JOURNAL OF FERMENTATION AND BIOENOINEERING Vol. 82, No. 3, 210-216. 1996

Aerobic Growth of and Activities of NADH Oxidase and NADH Peroxidase in Lactic Acid Bacteria

MITSUO SAKAMOTO* AND KAZUO KOMAGATA

Department of Agricultural Chemistry, Tokyo University of Agriculture, I-I-1 Sakuragaoka, Setagaya-ku, Tokyo 156, Japan

Received 4 March 1996IAccepted 7 June 1996

Aerobic growth of 22 strains of lactic acid bacteria of the genera Lactobacillus, Pediococcus, Leuconostoc, Streptococcus, and Enterococcus was investigated. Most strains grew well under aerobic conditions in parallel with an increase in the growth yield. Homofermentatives and heterofermentatives produced acetic acid under aerobic conditions with a decrease in lactic acid or ethanol in the culture medium. NADH oxidase and NADH peroxidase activities were detected in a wide variety of the bacteria. Most strains showed the activity of NADH oxidase that produced water, and some strains showed the activity of NADH oxidase that produced hydrogen peroxide. L. delbrueckii subsp. delbrueckii NRIC 1053 and L. fermenturn H3 and Y6 accumulated hydrogen peroxide in the culture medium, and these strains grew poorly under aerobic conditions, which might have been due to hydrogen peroxide toxicity. The hydrogen peroxide was eliminated by the addition of catalase to the culture medium. The activities of NADH oxidase and NADH peroxidase were enhanced by the addition of FAD to the reaction mixture. A possible role of FAD in the reaction was discussed.

[Key words: NADH oxidase, NADH peroxidase, lactic acid bacteria, homofermentative, heterofermentative]

Lactic acid bacteria are believed to be facultatively anaerobic or microaerophilic, obtain their energy through the Embden-Meyerhof-Parnas pathway or phos- phoketolase pathway, and grow poorly in the presence of oxygen. In addition, these bacteria do not utilize ox- ygen for energy production because they lack the respira- tory chain and catalase. However, some strains grow well under aerobic conditions (1, 2). Lactobacillus plantarum strains produce a large amount of acetic acid aerobically (I), and some heterofermentatives utilize oxygen and produce lactic acid and acetic acid as main end products from glucose (3). This indicates that some changes of carbohydrate metabolism of lactic acid bacteria occur under aerobic conditions.

Maintenance of a redox balance of NAD/NADH is important for the energy production and carbohydrate metabolism, and certain flavoproteins associate with the electron transport system of lactic acid bacteria (4). NADH oxidase [NADH: (oxygen-acceptor) oxidoreduc- tase, EC 1.6.991 is a flavoprotein, and reacts with oxy- gen to produce either water or hydrogen peroxide. In ad- dition, the hydrogen peroxide produced is broken down to water by NADH peroxidase (EC 1.11.1.1). Streptococ- cus (Enterococcus) faecaIis (5, 6), Streptococcus mutans (7), Lactobacillus plantarum (8), and Leuconostoc mesen- teroides (9, 10) contain NADH oxidase. Distribution of NADH oxidase and NADH peroxidase in various strep- tococci has been reported (11). However, the above-men- tioned metabolic change and the distributions of NADH oxidase and NADH peroxidase in a number of lactic acid bacteria have not yet been investigated in detail.

This paper deals with the aerobic growth and the dis- tributions of NADH oxidase and NADH peroxidase in lactic acid bacteria of industrial importance of the genera Lactobacillus, Pediococcus, Leuconostoc, Strep- tococcus, and Enterococcus.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains and growth conditions The strains used in our investigations are listed in Table 1. They were grown on glucose-yeast extract-peptone medium (GYP) consisting of glucose, lO.Og; yeast extract (Difco Laboratories, Detroit, Mich., USA), 10.0 g; peptone, lO.Og; sodium acetate, lO.Og; Tween 80, 0.25 g; MgS04. 7Hz0, 0.2 g; MnS04.4Hz0, 0.01 g; FeS04. 7H20, 0.01 g; NaCl, 0.01 g; and water to 1,000 ml; pH was adjusted to 6.8 with NaOH. The concentration of glucose was reduced to 4 g/l ,000 ml and sodium acetate was replaced with 0.6M potassium phosphate buffer (pH 6.8) for the analysis of end products.

The bacteria were aerobically cultivated in cotton- plugged Erlenmeyer flasks with shaking. Anaerobic con- ditions were produced by flushing anaerobic bottles with nitrogen gas and sealing them with tight-fitting rubber stoppers. In addition, 0.13 mM of titanium (III) citrate was added to the medium to lower the redox potential (12). Growth was monitored spectrophotometrically by reading the optical density at 660nm. Cultures were incubated at 30 or 37°C as shown in Table 1.

Analysis Lactic acid and acetic acid were deter- mined by HPLC. Acetoin was determined using a gas chromatograph (Shimadzu GC 9A; Shimadzu, Kyoto). Ethanol was determined using an F-kit ethanol (Boehrin- ger Mannheim Yamanouchi, Tokyo), and glucose was determined using a Glucose C II test Wako (Wako Pure Chemical Industries, Ltd., Osaka).

Dry weight determination Cells were collected by centrifugation, washed twice with 50mM potassium phosphate buffer (pH 7.0), and added to preweighed tubes. The cells were then dried to a constant weight by lyophilization and reweighed.

Preparation of cell-free extracts Cells were collect- ed at a late-log growth phase, washed twice with 50mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA (50mM PPB), and suspended in 50 mM PPB containing 1 mM phenylmethanesulfonylfluoride (PMSF).

210

VOL. 82, 1996 NADH OXIDASE AND NADH PEROXIDASE OF LACTIC ACID BACTERIA 211

TABLE 1. Bacterial strains used and effects of aeration on the growth yield

Species

Lactobacillus delbrueckii subsp. delbrueckii Lactobacillus delbrueckii subsp. lactis Lactobacillus acidophilus

Lactobacillus mali subsp. yamanashiensis Lactobacillus buchneri

Lactobacillus coryniformis subsp. coryniformis Lactobacillus casei subsp. casei Lactobacillus rhamnosus Pediococcus acidilactici

Strain numbera

NRIC 1053’ NRIC 1683r

H8 W6

NRIC 1076T NRIC 1040T NRIC 1684T NRIC 1067’

H3 Y6

NRIC 1638T NRIC 1042T NRIC 1043T NRIC O097T NRIC 1096

Culture temperature Growth yield (g dry weight/m01 glucose)

Anaerobic Aerobic

31°C 23.0 33.7 37OC 20.2 19.9 37°C 17.1 8.1 37°C 17.2 10.3 30°C 19.0 9.3 30°C 9.6 17.6

Lartobacillus brevis Lactobacillus plantarum Lactobacillus fermenturn

30°C 30°C 37°C 31°C 30°C 30°C 30°C 37OC 31°C 30°C 30°C 30°C 30°C 30°C 37OC 30°C

9.7 18.7 13.5 10.7 22.4 20.5 22.3 24.2 18.8 20.4 18.7 14.4 10.8 11.0

18.5 27.9 10.8 15.1 21.9 30.7 33.4 18.7 25.4 30.6 25.7 30.0 21.8 14.8 19.1 10.7

Pediococcus pentosaceus NRIC 0099T NRIC 1105

Leuconostoc lactis NRIC 1540T Leuconostoc mesenteroides subsp. cremoris NRIC 1538T Leuconostoc mesenteroides subsp. mesenteroides NRIC 1541T Streptococcus thermophilus NRIC 1747 Enterococcus faecium NRIC 1140

21.2 10.5

a T, Type strain; NRIC, NODAI Research Institute-Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan

The cells were then treated with lysozyme and N- acetylmuramidase SG (Seikagaku Kogyo Co., Tokyo) for 30-60 min, and disrupted by passing them twice through a French pressure cell (Aminco Instrument Co., Silver Spring, MD, USA) at 1,400 kg/cm2. The suspen- sion was centrifuged at 31,000 X g for 30 min and the pellet was discarded. The supernatant was recentrifuged at 187,000x g for 90min to obtain a cytoplasmic frac- tion.

Enzyme assays The activities of NADH oxidase and NADH peroxidase were assayed spectrophotometri- tally in 50 mM PPB containing 0.1 mM NADH with or without 50 /IM FAD. The activity of NADH peroxidase was determined independently from the activity of NADH oxidase by adding hydrogen peroxide to a reac- tion mixture to give a final concentration of 1 mM, replacing the atmosphere in a sealed cuvette with nitro- gen gas, and incubating it anaerobically. Assays of all enzyme activities were carried out at 30 or 37°C. One unit of enzyme activity was defined as the amount of enzyme (mg protein) which catalyzed the oxidation of 1 FM NADH min l. Protein was measured by the dye-binding method (13) using bovine serum albumin (BSA) as a standard.

Oxygen consumption and production of hydrogen peroxide The activity of NADH oxidase was also monitored in terms of oxygen consumption rate. Oxygen consumption was measured at 30 or 37°C with a Clark oxygen electrode (YSI model 5331 oxygen monitor; Yellow Springs Instrument Co., Yellow Springs, Ohio, USA). The production of hydrogen peroxide was deter- mined by adding catalase to the reaction mixture, which is a system similar to that used for the determination of oxygen consumption.

Accumulation of hydrogen peroxide The concentra- tion of hydrogen peroxide was determined every two hours. The culture broth was clarified by centrifugation and the supernatant was used for determining hydrogen peroxide concentration. Hydrogen peroxide concentra- tion was determined spectrophotometrically as follows:

100 ~1 of the supernatant, 200 1-11 of 6 mM 4-aminoantipy- rine, 200 1_t1 of 21 mM 3,5-dichlorobenzene-2-hydroxy sulfonate, and horseradish peroxidase were incubated at 30°C for 15 min, and the absorbance was measured at 546 nm spectrophotometrically. Catalase (Boehringer Mannheim Yamanouchi) was added (0.25 ml per 100 ml of culture medium) at the time of inoculation to the culture medium to eliminate hydrogen peroxide toxicity. L. delbrueckii subsp. delbrueckii NRIC 1053 and L. fer- mentum H3 and Y6 were investigated.

RESULTS

Effect of oxygen on growth Out of the 22 strains listed in Table 1, 15, L. delbrueckii subsp. luctis NRIC 1683, L. buchneri NRIC 1040, L. brevis NRIC 1684, L. plantarum NRIC 1067, L. coryniformis subsp. corynifor- mis NRIC 1638, L. casei subsp. cusei NRIC 1042, L. rhamnosus NRIC 1043, P. acidilactici NRIC 1096, P. pentosaceus NRIC 0099 and 1105, Leuconostoc lactis NRIC 1540, Leuconostoc mesenteroides subsp. cremoris NRIC 1538, Leuconostoc mesenteroides subsp. mesen- teroides NRIC 1541, S. thermophilus NRIC 1747, and E. faecium NRIC 1140, grew well under aerobic condi- tions. Growth patterns varied from strain to strain. Typi- cal patterns are shown in Fig. 1.

L. buchneri NRIC 1040, L. brevis NRIC 1684, L. plan- tarum NRIC 1067, L. cusei subsp. cusei NRIC 1042, L. rhamnosus NRIC 1043, P. acidilactici NRIC 1096, P. pentosaceus NRIC 0099 and 1105 (Fig. lA), Leu- conostoc lactis NRIC 1540, Leuconostoc mesenteroides subsp. cremoris NRIC 1538, and Leuconostoc mesen- teroides subsp. mesenteroides NRIC 1541 showed a higher specific growth rate and growth yield under aero- bic conditions than under anaerobic conditions.

L. delbrueckii subsp. luctis NRIC 1683 (Fig. lB), L. coryniformis subsp. coryniformis NRIC 1638, S. ther- mophilus NRIC 1747, and E. fuecium NRIC 1140 showed almost the same growth patterns and growth yields under aerobic and anaerobic conditions.

212 SAKAMOTO AND KOMAGATA J. FERMENT. BIOENG.,

2

z

g 1. .

2

0

000 r o.c 0 10 20 so 10 20 a0

J’J~o(~) rim(h))

10 20

nme(h)

w nme(h)

FIG. 1. Comparison of aerobic growth and anaerobic growth. (A) P. penfosaceus NRIC 1105; (B) L. delbrueckii subsp. lactis NRIC 16~33~; (C) L. delbrueckii subsp. delbrueckii NRIC 1053T; (D) L. fermenturn Y6. Symbols: 0, aerobic; l , anaerobic.

L. delbrueckii subsp. delbrueckii NRIC 1053 grew faster under aerobic conditions than under anaerobic conditions but eventually stopped growing under aerobic conditions (Fig. 1C). L. fermentum Y6 grew under aero- bic conditions as well as under anaerobic conditions until 6 h but the growth rate was gradually decreased under aerobic conditions (Fig. 1D). On the other hand, L. acidophilus H8 and W6, L. mali subsp. yamanashiensis NRIC 1076, L. fermentum H3, and P. acidilactici NRIC 0097 showed lower growth yields under aerobic condi- tions than under anaerobic conditions. These strains showed relatively poor growth under aerobic conditions.

homofermentatives produced acetic acid with a decrease in lactic acid in the culture medium under aerobic condi- tions. In addition, heterofermentatives produced acetic acid with a decrease in ethanol in the culture medium

Accumulation of hydrogen peroxide L. delbrueckii subsp. delbrueckii NRIC 1053, L. fermentum H3 and Y6, and Leuconostoc mesenteroides subsp. mesen- teroides NRIC 1541 accumulated hydrogen peroxide in proportion to the cell growth, and the concentration of hydrogen peroxide in the culture media of these strains reached 4.9, 1.8, 2.0, and 0.36mM, respectively, at the end of culture under aerobic conditions. No accumula- tion of hydrogen peroxide was detected the during cul- ture under anaerobic conditions. Other strains tested did not accumulate a detectable amount of hydrogen peroxide in the culture medium. Typical patterns of growth and accumulation of hydrogen peroxide are shown in Fig. 2. When catalase was added to the culture medium, L. delbrueckii subsp. delbrueckii NRIC 1053 and L. fermentum H3 and Y6 grew well under aerobic conditions (Fig. 3). No hydrogen peroxide was detected in the aerobic cultures of these three strains.

End products of glucose metabolism In general,

(Table 2). The homofermentatives, L. plantarum NRIC 1067, P.

acidilactici NRIC 1096, P. pentosaceus NRIC 0099, and S. thermophilus NRIC 1747 produced large amounts of acetic acid, 15.0, 22.8, 27.4, and 19.2mM, respectively, under aerobic conditions. On the other hand, L. acido- philus H8 and W6, P. pentosaceus NRIC 1105, and E. faecium NRIC 1140 did not produce acetic acid. In addition, some strains produced acetoin under aerobic conditions, and S. thermophilus NRIC 1747 produced 8.1 mM acetoin.

The heterofermentatives, L. buchneri NRIC 1040, L. brevis NRIC 1684, L. fermentum Y6, Leuconostoc mesenteroides subsp. cremoris NRIC 1538, and Leu- conostoc mesenteroides subsp. mesenteroides NRIC 1541 produced large amounts of acetic acid (12.8-24.5 mM) under aerobic conditions. No acetoin was detected in these cultures.

NADH oxidase and NADH peroxidase Out of the 22 strains, 20 showed NADH oxidase [NADH: (oxygen- acceptor) oxidoreductase] activity (Table 3). L. acidophi- lus H8 and L. acidophilus W6 showed no NADH oxi- dase activity. The cytoplasmic fraction of many strains showed NADH oxidase activity, and the membrane frac- tion of a few strains showed NADH oxidase activity but the NADH oxidase activity in the membrane fractions was negligibly low compared with that in the cytoplas-

VOL. 82, 1996 NADH OXIDASE AND NADH PEROXIDASE OF LACTIC ACID BACTERIA 213

4.0

Q a.0 1

4 2.0 =

0.0 0 10 20

rim(h))

I

2.0.

ii

75

6 1.0.

d

s a.0 E

z 2.0 I”

C i 6.0

0. 0.0 0 10 20

mm(h) 10 20

mm W

6.0

4.0

a.0 S

: 2.0 $

1.0

0.0

FIG. 2. Relationship between growth and the accumulation of hydrogen peroxide. (A) L. delbrueckii subsp. delbrueckii NRIC 1053r; (B) L. fermentum H3; (C) Leuconostoc mesenteroides subsp. mesenteroidesNRIC 1541T; (D) P. acidilacticiNRIC 1096. Symbols: 0, aerobic growth; 0, anaerobic growth; A, Hz02 (aerobic); A, Hz02 (anaerobic).

mic fractions (data not shown). The NADH oxidase was subsp. delbrueckii NRIC 1053 and 9 other strains. The classified into two types, based on the response to the presence of FAD. The activity of NADH oxidase type 1

activity of NADH oxidase type 2 was not enhanced by the addition of FAD to the reaction mixture. This was

was significantly enhanced by the addition of FAD to found in L. delbrueckii subsp. lactis NRIC 1683 and 9 the reaction mixture. This was found in L. delbrueckii other strains, as shown in Table 3.

8” A

0

I 0

0 r 2.0

I

B r 6 lo- d .

n-(h) am FIG. 3. Effect of catalase on growth. (A) L. delbrueckii subsp. delbrueckii NRIC 1053T; (B) L. fermentum H3; (C) L. fermentum Y6.

Symbols: 13, no catalase added; 0, catalase added.

214 SAKAMOTO AND KOMAGATA J. FERMENT. BIOENG.,

TABLE 2. Effects of aeration on the production of end products

Strains Condition Consumed sugar End product (mM) (mM) Lactate Acetate Ethanol Acetoin

L. delbrueckii subsp. delbrueckii

subsp. lactis

L. acidophilus

L. mali subsp. yamanashiensis

L. buchneri

L. brevis

L. plantarum

L. fermentum

L. coryniformis subsp. coryniformis

L. casei subsp. casei

L. rhamnosus

P. acidilactici

P. pentosaceus

Leuconostoc lactis

NRIC 1053T

NRIC 1683I

H8

W6

NRIC 1076T

NRIC 1040=

NRIC 1684T

NRIC 1067T

H3

Y6

NRIC 1638r

NRIC 1042r

NRIC 1043T

NRIC O097T

NRIC 1096

NRIC 0099T

NRIC 1105

NRIC 1540’

Leuconostoc mesenteroides subsp. cremoris NRIC 1538T

Leuconostoc mesenteroides subsp. mesenteroides NRIC 1541’

S. thermophilus NRIC 1747

E. faecium NRIC 1140

Aerobic 12.8 22.8 8.6 0 0 Anaerobic 22.2 46.1 0 0 0 Aerobic 18.9 25.5 8.8 0 0 Anaerobic 21.1 36.2 0 0 0 Aerobic 10.1 22.3 0 0 0 Anaerobic 21.0 41.2 0 0 0 Aerobic 13.1 23.0 0 0 0 Anaerobic 21.5 42.3 0 0 0

Aerobic 22.2 36.2 8.2 0 0 Anaerobic 22.2 43.7 0 0 0 Aerobic 22.1 19.5 24.5 2.1 0 Anaerobic 22.2 23.4 0 22.0 0 Aerobic 21.9 26.6 20.3 3.7 0 Anaerobic 22.2 21.7 0 23.1 0 Aerobic 20.3 27.8 15.0 5.0 0 Anaerobic 22.2 41.7 0 7.0 0 Aerobic 16.8 15.3 0 14.7 0 Anaerobic 21.5 21.3 0 23.4 0 Aerobic 21.0 20.5 15.5 6.1 0 Anaerobic 22.0 23.2 0 21.5 0

Aerobic 20.1 38.1 5.7 0 0 Anaerobic 22.1 43.2 0 0 0

Aerobic 22.1 36.1 6.3 0 0.4 Anaerobic 22.1 41.3 0 0 0 Aerobic 22.1 29.9 5.6 0 0.59 Anaerobic 22.1 39.1 0 0 0 Aerobic 22.2 33.0 7.2 0 0 Anaerobic 22.2 43.4 0 0 0 Aerobic 22.2 18.1 22.8 0 0.55 Anaerobic 22.2 46.4 0 0 0 Aerobic 22.1 12.8 27.4 0 0.9 Anaerobic 22.2 45.3 0 0 0 Aerobic 22.2 43.8 0 0 1.1 Anaerobic 22.2 45.8 0 0 0 Aerobic 22.2 23.1 4.1 21.0 0 Anaerobic 22.2 24.2 0 23.8 0

Aerobic 22.0 24.9 12.8 9.3 0 Anaerobic 22.2 23.7 0 22.5 0

Aerobic 21.9 19.1 15.3 3.3 0 Anaerobic 22.1 18.6 0 18.4 0 Aerobic 21.8 9.3 19.2 0 8.1 Anaerobic 22.2 45.2 0 0 0 Aerobic 22.1 43.6 0 0 0 Anaerobic 22.1 45.1 0 0 0

Out of the 20 strains with NADH oxidase activity, 17 possessed an HzO-forming NADH oxidase, and 3 (L. del- brueckii subsp. delbrueckii NRIC 1053, L. fermentum H3 and Y6) possessed an HzOz-forming NADH oxidase. Eighteen strains showed NADH peroxidase activity, but L. fermentum H3 and Y6 did not.

DISCUSSION

In this study, we found that a large number of lactic acid bacteria grew well under aerobic conditions. The growth yields of P. acidilactici NRIC 1096 and P. pen- tosaceus NRIC 0099 were 25.4 and 30.6g of dry weight per mol of glucose metabolized, respectively, under aero- bic conditions. Assuming Y*Tp to be approximately 10.5,

the above data indicate that these two strains produced 2.4 and 2.9 mol of ATP per mol of glucose metabolized under aerobic conditions, and 1.8 and 1.9 mol of ATP per mol of glucose metabolized under anaerobic condi- tions, respectively. Consequently, these strains would have produced 0.6 and 1 .O mol more ATP under aerobic conditions than under anaerobic conditions. It is noteworthy that out of the I5 homofermentatives, 11 produced acetic acid in addition to lactic acid under aero- bic conditions, and out of the 7 heterofermentatives, 6 produced acetic acid instead of ethanol. The above- mentioned findings indicate that some metabolic changes occur in the production of energy under aerobic con- ditions in lactic acid bacteria.

Out of the 22 strains tested, 20 showed NADH oxi-

VOL. 82, 1996 NADH OXIDASE AND NADH PEROXIDASE OF LACTIC ACID BACTERIA 215

TABLE 3. NADH oxidase and NADH peroxidase activities of various strains

Specific activity (mU/mg)

Strains

-FAD

NADH oxidase NADH peroxidase

+ FAD end product -FAD + FAD

L. delbrueckii subsp. delbrueckii L. delbrueckii subsp. lactis L. acidophilus

L. mali subsp. yamanashiensis L. buchneri L. brevis L. plantarum L. fertnentum

L. coryniformis subsp. coryniformis L. casei subsp. casei L. rhamnosus P. acidilactici

P. pentosaceus

NRIC 1053T NRIC 1683T

H8 W6

NRIC 1076T NRIC 1040T NRIC 1684T NRIC 1067T

H3 Y6

NRIC 163ST NRIC 1042T NRIC 1043T NRIC 0097T NRIC 1096 NRIC 0099T NRIC 1105 NRIC 154Or

5.0 10.7

N.D. N.D. 64.7 93.8

223.7 55.6

1.4 1.4

10.7 42.3 63.6

3.1 234.3 N.D. 22.7 23.8

67.6 19.1

N.D. N.D. 59.3

226.6 210.5

37.1 9.4 9.6

19.1 303.0 411.0

18.6 397.1 249.4

70.9 42.9

H202

H20

H2O

H20

H2O

Hz0

H202

H202

Hz0

H20

H20

H20

Hz0

H2O

H20

H20

10.0 51.1 N.T. N.T. 11.1 10.3 23.4 24.6 N.D. N.D.

9.8 31.5 29.4

3.5 14.1 25.7

6.5 59.0

106.5 81.7

N.T. N.T. 20.3 22.1 10.4 21.1

N.D. N.D.

12.3 29.6 23.5

6.4 16.5 32.1

5.5 49.8

Leuconostoc mesenteroides subsp. cremoris NRIC 1538’ 93.0 240.3 Hi0 71.5 81.7 Leuconostoc mesenteroides subsp. mesenteroides NRIC 1541T 741.6 820.2 H20 41.6 83.3 S. thermophilus NRIC 1747 465.6 557.3 H20 32.5 44.3 E. faecium NRIC 1140 71.4 90.9 H20 33.9 28.3

dase activity, and 18 showed NADH peroxidase activity. This indicates that NADH oxidase and NADH peroxi- dase are present in a wide variety of lactic acid bacteria. L. delbrueckii subsp. delbrueckii NRIC 1053 might produce water through coupling of NADH oxidase with NADH peroxidase. Thus lactic acid bacteria may eliminate oxygen and produce water in the above-men- tioned ways. L. acidophilus H8 and W6 did not show NADH oxidase activity, and L. fermentum H3 and Y6 did not show NADH peroxidase activity. These four strains grew poorly under aerobic conditions. In general, the activities of NADH oxidase and NADH peroxidase were induced under aerobic conditions (data not shown). The above-mentioned findings demonstrate that NADH oxidase and NADH peroxidase play roles in aerobic growth of lactic acid bacteria.

NADH oxidases have been purified from several bac- terial strains and characterized as flavoproteins (6, 7, 10, 14-18). The activities of some NADH oxidases are en- hanced by the addition of FAD to the reaction mixture (7, 16, 17, 19). The presence of FAD-requiring oxidases and intracellular FAD in L. casei has been reported (20). Recently, excess FAD was reported to lower the K,,, value of the NADH oxidase of Amphibacillus xylanus for oxygen (19). A number of strains tested in the present study produced NADH oxidase, the activities of which were enhanced by the addition of FAD to the reaction mixture. In addition, several strains produced NADH peroxidases, the activities of which were en- hanced by the addition of FAD to the reaction mixture. FAD-requiring oxidases may be found in many lactic acid bacteria, and the intracellular FAD may play an im- portant role in aerobic growth. Elucidation of the detail of the roles of FAD for NADH oxidase and NADH peroxidase activities is left for further study.

L. delbrueckii subsp. delbrueckii NRIC 1053 showed NADH oxidase and NADH peroxidase activities, and accumulated hydrogen peroxide in the culture medium under aerobic conditions. Accumulation of the hydrogen

peroxide might be due to the overall activity of an H202- production system being greater than that of an Hz02- elimination system. The inhibition of the growth of L. delbrueckii subsp. delbrueckii NRIC 1053 might thus be due to hydrogen peroxide toxicity. L. fermentum H3 and Y6 accumulated hydrogen peroxide under aerobic conditions, but they did not show NADH peroxidase ac- tivity. It seems likely that the growth of these strains was inhibited by the hydrogen peroxide produced. However, when catalase was in the medium, hydrogen peroxide was split into water and oxygen, and L. delbrueckii subsp. delbrueckii NRIC 1053 and L. fermenturn H3 and Y6 grew well under aerobic conditions. This finding indicates that NADH peroxidase plays a role in detox- ification of hydrogen peroxide in lactic acid bacteria, as does catalase. Thus lactic acid bacteria might have gained a primitive respiratory system for adaptation to aerobic conditions during evolution.

ACKNOWLEDGMENTS

We thank Dr. Youichi Niimura, Department of Food Science and Technology, Tokyo University of Agriculture, Prof. Emeritus Kei Yamanaka, Kyoto University, and Dr. H. W. Doelle, Depart- ment of Microbiology, University of Queensland for helpful discus- sions.

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216 SAKAMOTO AND KOMAGATA

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