Internauonal Journal of Food Microbiology, 12 (1991) 133-140 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
133
FOOD 00350
Heine-dependent catalase activity of lactobacilli Gudrun
W o l f , A n g e l a S t r a h l , J u t t a M e i s e l a n d W a l t e r P. H a m m e s Umversitiit Hohenheim, Inxtitut fl~r Lebensrrutteltechnologie, Stuttgart, F.R.G. (Received 13 September 1989; accepted 31 July 1990)
The berne-dependent catalase in Lactobacdlus pentosus, I.. sake, L. delbruecku and Enterococcusfaecahs was studied. The catalase was formed by cells grown aerobically in the presence of hematm or for lactobacilli when grown without added hematin, after incubation of buffered cells in the presence of hematin. The kinetics of the production of catalas¢ revealed maximum activity for L. pentosus and E. faecalis at late stationary and late logarithmic growth phase, respectively. The physiological role of catalase was studied with L sake. The presence of hematin allows higher growth yields, since it protects the cells against hydrogen peroxide formed endogenously up to concentrations of 4.6 mmol/l. Key words: Lactic acid bacteria; Hematin; Catalase formation, kinetics
Introduction Hydrogen peroxide is a c o m m o n metabolite of lactic acid bacteria and can be accumulated during fermentation processes. If this accumulation takes place in foods, their sensory quality may be strongly affected (Niven and Evans, 1957). Therefore the presence of catalase activity is a desirable property for starter cultures used in food fermentations (Liicke, 1985). For example, in meat products the accumulation of hydrogen peroxide can lead to fat rancidity and to discolourations of meat products by attacking heme pigments. Therefore Micrococcaceae are used for the fermentation of raw sausages, mainly because of their catalase activity. On the other hand, lactic acid bacteria are generally considered as being devoid of catalase activity, since these organisms axe not able to synthesize heme c o m p o u n d s (Kandler and Weiss, 1986). Nevertheless, there are reports showing that certain species of lactic acid bacteria exhibit catalase activity e.g. Whittenbury (1964) and Wolf and H a m m e s (1988) demonstrated, that the addition of hematin induced the production of catalase in strains of Lactobaciilus plantarum, L. brevis, L. acidilactici, L. viridescens, L. casei, L. delbrueckii, L. pentosus, L sake, L. bavaricus, Leuconostoc mesenteroides and Enterococcus faecalis. In addition, a heme independent catalase or pseudocatalase was described for strains of Pediococcus, Lactobacil-
Correspondence address: Dr. G. Wolf. Universifiit Hohenheim, Inst. f. Lebensmitteitechnologie,Oarbenstrasse 25, D-7000 Stuttgart 70, F.R.G.
134 lus. Leuconostoc and Emerococcus (Delwiche. 1961). This enzyme is insensitive to azide and cyanide and contains manganese (Kono and Fridovich. 1983). The various substrates for food fermentation may contain hematin and therefore. for the selection of the appropriate starter organism, the characterization of catalase activity and their possible induction is of major interest. The purpose of this paper was to extend our knowledge with regard to these properties of the heme-dependent catalases.
Materials and Methods
Mtcroorgamsms and growth conditions The following organisms were used in this study: L. pentosus Deutsche Sammlung von Mikroorganismen (DSM) 20314, L. sake 450 (Kagermeier, 1981), L. delbrueckii ssp. lactis National Collection of Dairy Organisms (NCDO) 280, Enterococcus faecalis National Collection of Industrial Bacteria (NCIB) 2131. The organisms were maintained and grown as described by Wolf and Hammes (1988). The strains were maintained in MRS stab cultures (de Man et al.. 1960) at 4 ° C and transferred monthly. For study of catalase, cells were grown aerobically in 250 ml Erlenmeyer flasks containing 50 ml medium at 30 °C on an oscillatory shaker. The medium was modified MRS, containing the following compounds per litre: 20 g glucose, 2 g KNO 3, 10 g Lab Lemco Powder. 5 g yeast extract, 10 g tryptone, 1 ml Tween 80, 0.05 g MnSO 4 • H20, 24.2 mg Na2MoO 4, 5 g sodium acetate. 3 H20, 0.2 g MgSO 4 • 7 H20, 5.4 mg FeC13, 2 g K2HPO ,. Where indicated 30 /tmol hematin were added (5 mg/ml solution in 0.2 m ol / l KOH, sterilized by filtration). The cells were harvested by centrifugation (Beckman J2-21, 25 000 × g, 10 min) and washed with phosphate buffer (0.05 mol/l, pH 7.0). Chemicals The following chemicals were obtained from Oxoid (Wesel, F.R.G.): Tryptone, Lab Lemco Powder, yeast extract and purified agar; from Sigma (Miinchen, F.R.G.): hematin, hemoglobin, chlorophyllin, protoporphyrin IX and catalase (bovine liver); from Boehringer (Mannheim, F.R.G.): ABTS (2,2'-azino-di-(3-ethylbenzthiazolin-6-sulfonate) and horseradish peroxidase; and from Merck (Darmstadt, F.R.G.) all other chemicals. Detection of catalase activity Catalase activity was detected with intact bacteria by the method of Rorth and Jensen (1967) using a WTW oxygraph (Oxi 530) equipped with a Clark electrode (Trioxmatic ® EO 200), both obtained from Wissenschaftlich-technische Werkstiitten, Weilheim, F.R.G. The oxygen electrode was calibrated with the Oxical ® calibration vessel (PE/OXI from WTW, Wissenschaftlich-technische Werkst~itten, Weilheim, F.R.G.). The assays were performed at 30°C in a stirred 30-ml flask with two side arms containing 25 ml H 2 0 : b u f f e r (0.01 m ol/ l H202 in 0.05 m ol / l phosphate
135 buffer, p H 7.0). For removing 02, the buffer was sparged with N 2 until it did not contain more than 0.1 mg O2/1. For controls the O2-production from H 2 0 : b u f f e r was observed with and without hematin. The reaction was started by the addition of whole cells or enzyme.
Induction of catalase activity in bacteria after their harvest by addition of hematm Growth was monitored by measurement of OD578 with a Beckman spectrophotometer or by counting cell numbers by the spread-plate technique (Koch, 1981). Cultures were harvested at the times indicated, washed with phosphate buffer (0.05 m o l / 1 , p H 7.0), resuspended in the same buffer and incubated with hematin, 30 ~tmol/1 at 30°C.
Determinanon of hydrogen peroxide Hydrogen peroxide was determined according to the method of Carter (1980). Aliquots (50 #1) of the culture broth were added to 2.5 ml of ABTS-peroxidase reagent (250 ml phosphate buffer, 0.1 m o l / l p H 7.0, containing 2.5 mg horseradish peroxidase and 250 mg ABTS). The mixture was incubated for 5 min at 25°C and the extinction was measured at 420 nm. All experiments were repeated at least three times. It was observed that, depending on the preparation, the cells exhibited varying amounts of activity between 0.2 and 0.8 mmol O2/1 × rain × 109 cfu for lactobacilli and up to 5 mmol O2/1 × rain x 109 cfu for E. faecalis.
Results and Discussion The conditions for production of catalase activity by L. pentosus were investigated. Addition of a n u m b e r of compounds was studied. As shown in Table I, hematin and hemoglobin were the only effective compounds. The dependence of catalase activity on the concentration of hematin was determined. As shown in Fig. 1, the catalase activity of aerobically grown cells showed a m a x i m u m in the range of 30-50 /~mol of hematin per litre of growth medium. Cultures grown in the presence of 30 /~mol/1 of hematin showed their highest
TABLE I Activity of catalase of L. pentosus, grown with various addtuons Compound None Hematin Hemoglobin Chlorophylhn Protoporphyrin IX protoporphyrin IX + FeCI3
Concentrauon (/xmol/l) 0 30 30 30 30 30
Enzyme activity (~) 0 100 100 0 0 0
An activity of 100% corresponds to the production of 189.14 ~tmol 02 1-1 n~n-i 10-9 cfu.
136 200 u
m
~.~_ .>- E 100
0
0.01
0.1
I
I0
I(30
1000
[ H e m a t m ] (,umol/I) Fig. 1. Catalas¢ activity of L penwsus grown aerobically in the presence of vanous concentratmns of hematin.
activity per cell unit at late stationary growth phase as shown in Fig. 2. The omission of hematin resulted in lower growth yield. Cells from this culture apparently produced catalase apoprotein, as catalase activity could be induced after addition of hematin to cells washed and suspended in phosphate buffer. This catalase activity also had a maximum at the late stationary phase and it was dependent on the length of incubation with hematin as depicted in Fig. 3. The activity was highest after incubation for 1-2 h at 30°C. As shown in Fig. 4, the catalase activity of cells of L. pemosus is as independent of the temperature within the range investigated as that of bovine liver catalase. The catalase activity was studied for different species of lactobacilli grown to late logarithmic growth phase with and without added hematin. As shown in Fig. 5, L. delbrueckii and L sake resembled L pentosus, i.¢. catalase activity was observed
2.0 0 l0
=I' ~
-=
05
c.,
)5
3~ 01
2
4
6
8
10
12
T,me (h) Fig. 2. The influence of catalase activity on growth of L pentosus. (Opucal densRy and catalase activity of cultures grown aerobically with (o, II) and without hematm ( o , E]).
137 100 ~ ~
w
~ - 50 0
r,..}~ 0--
I
I
0
I
2
Time of pre,ncubot~on (h)
Fig. 3. Catalas¢ activityfor cells of L pentosus harvested after 5 h of aerobic growth at 30°C, washed and suspendedin phosphatebufferwith added 30/~mol/1hcmatinfor the lengthof timesindicated. and it was virtually independent of whether hematin was added to the growth medium or to washed cells suspended in buffer. However, with E. faecalis a poor catalase activity was observed when hematin was added to cell suspensions grown in the absence of hematin. The kinetics of growth and acquisition of catalase activity revealed that this organism differed from /- pentosus in that the maximum activity is obtained cartier and further that cells of cultures grown in the absence of hematin not only exhibit higher activity but lose this activity dramatically during extended incubation (Fig. 6). The observed increase in growth yield for L pentosus when hematin was added, was also found for I_ sake. To study whether the increase was dependent on an active catalase, cultures were grown with and without hematin and in the presence of added catalase. As shown in Fig. 7, hydrogen peroxide accumulated up to concentrations surpassing 4.6 m m o l / l in cultures without hematin or added catalase. No accumulation was detected when hematin or catalase was added. In the
i
i
i
,
'
2'0
'
~'0
120
~=
so
0
0
Temperature ('C) Fig. 4. Influence of the temperature on activity of catalasc from bovine fiver (o) (7 units) and of cells of L pentosu~
(o) (8.0.l0 s cfu).
138
i L perWosus 20
I00
L SQ;(e
I delDruecku
E foecohs
314
~
~
,
!
.~
l
I :b U
o
50-
0
=
0 0
0 .
.
.
.
.
.
.
m,mm~
Fig. 5. Comparison of catalase acuvaty for various lactic acid bacteria grown aerobically to stauonary phase. (ra growth with 30 ~ m o l / l hematin, • growth without hematin, with subsequent incubation of washed cells in phosphate buffer at 30"C for 90 rain wtth 30 ~mol/I hematin). 100% catalase activity corresponds to the activity when hematin is added to the growth medium.
culture with accumulated hydrogen peroxide a lower growth yield was observed. Thus, the presence of an active catalase protects the organism and allows higher growth yields. The physiological activity of heine-dependent catalase of lactic acid bacteria exhibits properties similar to those described for true catalases (Aebi, 1983). For
i
1
,
,
2u %
30
60
~" E
iO 1-o ~n
c3 o
°~ ~ E
E 05
"6 20
o
o (..)
01~
'
A
2
z.
,
,
6 T,me
8
, 10
0
(h)
Fig. 6. Catalase activity during growth of E. faecahs. (Optical denstty and catalase activity of cell cultures grown aerobically with hematin (o, B) and without hematm (o, ra).
139
A
a
//A
108
= o
fi
=
e
E (-}
I 10 7
"~ 2 0
i 10l 0
I
*
2
~
~,J 0
"
6
; '[,me
"
~
"
[
"
~
"
8
(h)
Fig. 7. The effect of hematin and catalasc on growth (A) and H~O 2 accumulation (B) by /.. sake. The cells were grown at 30°C in MRS broth with 1.25 g/I glucose and the following additions: I , 5 mg/I hematin and 5mg/l bovine hver catalase; e, 5 mg/l hematm: v, 5 mg/l bovine hver catalase; A, no addition.
example, it was observed that the Q10 value was as low as 1.05-1.12, and the activity could be inhibited by 50% at 35 m m o l / l cyanide and 1 m m o l / l azide (unpublished results). The present results provide for a strategy of composing starter cultures with lactobacilli exhibiting catalase activity to minimize deleterious effects of hydrogen peroxide in fermenting food.
Aknowledgements We thank Heidi Griinenwald and Elisabeth Lindemann for expert technical assistance. This study was supported by Deutsche Forschungsgemeinschaft.
References Aebl, H.E. (1983) Catalase. in: H.U. Bergmeyer (Ed.). Methods of Enzymatic Analysis, Vol Ill., Verlag Chemie, Wemheim. Carter, R.S. 0980) The Deactivation Behavior of immobihzed Glucose Oxldase/Catalase on Hydrogen Peroxide Decomposing Supports, Thesis, ETH, Zimch. Delwiche, E.A. (1961) Catalase of PedJococcus cerev~sme. J. Bacteriol. 81,416-418. De Man. J.C., Rogosa, M.. Sharpe, M.E. (1960) A medmm for the cultivation of lactobacilh. J. Appl. Bactenol. 23, 130-135. Kagermeler, A. (1981) Taxonomie und Vorkommen yon Milchs~urebakterien in Fleischprodukten, Thesis, Universit~it Miinchen.
140 Kandler, O. and Weiss, N. (1986) Regular, nonsporing gram-posiuve rods. In: P.H.A. Sneath. N.S. Mawr, M.E. Sharpe. J.G. Holt (Eds.). Bergey's Manual of Systematic Bacteriology. Vol 2., W d h a m s & Wilkms. Balumore. MD. Koch, A.L. (1981) Growth measurements. In. P. Gerhardt, R.G.E. Murray. R.N. Costdow. E.W. Nester, W.A. Wood, N.R. Krieg and G.B. Phdhps (Eds.), Manual of Methods for General Bactenology pp. 179-207. American Society for Microbiology. Wasbangton, DC. Kono, Y and Fndov~ch, I. (1983) Isolauon and charactenzauon of the pseudocatalase of Lactobacdlus plantarum. J. Biol. Chem. 258, 6015-6019. Liacke. F.-K. (1985) Fermented sausages. In: B.J.B. Wood (Ed.), Microbiology of Fermented Foods. Vol. 2. pp. 41-83. Elsevier, London, New York. Niven, C.F. and Evans, J.B. (1957) Lactobacdlus vtr~descens nov. spec. A heterofermentatlve species that produces a green d~scolouratlon of cured meat p~gments. J. Bactenol. 73. 758-759. Rorth, M. and Jensen, P.K. (1967) Determination of eatalase activity by means of the Clark oxygen electrode. B~ochim. Biophys. Acta 139, 171-173. Whlttenbury, R. (1964) Hydrogen peroxade formation and catalase activity m the lacuc acid bacteria. J Gen. Microbiol. 35. 13-26. Wolf, G. and Hammes, W.P. (1988) Effect of h e m a t m on the a c u w u e s of m m t e reductase and catalase m lactobacilh. Arch. Microbiol. 149, 220-224.
133
FOOD 00350
Heine-dependent catalase activity of lactobacilli Gudrun
W o l f , A n g e l a S t r a h l , J u t t a M e i s e l a n d W a l t e r P. H a m m e s Umversitiit Hohenheim, Inxtitut fl~r Lebensrrutteltechnologie, Stuttgart, F.R.G. (Received 13 September 1989; accepted 31 July 1990)
The berne-dependent catalase in Lactobacdlus pentosus, I.. sake, L. delbruecku and Enterococcusfaecahs was studied. The catalase was formed by cells grown aerobically in the presence of hematm or for lactobacilli when grown without added hematin, after incubation of buffered cells in the presence of hematin. The kinetics of the production of catalas¢ revealed maximum activity for L. pentosus and E. faecalis at late stationary and late logarithmic growth phase, respectively. The physiological role of catalase was studied with L sake. The presence of hematin allows higher growth yields, since it protects the cells against hydrogen peroxide formed endogenously up to concentrations of 4.6 mmol/l. Key words: Lactic acid bacteria; Hematin; Catalase formation, kinetics
Introduction Hydrogen peroxide is a c o m m o n metabolite of lactic acid bacteria and can be accumulated during fermentation processes. If this accumulation takes place in foods, their sensory quality may be strongly affected (Niven and Evans, 1957). Therefore the presence of catalase activity is a desirable property for starter cultures used in food fermentations (Liicke, 1985). For example, in meat products the accumulation of hydrogen peroxide can lead to fat rancidity and to discolourations of meat products by attacking heme pigments. Therefore Micrococcaceae are used for the fermentation of raw sausages, mainly because of their catalase activity. On the other hand, lactic acid bacteria are generally considered as being devoid of catalase activity, since these organisms axe not able to synthesize heme c o m p o u n d s (Kandler and Weiss, 1986). Nevertheless, there are reports showing that certain species of lactic acid bacteria exhibit catalase activity e.g. Whittenbury (1964) and Wolf and H a m m e s (1988) demonstrated, that the addition of hematin induced the production of catalase in strains of Lactobaciilus plantarum, L. brevis, L. acidilactici, L. viridescens, L. casei, L. delbrueckii, L. pentosus, L sake, L. bavaricus, Leuconostoc mesenteroides and Enterococcus faecalis. In addition, a heme independent catalase or pseudocatalase was described for strains of Pediococcus, Lactobacil-
Correspondence address: Dr. G. Wolf. Universifiit Hohenheim, Inst. f. Lebensmitteitechnologie,Oarbenstrasse 25, D-7000 Stuttgart 70, F.R.G.
134 lus. Leuconostoc and Emerococcus (Delwiche. 1961). This enzyme is insensitive to azide and cyanide and contains manganese (Kono and Fridovich. 1983). The various substrates for food fermentation may contain hematin and therefore. for the selection of the appropriate starter organism, the characterization of catalase activity and their possible induction is of major interest. The purpose of this paper was to extend our knowledge with regard to these properties of the heme-dependent catalases.
Materials and Methods
Mtcroorgamsms and growth conditions The following organisms were used in this study: L. pentosus Deutsche Sammlung von Mikroorganismen (DSM) 20314, L. sake 450 (Kagermeier, 1981), L. delbrueckii ssp. lactis National Collection of Dairy Organisms (NCDO) 280, Enterococcus faecalis National Collection of Industrial Bacteria (NCIB) 2131. The organisms were maintained and grown as described by Wolf and Hammes (1988). The strains were maintained in MRS stab cultures (de Man et al.. 1960) at 4 ° C and transferred monthly. For study of catalase, cells were grown aerobically in 250 ml Erlenmeyer flasks containing 50 ml medium at 30 °C on an oscillatory shaker. The medium was modified MRS, containing the following compounds per litre: 20 g glucose, 2 g KNO 3, 10 g Lab Lemco Powder. 5 g yeast extract, 10 g tryptone, 1 ml Tween 80, 0.05 g MnSO 4 • H20, 24.2 mg Na2MoO 4, 5 g sodium acetate. 3 H20, 0.2 g MgSO 4 • 7 H20, 5.4 mg FeC13, 2 g K2HPO ,. Where indicated 30 /tmol hematin were added (5 mg/ml solution in 0.2 m ol / l KOH, sterilized by filtration). The cells were harvested by centrifugation (Beckman J2-21, 25 000 × g, 10 min) and washed with phosphate buffer (0.05 mol/l, pH 7.0). Chemicals The following chemicals were obtained from Oxoid (Wesel, F.R.G.): Tryptone, Lab Lemco Powder, yeast extract and purified agar; from Sigma (Miinchen, F.R.G.): hematin, hemoglobin, chlorophyllin, protoporphyrin IX and catalase (bovine liver); from Boehringer (Mannheim, F.R.G.): ABTS (2,2'-azino-di-(3-ethylbenzthiazolin-6-sulfonate) and horseradish peroxidase; and from Merck (Darmstadt, F.R.G.) all other chemicals. Detection of catalase activity Catalase activity was detected with intact bacteria by the method of Rorth and Jensen (1967) using a WTW oxygraph (Oxi 530) equipped with a Clark electrode (Trioxmatic ® EO 200), both obtained from Wissenschaftlich-technische Werkstiitten, Weilheim, F.R.G. The oxygen electrode was calibrated with the Oxical ® calibration vessel (PE/OXI from WTW, Wissenschaftlich-technische Werkst~itten, Weilheim, F.R.G.). The assays were performed at 30°C in a stirred 30-ml flask with two side arms containing 25 ml H 2 0 : b u f f e r (0.01 m ol/ l H202 in 0.05 m ol / l phosphate
135 buffer, p H 7.0). For removing 02, the buffer was sparged with N 2 until it did not contain more than 0.1 mg O2/1. For controls the O2-production from H 2 0 : b u f f e r was observed with and without hematin. The reaction was started by the addition of whole cells or enzyme.
Induction of catalase activity in bacteria after their harvest by addition of hematm Growth was monitored by measurement of OD578 with a Beckman spectrophotometer or by counting cell numbers by the spread-plate technique (Koch, 1981). Cultures were harvested at the times indicated, washed with phosphate buffer (0.05 m o l / 1 , p H 7.0), resuspended in the same buffer and incubated with hematin, 30 ~tmol/1 at 30°C.
Determinanon of hydrogen peroxide Hydrogen peroxide was determined according to the method of Carter (1980). Aliquots (50 #1) of the culture broth were added to 2.5 ml of ABTS-peroxidase reagent (250 ml phosphate buffer, 0.1 m o l / l p H 7.0, containing 2.5 mg horseradish peroxidase and 250 mg ABTS). The mixture was incubated for 5 min at 25°C and the extinction was measured at 420 nm. All experiments were repeated at least three times. It was observed that, depending on the preparation, the cells exhibited varying amounts of activity between 0.2 and 0.8 mmol O2/1 × rain × 109 cfu for lactobacilli and up to 5 mmol O2/1 × rain x 109 cfu for E. faecalis.
Results and Discussion The conditions for production of catalase activity by L. pentosus were investigated. Addition of a n u m b e r of compounds was studied. As shown in Table I, hematin and hemoglobin were the only effective compounds. The dependence of catalase activity on the concentration of hematin was determined. As shown in Fig. 1, the catalase activity of aerobically grown cells showed a m a x i m u m in the range of 30-50 /~mol of hematin per litre of growth medium. Cultures grown in the presence of 30 /~mol/1 of hematin showed their highest
TABLE I Activity of catalase of L. pentosus, grown with various addtuons Compound None Hematin Hemoglobin Chlorophylhn Protoporphyrin IX protoporphyrin IX + FeCI3
Concentrauon (/xmol/l) 0 30 30 30 30 30
Enzyme activity (~) 0 100 100 0 0 0
An activity of 100% corresponds to the production of 189.14 ~tmol 02 1-1 n~n-i 10-9 cfu.
136 200 u
m
~.~_ .>- E 100
0
0.01
0.1
I
I0
I(30
1000
[ H e m a t m ] (,umol/I) Fig. 1. Catalas¢ activity of L penwsus grown aerobically in the presence of vanous concentratmns of hematin.
activity per cell unit at late stationary growth phase as shown in Fig. 2. The omission of hematin resulted in lower growth yield. Cells from this culture apparently produced catalase apoprotein, as catalase activity could be induced after addition of hematin to cells washed and suspended in phosphate buffer. This catalase activity also had a maximum at the late stationary phase and it was dependent on the length of incubation with hematin as depicted in Fig. 3. The activity was highest after incubation for 1-2 h at 30°C. As shown in Fig. 4, the catalase activity of cells of L. pemosus is as independent of the temperature within the range investigated as that of bovine liver catalase. The catalase activity was studied for different species of lactobacilli grown to late logarithmic growth phase with and without added hematin. As shown in Fig. 5, L. delbrueckii and L sake resembled L pentosus, i.¢. catalase activity was observed
2.0 0 l0
=I' ~
-=
05
c.,
)5
3~ 01
2
4
6
8
10
12
T,me (h) Fig. 2. The influence of catalase activity on growth of L pentosus. (Opucal densRy and catalase activity of cultures grown aerobically with (o, II) and without hematm ( o , E]).
137 100 ~ ~
w
~ - 50 0
r,..}~ 0--
I
I
0
I
2
Time of pre,ncubot~on (h)
Fig. 3. Catalas¢ activityfor cells of L pentosus harvested after 5 h of aerobic growth at 30°C, washed and suspendedin phosphatebufferwith added 30/~mol/1hcmatinfor the lengthof timesindicated. and it was virtually independent of whether hematin was added to the growth medium or to washed cells suspended in buffer. However, with E. faecalis a poor catalase activity was observed when hematin was added to cell suspensions grown in the absence of hematin. The kinetics of growth and acquisition of catalase activity revealed that this organism differed from /- pentosus in that the maximum activity is obtained cartier and further that cells of cultures grown in the absence of hematin not only exhibit higher activity but lose this activity dramatically during extended incubation (Fig. 6). The observed increase in growth yield for L pentosus when hematin was added, was also found for I_ sake. To study whether the increase was dependent on an active catalase, cultures were grown with and without hematin and in the presence of added catalase. As shown in Fig. 7, hydrogen peroxide accumulated up to concentrations surpassing 4.6 m m o l / l in cultures without hematin or added catalase. No accumulation was detected when hematin or catalase was added. In the
i
i
i
,
'
2'0
'
~'0
120
~=
so
0
0
Temperature ('C) Fig. 4. Influence of the temperature on activity of catalasc from bovine fiver (o) (7 units) and of cells of L pentosu~
(o) (8.0.l0 s cfu).
138
i L perWosus 20
I00
L SQ;(e
I delDruecku
E foecohs
314
~
~
,
!
.~
l
I :b U
o
50-
0
=
0 0
0 .
.
.
.
.
.
.
m,mm~
Fig. 5. Comparison of catalase acuvaty for various lactic acid bacteria grown aerobically to stauonary phase. (ra growth with 30 ~ m o l / l hematin, • growth without hematin, with subsequent incubation of washed cells in phosphate buffer at 30"C for 90 rain wtth 30 ~mol/I hematin). 100% catalase activity corresponds to the activity when hematin is added to the growth medium.
culture with accumulated hydrogen peroxide a lower growth yield was observed. Thus, the presence of an active catalase protects the organism and allows higher growth yields. The physiological activity of heine-dependent catalase of lactic acid bacteria exhibits properties similar to those described for true catalases (Aebi, 1983). For
i
1
,
,
2u %
30
60
~" E
iO 1-o ~n
c3 o
°~ ~ E
E 05
"6 20
o
o (..)
01~
'
A
2
z.
,
,
6 T,me
8
, 10
0
(h)
Fig. 6. Catalase activity during growth of E. faecahs. (Optical denstty and catalase activity of cell cultures grown aerobically with hematin (o, B) and without hematm (o, ra).
139
A
a
//A
108
= o
fi
=
e
E (-}
I 10 7
"~ 2 0
i 10l 0
I
*
2
~
~,J 0
"
6
; '[,me
"
~
"
[
"
~
"
8
(h)
Fig. 7. The effect of hematin and catalasc on growth (A) and H~O 2 accumulation (B) by /.. sake. The cells were grown at 30°C in MRS broth with 1.25 g/I glucose and the following additions: I , 5 mg/I hematin and 5mg/l bovine hver catalase; e, 5 mg/l hematm: v, 5 mg/l bovine hver catalase; A, no addition.
example, it was observed that the Q10 value was as low as 1.05-1.12, and the activity could be inhibited by 50% at 35 m m o l / l cyanide and 1 m m o l / l azide (unpublished results). The present results provide for a strategy of composing starter cultures with lactobacilli exhibiting catalase activity to minimize deleterious effects of hydrogen peroxide in fermenting food.
Aknowledgements We thank Heidi Griinenwald and Elisabeth Lindemann for expert technical assistance. This study was supported by Deutsche Forschungsgemeinschaft.
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