JOURNAL. OF FERMENTATION AND BIOENGINEERING Vol. 85, No. 6, 627-629. 1998

Aerobic Growth of Some Lactic Acid Bacteria Enabled by the External Addition of Peroxidase (Horseradish) to the Culture Medium

MITSUO SAKAMOTO,* YUKIO TANO, TAI UCHIMURA, AND KAZUO KOMAGATA

Department of Agricultural Chemistry, Tokyo University of Agriculture, 1-I-l Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received 5 December 1997/Accepted 2 March 1998

The growth of Lactobacillus fermenturn H3, L. fermenturn Y6, and L. delbrueckii subsp. delbrueckii NRIC 1053T, which do not grow well and accumulate hydrogen peroxide in the culture medium under ordinarily aerobic conditions, was gradually enhanced by increasing the concentration of peroxidase up to 100 U. In addition, the concentration of hydrogen peroxide accumulated was decreased by adding 100 U of peroxidase to the culture medium. These findings indicate that peroxidase efficiently catalyzes the decomposition of hydrogen peroxide accumulated during aerobic growth of the strains tested. A physiological role for peroxidase, for example NADH peroxidase, in the aerobic growth of lactic acid bacteria is discussed.

[Key words: peroxidase, catalase, hydrogen peroxide, lactic acid bacteria, aerobic growth]

In a previous paper (l), we reported the good growth under aerobic conditions of a wide variety of lactic acid bacteria. However, out of the 22 strains tested, excep- tionally Lactobacillus fermentum H3, L. fermenturn Y6, and L. delbrueckii subsp. delbrueckii NRIC 1053T grew well under anaerobic conditions but poorly aerobically. In addition, these three strains accumulated a high con- centration of hydrogen peroxide in the culture medium under aerobic conditions, which caused the growth to cease due to its toxicity. However, the hydrogen peroxide produced was eliminated by the external addi- tion of catalase to the culture medium, leading to growth recovery. In cultures that grew well aerobically, no effect was observed when catalase was added to the culture medium. Generally, these findings appear to indi- cate the existence of some form of rational mechanism that enables lactic acid bacteria to eliminate hydrogen peroxide because these bacteria do not have catalase, which is widely distributed in aerobic bacteria.

Hydrogen peroxide is the product of certain flavo- protein oxidases of lactic acid bacteria (l-3). Hydrogen peroxide produced by HzOz-forming NADH oxidase or other oxidases is broken down to water by NADH peroxidase. Thus, NADH peroxidase is considered to be concerned in the aerobic growth of and elimination of hydrogen peroxide by lactic acid bacteria (1). NADH oxidase and NADH peroxidase have been reported to be widely distributed in lactic acid bacteria, but the activity of NADH peroxidase is known to be weak compared with that of NADH oxidase (1, 4-6). Out of the 22 strains tested, only the three above-mentioned strains, L. fermentum H3, L. fermentum Y6, and L. delbrueckii subsp. delbrueckii NRIC 1053T showed HzOz-forming NADH oxidase activity and no or only weak NADH peroxidase activity (1). In addition, the HzOz-forming NADH oxidase activities of these strains were induced under aerobic conditions, while the NADH peroxidase activity of L. delbrueckii subsp. delbrueckii NRIC 1053= was low in aerobically grown cells in comparison with

* Corresponding author. Present address: Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan.

that in cells grown anaerobically (unpublished data). Since little is known as to whether NADH peroxidase efficiently catalyzes the decomposition of hydrogen peroxide in vivo, investigating the elimination of hydro- gen peroxide accumulated in the culture medium will be useful in improving our understanding of the aer- obic growth of lactic acid bacteria. Here, we report on the effect of the external addition of peroxidase (horse- radish) on the aerobic growth of some lactic acid bac- teria that do not grow well under ordinarily aerobic conditions.

L. fermentum H3, L. fermentum Y6, and L. del- brueckii subsp. delbrueckii NRIC 1053* were used in the study. Pediococcus pentosaceus NRIC 1105 was em- ployed as a control because this strain was found to grow well under anaerobic and aerobic conditions, and not to accumulate hydrogen peroxide in the culture me- dium (1). L. fermentum H3, L. fermentum Y6, and L. delbrueckii subsp. delbrueckii NRIC 1053T were cultured at 37°C; P. pentosaceus NRIC 1105 was grown at 30°C. All cultures were aerobically grown as previously described (l), using MRS broth (Difco Laboratories, Detroit, MI, USA). Peroxidase (horseradish, Wako Pure Chemical Industries, Osaka) was added at a concentra- tion of 100mU or 1, 10, 100, or 1OOOU per lOOmI of the culture medium at the time of inoculation. Growth was monitored spectrophotometrically by reading the optical density at 660 nm. The concentration of hydrogen peroxide accumulated in the culture medium was deter- mined as previously described (1).

The growth of L. fermenturn H3 and L. fermentum Y6 was gradually enhanced by increasing the concentra- tion of peroxidase up to 1OOOU (Fig. 1). The growth of L. delbrueckii subsp. delbrueckii NRIC 1053= was stimu- lated by adding peroxidase to the culture medium but was inhibited at a high concentration of peroxidase (Fig. 1). The growth of P. pentosaceus NRIC 1105 was not affected by the addition of peroxidase. The concentra- tion of hydrogen peroxide in the culture medium was low in the presence of peroxidase compared with that in its absence (Fig. 2).

In this study, we found that the external addition of peroxidase enabled some lactic acid bacteria to grow

627

628 SAKAMOTO ET AL. J. FERMENT. BIOENG..

2.5 A

E 1.5-

2 z Ci

d l.O-

0.5 -

0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25

Time (h) Time (h) Time (h)

2.5

FIG. 1. Effect of concentration of peroxidase on growth. (A) L. delbrueckii subsp. de&rue&ii NRIC 1053T; (B) L. fermenlum H3; (C) L. fermenturn Y6. Symbols: q , 0 mU; A, 100 mu; 0, 1 U; n , 10 U; 0, 100 U; A, 1006 U.

aerobically, and that the concentration of hydrogen peroxide accumulated in the culture medium was decreased by adding peroxidase. These findings indicate that peroxidase efficiently catalyzes the decomposition of hydrogen peroxide accumulated during the aerobic growth of the strains tested. In contrast, the growth of P. pentosaceus NRIC 1105 was not affected by the exter- nal addition of peroxidase. This strain did not accumu- late hydrogen peroxide in the culture medium under anaerobic and aerobic conditions, and showed NADH peroxidase activity (1). Dobrogosz reported that P. pentosaceus strains possessed catalase and that external cataiase did not stimulate their growth (7). This might

be due to no accumulation of hydrogen peroxide by the strains. In general, catalase is not found in lactic acid bacteria; although the production of a pseudocatalase has been reported, this is produced under rather limited conditions (8).

On the basis of the above, it is reasonable to conclude that peroxidase, probably NADH peroxidase, plays a role in vivo in eliminating hydrogen peroxide produced during the aerobic growth of lactic acid bacteria. In addi- tion, H202-forming NADH oxidases of Amphibacillus xylanus and Sporolactobacillus inulinus have recently been reported to show extremely high peroxide reductase activity for both hydrogen peroxide and alkyl hydro-

8 2.5

7

2.0 6

I 0

8 2.5

6

.-O 5 10 15 20 25 30 -

Time (h) Time (h)

0 5 10 15 20 25 30

Time (h)

8

7

6

FIG. 2. Relationship between growth and the accumulation of hydrogen peroxide. (A) L. delbmeckii subsp. delbrueckii NRIC 1053T; (B) L. fermenturn H3; (C) L. fermenturn Y6. Symbols: 0, growth (no peroxidase added); 0, growth (100 U of peroxidase added); A, Hz02 (no peroxidase added); A, Hz02 (100 U of peroxidase added).

VOL. 85. 1998 NOTES 629

peroxide in the presence of a 22-kDa protein component (AhpC), and the AhpC-NADH oxidase system is con- sidered to function as a scavenging system for hydro- gen peroxide produced by such bacteria grown under aerobic conditions (9, 10). However, it is not clear whether such a system functions in the strains tested in this study. From a physiological point of view, it would appear important for lactic acid bacteria growing under aerobic conditions to have a mechanism that can func- tion instead of catalase to eliminate hydrogen peroxide and that the mechanism should function efficiently. The aerobic growth of a wide variety of lactic acid bacteria despite their lack of cataiase can be accounted for by the presence of such an appropriate mechanism to eliminate hydrogen peroxide and other peroxides. This mech- anism(s) will elucidate further our knowledge on the growth of lactic acid bacteria in various natural environ- ments, under both aerobic and anaerobic conditions.

REFERENCES

Sakamoto, M. and Komagata, K.: Aerobic growth of and activ- ities of NADH oxidase and NADH peroxidase in lactic acid bacteria. J. Ferment. Bioeng., 82, 210-216 (1996). Condon, S.: Responses of lactic acid bacteria to oxygen. FEMS Microbial. Rev.. 46. 269-280 (1987). Sakamoto, M., U&m&a, T., and Kimagata, K.: Comparison of HzO-forming NADH oxidase from Leuconostoc mesen- teroides subsp. mesenteroides NRIC 1541’ and HzOz-forming

4.

5.

6.

I.

8.

9.

10.

NADH oxidase from Sporolactobacillus inulinus NRlC I 133’. J. Ferment. Bioeng., 82, 531-537 (1996). Brown, J. P. and van Demark, P. J.: Respiration of Lacto- bacillus casei. Can. J. Microbial., 14, 829-835 (1968). Anders, R. F., Hogg, D. M., and Jago, G. R.: Formation of hydrogen peroxide by group N streptococci and its effect on their growth and metabolism. Anul. Microbial., 19. 608-612 (197Oj:

. .

Smart, J.B. and Thomas, T.D.: Effecl of oxygen on lactose metabolism in lactic streptococci. Appl. Environ. Microbial., 53, 533-541 (1987). Dobrogosz, W. J. and Stone, R. W.: Oxidative metabolism in

Pediococcus pentosaceus, I. Role of oxygen and catalase. J. Bacterial., 84, 716723 (1962). Engesser. D. M. and Hammes. W. P.: Non heme catalase activ- ity of lactic acid bacteria. System. Appl. Microbial., 17, 1 l-19 (1994). Niimura, Y., Poole, L. B., and Massey, V.: AmphibaciNus xylanus NADH oxidase and Salmonella typhimurium alkyl- hydroperoxide reductase flavoprotein components show ex- tremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component. J. Biol. Chem.. 270. 25645-25650 (1995). Nishiyama, i., Massey, V., Aniai, Y., Watanabe, T., Miyaji, T., Uchimura, T., Kozaki, M., Suzuki, H., and Niimura, Y.: Purification and characterization of Sporolactobacillus inulinus NADH oxidase and its physiological role in aerobic metabolism of the bacterium. J. Ferment. Bioeng., 84, 22-27 (1997).