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Non-Heme Catalase Activity of Lactic Acid Bacteria
System. Appl. Microbiol. 17, 11-19 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Non-Heme Catalase Activity of Lactic Acid Bacteria DORIS M. ENGESSER and WALTER P. HAMMES" Institute of Food Technology, Section General Food Technology and Food Microbiology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany Received July 1, 1993
Summary A number of 71 species of lactic acid bacteria (LAB) were investigated for non-heme and heme catalase activity and their potential to form hydrogen peroxide. Non-heme catalase activity was restricted to strains of Lactobacillus plantarum, Lactobacillus mali and Pediococcus pentosaceus whereas heme catalase activity was present in 21 species belonging to virtually all genera of LAB. P. pentosaceus LTH 416 exhibited maximum non-heme catalase activity when cultured aerobically at the stationary phase. The activity increased after glucose was exhausted. Transfer of cells from glucose minimal medium (0.05%) to media containing 0.2% and 1% glucose, respectively, caused reduction of non-heme catalase activity. The effect of glucose was also seen if it was added to cells growing under static pH conditions (pH 6.5). The non-heme catalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 were characterized with regard to environmental factors important in sausage fermentation such as pH, temperature, concentration of nitrate or nitrite and water activity. The results permit an estimation of the non-heme catalase activity in the two strains during the fermentation process.
Key words: lactic acid bacteria - non-heme catalase activity - heme catalase activity - hydrogen peroxide formation - sausage fermentation
Introduction Lactic acid bacteria (LAB) are commonly regarded as being devoid of catalase activity. However certain species of LAB exhibited catalase activity in heme-containing media (Whittenbury, 1960; Whitten bury, 1964; Wolf and Hammes, 1988). In addition, the presence of a non-heme catalase or "pseudocatalase" was described for strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc and Enterococcus (Whittenbury, 1960; Whittenbury, 1964; Delwiche, 1961; Johnston and Delwiche, 1962; Johnston and Delwiche, 1965 alb; Jones et al., 1964). In Lactobacillus plantarum T-1403-5 (ATCC 14431) a very active non-heme catalase was detected and partially purified by Johnston and Delwiche (1965 alb) and subsequently characterized as a homopentameric manganese enzyme (Kono and Fridovich, 1983; Beyer and Fridovich, 1985). This enzyme is not a heme protein and is, thus, insensitive to heme poisons e. g. azide or cyanide. The potential of LAB to exhibit catalase activity is a desirable property for starter cultures used in food tech-
* Corresponding author
no logy to minimize deleterious effects of hydrogen peroxide. During food fermentation processes H 2 0 2 may be formed by microbial reduction of oxygen leading to an accumulation of oxygenated metabolites which may affect the sensory quality of the product by oxidative spoilage. In some products H 2 0 2 may also exert protective effects, since it contributes to the inhibition of undesired microorgamsms. A regulatory effect of glucose on the biosynthesis of heme catalase was described for Escherichia coli, Saccharomyces cerevisiae and Bacteroides fragilis (Sulebele and Rege, 1968; Yoshpe-Purer et al., 1977; Hassan and Fridovich, 1978), whereas only little information is available about the effect of glucose an non-heme catalase activity. As reported by Felton et al. (1953) and Whittenbury (1964) the non-heme catalase activity was enhanced in media containing low concentrations of glucose and the pH was suggested to be the regulatory factor (Gutekunst et al., 1957). The application of LAB as starter cultures in sausage fermentation requires a good knowledge of their potential
12
D. M. Engesser and W. P. Hammes
to form or to degrade hydrogen peroxide. It was the purpose of this study to investigate the occurrence of nonheme catalase in LAB and to characterize the non-heme catalase actlvltles of Lactobacillus plantarum and Pediococcus pentosaceus. Strains of these species are components of starter cultures and have to perform under various ecological conditions such as pH, temperature, water activity, concentration of nitrate or nitrite and glucose. Therefore, special attention was paid to the effect of these factors on non-heme catalase activity.
Materials and Methods Bacterial strains. The following organisms were used: Lactobacillus plantarum ATCC 14431; 1. mali ATCC 27053, 27054 and 27055; 1. yamanashiensis LTH 2227-2241 (LTH, Stammsammlung Institut fiir Allgemeine Lebensmitteltechnologie Hohenheim); Pediococcus pentosaceus DSM 20206, P. pent~saceus LTH 416 and LTH 2887. Strains included in the screemng for catalase activity and hydrogen peroxide formation are compiled in Table 1. Carnobacteria, enterococci and vagococci were not examined since they are not applicable in starter cultures for food fermentations. Culture conditions, media, and cell preparation. For study of catalase activity, the strains were grown aerobically at 30°C in 300 ml Erlenmeyer flasks containing 100 ml medium. The cultures were incubated on an oscillatory shaker at 150 rpm. The investigation of the kinetics of the synthesis of non-heme catalase was performed in a fermenter vessel (Infors, Miinchen, Germany) containing 1.8 I basal medium, pH 6.5. Where indicated, the pH was kept constant during fermentation (automatic pH control, pH 6.5). For aerobic and anaerobic growth, the vessel was sparged with sterile air and with sterile N 2, respectively. Foammg was prevented by addition of silicon antifoam. Cells of P. pentosaceus LTH 416 were grown anaerobically in MRS medium, harvested by centrifugation, washed with phosphate buffer (0.1 M, pH 7.0), resuspended and used as inoculum. When the effect of glucose on the non-heme catalase synthesis was exam!ned,. glucose was added aseptically through a septum at the time mdlcated. During fermentation changes in turbidity (OD S78 ), viable cells, pH and non-heme catalase activity were followed. For the determination of non-heme catalase activity, aliquots of the culture were centrifuged, resuspended in 0.05 M phosphate buffer, pH 7.0, and chilled on ice. The basal mediuom contained 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.5 Yo sodIUm Citrate, 0.05%-1 % glucose, 0.2% KH 2P0 4, 0.02% MgS04 x 7 H 20, 0.015% MnS04 x H 20, 0.00054% FeS04 x 7 H 2 0, pH 6.5. Cells used for the investigations of the heme catalase were grown in MRS medium (de Man et aI., 1960), supplemented with hematin (0.5 gil in 0.2 M KOH, sterilized by filtration) to obtain a final concentration of 31.5 flM (modified MRS). Growth of the cultures was monitored in all experiments by determination of OD S78 with a spectrophotometer or by counting viable cells employing the spread-plate technique. Cell-free extracts were prepared from overnight cultures (17 h). The cells were harvested by centrifugation, washed, resuspended in 700 fll of 0.05 M phosphate buffer, pH 7.0 and transfered to 5 ml screw cap tubes containing 0.7 mg glass beads of 0.5 mm III diameter. After diSruption of the cells in a cell mill (Biihler, Tiibingen, Germany) for 5 min at O°C, cell debris were removed by centnfugatlon and the . supernatant fraction was stored at - 20°C. Screening for catalase activity and hydrogen peroXIde formation. Cells were grown overnight in MRS broth, harvested, washed and resuspended in 0.1 M phosphate buffer, pH 7.0.
Tubes containing basal medium with 0.05% and 1 % glucose, respectively, or modified MRS were inoculated with 1 ml of cell suspension and incubated aerobically on an OSCillatory shaker (150 rpm). After harvesting the cells by centrifugation non-heme and heme catalase activity was recognized visually by effervescence on adding hydrogen peroxide (3%) onto the cell pellets. Hydrogen peroxide formation was tested by streaking ~he strains o~to ABTS agar. This agar consisted of MRS medIUm contammg 0.2% glucose and was supplemented with ABTS (~,2' -azino-.di-3ethyl-benzthiazolin-6-sulfonate) and horseradish peroXidase (HRP) to obtain a final concentration of 0.5 mM and 300 U/ml, respectively (both reagents were solved in distilled water and sterilized by filtration). Plates were mcubated for 48 h m an atmosphere of COzlN 2 and subsequently aerobically for ~-6 h. Hydrogen peroxide formation was indicated by the formatIOn of violet halos around the colonies. Test for catalase activity. Quantitative analysis of catalase activities was performed with either intact bacteria or cell-free extract by following oxygen production using a WTW oxigraph (Oxi 2000) equipped with a Clark electrode (TriOxmatic EO 200), both obtained from WTW (Weilheim, Germany). Calibration was done by means of the Oxical calibration vessel (WTW). The assays were performed at 30°C in a stirred 30 ml flask containing 25 ml reaction mixture (0.01 M H 20 2 in 0.05 M phosphate buffer, pH 7.0). The dependence of the catalase activity on hydrogen ion concentration was measured at a pH range between 2.6 and 10.0. The reaction mixture was buffered With Na2HP04/Citric acid (pH 2.6-7.6), Gomoris-Tris (pH 7.2-9.0), and Glycin-NaOH (pH 8.6-10.0). For removing oxygen, the solution was sparged with N2 until the concentration fell below 0.1 mg 0 2 /1. The reaction was run for 2-3 min and the initial linear rate was used for calculation of the activity. Assays. The protein content of crude cell extracts was determined according to the method of Bradford (1976) using the Biorad Protein Assay (Biorad, Miinchen, Germany) with bovine plasma albumin as a standard. Analysis of soluble proteins was performed according to the method of Moore et al. (1980). The total soluble protein was isolated as described under cell preparations. The proteins in 25 fll of the supernatant were separated on 7.5% nondenaturing polyacrylamide gels and the electrophoresIs was performed at ambient temperature. Activity staining of catalase was performed according to Clare et al. (1984). Glucose was determined enzymatically by means of the glucose test kit of Boehringer Mannheim (Tutzing, Germany) or by HPLC according to the method of Hamad et al. (1992). Treatment with ethanol/chloroform. The crude cell extract was shaken for 10 min at room temperature with the organic solvents (Nadler et aI., 1986) using a 101513 (v/v) ratio of cell extractlethanol (95%)/chloroform, followed by centrifugation at 13 000 x g for 5 min. The upper liquid phase, containing the ps~udocatalase, was assayed for non-heme catalase activity. Chemicals. Tryptone, Lab Lemco Powder and yeast extract were purchased from Oxoid (Wesel, Germany). Hematin, ABTS and HRP from Boehringer Mannheim. 3,3-diaminobenzidine was purchased from Sigma (Deisenhofen, Germany), all other chemicals were obtained from Merck (Darmstadt, Germany). Results
Screening of LAB for Catalase Activity and Hydrogen Peroxide Formation
A number of 71 species of LAB were investigated for non-heme and heme catalase activity and the formation of hydrogen peroxide. The results are illustrated in Table 1.
Non-Heme Catalase Activity of Lactic Acid Bacteria
13
Table 1. Species tested for catalase activity and hYdrogen peroxide formation Strain
Non-heme Heme catalase catalase
Strain
Lactobacillus sp.
I. Obligately homo fermentative L. acidophilus L. amylophilus L. amylovorus L.animalis L. delbrueckii ssp. delbrueckii L. delbrueckii ssp. bulgaricus L. delbrueckii ssp. lactis L. farciminis L.gasseri L. helveticus L.jensenii L. mali L. yamanashiensis L. sharpeae L. vitulinus
+ +
(+) (+)
+ +
(+)
(+) (+ )
+
(+) (+ ) (+ )
L. acetotolerans L. agilis L. alimentarius L. bavaricus t L. casei spp. casei L. casei spp. alactosus L. casei spp. pseudoplantarum L. casei spp. rhamnosus L. casei spp. tolerans L. coryniformis spp. coryniformis L. coryniformis spp. torquens L. curvatus L.graminis L. homohiochii L. maltaromicus L.murinus L.pentosus L. plantarum L. plantarum# L. sake
+
(+)
Leuconostoc sp.
+ +
+ + +
+
+
+
+
+
+ +
+
(+ )
+
+ + + +
+ +
III. Obligately heterofermentative
L. bifermentans L. brevis L. buchneri L. cellobiosus L. confusus L. fermentum L. fructivorans L. fructosus L. halotolerans L. hilgardii
+
+
(+)
+
+
(+)
+
+ +
+
(+)
(+)
+
(+)
+
+ +
(+)
+
(+)
(+) +
Lactococcus sp.
(+)
(+)
+
L. kandleri L. kefir L. malefermentans L. minor L. oris L. parabuchneri L. reuteri L. sanfrancisco L. suebicus L. vaccinostercus L. viridescens
L.garvieae L.lactis spp. lactis L. lactis spp. cremoris L. raffinolactis
II. Facultatively heterofermentative
Non-heme Heme catalase catalase
(+) (+)
L.lactis L. mesenteroides spp. mesenteroides L. mesenteroides spp. cremoris L.mesenteroides spp. dextranicum t L. oenos L. paramesenteroides
(+)
(+) (+) (+)
(+)
Pediococcus sp. P. acidilactici P. acidilactici## P. dextrinicus P. inopinatus P.parvulus P. pentosaceus
+
+
(+) (+)
+
(+)
+
+
Streptococcus sp.
S. salivarius t ssp. thermophilus
+
Positive (immediate evolution of oxygen bubbles). Negative (+) Weak positive reaction (delayed evolution of oxygen bubbles). t Strains obtained from LTH (Lactobacillus bavaricus LTH 2071, Leuconostoc mesenteroides ssp. dextranicum LTH 1558; Streptococcus salivarius ssp. thermophilus LTH 1276); all other strains represent the type strains of the respective species and were obtained from DSM. # ATCC 14431, reference strain exhibiting non-heme catalase activity. ## DSM 20284; an opinion is requested proposing DSM 20284 as neotype strain of the species P. acidilactici because of the very high DNA homology of the type strain DSM 20333 with the type strain of P. pentosaceus DSM 20336.
14
D. M. Engesser and W. P. Hammes
Non-heme catalase activity was restricted to strains of Lactobacillus plantarum, Lactobacillus mali and Pediococcus pentosaceus, whereas heme catalase activity was present in 21 species. Simultaneous Occurrence of Non-Heme and Heme Catalase in One Strain
culture. When grown anaerobically, a low level of nonheme catalase was detected. The high initial level of activity was due to a carryover from the inoculum, which was not prepared under strictly anaerobic conditions. Effect of Glucose on the Activity of Non-heme Catalase
Johnston and Delwiche (1965a) examined various pseudocatalase positive strains of LAB for their capacity to produce an additional heme catalase during growth in media containing heated blood or hematin. They observed that all strains (including L. plantarum T-1403-5 ) showed simultaneous formation of non-heme and heme catalase under these conditions. To investigate if the simultaneous presence of non-heme and heme catalase is a characteristic of all LAB exhibiting non-heme catalase activity, cells of L. mali (ATCC 27053, 27054, 27055 ) and P. pentosaceus (LTH 416, LTH 2887, DSM 20206 ) grown aerobically in medium supplemented with hematin were examined for the presence of additional heme catalase activity. L. plantarum ATCC 14431 (= T-1403-5 ) was included as a control. In no case we had been able to obtain evidence for the simultaneous expression of non-heme and heme catalase in one strain. Addition of 1 mM hydroxylamine completely suppressed catalase activity and the activity was not affected by 100 mM azide. Polyacrylamide electrophoresis followed by activity staining of catalase revealed one single band of activity for each strain which resembled the non-heme catalase activity (data not shown ).
The effect of glucose on the non-heme catalase activity was studied with P. pentosacues LTH 416. The cells were grown aerobically to late log phase in basal media containing 0.05% glucose (glucose minimal medium). These cultures were then suspended into fresh media (adjusted to equal optical density) containing the indicated concentrations of glucose (i. e. 0.05% , 0.2% and 1 % glucose). Samples were taken after 0, 2 and 4 h of incubation and assayed immediately for catalase activity. As shown in Fig. 2, exposure to increased glucose concentrations (0.2 or 1 % glucose) caused reduction of non-heme catalase activity.
100
.~ ~
~
uCII QI 411 CII
Kinetics of the Synthesis of Non-heme Catalase
Fig. 1 shows the formation of non-heme catalase during aerobic and anaerobic grown of P. pentosaceus LTH 416 in basal medium with 0.2 % glucose. The experiments were performed in a fermenter vessel. Maximum catalase activity was exhibited at the stationary phase of aerobic
..
50
iii CII
U
0 L-_'--'..L..D...>..lL._ _ _-i..-L...'-'-"-'-_ _ _-'--.........L.J..J'----' 2 4 o time (h)
~
0 0
10
14
0 0.05% E'L1 0.2% ~ 1%
)( 12
c
~ 10
9
Oi
E 8
E ...... ::I
Q:
U CI
~
~
8
.,
'0
oS!
.,., GI
iii
cau
Fig. 2. Effect of shifting cells from glucose minimal medium (0.05 % glucose) to media containing various concentrations of glucose (0.05 %, 0.2% and 1% ) on the activity of non-heme catalase of Pediococcus pentosaceus LTH 416. An activity of 100% corresponds to 21.4 mg 0 2/1x min x OD.
7
0 0
8
4
12
16
20
24
time (h)
... aerobic growth
-I:r
anaerobic growth --- cat aerobic
-0-
cat anaerobic
Fig. 1. Kinetics of growth and formation of non-heme catalase (cat) by Pediococcus pentosaceus LTH 416 in basal medium.
Effect of NaCl Concentration on Non-Heme Catalase Activity
The influence of water activity (a w ) on non-heme catalase activity of L. plantarum ATCC 14431 is illustrated in Fig. 6. Concentrations of 0-16.3% NaCI, corresponding to an aw range of 1-0.88 were employed. The pH of the reaction mixtures were adjusted to pH 7.0 before measuring the activities. In the presence of NaCl catalase activity clearly decreased,e. g. a NaCI concentration of 16.3% resulted in a 55 % inhibition of activity.
Non-Heme Catalase Activity of Lactic Acid Bacteria 5"
12
10
glucose
0
><
c:
~
10
g
'E
9
><
40 8 ;;J E 0. 8 u 30
:::::. 8 CI E
e:
OJ III
2
6
~
~
">
....
20
t;
III
i
u
The effects of a w , nitrate/nitrite and pH in combination were also examined. These experiments should simulate the conditions in fermented sausages. Therefore a reaction mixture containing 6.5% NaCl, 150 ppm nitrite or 300 ppm nitrate was tested at 3 different pH values (pH 4.8, 5.2 and 5.6). As shown in Fig. 7 the pseudocatalase activity of L. plantarum ATCC 14431 was strongly diminished to a level of 6-12% whereas that one of P. pentosaceus LTH 416 retained 80-94% of its original activity.
7
1'! co
0
0
4
8
12
16
20
J6
Effect of Hydroxylamine on Non-Heme Catalase Activity
10 0
24
15
Cells of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 grown aerobically in basal medium were col-
time (h) -o- calal.se acllvlly "cell counl [il glucose
~100 r-------------------------------'
Fig. 3. Effect of the addition of glucose on the kinetics of growth Q and formation of non-heme catalase in Pediococcus pentasaceus 0)( LTH 416. The arrow indicates the addition of glucose. ~
a 80
)(
The effect of glucose was also seen if it was added to cells of P. pentosaceus LTH 416 growing in basal medium (5 mM glucose) under static pH conditions (pH 6.5) in a fermenter vessel. At the time indicated, glucose was added to a final concentration of 40 mM. Immediately after exposure of the culture to increased glucose concentration the non-heme catalase activity decreased slowly (Fig. 3).
~
60
n
9_ -
f
40
ti
III
:: 20 iii
pH Dependence of Non-Heme Catalase Activity
'iu
O ~~~'---r-~--~-----.--------~
A characteristic of heme catalase is the high activity within a broad pH range (Schonbaum and Chance, 1976). The influence of pH on the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 was examined. The pseudocatalases of both strains exhibited maximal activity between pH 5.5 and 9 (Fig.4a/b).
2
1
3
4
5
6
7
8
9
10
11
pH
-+- L. plan/arum ~20 r---------------------------------' Q
Effect of Temperature on Non-Heme Catalase Activity 0
The effect of temperature on the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 was studied. The activities were not affected between 15 and 45 De. When cell extracts were exposed to higher temperatures between 45 and 80 DC both activities showed moderate stability (Fig. 5). The pseudocatalase activities were also stable to freezing. No loss of activity was found during 40 days at -20 De.
c
E 15
aE)(
9: 10 l:
~ ti
III GI 1/1 III
Effect of Nitrate and Nitrite on Non-Heme Catalase iii 'i Activity u
Nitrate and nitrite are used as curing aids for the production of fermented sausages. We observed that the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 remained constant up to 300 ppm nitrate and 150 ppm nitrite and retained 90-100% of their activity during 60 min of incubation (data not shown).
b
)(
5
O r--'r--'---'---.---.---'---r---r---.--~ 7 9 10 11 1 2 4 8 6 3 5 pH
-.- P. penlosaceus
Fig.4alb. Effect of pH on the activity of non-heme catalase in Lactobacillus plantarum ATCC 14431 (4a) and Pediococcus pentosaceus LTH 416 (4b) .
16
D.M. Engesser and W.P. Hammes
the viability of the cells. Samples were taken before and after 30 min of treatment with hydrogen peroxide, diluted, and plated for counting of viable organisms. As depicted in Fig. 8, suppression of non-heme catalase activity by hydroxylamine rendered the cells sensitive to hydrogen peroxide.
100 ~
~
::
:~
'0 ctI
C1J
(I)
ctI ctI
50
fti0
P. pen/osaceus
100
1\
l
1\ 1\ 1\
::
40
50
60
70
">
;:;
80
CII
temperature (OC)
GI
III
ctI
o L. plantarum !7] P. pentosaceus
50
iii ctI
Fig. 5. Thermostability of the non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. Crude cell extracts were exposed for 10 min at the indicated temperatures, chilled on ice and assayed for residual catalase activity. Catalase activity is expressed as a percentage of control values.
1\ 1\
1\ 1\ 1\
1\ 1\
1\ 1\
1\ 1\
1\ 1\
5,2
5,6
1\
0
U
L. plan/arum
m
o m
4,8
5,2
m
5,6
4,8
1\
1\
1\
1\
pH
o nitrate ISJ
nitrite
Fig. 7. Effect of aw , nitrate or nitrite and pH on the non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. An activity of 100% corresponds to 71.3 mg O 2/1 X min X OD for Lactobacillus plantarum and 19.8 mg O 2/1 X min X OD for Pediococcus pentosaceus, respectively.
100
~ :: ~
'0
III GI 1/1 III
50
L. plantarum
iii iii
100
0
rr-
,
:",
*... 0,88
0,9
0,92
0,94
0,96
0,98
1
water activity
:-
o
,-,-
..;
:l
..,0
C1J :cIII 50
.:;
>
I~ t,
Fig. 6. Dependence of non-heme catalase activity of Lactobacillus plantarum ATCC 14431 on water activity (a w ) . An activity of 100% corresponds to 70.9 mg O 2/1x min X OD.
:... I~
~
I'
t--
o lected by centrifugation and washed with NaCI/tryptone solution (0.85%/0.1 %). The cells were then resuspended and either hydrogen peroxide (7 mM) or hydrogen peroxidelhydroxylamine (7mMllmM) were added. Hydroxylamine (adjusted to pH 6.0) was added 5 min prior to hydrogen peroxide treatment to inhibit the pseudo catalase activity of the cells. The employed concentration of hydroxylamine (1 mM) was found to be sufficient to suppress non-heme catalase activity and does not influence
r0-
:~
c:
o ~--------~----~----------~--~
P. pentosaceus
,.....-
o
o
30
30
time (minI
o HA 0
hydrogen peroxide
0
HA/hydrogen peroxide
Fig. 8. Effect of hydroxylamine (HA) on non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. Cells were incubated with either H 2 0 2 or HAlH 2 0 2 and viable organisms were counted before and after treatment. The effect of hydroxylamine on cell viability is included as a control.
Non-Heme Catalase Activity of Lactic Acid Bacteria Activity staining of Non-Heme Catalase Cell-free extracts of L. plantarum ATCC 14431, P. pentosaceus DSM 20206, LTH 416, LTH 2887, L. mali ATCC 27053, ATCC 27054 and ATCC 27055 were subjected to native polyacrylamide gel electrophoresis (PAGE) followed by activity staining of catalase. For each of the three species the activity was restricted to a single band of a distinct R f (Fig. 9). Strains belonging to the same species exhibited bands with similar R f values (data not shown).
2
3
Fig. 9. Activity staining of non-heme catalase after native polyacrylamide gel electrophoresis (native PAGE) of cell-free extracts of Lactobacillus mali ATCC 27053 (Lane 1), Lactobacillus plantarum ATCC 14431 (Lane 2) and Pediococcus pentosaceus LTH 416 (Lane 3). The preparation of cell-free extracts, the electrophoresis and the activity staining of non-heme catalase were performed as described under Materials and Methods.
Treatment of Crude Cell Extract with Ethanol! Chloroform The stability to ethanoVchloroform was found to be characteristic of "typical" catalases (Nadler et aI., 1986). Cell extracts of L. plantarum ATCC 14431, P. pentosaceus LTH 416 and L. mali ATCC 27053 were shaken with a mixture of ethanoVchloroform and afterwards tested for non-heme catalase activity. The non-heme catalase activities of the three strains showed moderate stability to the treatment with ethanoVchloroform (data not shown) and behaved comparably to "typical" catalases. Discussion Our results indicate that formation of non-heme catalase activity is a quite rare property among LAB, restricted to strains of L. plantarum, L. mali and P. pentosaceus. These species phylogenetic ally belong to the L. casei Pediococcus group and are evolutionary related (Hammes and Vogel, in press). On the other hand, heme catalase is present in numerous species which belong to virtually all genera of LAB. For L. mali we have used the former epithets mali and yamanashiensis to differentiate between 2 SYStem. Appl. Microbiol. Vol. 17/1
17
the two former subspecies (Kaneuchi et aI., 1988; Moore and Moore, 1989). It is remarkable that the three available strains of L. mali (ATCC 27053, 27054 and 27055) exhibited non-heme catalase activity, whereas no activity was present in L. yamanashiensis (ATCC 27304) and a number of 15 isolates (LTH 2227-2241) from apple, pear and plum mashes, which resembled L. yamanashiensis. Johnston and Delwiche (1965a) reported the simultaneous presence of non-heme and heme catalase activities in L. plantarum T-1403-5 (ATCC 14431) and in a few other strains of LAB. In contrast to these findings, we were not able to obtain evidence for the formation of an additional heme catalase activity in cells of L. plantarum ATCC 14431 grown in MRS medium supplemented with hematin. The activity of these cells was suppressed by 1 mM hydroxylamine and not affected by 100 mM azide. Polyacrylamide gel electrophoresis followed by activity staining resulted in one single band of catalase activity, which resembled the non-heme catalase of this strain. Furthermore, no simultaneous occurrence of both catalase activities was found in any of the non-heme catalase positive strains. Hydrogen peroxide is formed during aerobic metabolism in reactions catalyzed by oxidases, e. g. NADH oxidase or pyruvate oxidase, transferring two electrons to one molecule of oxygen. Additionally, the superoxide radical (0 2 -) can dismute either spontaneously, catalyzed by superoxide dismutase or high intracellular Mn2+. Both, superoxide and hydrogen peroxide are toxic to the living cell and it has been suggested that HzO z and Oz - can react under formation of OH radicals which constitute the most harmful metabolites (Fridovich, 1978). The accumulation of hydrogen peroxide is a widespread property of LAB. Our study revealed most species to be capable of HzO z formation. Several strains did neither express catalase activity nor accumulate HzO z. This observation might be explained by the presence of peroxidase activity in these strains. A flavoprotein NADH peroxidase was firstly demonstrated by Dolin (1957) for E. faecalis and its presence has since been confirmed for many additional strains of LAB (reviewed in Condon, 1987). P. pentosaceus LTH 416 grew under aerobic conditions in glucose limited medium more rapidly and to a higher yield then it was observed anaerobically. This increase is consistent with the formation of acetate rather than lactate under these conditions (data not shown). The switch from lactate to acetate generation results in additional ATP synthesis. The formation of non-heme catalase activity by cells of P. pentosaceus in the presence of oxygen exerts a protective mechanism against the accumulation of hydrogen peroxide and enables the cells to benefit from the availability of the extra ATP. The regulatory effect of glucose on the non-heme catalase synthesis in P. pentosaceus LTH 416 is not unique in LAB, but appears to be a more general effect also described for other enzymes involved in aerobic metabolism, e. g., NADH oxidases or pyruvate oxidase (Grufferty and Condon, 1983; Murphy and Condon 1984; Brosnan, 1984). In sausage fermentation glucose is applied at about 0.5%. This concentration might already exert an inhibit-
18
D. M. Engesser and W. P. Hammes
ory effect on non-heme catalase activity. However, since glucose inhibits also H 2 0 2 generation, the non-heme catalase activity can exert its protective effect after glucose is exhausted, just at the time when it is required. For the application of starter cultures in sausage fermentation it is essential to know the effects of ecological factors on non-heme catalase activity affecting the transformation of the raw materials into the desired end-product. In experiments which simulated the conditions in fermented sausages (i. e. 6.5% NaCl, 150 ppm nitrite or 300 ppm nitrate, pH 4.8-5.6) P. pentosaceus retained 80-94% of its original non-heme catalase activity whereas the activity of L. plantarum was strongly diminished to a level of 6-12% . Thus, although L. plantarum exhibited the higher activity when tested under optimal conditions (71 mg 0 2/1 X min X OD for L. plantarum as compared to 20 mg 0 2 /1 X min X OD for P. pentosaceus) P. pentosaceus appears to be a more suitable candidate for further investigations aiming at the application of starter cultures in sausage fermentation exhibiting catalase activity to minimize deleterious effects of hydrogen peroxide. Acknowledgements. The authors are indebted to Miss E. Zimmermann for her skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. The authors are responsible for the content of this publication.
References Beyer, W. F. Jr., Fridovich, I.: Pseudocatalase from Lactobacillus plantarum: evidence for a homopentameric structure containing two atoms of manganese per subunit. Biochem. 24, 6460-6467 (1985) Bradford, M. M.: A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976) Brosnan, C A .: Regulation of oxygen-dependent lactate metabolism by Lactobacillus plantarum. M. Sc. Thesis, University College, Cork, Ireland (1984) Clare, D. A., Duong, M. N., Darr, D., Archibald, F., Fridovich, I.: Effects of molecular oxygen on the detection of superoxide radical with nitroblue tetrazolium and an activity stain for catalase. Anal. Biochem. 140,532-537 (1984 ) Col/ins, M. D., Rodrigues, U., Ash, C, Aguirre, M., Farrow, J. A . E., Martinez-Murcia, A., Phillips, B. A., Williams, A. M., Wallbanks, S.: Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s rRNA. FEMS Microbiol. Lett. 77,5-12 (1991 ) Condon, S.: Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46, 269-280 (1987) De/wiehe, E. A.: Catalase of Pedioeoeeus eerevisiae. J. Bacteriol. 81,416-418 (1961) De Man, j. C, Rogosa, M., Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135 (1960) Dolin, M. I. The Streptococcus faecalis oxidases for reduced diphosphopyridine nucleotide. III. Isolation and properties of a flavin peroxidase for reduced diphosphopyridine nucleotide. J. BioI. Chern. 225, 557-573 (1957) Felton, E. A., Evans, j. B., Niven, C F. Jr.: Production of catalase by pediococci. J. Bacteriol. 65, 481-482 (1953)
Fridovich, I.: Oxygen is toxic! A review of the chemistry of toxic oxygen products and the enzymes that destroy them. Bioscience 27, 462-466 (1977) Gregory, E. M., Veltri, B. j., Wagner, D. L., Wilkins, T. D.: Carbohydrate repression of catalase synthesis in Bacteroides fragilis. J. Bacteriol. 129, 534-535 (1977) Grufferty, R. C, Condon, S.: Effect of fermentation sugar on hydrogen peroxide accumulation by Streptococcus lactis CI0. J. Dairy Res. 50, 481-489 (1983) Gutekunst, R., De/wiehe, E. A., Seeley, H. W.: Catalase activity in Pediococcus cerevisiae as related to hydrogen ion activity. J. Bacteriol. 74, 694-695 (1957) Hamad, S. H., Boeker, G., Vogel, R. F., Hammes, W. P.: Microbiological and chemical analysis of fermented sorghum dough for Kisra production. Appl. Microbiol. and Biotechnol. 37, 728-731 (1992) Hammes, W. P., Vogel, R. F.: The genera of lactic acid bacteria. In: The lactic acid bacteria, Vol. II, (Wood, B. j. B., Holzapfel, W. H., eds.). In press Hassan, H. M., Fridovich, I.: Regulation of the synthesis of catalase and peroxidase in Escherichia coli. J. BioI. Chern. 253, 6445-6450 (1978) Johnston, M. A., Delwiche, E. A.: Catalase of the Lactobacillaceae. J. Bacteriol. 83, 936-938 (1962) Johnston, M. A., Delwiehe, E. A.: Distribution and characteristics of the catalases of Lactobacillaceae. J. Bacteriol. 90, 347-351 (1965a) Johnston, M. A., Delwiche, E. A.: Isolation and characterization of the cyanide-resistant and azide-resistant catalase of Lactobacillus plantarum. J. Bacteriol. 90, 352-356 (1965b) Jones, D., Deibel, R. H., Niven, C F. Jr.: Catalase activity of two Streptococcus faecalis strains and its enhancement by aerobiosis and added cations. J. Bacteriol. 88, 602-610 (1964) Kaneuchi, C, Masako, S., Komagata, K.: Taxonomic study of Lactobacillus mali Carr and Davies 1970 and related strains: Validation of Lactobacillus mali Carr and Davies 1970 over Lactobacillus yamanashiensis Nonomura 1983. J. Sys. Bacteriol. 38, 269-272 (1988) Kono, Y., Fridovich, I.: Isolation and characterization of the pseudocatalase of Lactobacillus plantarum. J. BioI. Chern. 258, 6015-6019 (1983) Moore, W. E. C, Hash, D. E., Holderman, L. V., Eato, E. P.: Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. Environ. Microbiol. 39, 900-907 (1980) Moore, W. E. C, Moore, L. V. H.: Index of bacterial and yeast nomenclature changes published in the International Journal of Systematic Bacteriology. American Society for Microbiology, Washington, D. C. (1989) Murphy, M. G., Condon, S.: Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium. Arch. Microbiol. Murphy, M. G., Condon, S. : Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium. Arch. Microbiol. 138,49-53 (1984) Nadler, V., Goldberg, I., Hochmann, A.: Comparative study of bacterial catalases. Biochim. Biophys. Acta 882, 234-242 (1986) Schonbaum, G. R., Chance, B.: pp.363-408. In: The enzymes (Boyer, P. D., ed.) 13, 2nd ed., Academic Press, New York (1976) Sulebele, G. A., Rege, D. V.: The nature of the glucose effect on the induced synthesis of catalase in Saccharomyces cerevisiae. Enzymologia 35, 321-334 (1968) Whitten bury, R.: Two types of catalase-like activity in lactic acid bacteria. Nature, 187, 433-434 (1960)
Non-Heme Catalase Activity of Lactic Acid Bacteria Whitten bury, R.: Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. J. Gen. Microbiol. 35, 12-26 (1964) Wolf, G., Hammes, W. P.: Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Arch. Microbiol. 149, 220-224 (1988)
19
Yoshpe-Purer, Y., Henis, Y., Yashphe, J.: Regulation of catalase level in Escherichia coli K12 . Can. J. Microbiol. 23, 84-91 (1977)
Prof. Dr. W. P. Hammes, Institute of Food Technology, Section General Food Technology and Food Microbiology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany
Non-Heme Catalase Activity of Lactic Acid Bacteria DORIS M. ENGESSER and WALTER P. HAMMES" Institute of Food Technology, Section General Food Technology and Food Microbiology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany Received July 1, 1993
Summary A number of 71 species of lactic acid bacteria (LAB) were investigated for non-heme and heme catalase activity and their potential to form hydrogen peroxide. Non-heme catalase activity was restricted to strains of Lactobacillus plantarum, Lactobacillus mali and Pediococcus pentosaceus whereas heme catalase activity was present in 21 species belonging to virtually all genera of LAB. P. pentosaceus LTH 416 exhibited maximum non-heme catalase activity when cultured aerobically at the stationary phase. The activity increased after glucose was exhausted. Transfer of cells from glucose minimal medium (0.05%) to media containing 0.2% and 1% glucose, respectively, caused reduction of non-heme catalase activity. The effect of glucose was also seen if it was added to cells growing under static pH conditions (pH 6.5). The non-heme catalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 were characterized with regard to environmental factors important in sausage fermentation such as pH, temperature, concentration of nitrate or nitrite and water activity. The results permit an estimation of the non-heme catalase activity in the two strains during the fermentation process.
Key words: lactic acid bacteria - non-heme catalase activity - heme catalase activity - hydrogen peroxide formation - sausage fermentation
Introduction Lactic acid bacteria (LAB) are commonly regarded as being devoid of catalase activity. However certain species of LAB exhibited catalase activity in heme-containing media (Whittenbury, 1960; Whitten bury, 1964; Wolf and Hammes, 1988). In addition, the presence of a non-heme catalase or "pseudocatalase" was described for strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc and Enterococcus (Whittenbury, 1960; Whittenbury, 1964; Delwiche, 1961; Johnston and Delwiche, 1962; Johnston and Delwiche, 1965 alb; Jones et al., 1964). In Lactobacillus plantarum T-1403-5 (ATCC 14431) a very active non-heme catalase was detected and partially purified by Johnston and Delwiche (1965 alb) and subsequently characterized as a homopentameric manganese enzyme (Kono and Fridovich, 1983; Beyer and Fridovich, 1985). This enzyme is not a heme protein and is, thus, insensitive to heme poisons e. g. azide or cyanide. The potential of LAB to exhibit catalase activity is a desirable property for starter cultures used in food tech-
* Corresponding author
no logy to minimize deleterious effects of hydrogen peroxide. During food fermentation processes H 2 0 2 may be formed by microbial reduction of oxygen leading to an accumulation of oxygenated metabolites which may affect the sensory quality of the product by oxidative spoilage. In some products H 2 0 2 may also exert protective effects, since it contributes to the inhibition of undesired microorgamsms. A regulatory effect of glucose on the biosynthesis of heme catalase was described for Escherichia coli, Saccharomyces cerevisiae and Bacteroides fragilis (Sulebele and Rege, 1968; Yoshpe-Purer et al., 1977; Hassan and Fridovich, 1978), whereas only little information is available about the effect of glucose an non-heme catalase activity. As reported by Felton et al. (1953) and Whittenbury (1964) the non-heme catalase activity was enhanced in media containing low concentrations of glucose and the pH was suggested to be the regulatory factor (Gutekunst et al., 1957). The application of LAB as starter cultures in sausage fermentation requires a good knowledge of their potential
12
D. M. Engesser and W. P. Hammes
to form or to degrade hydrogen peroxide. It was the purpose of this study to investigate the occurrence of nonheme catalase in LAB and to characterize the non-heme catalase actlvltles of Lactobacillus plantarum and Pediococcus pentosaceus. Strains of these species are components of starter cultures and have to perform under various ecological conditions such as pH, temperature, water activity, concentration of nitrate or nitrite and glucose. Therefore, special attention was paid to the effect of these factors on non-heme catalase activity.
Materials and Methods Bacterial strains. The following organisms were used: Lactobacillus plantarum ATCC 14431; 1. mali ATCC 27053, 27054 and 27055; 1. yamanashiensis LTH 2227-2241 (LTH, Stammsammlung Institut fiir Allgemeine Lebensmitteltechnologie Hohenheim); Pediococcus pentosaceus DSM 20206, P. pent~saceus LTH 416 and LTH 2887. Strains included in the screemng for catalase activity and hydrogen peroxide formation are compiled in Table 1. Carnobacteria, enterococci and vagococci were not examined since they are not applicable in starter cultures for food fermentations. Culture conditions, media, and cell preparation. For study of catalase activity, the strains were grown aerobically at 30°C in 300 ml Erlenmeyer flasks containing 100 ml medium. The cultures were incubated on an oscillatory shaker at 150 rpm. The investigation of the kinetics of the synthesis of non-heme catalase was performed in a fermenter vessel (Infors, Miinchen, Germany) containing 1.8 I basal medium, pH 6.5. Where indicated, the pH was kept constant during fermentation (automatic pH control, pH 6.5). For aerobic and anaerobic growth, the vessel was sparged with sterile air and with sterile N 2, respectively. Foammg was prevented by addition of silicon antifoam. Cells of P. pentosaceus LTH 416 were grown anaerobically in MRS medium, harvested by centrifugation, washed with phosphate buffer (0.1 M, pH 7.0), resuspended and used as inoculum. When the effect of glucose on the non-heme catalase synthesis was exam!ned,. glucose was added aseptically through a septum at the time mdlcated. During fermentation changes in turbidity (OD S78 ), viable cells, pH and non-heme catalase activity were followed. For the determination of non-heme catalase activity, aliquots of the culture were centrifuged, resuspended in 0.05 M phosphate buffer, pH 7.0, and chilled on ice. The basal mediuom contained 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.5 Yo sodIUm Citrate, 0.05%-1 % glucose, 0.2% KH 2P0 4, 0.02% MgS04 x 7 H 20, 0.015% MnS04 x H 20, 0.00054% FeS04 x 7 H 2 0, pH 6.5. Cells used for the investigations of the heme catalase were grown in MRS medium (de Man et aI., 1960), supplemented with hematin (0.5 gil in 0.2 M KOH, sterilized by filtration) to obtain a final concentration of 31.5 flM (modified MRS). Growth of the cultures was monitored in all experiments by determination of OD S78 with a spectrophotometer or by counting viable cells employing the spread-plate technique. Cell-free extracts were prepared from overnight cultures (17 h). The cells were harvested by centrifugation, washed, resuspended in 700 fll of 0.05 M phosphate buffer, pH 7.0 and transfered to 5 ml screw cap tubes containing 0.7 mg glass beads of 0.5 mm III diameter. After diSruption of the cells in a cell mill (Biihler, Tiibingen, Germany) for 5 min at O°C, cell debris were removed by centnfugatlon and the . supernatant fraction was stored at - 20°C. Screening for catalase activity and hydrogen peroXIde formation. Cells were grown overnight in MRS broth, harvested, washed and resuspended in 0.1 M phosphate buffer, pH 7.0.
Tubes containing basal medium with 0.05% and 1 % glucose, respectively, or modified MRS were inoculated with 1 ml of cell suspension and incubated aerobically on an OSCillatory shaker (150 rpm). After harvesting the cells by centrifugation non-heme and heme catalase activity was recognized visually by effervescence on adding hydrogen peroxide (3%) onto the cell pellets. Hydrogen peroxide formation was tested by streaking ~he strains o~to ABTS agar. This agar consisted of MRS medIUm contammg 0.2% glucose and was supplemented with ABTS (~,2' -azino-.di-3ethyl-benzthiazolin-6-sulfonate) and horseradish peroXidase (HRP) to obtain a final concentration of 0.5 mM and 300 U/ml, respectively (both reagents were solved in distilled water and sterilized by filtration). Plates were mcubated for 48 h m an atmosphere of COzlN 2 and subsequently aerobically for ~-6 h. Hydrogen peroxide formation was indicated by the formatIOn of violet halos around the colonies. Test for catalase activity. Quantitative analysis of catalase activities was performed with either intact bacteria or cell-free extract by following oxygen production using a WTW oxigraph (Oxi 2000) equipped with a Clark electrode (TriOxmatic EO 200), both obtained from WTW (Weilheim, Germany). Calibration was done by means of the Oxical calibration vessel (WTW). The assays were performed at 30°C in a stirred 30 ml flask containing 25 ml reaction mixture (0.01 M H 20 2 in 0.05 M phosphate buffer, pH 7.0). The dependence of the catalase activity on hydrogen ion concentration was measured at a pH range between 2.6 and 10.0. The reaction mixture was buffered With Na2HP04/Citric acid (pH 2.6-7.6), Gomoris-Tris (pH 7.2-9.0), and Glycin-NaOH (pH 8.6-10.0). For removing oxygen, the solution was sparged with N2 until the concentration fell below 0.1 mg 0 2 /1. The reaction was run for 2-3 min and the initial linear rate was used for calculation of the activity. Assays. The protein content of crude cell extracts was determined according to the method of Bradford (1976) using the Biorad Protein Assay (Biorad, Miinchen, Germany) with bovine plasma albumin as a standard. Analysis of soluble proteins was performed according to the method of Moore et al. (1980). The total soluble protein was isolated as described under cell preparations. The proteins in 25 fll of the supernatant were separated on 7.5% nondenaturing polyacrylamide gels and the electrophoresIs was performed at ambient temperature. Activity staining of catalase was performed according to Clare et al. (1984). Glucose was determined enzymatically by means of the glucose test kit of Boehringer Mannheim (Tutzing, Germany) or by HPLC according to the method of Hamad et al. (1992). Treatment with ethanol/chloroform. The crude cell extract was shaken for 10 min at room temperature with the organic solvents (Nadler et aI., 1986) using a 101513 (v/v) ratio of cell extractlethanol (95%)/chloroform, followed by centrifugation at 13 000 x g for 5 min. The upper liquid phase, containing the ps~udocatalase, was assayed for non-heme catalase activity. Chemicals. Tryptone, Lab Lemco Powder and yeast extract were purchased from Oxoid (Wesel, Germany). Hematin, ABTS and HRP from Boehringer Mannheim. 3,3-diaminobenzidine was purchased from Sigma (Deisenhofen, Germany), all other chemicals were obtained from Merck (Darmstadt, Germany). Results
Screening of LAB for Catalase Activity and Hydrogen Peroxide Formation
A number of 71 species of LAB were investigated for non-heme and heme catalase activity and the formation of hydrogen peroxide. The results are illustrated in Table 1.
Non-Heme Catalase Activity of Lactic Acid Bacteria
13
Table 1. Species tested for catalase activity and hYdrogen peroxide formation Strain
Non-heme Heme catalase catalase
Strain
Lactobacillus sp.
I. Obligately homo fermentative L. acidophilus L. amylophilus L. amylovorus L.animalis L. delbrueckii ssp. delbrueckii L. delbrueckii ssp. bulgaricus L. delbrueckii ssp. lactis L. farciminis L.gasseri L. helveticus L.jensenii L. mali L. yamanashiensis L. sharpeae L. vitulinus
+ +
(+) (+)
+ +
(+)
(+) (+ )
+
(+) (+ ) (+ )
L. acetotolerans L. agilis L. alimentarius L. bavaricus t L. casei spp. casei L. casei spp. alactosus L. casei spp. pseudoplantarum L. casei spp. rhamnosus L. casei spp. tolerans L. coryniformis spp. coryniformis L. coryniformis spp. torquens L. curvatus L.graminis L. homohiochii L. maltaromicus L.murinus L.pentosus L. plantarum L. plantarum# L. sake
+
(+)
Leuconostoc sp.
+ +
+ + +
+
+
+
+
+
+ +
+
(+ )
+
+ + + +
+ +
III. Obligately heterofermentative
L. bifermentans L. brevis L. buchneri L. cellobiosus L. confusus L. fermentum L. fructivorans L. fructosus L. halotolerans L. hilgardii
+
+
(+)
+
+
(+)
+
+ +
+
(+)
(+)
+
(+)
+
+ +
(+)
+
(+)
(+) +
Lactococcus sp.
(+)
(+)
+
L. kandleri L. kefir L. malefermentans L. minor L. oris L. parabuchneri L. reuteri L. sanfrancisco L. suebicus L. vaccinostercus L. viridescens
L.garvieae L.lactis spp. lactis L. lactis spp. cremoris L. raffinolactis
II. Facultatively heterofermentative
Non-heme Heme catalase catalase
(+) (+)
L.lactis L. mesenteroides spp. mesenteroides L. mesenteroides spp. cremoris L.mesenteroides spp. dextranicum t L. oenos L. paramesenteroides
(+)
(+) (+) (+)
(+)
Pediococcus sp. P. acidilactici P. acidilactici## P. dextrinicus P. inopinatus P.parvulus P. pentosaceus
+
+
(+) (+)
+
(+)
+
+
Streptococcus sp.
S. salivarius t ssp. thermophilus
+
Positive (immediate evolution of oxygen bubbles). Negative (+) Weak positive reaction (delayed evolution of oxygen bubbles). t Strains obtained from LTH (Lactobacillus bavaricus LTH 2071, Leuconostoc mesenteroides ssp. dextranicum LTH 1558; Streptococcus salivarius ssp. thermophilus LTH 1276); all other strains represent the type strains of the respective species and were obtained from DSM. # ATCC 14431, reference strain exhibiting non-heme catalase activity. ## DSM 20284; an opinion is requested proposing DSM 20284 as neotype strain of the species P. acidilactici because of the very high DNA homology of the type strain DSM 20333 with the type strain of P. pentosaceus DSM 20336.
14
D. M. Engesser and W. P. Hammes
Non-heme catalase activity was restricted to strains of Lactobacillus plantarum, Lactobacillus mali and Pediococcus pentosaceus, whereas heme catalase activity was present in 21 species. Simultaneous Occurrence of Non-Heme and Heme Catalase in One Strain
culture. When grown anaerobically, a low level of nonheme catalase was detected. The high initial level of activity was due to a carryover from the inoculum, which was not prepared under strictly anaerobic conditions. Effect of Glucose on the Activity of Non-heme Catalase
Johnston and Delwiche (1965a) examined various pseudocatalase positive strains of LAB for their capacity to produce an additional heme catalase during growth in media containing heated blood or hematin. They observed that all strains (including L. plantarum T-1403-5 ) showed simultaneous formation of non-heme and heme catalase under these conditions. To investigate if the simultaneous presence of non-heme and heme catalase is a characteristic of all LAB exhibiting non-heme catalase activity, cells of L. mali (ATCC 27053, 27054, 27055 ) and P. pentosaceus (LTH 416, LTH 2887, DSM 20206 ) grown aerobically in medium supplemented with hematin were examined for the presence of additional heme catalase activity. L. plantarum ATCC 14431 (= T-1403-5 ) was included as a control. In no case we had been able to obtain evidence for the simultaneous expression of non-heme and heme catalase in one strain. Addition of 1 mM hydroxylamine completely suppressed catalase activity and the activity was not affected by 100 mM azide. Polyacrylamide electrophoresis followed by activity staining of catalase revealed one single band of activity for each strain which resembled the non-heme catalase activity (data not shown ).
The effect of glucose on the non-heme catalase activity was studied with P. pentosacues LTH 416. The cells were grown aerobically to late log phase in basal media containing 0.05% glucose (glucose minimal medium). These cultures were then suspended into fresh media (adjusted to equal optical density) containing the indicated concentrations of glucose (i. e. 0.05% , 0.2% and 1 % glucose). Samples were taken after 0, 2 and 4 h of incubation and assayed immediately for catalase activity. As shown in Fig. 2, exposure to increased glucose concentrations (0.2 or 1 % glucose) caused reduction of non-heme catalase activity.
100
.~ ~
~
uCII QI 411 CII
Kinetics of the Synthesis of Non-heme Catalase
Fig. 1 shows the formation of non-heme catalase during aerobic and anaerobic grown of P. pentosaceus LTH 416 in basal medium with 0.2 % glucose. The experiments were performed in a fermenter vessel. Maximum catalase activity was exhibited at the stationary phase of aerobic
..
50
iii CII
U
0 L-_'--'..L..D...>..lL._ _ _-i..-L...'-'-"-'-_ _ _-'--.........L.J..J'----' 2 4 o time (h)
~
0 0
10
14
0 0.05% E'L1 0.2% ~ 1%
)( 12
c
~ 10
9
Oi
E 8
E ...... ::I
Q:
U CI
~
~
8
.,
'0
oS!
.,., GI
iii
cau
Fig. 2. Effect of shifting cells from glucose minimal medium (0.05 % glucose) to media containing various concentrations of glucose (0.05 %, 0.2% and 1% ) on the activity of non-heme catalase of Pediococcus pentosaceus LTH 416. An activity of 100% corresponds to 21.4 mg 0 2/1x min x OD.
7
0 0
8
4
12
16
20
24
time (h)
... aerobic growth
-I:r
anaerobic growth --- cat aerobic
-0-
cat anaerobic
Fig. 1. Kinetics of growth and formation of non-heme catalase (cat) by Pediococcus pentosaceus LTH 416 in basal medium.
Effect of NaCl Concentration on Non-Heme Catalase Activity
The influence of water activity (a w ) on non-heme catalase activity of L. plantarum ATCC 14431 is illustrated in Fig. 6. Concentrations of 0-16.3% NaCI, corresponding to an aw range of 1-0.88 were employed. The pH of the reaction mixtures were adjusted to pH 7.0 before measuring the activities. In the presence of NaCl catalase activity clearly decreased,e. g. a NaCI concentration of 16.3% resulted in a 55 % inhibition of activity.
Non-Heme Catalase Activity of Lactic Acid Bacteria 5"
12
10
glucose
0
><
c:
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9
><
40 8 ;;J E 0. 8 u 30
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OJ III
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i
u
The effects of a w , nitrate/nitrite and pH in combination were also examined. These experiments should simulate the conditions in fermented sausages. Therefore a reaction mixture containing 6.5% NaCl, 150 ppm nitrite or 300 ppm nitrate was tested at 3 different pH values (pH 4.8, 5.2 and 5.6). As shown in Fig. 7 the pseudocatalase activity of L. plantarum ATCC 14431 was strongly diminished to a level of 6-12% whereas that one of P. pentosaceus LTH 416 retained 80-94% of its original activity.
7
1'! co
0
0
4
8
12
16
20
J6
Effect of Hydroxylamine on Non-Heme Catalase Activity
10 0
24
15
Cells of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 grown aerobically in basal medium were col-
time (h) -o- calal.se acllvlly "cell counl [il glucose
~100 r-------------------------------'
Fig. 3. Effect of the addition of glucose on the kinetics of growth Q and formation of non-heme catalase in Pediococcus pentasaceus 0)( LTH 416. The arrow indicates the addition of glucose. ~
a 80
)(
The effect of glucose was also seen if it was added to cells of P. pentosaceus LTH 416 growing in basal medium (5 mM glucose) under static pH conditions (pH 6.5) in a fermenter vessel. At the time indicated, glucose was added to a final concentration of 40 mM. Immediately after exposure of the culture to increased glucose concentration the non-heme catalase activity decreased slowly (Fig. 3).
~
60
n
9_ -
f
40
ti
III
:: 20 iii
pH Dependence of Non-Heme Catalase Activity
'iu
O ~~~'---r-~--~-----.--------~
A characteristic of heme catalase is the high activity within a broad pH range (Schonbaum and Chance, 1976). The influence of pH on the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 was examined. The pseudocatalases of both strains exhibited maximal activity between pH 5.5 and 9 (Fig.4a/b).
2
1
3
4
5
6
7
8
9
10
11
pH
-+- L. plan/arum ~20 r---------------------------------' Q
Effect of Temperature on Non-Heme Catalase Activity 0
The effect of temperature on the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 was studied. The activities were not affected between 15 and 45 De. When cell extracts were exposed to higher temperatures between 45 and 80 DC both activities showed moderate stability (Fig. 5). The pseudocatalase activities were also stable to freezing. No loss of activity was found during 40 days at -20 De.
c
E 15
aE)(
9: 10 l:
~ ti
III GI 1/1 III
Effect of Nitrate and Nitrite on Non-Heme Catalase iii 'i Activity u
Nitrate and nitrite are used as curing aids for the production of fermented sausages. We observed that the pseudocatalase activities of L. plantarum ATCC 14431 and P. pentosaceus LTH 416 remained constant up to 300 ppm nitrate and 150 ppm nitrite and retained 90-100% of their activity during 60 min of incubation (data not shown).
b
)(
5
O r--'r--'---'---.---.---'---r---r---.--~ 7 9 10 11 1 2 4 8 6 3 5 pH
-.- P. penlosaceus
Fig.4alb. Effect of pH on the activity of non-heme catalase in Lactobacillus plantarum ATCC 14431 (4a) and Pediococcus pentosaceus LTH 416 (4b) .
16
D.M. Engesser and W.P. Hammes
the viability of the cells. Samples were taken before and after 30 min of treatment with hydrogen peroxide, diluted, and plated for counting of viable organisms. As depicted in Fig. 8, suppression of non-heme catalase activity by hydroxylamine rendered the cells sensitive to hydrogen peroxide.
100 ~
~
::
:~
'0 ctI
C1J
(I)
ctI ctI
50
fti0
P. pen/osaceus
100
1\
l
1\ 1\ 1\
::
40
50
60
70
">
;:;
80
CII
temperature (OC)
GI
III
ctI
o L. plantarum !7] P. pentosaceus
50
iii ctI
Fig. 5. Thermostability of the non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. Crude cell extracts were exposed for 10 min at the indicated temperatures, chilled on ice and assayed for residual catalase activity. Catalase activity is expressed as a percentage of control values.
1\ 1\
1\ 1\ 1\
1\ 1\
1\ 1\
1\ 1\
1\ 1\
5,2
5,6
1\
0
U
L. plan/arum
m
o m
4,8
5,2
m
5,6
4,8
1\
1\
1\
1\
pH
o nitrate ISJ
nitrite
Fig. 7. Effect of aw , nitrate or nitrite and pH on the non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. An activity of 100% corresponds to 71.3 mg O 2/1 X min X OD for Lactobacillus plantarum and 19.8 mg O 2/1 X min X OD for Pediococcus pentosaceus, respectively.
100
~ :: ~
'0
III GI 1/1 III
50
L. plantarum
iii iii
100
0
rr-
,
:",
*... 0,88
0,9
0,92
0,94
0,96
0,98
1
water activity
:-
o
,-,-
..;
:l
..,0
C1J :cIII 50
.:;
>
I~ t,
Fig. 6. Dependence of non-heme catalase activity of Lactobacillus plantarum ATCC 14431 on water activity (a w ) . An activity of 100% corresponds to 70.9 mg O 2/1x min X OD.
:... I~
~
I'
t--
o lected by centrifugation and washed with NaCI/tryptone solution (0.85%/0.1 %). The cells were then resuspended and either hydrogen peroxide (7 mM) or hydrogen peroxidelhydroxylamine (7mMllmM) were added. Hydroxylamine (adjusted to pH 6.0) was added 5 min prior to hydrogen peroxide treatment to inhibit the pseudo catalase activity of the cells. The employed concentration of hydroxylamine (1 mM) was found to be sufficient to suppress non-heme catalase activity and does not influence
r0-
:~
c:
o ~--------~----~----------~--~
P. pentosaceus
,.....-
o
o
30
30
time (minI
o HA 0
hydrogen peroxide
0
HA/hydrogen peroxide
Fig. 8. Effect of hydroxylamine (HA) on non-heme catalase activities of Lactobacillus plantarum ATCC 14431 and Pediococcus pentosaceus LTH 416. Cells were incubated with either H 2 0 2 or HAlH 2 0 2 and viable organisms were counted before and after treatment. The effect of hydroxylamine on cell viability is included as a control.
Non-Heme Catalase Activity of Lactic Acid Bacteria Activity staining of Non-Heme Catalase Cell-free extracts of L. plantarum ATCC 14431, P. pentosaceus DSM 20206, LTH 416, LTH 2887, L. mali ATCC 27053, ATCC 27054 and ATCC 27055 were subjected to native polyacrylamide gel electrophoresis (PAGE) followed by activity staining of catalase. For each of the three species the activity was restricted to a single band of a distinct R f (Fig. 9). Strains belonging to the same species exhibited bands with similar R f values (data not shown).
2
3
Fig. 9. Activity staining of non-heme catalase after native polyacrylamide gel electrophoresis (native PAGE) of cell-free extracts of Lactobacillus mali ATCC 27053 (Lane 1), Lactobacillus plantarum ATCC 14431 (Lane 2) and Pediococcus pentosaceus LTH 416 (Lane 3). The preparation of cell-free extracts, the electrophoresis and the activity staining of non-heme catalase were performed as described under Materials and Methods.
Treatment of Crude Cell Extract with Ethanol! Chloroform The stability to ethanoVchloroform was found to be characteristic of "typical" catalases (Nadler et aI., 1986). Cell extracts of L. plantarum ATCC 14431, P. pentosaceus LTH 416 and L. mali ATCC 27053 were shaken with a mixture of ethanoVchloroform and afterwards tested for non-heme catalase activity. The non-heme catalase activities of the three strains showed moderate stability to the treatment with ethanoVchloroform (data not shown) and behaved comparably to "typical" catalases. Discussion Our results indicate that formation of non-heme catalase activity is a quite rare property among LAB, restricted to strains of L. plantarum, L. mali and P. pentosaceus. These species phylogenetic ally belong to the L. casei Pediococcus group and are evolutionary related (Hammes and Vogel, in press). On the other hand, heme catalase is present in numerous species which belong to virtually all genera of LAB. For L. mali we have used the former epithets mali and yamanashiensis to differentiate between 2 SYStem. Appl. Microbiol. Vol. 17/1
17
the two former subspecies (Kaneuchi et aI., 1988; Moore and Moore, 1989). It is remarkable that the three available strains of L. mali (ATCC 27053, 27054 and 27055) exhibited non-heme catalase activity, whereas no activity was present in L. yamanashiensis (ATCC 27304) and a number of 15 isolates (LTH 2227-2241) from apple, pear and plum mashes, which resembled L. yamanashiensis. Johnston and Delwiche (1965a) reported the simultaneous presence of non-heme and heme catalase activities in L. plantarum T-1403-5 (ATCC 14431) and in a few other strains of LAB. In contrast to these findings, we were not able to obtain evidence for the formation of an additional heme catalase activity in cells of L. plantarum ATCC 14431 grown in MRS medium supplemented with hematin. The activity of these cells was suppressed by 1 mM hydroxylamine and not affected by 100 mM azide. Polyacrylamide gel electrophoresis followed by activity staining resulted in one single band of catalase activity, which resembled the non-heme catalase of this strain. Furthermore, no simultaneous occurrence of both catalase activities was found in any of the non-heme catalase positive strains. Hydrogen peroxide is formed during aerobic metabolism in reactions catalyzed by oxidases, e. g. NADH oxidase or pyruvate oxidase, transferring two electrons to one molecule of oxygen. Additionally, the superoxide radical (0 2 -) can dismute either spontaneously, catalyzed by superoxide dismutase or high intracellular Mn2+. Both, superoxide and hydrogen peroxide are toxic to the living cell and it has been suggested that HzO z and Oz - can react under formation of OH radicals which constitute the most harmful metabolites (Fridovich, 1978). The accumulation of hydrogen peroxide is a widespread property of LAB. Our study revealed most species to be capable of HzO z formation. Several strains did neither express catalase activity nor accumulate HzO z. This observation might be explained by the presence of peroxidase activity in these strains. A flavoprotein NADH peroxidase was firstly demonstrated by Dolin (1957) for E. faecalis and its presence has since been confirmed for many additional strains of LAB (reviewed in Condon, 1987). P. pentosaceus LTH 416 grew under aerobic conditions in glucose limited medium more rapidly and to a higher yield then it was observed anaerobically. This increase is consistent with the formation of acetate rather than lactate under these conditions (data not shown). The switch from lactate to acetate generation results in additional ATP synthesis. The formation of non-heme catalase activity by cells of P. pentosaceus in the presence of oxygen exerts a protective mechanism against the accumulation of hydrogen peroxide and enables the cells to benefit from the availability of the extra ATP. The regulatory effect of glucose on the non-heme catalase synthesis in P. pentosaceus LTH 416 is not unique in LAB, but appears to be a more general effect also described for other enzymes involved in aerobic metabolism, e. g., NADH oxidases or pyruvate oxidase (Grufferty and Condon, 1983; Murphy and Condon 1984; Brosnan, 1984). In sausage fermentation glucose is applied at about 0.5%. This concentration might already exert an inhibit-
18
D. M. Engesser and W. P. Hammes
ory effect on non-heme catalase activity. However, since glucose inhibits also H 2 0 2 generation, the non-heme catalase activity can exert its protective effect after glucose is exhausted, just at the time when it is required. For the application of starter cultures in sausage fermentation it is essential to know the effects of ecological factors on non-heme catalase activity affecting the transformation of the raw materials into the desired end-product. In experiments which simulated the conditions in fermented sausages (i. e. 6.5% NaCl, 150 ppm nitrite or 300 ppm nitrate, pH 4.8-5.6) P. pentosaceus retained 80-94% of its original non-heme catalase activity whereas the activity of L. plantarum was strongly diminished to a level of 6-12% . Thus, although L. plantarum exhibited the higher activity when tested under optimal conditions (71 mg 0 2/1 X min X OD for L. plantarum as compared to 20 mg 0 2 /1 X min X OD for P. pentosaceus) P. pentosaceus appears to be a more suitable candidate for further investigations aiming at the application of starter cultures in sausage fermentation exhibiting catalase activity to minimize deleterious effects of hydrogen peroxide. Acknowledgements. The authors are indebted to Miss E. Zimmermann for her skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. The authors are responsible for the content of this publication.
References Beyer, W. F. Jr., Fridovich, I.: Pseudocatalase from Lactobacillus plantarum: evidence for a homopentameric structure containing two atoms of manganese per subunit. Biochem. 24, 6460-6467 (1985) Bradford, M. M.: A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976) Brosnan, C A .: Regulation of oxygen-dependent lactate metabolism by Lactobacillus plantarum. M. Sc. Thesis, University College, Cork, Ireland (1984) Clare, D. A., Duong, M. N., Darr, D., Archibald, F., Fridovich, I.: Effects of molecular oxygen on the detection of superoxide radical with nitroblue tetrazolium and an activity stain for catalase. Anal. Biochem. 140,532-537 (1984 ) Col/ins, M. D., Rodrigues, U., Ash, C, Aguirre, M., Farrow, J. A . E., Martinez-Murcia, A., Phillips, B. A., Williams, A. M., Wallbanks, S.: Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s rRNA. FEMS Microbiol. Lett. 77,5-12 (1991 ) Condon, S.: Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46, 269-280 (1987) De/wiehe, E. A.: Catalase of Pedioeoeeus eerevisiae. J. Bacteriol. 81,416-418 (1961) De Man, j. C, Rogosa, M., Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135 (1960) Dolin, M. I. The Streptococcus faecalis oxidases for reduced diphosphopyridine nucleotide. III. Isolation and properties of a flavin peroxidase for reduced diphosphopyridine nucleotide. J. BioI. Chern. 225, 557-573 (1957) Felton, E. A., Evans, j. B., Niven, C F. Jr.: Production of catalase by pediococci. J. Bacteriol. 65, 481-482 (1953)
Fridovich, I.: Oxygen is toxic! A review of the chemistry of toxic oxygen products and the enzymes that destroy them. Bioscience 27, 462-466 (1977) Gregory, E. M., Veltri, B. j., Wagner, D. L., Wilkins, T. D.: Carbohydrate repression of catalase synthesis in Bacteroides fragilis. J. Bacteriol. 129, 534-535 (1977) Grufferty, R. C, Condon, S.: Effect of fermentation sugar on hydrogen peroxide accumulation by Streptococcus lactis CI0. J. Dairy Res. 50, 481-489 (1983) Gutekunst, R., De/wiehe, E. A., Seeley, H. W.: Catalase activity in Pediococcus cerevisiae as related to hydrogen ion activity. J. Bacteriol. 74, 694-695 (1957) Hamad, S. H., Boeker, G., Vogel, R. F., Hammes, W. P.: Microbiological and chemical analysis of fermented sorghum dough for Kisra production. Appl. Microbiol. and Biotechnol. 37, 728-731 (1992) Hammes, W. P., Vogel, R. F.: The genera of lactic acid bacteria. In: The lactic acid bacteria, Vol. II, (Wood, B. j. B., Holzapfel, W. H., eds.). In press Hassan, H. M., Fridovich, I.: Regulation of the synthesis of catalase and peroxidase in Escherichia coli. J. BioI. Chern. 253, 6445-6450 (1978) Johnston, M. A., Delwiche, E. A.: Catalase of the Lactobacillaceae. J. Bacteriol. 83, 936-938 (1962) Johnston, M. A., Delwiehe, E. A.: Distribution and characteristics of the catalases of Lactobacillaceae. J. Bacteriol. 90, 347-351 (1965a) Johnston, M. A., Delwiche, E. A.: Isolation and characterization of the cyanide-resistant and azide-resistant catalase of Lactobacillus plantarum. J. Bacteriol. 90, 352-356 (1965b) Jones, D., Deibel, R. H., Niven, C F. Jr.: Catalase activity of two Streptococcus faecalis strains and its enhancement by aerobiosis and added cations. J. Bacteriol. 88, 602-610 (1964) Kaneuchi, C, Masako, S., Komagata, K.: Taxonomic study of Lactobacillus mali Carr and Davies 1970 and related strains: Validation of Lactobacillus mali Carr and Davies 1970 over Lactobacillus yamanashiensis Nonomura 1983. J. Sys. Bacteriol. 38, 269-272 (1988) Kono, Y., Fridovich, I.: Isolation and characterization of the pseudocatalase of Lactobacillus plantarum. J. BioI. Chern. 258, 6015-6019 (1983) Moore, W. E. C, Hash, D. E., Holderman, L. V., Eato, E. P.: Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. Environ. Microbiol. 39, 900-907 (1980) Moore, W. E. C, Moore, L. V. H.: Index of bacterial and yeast nomenclature changes published in the International Journal of Systematic Bacteriology. American Society for Microbiology, Washington, D. C. (1989) Murphy, M. G., Condon, S.: Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium. Arch. Microbiol. Murphy, M. G., Condon, S. : Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium. Arch. Microbiol. 138,49-53 (1984) Nadler, V., Goldberg, I., Hochmann, A.: Comparative study of bacterial catalases. Biochim. Biophys. Acta 882, 234-242 (1986) Schonbaum, G. R., Chance, B.: pp.363-408. In: The enzymes (Boyer, P. D., ed.) 13, 2nd ed., Academic Press, New York (1976) Sulebele, G. A., Rege, D. V.: The nature of the glucose effect on the induced synthesis of catalase in Saccharomyces cerevisiae. Enzymologia 35, 321-334 (1968) Whitten bury, R.: Two types of catalase-like activity in lactic acid bacteria. Nature, 187, 433-434 (1960)
Non-Heme Catalase Activity of Lactic Acid Bacteria Whitten bury, R.: Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. J. Gen. Microbiol. 35, 12-26 (1964) Wolf, G., Hammes, W. P.: Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Arch. Microbiol. 149, 220-224 (1988)
19
Yoshpe-Purer, Y., Henis, Y., Yashphe, J.: Regulation of catalase level in Escherichia coli K12 . Can. J. Microbiol. 23, 84-91 (1977)
Prof. Dr. W. P. Hammes, Institute of Food Technology, Section General Food Technology and Food Microbiology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany