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Influence of pH, salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria
ELSEVIER
InternationalJournal of Food Microbiology24 (1994) 191-198
International Journal of FoodMicrobiology
Influence of pH, salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria A. Mares, K. Neyts, J. Debevere * Department of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, University of Ghent, Coupure Links 653, 9000 Gent, Belgium
Abstract
A screening of commercial starter cultures used for the production of dry sausage showed a maximum heme-dependent catalase activity in the range of 60 /,mol/1 hematin for Lactobacillus sake, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus acidilactici. Pseudocatalase activity was not detected. In standard dry sausage production, 2-3% (w/w) nitrite salt (0.6% sodium nitrite per 100 g NaC1) is normally added, which corresponds to 4-6% salt in the water phase. In vitro experiments with L. sake and L. plantarum have shown that such a high concentration of salt caused a significant reduction of catalase activity and bacterial growth. In the case of P. acidilactici, the catalase activity remained constant at a salt concentration up to 6% (w/w); at 7% (w/w) the activity decreased sharply. The pH also affected the catalase activity, which remained constant up to pH 5.1 and decreased dramatically at lower values. The effect of nitrite has also been investigated. L. pentosus and P. acidilactici were not affected by the addition of 160 ppm nitrite (NO2); L. plantarum, on the other hand, showed a significantly reduced catalase activity. In practice, optimum fermentation characteristics combined with an optimum catalase activity which are not inhibited by salt concentrations higher than 6% (w/v) and a residual nitrite content of about 160 ppm (w/v), are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures.
Keywords: pH; Salt; Nitrite; Heme-dependent catalase activity; Lactic acid bacteria; Dry sausage production
* Corresponding author. Tel. 32-9-264 6l 64. Fax 32-9-225 55 10. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00112-X
192
A. Mares et al. / International Journal of Food Microbiology 24 (1994) 191-198
1. Introduction
Starter cultures used for the fermentation of dry sausage consist mostly of lactic acid bacteria and micrococci. Dry sausage production is a method of conservation based on a combination of acidification by starter cultures, salting and drying, to obtain a stable product. Lactic acid bacteria are facultatively anaerobic (micro-aerophilic) microorganisms. Under anaerobic conditions, the pyruvate formed acts as a hydrogen acceptor in the fermentation of glucose to lactic acid; in aerobiosis, electrons are transported via flavoprotein to oxygen as final electron acceptor, producing H 2 0 or H202, depending on the species (Collins and Koichiro, 1980; Whittenbury, 1964). The accumulation of H 2 0 2 in meat products such as fermented sausage can have serious consequences for the sensorial quality: hydrogen peroxide formation can lead to fat rancidity and discolourations of the meat product by attacking heine pigments (Raccach et al., 1978; Wolf et al., 1991). This accumulation of H20 2 during fermentation is normally inhibited by adding Micrococcaceae because of their catalase activity. Lactic acid bacteria are generally considered to be devoid of catalase activity, since they are not able to synthesize heme compounds (Kandler and Weiss, 1986). Nevertheless, certain species of lactic acid bacteria exhibit catalase activity. Two types of catalase are described (Delwiche, 1961; Whittenbury, 1964; Wolf and Hammes, 1988): (1) a heme-dependent catalase, induced by the addition of heme compounds and produced by some LactobaciUus, Pediococcus, Enterococcus and Leuconostoc strains; (2) a heme-independent catalase or pseudocatalase, produced by some Leuconostoc, Pediococcus, Enterococcus and Lactobacillus plantarum strains. The characterization of catalase activity and the influence of different factors, such as pH, salt and nitrite on the catalase activity are of major interest for the screening and selection of appropriate starter organisms used for the production of dry sausage.
2. Materials and methods
Microorganisms. The microorganisms used in this study were all isolated from commercially available starter cultures: Lactobacillus sake, isolated from Lyofore 2M (LactoLabo, Groupe Rh6ne-Poulenc, Dang6, Saint-Romain, France); Lactobacillus plantarum (strain a), isolated from Duploferment H80 (Rudolf Mtiller & Co., Giessen, Germany); Lactobacillus plantarum L32 (strain b) Deutsche Sammlung von Mikroorganismen (DSM) 1966; Lactobacillus plantarum L74 (strain c) Deutsche Sammlung von Mikroorganismen (DSM) 1954; Lactobacillus pentosus O3a Deutsche Sammlung von Mikroorganismen (DSM) 3402; Pediococcus acidilactici (strain a), isolated from Schneiderferment (Switzerland) and Pediococcus acidilactici (strain b) Deutsche Sammlung von Mikroorganismen (DSM) 2536. All strains were catalase-positive in the presence of 30/xmol/l hematin, the optimum
193
A. Mares et aL /International Journal of Food Microbiology 24 (1994) 191-198
ppm 02/30s 200 !
+ L. p e n t o s u s
- - L. s a k e
I
R a c i d i l a c t i c i (strain b) ÷ L. p l a n t a r u m (strain
150
~
100
j~
/
I "~
o.i 0
/
- --
II
P It
/
! t/
10
20
30
40 50 60 70 80 90 ppm hematin Fig. 1. Catalase activity (expressed as the oxygen produced after 30 s from added H202) after 1 day incubation at 30°C in supplemented MRS Broth as a function of hematin concentration. concentration for Lactobacillus pentosus according to Wolf et al. (1991). Pseudocatalase activity was not detected (unpublished results). The strains were isolated on MRS agar plates (Lab m, Lab 93, International Medical, Amersham, Bury, England) and maintained in MRS stab cultures at 6°C. Media and growth conditions. For study of the catalase activity, cells were grown
aerobically without shaking at 30°C in 250 ml Schott flasks containing 50 ml MRS Broth (Lab m, Lab 94, International Medical, Amersham, Bury, England) or Tryptone Soy Broth (Lab m, Lab 4, International Medical, Amersham, Bury, England). In order to have conditions comparable to those in dry sausage, the media were supplemented with glucose (Sigma, G-8270, St. Louis, MO), salt (UCB, 8605, VEL, Leuven, Belgium) and sodium ascorbate (UCB, 9246, VEL, Leuven, Belgium) to obtain a final concentration (w/v) of 2% glucose, 5% NaC1 and 0.2% sodium ascorbate. After the determination of the optimum hematin concentration for catalase activity (see Fig. 1), the culture media for the further tests were supplemented with 60/zmol hematin (hematin from bovine blood, Sigma, H-3505, St. Louis, MO) per litre. The p H of the media was adjusted with acetic acid (UCB, 1005, VEL, Leuven, Belgium) to 5.8, the initial pH of a dry sausage. To determine the optimum hematin concentration for catalase production, the microorganisms were grown in supplemented MRS Broth with a hematin content
194
A. Mares et al. /International Journal o f Food Microbiology 24 (1994) 191-198
varying from 3 to 95 ppm. The catalase activity was measured after 1 day incubation at 30°C. To study the influence of salt, the organisms were grown in the supplemented MRS Broth with adjusted salt content (0%, 2.5%, 4%, 5%, 6% and 7% (w/v)). The catalase activity and the growth were examined after an incubation of 48 h at 30°C. To study the influence of pH, the organisms were grown in the supplemented MRS Broth at different pH values (4.8, 5.1, 5.4, 5.7, 6.0). The pH was adjusted with lactic acid (CCA Biochem, Gorinchem, The Netherlands). The catalase activity and the final pH of the broth were examined after an incubation of 2 days at 30°C. To study the influence of nitrite, the organisms were inoculated in the supplemented Trypton Soy Broth completed with a filter-sterilised nitrite (UCB, 1759, VEL, Leuven, Belgium) solution to obtain a final concentration of 160 ppm nitrite. The catalase activity and the growth were measured during an incubation period of 14 days at 30°C, in the presence and absence of nitrite.
Determination of growth. The number of cells present in one ml culture medium after the incubation period, was determined by counting colonies in MRS agar after an incubation of 2-3 days at 30°C, applying the pour-plate technique. A top agar layer was added to create micro-aerophilic growth conditions. Measurement ofpH. The pH was measured with a microprocessor-controlled pH meter, (Knick, type 763, Berlin, Germany). Detection of catalase activity. Catalase activity was determined by adding 0.5 ml volume of 30% H 2 0 2 (Belgolabo, 8597, Merck, Darmstadt, Germany) to 10 ml of the culture grown aerobically in the supplemented MRS or TSB medium. After 30 s, the oxygen produced was measured with a membrane-covered, amperometric oxygen electrode (Type S-OM8, Endress and Hauser S.A., Brussels, Belgium). 3. Results and discussion
3.1. Optimum hematin concentration The catalase activity of aerobically grown L. pentosus, P. acidilactici (strain b), L. sake and L. plantarum (strain c) cells was measured as a function of hematin concentration. For L. plantarum (strain c), L. sake, P. acidilactici (strain b) and L. pentosus, Fig. 1 shows the catalyse activity in the presence of up to 90 ppm hematin. Maxima are indicated but further experiments and statistical analyses are required to make a clear distinction between the effect of 20-90 ppm hematin. According to Fig. 1, the addition of 40 ppm (60 ~zmol) hematin per litre of growth medium was chosen as an average value to obtain a high to optimum catalase activity of the organisms tested.
195
A. Mares et al. / International Journal of Food Microbiology 24 (1994) 191-198 3.2. Influence of salt
In standard dry sausage production, 2 - 3 % ( w / w ) nitrite salt (0.6 g sodium nitrite per 100 g salt) is added, which corresponds to a salt content of 4 - 6 % in the water phase. The influence of various salt concentrations on the growth and on the catalase activity of L. sake, L. plantarum (strain a) and P. acidilactici (strain a) was studied. The organisms were inoculated (10 3 cells per ml) in supplemented MRS Broth with increasing salt concentrations. As shown in Fig. 2, the catalase activity of L. sake and L. plantarum (strain a) remained constant up to a salt concentration of 5% (w/w). Higher concentrations caused a significant reduction in catalase activity. However, it was shown that growth also decreased remarkably in the presence of salt at concentrations higher than 5% (data not included). The catalase activity of P. acidilactici (strain a) remained constant at a salt concentration up to 6%; at 7% the activity decreased sharply (Fig. 2). It was shown that a content of 6% salt had no effect on the growth of P. acidilactici (strain a), whereas a content of 7% caused a slight decrease in growth (data not included). It can be concluded that the salt content of dry sausage has an influence on the catalase activity of lactic acid bacteria. A level of 6% salt in the water phase results in a lower catalase activity of the lactic acid bacteria. Also in dry sausage with a
[] L. sake [] L. plantarum(strain a) l [] R acidilactici(straina) ]
ppm 02/30s/Iog(cells/ml)
:
.
30 ~
- - - ~ ~ - 25
t 30
1 25
i
20 20 15 15 10 10 5 0
/ 0
2.5
4
5
6
7
% (W/V) NaCl
Fig. 2. Catalase activity (expressed as the oxygen produced after 30 s from added H202 per log cells per ml) after 2 days incubation at 30°C in supplemented MRS Broth as a function of salt concentration•
196
A. Mares et al. /International Journal of Food Microbiology 24 (1994) 191-198
~ L. sake L. plantarum(strain
ppm 02/30s
a)
200
200
150 150 ~!"i ii
1 O0 1 O0
50 50!
4.8
5.1
5.4
5.7
6
Initial
pH
Fig. 3. Catalase activity (expressed as the oxygen produced after 30 s from added H 2 0 2) after 2 days incubation at 30°C in supplemented MRS Broth as a function of initial pH.
high fat content (corresponding to a proportionally lower water content) the catalase activity can be reduced due to the relatively high salt content in the water phase. However, P. acidilactici (strain a) was able to grow well in the presence of 6% salt, whereas the lactobacilli tested (L. sake and L. plantarum (strain a)) showed a dramatically reduced growth (data not shown).
3.3. Influence of pH The pH of dry sausage decreases as a result of lactic acid fermentation. To study the influence of pH on the catalase activity of lactic acid bacteria during the fermentation proces, L. sake, L. plantarum (strain a) and P. acidilactici (strain a) were inoculated (103 cells per ml) in the supplemented MRS Broth with different pH values. As Fig. 3 shows, the catalase activity seemed not to be influenced by the pH down to 5.1. However, at pH values lower than 5.1, a decrease in activity could be observed, as previously reported by Whittenbury (1960). L. plantarum (strain a) caused a strong pH decrease (results not shown), which explains the low catalase activity of this organism.
A. Mares et aL / International Journal of Food Microbiology 24 (1994) 191-198
ppm 02/30s
log (cells/ml) 10
160[
140 -
~
catalase -m-catalase+ nitrite +growth + growth + nitrite
_
120 100 \
t'
\
//
"~ . . . .
,
40 '// 20
9 -8
80
60
197
41- . . . . .
;7
41,
/
6
/
i
II
0
2
4
6
8
10
12
14 Time (days)
Fig. 4. Catalase activity(expressed as the oxygenproduced after 30 s from added H20 2) and number of L. plantarum (strain b) cells during incubation at 30°C in supplemented TSB in the presence and the absence of 160 ppm nitrite. 3.4. Influence o f nitrite
In standard dry sausage production, 2 - 3 % nitrite salt (containing 0.012-0.018% N a N O 2) is added, which corresponds to 240-360 p p m N a N O 2 or 160-240 p p m N O ~ in the water phase. In previous studies it was observed that in extreme cases a residual content of 80 p p m N O ~ could be detected. In this work the influence of a residual content of 80 p p m nitrite - corresponding to 160 p p m N O y in the water phase - on growth and catalase activity of L. plantarum (strain b), L. pentosus and P. acidilactici (strain b), inoculated ( 1 0 6 / m l ) in the supplemented TSB was studied. Fig. 4 shows a dramatical decrease in the catalase activity of L. plantarum (strain b). However, there was no difference between the growth (lag and log phase) of the organism in the presence or absence of nitrite. In similar trials no significant decrease of growth and catalase activity for L. pentosus and P. acidilactici could be observed; however, the optimum activity was reached later (results not given).
4. Conclusions O p t i m u m fermentation characteristics combined with an optimum catalase activity which is not inhibited by salt concentrations up to 6% and a nitrite content
198
A. Mares et al. / International Journal of Food Microbiology 24 (1994) 191-198
of about 160 ppm are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures. From the screened lactic acid bacteria, P. acidilactici seemed to be the most suitable organism: (a) a catalase activity of 100-120 ppm 0 2 / 3 0 s in the presence of 20-90 ppm hematin and after an incubation of 1 day in supplemented MRS Broth at 30°C; (b) no inhibition of the catalase activity by about 160 ppm nitrite; (c) no decrease of the catalase activity by salt concentrations up to 6% (w/v). However, the fact that P. acidilactici acidifies the medium more slowly than the lactobacilli is a significant disadvantage.
References Collins, E.B. and Kiochiro, A. (1980). Production of hydrogen peroxide by Lactobacillus acidophilus. J. Dairy Sci. 63, 353-357. Delwiche, E.A. (1961) Catalase of Pediococcus cerecisiae. J. Bacteriol. 8l, 416-418. Kandler, O. and Weiss, N. (1986) Regular, nonsporing gram-positive rods. In: P.H.A. Sneath, N.S. Mair, M,E. Sharpe, J.G. Holt (Editors) Bergey's Manual of Systematic Bacteriology, 2. Williams & Wilkins, Baltimore, MD, pp. 1208-1234. Raccach, M. and Baker, R.C. (1978) Formation of hydrogen peroxide by meat starter cultures. J. Food Prot. 41, 798-799. Whittenbury, R. (1960) Two types of catalase-like activity in lactic acid bacteria. Nature 167, 433-434. Whittenbury, R. (1964) Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. J. Gen. Microbiol. 35, 13-26. Wolf, G. and Hammes, W.P. (1988) Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Arch. Microbiol. 149, 220-224. Wolf, G., Strahl, A., Meisel, J. and Hammes, W.P. (1991) Heme-dependent catalase activity of lactobacilli. Int. J. Food Microbiol. 12, 133-140.
InternationalJournal of Food Microbiology24 (1994) 191-198
International Journal of FoodMicrobiology
Influence of pH, salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria A. Mares, K. Neyts, J. Debevere * Department of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, University of Ghent, Coupure Links 653, 9000 Gent, Belgium
Abstract
A screening of commercial starter cultures used for the production of dry sausage showed a maximum heme-dependent catalase activity in the range of 60 /,mol/1 hematin for Lactobacillus sake, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus acidilactici. Pseudocatalase activity was not detected. In standard dry sausage production, 2-3% (w/w) nitrite salt (0.6% sodium nitrite per 100 g NaC1) is normally added, which corresponds to 4-6% salt in the water phase. In vitro experiments with L. sake and L. plantarum have shown that such a high concentration of salt caused a significant reduction of catalase activity and bacterial growth. In the case of P. acidilactici, the catalase activity remained constant at a salt concentration up to 6% (w/w); at 7% (w/w) the activity decreased sharply. The pH also affected the catalase activity, which remained constant up to pH 5.1 and decreased dramatically at lower values. The effect of nitrite has also been investigated. L. pentosus and P. acidilactici were not affected by the addition of 160 ppm nitrite (NO2); L. plantarum, on the other hand, showed a significantly reduced catalase activity. In practice, optimum fermentation characteristics combined with an optimum catalase activity which are not inhibited by salt concentrations higher than 6% (w/v) and a residual nitrite content of about 160 ppm (w/v), are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures.
Keywords: pH; Salt; Nitrite; Heme-dependent catalase activity; Lactic acid bacteria; Dry sausage production
* Corresponding author. Tel. 32-9-264 6l 64. Fax 32-9-225 55 10. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00112-X
192
A. Mares et al. / International Journal of Food Microbiology 24 (1994) 191-198
1. Introduction
Starter cultures used for the fermentation of dry sausage consist mostly of lactic acid bacteria and micrococci. Dry sausage production is a method of conservation based on a combination of acidification by starter cultures, salting and drying, to obtain a stable product. Lactic acid bacteria are facultatively anaerobic (micro-aerophilic) microorganisms. Under anaerobic conditions, the pyruvate formed acts as a hydrogen acceptor in the fermentation of glucose to lactic acid; in aerobiosis, electrons are transported via flavoprotein to oxygen as final electron acceptor, producing H 2 0 or H202, depending on the species (Collins and Koichiro, 1980; Whittenbury, 1964). The accumulation of H 2 0 2 in meat products such as fermented sausage can have serious consequences for the sensorial quality: hydrogen peroxide formation can lead to fat rancidity and discolourations of the meat product by attacking heine pigments (Raccach et al., 1978; Wolf et al., 1991). This accumulation of H20 2 during fermentation is normally inhibited by adding Micrococcaceae because of their catalase activity. Lactic acid bacteria are generally considered to be devoid of catalase activity, since they are not able to synthesize heme compounds (Kandler and Weiss, 1986). Nevertheless, certain species of lactic acid bacteria exhibit catalase activity. Two types of catalase are described (Delwiche, 1961; Whittenbury, 1964; Wolf and Hammes, 1988): (1) a heme-dependent catalase, induced by the addition of heme compounds and produced by some LactobaciUus, Pediococcus, Enterococcus and Leuconostoc strains; (2) a heme-independent catalase or pseudocatalase, produced by some Leuconostoc, Pediococcus, Enterococcus and Lactobacillus plantarum strains. The characterization of catalase activity and the influence of different factors, such as pH, salt and nitrite on the catalase activity are of major interest for the screening and selection of appropriate starter organisms used for the production of dry sausage.
2. Materials and methods
Microorganisms. The microorganisms used in this study were all isolated from commercially available starter cultures: Lactobacillus sake, isolated from Lyofore 2M (LactoLabo, Groupe Rh6ne-Poulenc, Dang6, Saint-Romain, France); Lactobacillus plantarum (strain a), isolated from Duploferment H80 (Rudolf Mtiller & Co., Giessen, Germany); Lactobacillus plantarum L32 (strain b) Deutsche Sammlung von Mikroorganismen (DSM) 1966; Lactobacillus plantarum L74 (strain c) Deutsche Sammlung von Mikroorganismen (DSM) 1954; Lactobacillus pentosus O3a Deutsche Sammlung von Mikroorganismen (DSM) 3402; Pediococcus acidilactici (strain a), isolated from Schneiderferment (Switzerland) and Pediococcus acidilactici (strain b) Deutsche Sammlung von Mikroorganismen (DSM) 2536. All strains were catalase-positive in the presence of 30/xmol/l hematin, the optimum
193
A. Mares et aL /International Journal of Food Microbiology 24 (1994) 191-198
ppm 02/30s 200 !
+ L. p e n t o s u s
- - L. s a k e
I
R a c i d i l a c t i c i (strain b) ÷ L. p l a n t a r u m (strain
150
~
100
j~
/
I "~
o.i 0
/
- --
II
P It
/
! t/
10
20
30
40 50 60 70 80 90 ppm hematin Fig. 1. Catalase activity (expressed as the oxygen produced after 30 s from added H202) after 1 day incubation at 30°C in supplemented MRS Broth as a function of hematin concentration. concentration for Lactobacillus pentosus according to Wolf et al. (1991). Pseudocatalase activity was not detected (unpublished results). The strains were isolated on MRS agar plates (Lab m, Lab 93, International Medical, Amersham, Bury, England) and maintained in MRS stab cultures at 6°C. Media and growth conditions. For study of the catalase activity, cells were grown
aerobically without shaking at 30°C in 250 ml Schott flasks containing 50 ml MRS Broth (Lab m, Lab 94, International Medical, Amersham, Bury, England) or Tryptone Soy Broth (Lab m, Lab 4, International Medical, Amersham, Bury, England). In order to have conditions comparable to those in dry sausage, the media were supplemented with glucose (Sigma, G-8270, St. Louis, MO), salt (UCB, 8605, VEL, Leuven, Belgium) and sodium ascorbate (UCB, 9246, VEL, Leuven, Belgium) to obtain a final concentration (w/v) of 2% glucose, 5% NaC1 and 0.2% sodium ascorbate. After the determination of the optimum hematin concentration for catalase activity (see Fig. 1), the culture media for the further tests were supplemented with 60/zmol hematin (hematin from bovine blood, Sigma, H-3505, St. Louis, MO) per litre. The p H of the media was adjusted with acetic acid (UCB, 1005, VEL, Leuven, Belgium) to 5.8, the initial pH of a dry sausage. To determine the optimum hematin concentration for catalase production, the microorganisms were grown in supplemented MRS Broth with a hematin content
194
A. Mares et al. /International Journal o f Food Microbiology 24 (1994) 191-198
varying from 3 to 95 ppm. The catalase activity was measured after 1 day incubation at 30°C. To study the influence of salt, the organisms were grown in the supplemented MRS Broth with adjusted salt content (0%, 2.5%, 4%, 5%, 6% and 7% (w/v)). The catalase activity and the growth were examined after an incubation of 48 h at 30°C. To study the influence of pH, the organisms were grown in the supplemented MRS Broth at different pH values (4.8, 5.1, 5.4, 5.7, 6.0). The pH was adjusted with lactic acid (CCA Biochem, Gorinchem, The Netherlands). The catalase activity and the final pH of the broth were examined after an incubation of 2 days at 30°C. To study the influence of nitrite, the organisms were inoculated in the supplemented Trypton Soy Broth completed with a filter-sterilised nitrite (UCB, 1759, VEL, Leuven, Belgium) solution to obtain a final concentration of 160 ppm nitrite. The catalase activity and the growth were measured during an incubation period of 14 days at 30°C, in the presence and absence of nitrite.
Determination of growth. The number of cells present in one ml culture medium after the incubation period, was determined by counting colonies in MRS agar after an incubation of 2-3 days at 30°C, applying the pour-plate technique. A top agar layer was added to create micro-aerophilic growth conditions. Measurement ofpH. The pH was measured with a microprocessor-controlled pH meter, (Knick, type 763, Berlin, Germany). Detection of catalase activity. Catalase activity was determined by adding 0.5 ml volume of 30% H 2 0 2 (Belgolabo, 8597, Merck, Darmstadt, Germany) to 10 ml of the culture grown aerobically in the supplemented MRS or TSB medium. After 30 s, the oxygen produced was measured with a membrane-covered, amperometric oxygen electrode (Type S-OM8, Endress and Hauser S.A., Brussels, Belgium). 3. Results and discussion
3.1. Optimum hematin concentration The catalase activity of aerobically grown L. pentosus, P. acidilactici (strain b), L. sake and L. plantarum (strain c) cells was measured as a function of hematin concentration. For L. plantarum (strain c), L. sake, P. acidilactici (strain b) and L. pentosus, Fig. 1 shows the catalyse activity in the presence of up to 90 ppm hematin. Maxima are indicated but further experiments and statistical analyses are required to make a clear distinction between the effect of 20-90 ppm hematin. According to Fig. 1, the addition of 40 ppm (60 ~zmol) hematin per litre of growth medium was chosen as an average value to obtain a high to optimum catalase activity of the organisms tested.
195
A. Mares et al. / International Journal of Food Microbiology 24 (1994) 191-198 3.2. Influence of salt
In standard dry sausage production, 2 - 3 % ( w / w ) nitrite salt (0.6 g sodium nitrite per 100 g salt) is added, which corresponds to a salt content of 4 - 6 % in the water phase. The influence of various salt concentrations on the growth and on the catalase activity of L. sake, L. plantarum (strain a) and P. acidilactici (strain a) was studied. The organisms were inoculated (10 3 cells per ml) in supplemented MRS Broth with increasing salt concentrations. As shown in Fig. 2, the catalase activity of L. sake and L. plantarum (strain a) remained constant up to a salt concentration of 5% (w/w). Higher concentrations caused a significant reduction in catalase activity. However, it was shown that growth also decreased remarkably in the presence of salt at concentrations higher than 5% (data not included). The catalase activity of P. acidilactici (strain a) remained constant at a salt concentration up to 6%; at 7% the activity decreased sharply (Fig. 2). It was shown that a content of 6% salt had no effect on the growth of P. acidilactici (strain a), whereas a content of 7% caused a slight decrease in growth (data not included). It can be concluded that the salt content of dry sausage has an influence on the catalase activity of lactic acid bacteria. A level of 6% salt in the water phase results in a lower catalase activity of the lactic acid bacteria. Also in dry sausage with a
[] L. sake [] L. plantarum(strain a) l [] R acidilactici(straina) ]
ppm 02/30s/Iog(cells/ml)
:
.
30 ~
- - - ~ ~ - 25
t 30
1 25
i
20 20 15 15 10 10 5 0
/ 0
2.5
4
5
6
7
% (W/V) NaCl
Fig. 2. Catalase activity (expressed as the oxygen produced after 30 s from added H202 per log cells per ml) after 2 days incubation at 30°C in supplemented MRS Broth as a function of salt concentration•
196
A. Mares et al. /International Journal of Food Microbiology 24 (1994) 191-198
~ L. sake L. plantarum(strain
ppm 02/30s
a)
200
200
150 150 ~!"i ii
1 O0 1 O0
50 50!
4.8
5.1
5.4
5.7
6
Initial
pH
Fig. 3. Catalase activity (expressed as the oxygen produced after 30 s from added H 2 0 2) after 2 days incubation at 30°C in supplemented MRS Broth as a function of initial pH.
high fat content (corresponding to a proportionally lower water content) the catalase activity can be reduced due to the relatively high salt content in the water phase. However, P. acidilactici (strain a) was able to grow well in the presence of 6% salt, whereas the lactobacilli tested (L. sake and L. plantarum (strain a)) showed a dramatically reduced growth (data not shown).
3.3. Influence of pH The pH of dry sausage decreases as a result of lactic acid fermentation. To study the influence of pH on the catalase activity of lactic acid bacteria during the fermentation proces, L. sake, L. plantarum (strain a) and P. acidilactici (strain a) were inoculated (103 cells per ml) in the supplemented MRS Broth with different pH values. As Fig. 3 shows, the catalase activity seemed not to be influenced by the pH down to 5.1. However, at pH values lower than 5.1, a decrease in activity could be observed, as previously reported by Whittenbury (1960). L. plantarum (strain a) caused a strong pH decrease (results not shown), which explains the low catalase activity of this organism.
A. Mares et aL / International Journal of Food Microbiology 24 (1994) 191-198
ppm 02/30s
log (cells/ml) 10
160[
140 -
~
catalase -m-catalase+ nitrite +growth + growth + nitrite
_
120 100 \
t'
\
//
"~ . . . .
,
40 '// 20
9 -8
80
60
197
41- . . . . .
;7
41,
/
6
/
i
II
0
2
4
6
8
10
12
14 Time (days)
Fig. 4. Catalase activity(expressed as the oxygenproduced after 30 s from added H20 2) and number of L. plantarum (strain b) cells during incubation at 30°C in supplemented TSB in the presence and the absence of 160 ppm nitrite. 3.4. Influence o f nitrite
In standard dry sausage production, 2 - 3 % nitrite salt (containing 0.012-0.018% N a N O 2) is added, which corresponds to 240-360 p p m N a N O 2 or 160-240 p p m N O ~ in the water phase. In previous studies it was observed that in extreme cases a residual content of 80 p p m N O ~ could be detected. In this work the influence of a residual content of 80 p p m nitrite - corresponding to 160 p p m N O y in the water phase - on growth and catalase activity of L. plantarum (strain b), L. pentosus and P. acidilactici (strain b), inoculated ( 1 0 6 / m l ) in the supplemented TSB was studied. Fig. 4 shows a dramatical decrease in the catalase activity of L. plantarum (strain b). However, there was no difference between the growth (lag and log phase) of the organism in the presence or absence of nitrite. In similar trials no significant decrease of growth and catalase activity for L. pentosus and P. acidilactici could be observed; however, the optimum activity was reached later (results not given).
4. Conclusions O p t i m u m fermentation characteristics combined with an optimum catalase activity which is not inhibited by salt concentrations up to 6% and a nitrite content
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of about 160 ppm are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures. From the screened lactic acid bacteria, P. acidilactici seemed to be the most suitable organism: (a) a catalase activity of 100-120 ppm 0 2 / 3 0 s in the presence of 20-90 ppm hematin and after an incubation of 1 day in supplemented MRS Broth at 30°C; (b) no inhibition of the catalase activity by about 160 ppm nitrite; (c) no decrease of the catalase activity by salt concentrations up to 6% (w/v). However, the fact that P. acidilactici acidifies the medium more slowly than the lactobacilli is a significant disadvantage.
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