Na-lactate broth, and completely pre-
vented in the presence of 15 or 30 ppm Cu. However, the
cell viability of the P131 culture was not practically lost
when copper was added to have 15 and 30 ppm in Na-
lactate broth (Fig. 1a). In this, the Propionibacterium
strain P131 resembled the strains Lc705 and GG of
L. rhamnosus (Table 3). When the strain P131 was grown
in MRS broth (Fig. 1c,d), copper had a quite similar
effect on the growth and viability of the strain P131 as in
the case of Na-lactate broth, except that there was a slight
drop in the viability in the presence of 30 ppm Cu in
MRS broth at 72 h (Fig. 1c). These results could suggest
that possible interactions of growth medium components
in MRS broth or Na-lactate broth with copper do not
play a significant role, and accordingly, the observed cop-
per effects on the strain P131 growth and viability could
be considered as direct effects on cellular functions and
not indirect medium-dependent effects.
Effects of copper supplement and dissolved oxygen in
M17L broth on growth and viability ofStreptococcus
thermophilus
Thermophilic S. thermophilus is the second essential spe-
cies component in the primary starter culture together
with thermophilic Lactobacillus species (typicallyL. helve-
ticus or L. delbrueckii ssp. lactis) for Emmental cheese
manufacture. The growth of S. thermophilus species is
known to be less oxygen tolerant than Lactobacillus spe-
cies employed in Emmental cheese starter cultures. This is
at least partially because of the lack of catalase or other
peroxidase activities (Condon 1987; Hols et al. 2005). For
this reason, the effect of dissolved oxygen in M17L broth
was studied together with the effects of copper supple-
ment in M17L broth. Accordingly, both aerobic and
anaerobic growth conditions were employed for culture
incubations at 37C. Streptococcus thermophilus dairy star-
ter strain T101, which is also known to be very sensitive
to inhibitory effects by antibiotics, was chosen for this
study. As shown in Fig. 2, the growth of the strain T101
was favoured by anaerobic growth conditions as indicated
by higher OD600 value and lower final pH value of the
control culture (no Cu) under anaerobic atmosphere
(Fig. 2b,c) compared with aerobic atmosphere (Fig. 2e,f).
Table 3 Counts of viable cells expressed in
log10 CFU ml)1 SD of Lactobacillus
rhamnosus strain cultures in MRS broth
supplemented with different copper
concentrations (030 ppm) and incubated at
37C in anaerobic atmosphere up to 72 h.
Samples were taken at times indicated, and
counts of viable cells were determined byplating in MRS agar as described in
Materials and methods
Strain T(h)
log10 CFU ml)1 SD
Copper supplemented in medium (ppm)
0 75 15 30
Lc705 0 555 023 555 023 555 023 555 023
24 903 023 575 026 328 003 308 054
48 864 0
35 6
93 0
21 5
68 0
03 5
53 0
29
72 815 013 781 002 624 020 594 014
13 0 635 007 635 007 635 007 635 007
24 927 017 345 013 310 014 325 021
48 892 001 841 043 287 018 335 007
72 865 013 835 025 489 018 147 001
GG 0 630 007 630 007 630 007 630 007
24 914 017 576 010 489 040 392 005
48 781 009 877 012 652 010 580 009
72 594 011 839 010 709 021 603 007
CFU, colony-forming units; MRS, de man Rogosa Sharpe.
Effects of copper on starters for Emmental cheese L. Mato Rodrguez and T. Alatossava
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Interestingly, the strain T101 was significantly more sensi-
tive to growth inhibition by copper in M17L broth under
anaerobic atmosphere (Fig. 2). In the presence of
25 ppm Cu in M17L broth, only slight reduction of
OD600 and colony counts could be observed without any
effect on the final pH under aerobic conditions (Fig. 2d,f)compared with the evident 24-h lag period in the growth
of the culture under anaerobic conditions (Fig. 2a,c). In
addition, identical amount of supplemented copper (e.g.
10 ppm) affects the cell viability of the culture more dras-
tically under anaerobic conditions (Fig. 2a,d).
Discussion
Our study revealed the effects of supplemented copper on
the growth and cell viability of strains used as Emmental
cheese starters and adjunct cultures. In the literature,
there are very limited data available on this subject,
although from the Emmental cheese technology point of
view, copper and its biochemical and microbial reactions
in milk and cheese matrix are of great interest. Our study
focussed on the microbial effects in specific growth
media, rather than in milk or cheese matrix. Despite these
limitations, some conclusions can be drawn from the
results obtained. Among the bacterial species studied,
S. thermophilus was the most sensitive to copper, with
25 ppm Cu in M17L broth being sufficient to inhibit
bacterial growth and to reduce cell viability of the culture
under anaerobic conditions (Fig. 2a,c). However, this
conclusion is based on the assumption that the broths
used in this study (MRS, Na-lactate and M17L) did not
differ significantly in their copper interaction properties.
However, Ramamoorthy and Kushner (1975) have forexample demonstrated that copper is able to bind to dif-
ferent media components. On the other hand, in this
study, we demonstrated that at least Na-lactate broth and
MRS broth did not differ drastically in their possible cop-
per-binding capacities; similar copper effects on the
growth and viability of strain P. freudenreichii ssp. freud-
enreichii P131 could be observed with similar copper
supplements (Fig. 1).
General mechanisms of microbial metal resistance
could include biotransformation of a metal cation to a
less toxic form, and decreased accumulation owing to
efflux or exclusion mechanisms. Exclusion of copper ions
from the microbial cell appears to be the main mode of
bacterial copper resistance. The copper resistance mecha-
nism in E. coli apparently involves copper efflux, and in
Pseudomonas syringae, resistance mechanism involves
copper sequestration. However, the copper-resistance
genes show a high degree of similarity in these two
species (OHalloran 1993). Copper resistance can also be
a plasmid-encoded property. Ishihara et al. (1978)
reported a temperature-sensitive, conjugative plasmid,
10
(a) (b)
(c) (d)
9
8
7
Log10
CFU
ml1
Log1
0CFU
ml1
6
5
4
3
10
9
8
7
6
5
4
3 0
0 72 0 24 48 72
Time (h)
02
0
02
01
03
04
05
04OD
(600nm)
OD
(600nm)
06
08
1
12
14
24 48
Time (h)
0 7224 48Time (h)
0 7224 48Time (h)
Figure 1 Effects of various copper concentra-
tions in (a, b) Na-lactate broth and in (c, d)
de man Rogosa Sharpe (MRS) broth on
growth and viability of Propionibacterium
freudenreichiissp. freudenreichistrain P131
when incubated at 30C in anaerobic atmo-
sphere and measured at indicated points of
incubation periods by the following parame-
ters: (a and c) log10 CFU ml)1 and (b and d)
OD600of the culture. (r) Na-lactateMRS
broth without copper supplement; ( ) with
75 ppm of copper; ( ) with 15 ppm of cop-
per; or (d) with 30 ppm copper supplement.
Each value plotted is a mean of duplicate.
L. Mato Rodrguez and T. Alatossava Effects of copper on starters for Emmental cheese
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 10981106 1103
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Rts1, associated with Cu2+
resistance in E. coli host. Cop-per resistance in Ps. syringae p.v. tomato strains was con-
trolled by two conjugative plasmids (Bender and Cooksey
1986). Goodson and Rowbury (1986) suggested that the
presence of plasmids may affect other cellular functions
and indirectly alter the ability of the cell to tolerate cop-
per. In recent studies, it has been found that some lacto-
bacilli strains have the ability to chelate Fe2+ and Cu2+
ions, Lactobacillus plantarum KCTC 3099 and L. casei
3260 showed higher chelating activity for Fe2+ and Cu2+
when compared with other lactobacilli strains tested (Lee
et al. 2005a; b). This chelating ability can allow some
strains to withstand higher concentrations of copper
when compared with other strains. Our results from
Lactobacillus species and strains, which were studied in
the same growth medium (MRS) under anaerobic atmo-
sphere, demonstrated both species- and strain-dependent
differences in copper resistance (Tables 1)3). Among the
three Lactobacillus species considered, the order of
increasing copper resistance was: L. rhamnosus, L. helveti-
cus and L. delbrueckii. In addition, inside each species,
there was a considerable variation at strain level in
copper-induced growth inhibition and loss of cell viabil-ity, as measured by CFU ml)1 during prolonged incuba-
tion of the culture. Both species- and strain-specific
differences ofLactobacillus in copper resistance could be
at least partially explained by structural and composi-
tional variation of negatively charged cell-wall bound or
excreted biopolymers, such as certain types of capsular or
free exopolysaccharides (CPS, EPS) and teichoic acids
(TA), including lipoteichoic acids (LTA) and wall teichoic
acids (WTA). For example, it is known that there are
differences in the LTA structures of some L. delbrueckii
ssp. lactis strains included in this study (Raisanen et al.
2007). In addition, L. delbrueckii strains, and to a certain
extent, L. helveticus strains, but contrary to L. rhamnosus
strains, have been found to secrete or release LTA-type
structures into MRS broth as free forms (Viitanen et al.,
unpublished results). These results could suggest that
varying copper-bounding capacities by free and
cell-bound LTA of Lactobacillus could explain at least
partially the observed differences in copper resistance
both between strains inside the same species and between
the species of LAB. Interestingly, S. thermophilus, which
9
10(a) (b) (c)
(d) (e) (f)
8
7
6
5
43
2
1
00 24 48 72
Time (h)0
0
0
OD
(600nm)
OD
(600nm)
04
04
08
08
1
06
02
12
16
2
24 48 72Time (h)
04
45
5
55
6pH
65
7
8
75
4
45
555
6pH
65
7
8
75
24 48 72Time (h)
0 24 48 72
Time (h)
0 24 48 72
Time (h)
0 24 48 72
Time (h)
Log10C
FU
ml1
9
10
8
7
6
5
4
3
2
1
0
Log10CFU
ml1
Figure 2 Effects of various copper concentrations in M17L broth on growth and viability of Streptococcus thermophilus strain T101 when
incubated at 37C in (a)c) anaerobic atmosphere or in (d)f) aerobic atmosphere, and measured at indicated points of incubation periods by the
following parameters: (a and d) log10 CFU ml)1; (b and e) OD 600; and (c and f) pH of the culture. (r) M17L broth without copper supplement;
( ) with 25 ppm of copper; ( ) with 5 ppm of copper; (h) with 75 ppm of copper; (d) with 10 ppm of copper; or (e) with 15 ppm copper
supplement. Each value plotted is a mean of duplicate.
Effects of copper on starters for Emmental cheese L. Mato Rodrguez and T. Alatossava
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in this study turned out to be the most sensitive to cop-
per under anaerobic atmosphere, lacks the gene cluster
involved in TA biosynthesis (Hols et al. 2005), and
accordingly, is lacking TA among its cell-wall compo-
nents. Maurer et al. (1975) have also reported varying tol-
erance for copper among different groups of bacteria, and
S. thermophilus strains turned out to be the most sensitiveones which agreed with our results. Kiermeier et al.
(1961) also found that 5 ppm Cu in milk has an inhibi-
tory effect in S. thermophilus and propionic acid bacteria,
but Lactobacillus fermenti still was able to grow and pro-
duce acid in the presence of 20 ppm Cu in milk.
The toxicity of copper to bacteria can be influenced by
many environmental factors, such as pH, redox potential,
moisture, temperature, copper binding to environmental
constituents and interactions with other ions (Gadd and
Griffiths 1978; Babich and Stotzky 1980). In addition, toxic
effects of copper can vary depending on the ways copper is
able to bind different cellular components and on the
mechanisms that affect the essential physiological functions
in various bacterial cells. The toxic effect of copper is gener-
ally attributed to Cu2+ (Summers and Silver 1978), but the
conversion of Cu2+ to Cu1+ under anaerobic conditions
can be responsible for the decreased survival of bacterial
species (Beswick et al. 1976). Redox recycling between
Cu2+ and Cu1+ can catalyse the production of highly toxic
hydroxyl radicals, with subsequent damage to lipids,
proteins, DNA and other biomolecules (Harrison et al.
2000). Our results were in agreement with this view, as
25 ppm copper supplemented in M17L broth produced
stronger growth inhibition and cell viability reducing
effects on the S. thermophilus T101 strain under anaerobicatmosphere (Fig. 2a,d). During cheese manufacture, after
the moulding and pressing steps, the presence of oxygen is
much more limited inside the cheese mass, and conse-
quently, anaerobic environment begins to dominate and
influence the copper ions inside the cheese.
Our study has shown that the presence of supple-
mented copper, at concentrations that starters can face in
the Emmental cheese manufacture, can influence growth,
acid production and cell viability of starter and adjunct
culture strains in species- and strain-dependent manner.
Further studies are required by using cheese milk and
Emmental cheese with and without supplemented copper
in order to reveal both microbiological and biochemical
effects of supplemented copper in Emmental cheese itself.
However, in practice, experiments like those included in
this study would not be possible to perform in milk with
all 13 strains considered, as some of these strains are not
able to grow alone in milk and some strains have a poor
growth. Accordingly, the experiments performed in this
study were more or less an obligatory phase prior to the
experiments with milk and cheese systems.
Acknowledgements
This study was supported by the research grant from the
Ministry of Agriculture and Forestry in Finland (project
310075) and by the personal grant from the Finnish Cul-
tural Foundation to L.M.R.
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