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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


2/9

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


3/9

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



4/9

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


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.


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


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


*Significant at P 005.


2008 The Authors



5/9

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).


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


Strain T(h)

log10 CFU ml)1 SD


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





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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.


2008 The Authors



7/9

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

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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.




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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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