1161 Journal o f Food Protection, Vol. 77, No. 7, 2014, Pages 1161-1167 doi:10.4315/0362-028X.JFP-13-475 Copyright © , International Association for Food Protection

Altered Superoxide Dismutase Activity by Carbohydrate Utilization in a Lactococcus lactis Strain H. KIMOTO-NIRA,* N. MORIYA, H. OHMORI,

and

C. SUZUKI

NARO Institute o f Livestock and Grassland Science, Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan MS 13-475: Received 23 October 2013/Accepted 7 March 2014

A BSTR A CT Reactive oxygen species, such as superoxide, can damage cellular components, such as proteins, lipids, and DNA. Superoxide dismutase (SOD) enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and dioxygen. SOD is present in most lactococcal bacteria, which are commonly used as starters for manufacturing fermented dairy products and may have health benefits when taken orally. We assessed the effects of carbohydrate use on SOD activity in lactococci. In Lactococcus lactis ssp. lactis G50, the SOD activity of cells grown on lactose and galactose was higher than that on glucose; in Lactococcus lactis ssp. cremoris H61, SOD activity was independent of the type of carbohydrate used. We also investigated the activity of NADH oxidase, which is related to the production of superoxide in strains G50 and H61. Activity was highest in G50 cells grown on lactose, lower on galactose, and lowest on glucose, whereas activity in H61 cells did not differ with the carbohydrate source used. The SOD and NADH oxidase activities of strain G50 in three carbohydrates were linked. Strain G50 fermented lactose and galactose to lactate, acetate, formate, and ethanol (mixed-acid fermentation) and fermented glucose to mainly lactate (homolactic fermentation). Strain H 61 fermented glucose, lactose, and galactose to mainly lactate (homolactic fermentation). In strain G50, when growth efficiency was reduced by adding a metabolic inhibitor to the growth medium, SOD activity was higher than in the control; however, the metabolism was homofermentative. Aerobic conditions, but not glucose-limited conditions, increased SOD activity, and mixed-acid fermentation occurred. We conclude that the effect of carbohydrate on SOD activity in lactococci is strain dependent and that the activity of commercial lactococci can be enhanced through carbohydrate selection for mixed-acid fermentation or by changing the energy distribution, thus enhancing the value of the starter and the resulting dairy products.

Lactic acid bacteria play essential roles in the manufacture of many fermented products (cheese, yogurt, etc.), and their probiotic possibilities has recently attracted considerable interest. The Food and Agriculture Organiza­ tion/World Health Organization defines probiotics as “ live microorganisms which when administered in adequate amounts confer a health benefit to the host.” Reactive oxygen species damage cellular components such as protein, lipid, and DNA molecules. High concentrations of reactive oxygen species, such as superoxide radicals, can induce the intestinal damage seen in patients with inflammatory bowel disease ( 16 , 21 ) and Crohn’s disease ( 1). Therefore, treatments that reduce the concentrations of oxidizing compounds represent a promising therapeutic approach to such diseases. Superoxide dismutase (SOD; EC 1.15.1.1) enzymes, which catalyze dismutation of the superoxide radical (202' _ + 2H+ —» H20 2 + 0 2), are possible candidates among compounds in lactic acid bacteria that may have health benefits in humans. The use of SOD enzymes in the treatment of inflammatory diseases has been hampered by the short circulatory half-life of these enzymes. However, Han et al. ( 11) found that SOD supplied * Author for correspondence. Tel: +81-29-838-8606; Fax: +81-29-838-8683; E-mail: [email protected].

by bacteria to the rat colon improved experimental colitis, suggesting that a bacterial vector harboring SOD might be effective in inflammatory bowel disease therapy. In addition, LeBlank et al. (23 ) reported that in a murine model of trinitrobenzenesulfonic acid-induced Crohn’s disease, mice that received SOD-producing lactic acid bacteria recovered faster from weight loss than did mice that did not receive the bacteria. Thus, clarification of the factors that influence SOD activity in lactic acid bacteria is important for the development of clinical protocols. The relationship between SOD activity and the conditions under which bacteria are cultured is not yet fully understood, although some studies indicate that SOD activity is influenced by factors in the growth condition, such as 0 2 concentration ( 12 , 32 ) and pH level (30 ). NADH oxidase, an enzyme that relates the production of superoxide radicals ( 10), might be linked with SOD. Here, we investigated various culture conditions and factors that affect the superoxide-scavenging activity of lactic acid bacteria, including the kinds and concentrations of carbon sources, the growth rates of bacterial populations, aerobic and batch conditions, patterns of sugar metabolism (including analysis of metabolites and energy production), and NADH oxidase activity. The dairy industry would benefit from the development of probiotic species of

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Lactococcus (a genus of lactic acid bacteria), which are used as starter bacteria for making fermented dairy products (15). Most strains of lactococci have manganese superoxide dismutase, which detoxifies reactive oxygen species (38). We, therefore, used Lactococcus lactis subsp. lactis strain G50 and Lactococcus lactis subsp. cremoris strain H61, because these strains are candidates for effective probiotic formulas and exhibit immunomodulatory and antiaging activity. For example, oral administration of strain G50 suppresses the production of immunoglobulin E antibodies in sera from antigen-immunized mice (17). Moreover, oral administration of strain H61 suppresses the reduction in bone density with increasing age in senescence-accelerated mice and improves the mice’s external skin status (19). This study expands our understanding of the superoxide­ scavenging activity of these potential probiotic strains; it also offers a new approach to developing dairy starters by exploiting the factors that affect this activity, thus enhancing the value of the starter and the resulting dairy products. MATERIALS AND METHODS Bacterial strains and growth conditions. L. lactis subsp. lactis G50 and L. lactis subsp. cremoris H61 were maintained in the International Patent Organism Depository (Tsukuba, Japan). The strains were maintained by subculturing 0.5% (vol/vol) inocula in tryptone-yeast extract (TY) broth comprising 0.5% tryptone (BD, Sparks, MD), 0.5% yeast extract (Wako Pure Chemical Industries, Osaka, Japan), 1% sodium succinate, and 1% sodium chloride, supplemented with 1% glucose (TYG); inocula were batch incubated at 30°C for 18 to 24 h. The glucose-limited TYG contained 0.1% glucose. The culture was stored at 4°C between transfers and was subcultured once before experimental use. To test the dependence of superoxide-scavenging activity on carbohydrate composition (and the formation of end-products in test strains), TY broth was dispensed in 4-ml aliquots and sterilized by autoclaving at 121°C for 15 min. A filter-sterilized (0.2 pm; Advantec, Tokyo, Japan) solution of 10% (wt/vol) glucose, lactose, or galactose was added to the TY broth to obtain a final concentration of 1.0% (vol/vol). The TY broth supplemented with carbohydrate was inoculated with 0.5% of a fresh overnight culture of the test strains and cultured at 30°C for 18 to 24 h in batch conditions; the pH value was then determined. To test the effects of metabolic inhibitors on superoxide­ scavenging activity and bacterial growth, 10 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Nacalai Tesque) in methanol or 20 mM N, V'-dicyclohexylcarbodiimide (DCCD; Nacalai Tesque, Kyoto, Japan) in ethanol was added to the TYG broth to obtain final concentrations of 10 and 100 pM, respectively. CCCP dissipates the proton motive force and inhibits bacterial growth (27). DCCD, a well-known inhibitor of F,-F0 ATPase (31), delays glycolysis by inhibiting the degradation of ATP and the transport of substrate. Methanol or ethanol alone (i.e., without inhibitors) served as a control. The broths were inoculated with 1.0% (vol/vol) of a fresh overnight culture of the test strains and cultured at 30°C for 24 h under batch conditions. To test the effect of aerobic conditions on superoxide­ scavenging activity and bacterial growth, TYG broth (20 ml) was established by culture in a 100-ml vessel with rotary shaking (200 rpm) by shaker (Innova 4330, New Brunswick Scientific Co., Inc. Enfield, CT). The broths were inoculated with 0.5% (vol/vol) of a fresh overnight culture of the test strains and cultured at 30°C for 18 to 24 h.

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Polyacrylamide gel electrophoresis (PAGE) and SOD activity detection in gels. To obtain cell-free extracts, station­ ary-phase bacterial cultures were centrifuged at 1,800 x g for 15 min. The pellets were washed twice with 0.85% NaCl and then mechanically disrupted with Fast Prep FP120 (ThermoSavant, Holbrook, NY) in 240 pi of suspension buffer (0.1 M KH2P 0 4, pH 7.0) and 100 pi of lysis buffer (0.3% sodium dodecyl sulfate [SDS] in 50 mM Tris-HCl, pH 8.0, or 0.05 M sodium phosphate, pH 7.0). Cell debris was removed by centrifugation at 8,000 x g for 10 min; the resulting supernatant was a cell-free extract. Protein concentrations in the cell-free extract were assayed by using a BCA Protein Assay Kit (Pierce, Rockford, IL) with bovine serum albumin as the standard. Total protein extract (10 pg) was separated by SDS-PAGE, according to the protocol of Laemmli (22). Nondenaturing PAGE was performed similarly, except that the SDS and mercaptoethanol were omitted. Polyacrylamide gels were stained with Coomassie brilliant blue for total protein detection. SOD activity in nondenaturing gels was determined by using the method of Beauchamp and Fridovich (5). Enzyme assay. Superoxide-scavenging activity in the cellfree extract was determined by using a calorimetric SOD assay kit (Dojindo Molecular Technologies, Kumamoto, Japan), in which a water-soluble tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2(4-nitrophenyl)-2//-5-tetrazolio]-l,3-benzene disulfonate sodium salt), is applied (36). The superoxide anion generated by xanthine/xanthine oxidase reduces the WST-1 to water-soluble formazan, which exhibits absorbance at a wavelength of 450 nm. The rate of reduction is linearly related to the xanthine oxidase activity, and reduction is inhibited by superoxide-scavenging substances. Superoxide-scavenging activity determined by using this method was estimated as SOD activity by using purified SOD from bovine erythrocytes (Sigma-Aldrich, St. Louis, MO) as the standard (3). The enzyme reaction was carried out at 37°C. NADH oxidase activity was assayed with a spectrophotom­ eter (DU-640, Beckman Coulter Inc., Brea, CA) to measure the absorbance at 340 nm of cell-free extract mixed with 0.26 mM NADH in 0.05 M sodium phosphate buffer, pH 7.0 (33). The enzyme reaction was carried out at 30°C. One unit of NADH oxidase activity was defined as the amount of enzyme (milligrams of protein) required to cause a 0.01/min change in absorbance (12). Determination of bacterial growth and molar growth yield. The effects of CCCP and DCCD on growth were determined by measuring the absorbance at 620 nm with a Spectronic 20 spectrophotometer (Bausch & Lomb, Rochester, NY). The maximum growth rates in the culture were calculated by use of linear regression from the rate of increase in the number of viable cells. Viable cells were counted by plating samples of the appropriate dilution in 0.85% NaCl solution in de Man Rogosa Sharpe (BD) supplemented with agar (1.6%) plate. All plates were then incubated at 30°C for 2 days. The growth yield of cells generated per mole of glucose invested (the molar growth yield for glucose, Yg) is widely used to assay the molecular energetics of biochemical pathways (24, 29). The molar growth yield was calculated by dividing the dry weight of cells by the molar mass of consumed glucose, as determined below. To measure dry weight, cells were collected by centrifu­ gation (1,800 x g, 20 min, 4°C), washed once with distilled water, and dried at 105°C for 4 h. Analyses of carbohydrates and fermentation end-products. Residual glucose in the broth was measured enzymatically with a

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CARBOHYDRATE EFFECTS ON SOD ACTIVITY IN LACTOCOCCI

(A)

(B) 150

Glc

Lac

Gal

FIGURE 1. (A) SOD and (B) NADH oxidase activities o f Lactococcus lactis strains G50 (black bars) and H61 (gray bars) as a function o f the carbon source used (Glc, glucose; Lac, lactose; Gal, galactose). Results are mean values from triplicate samples, with standard errors represented by vertical bars. Mean values with different letters within each strain are significantly different ("P < 0.05). Glucose CII test kit (Wako); residual lactose or galactose was measured with an F-kit for lactose/galactose (Boehringer, Mannheim, Germany). Lactate, acetate, and formate in the broth were measured with a Boehringer test kit or were quantified with a postcolumn high-performance liquid chromatography system (LC-VP, Shimadzu, Tokyo, Japan) equipped with a Shim-pack SCR-102H Column (8 by 300 mm; Shimadzu) fitted with an SCR-102H guard column and a CDD-lOAvp Conductivity Detector. The mobile phase contained 5 mM p-toluenesulfonic acid. The postcolumn buffer solution was composed of 5 mM ptoluenesulfonic acid, 100 pM EDTA, and 20 mM Bis-Tris. Ethanol in the broth was measured by using a Boehringer test kit. Acetoin was measured under aerobic conditions by using the method of Westerfeld (37). The percentage of lactate was calculated as follows (28): 100 x (g of lactate) / (g of lactate + g of acetate + g of formate + g of ethanol)

(1)

Statistical analyses. Data are expressed as means or means ± standard errors of three culture samples. The effects of carbon source on the activities of SOD and NADH oxidase and on the culture pH and lactate were analyzed by using a general linear model procedure (SAS version 9.1, SAS Institute, Cary, NC). Results were compared by applying the Tukey test. Statistical analyses were performed by applying Student’s t test or the Dunnett test to the differences between cells cultured in TYG broth under batch conditions and those cultured with or without the different treatments and growth conditions; P < 0.05 was considered statistically significant, and P values < 0 . 1 indicated trends. RESULTS SOD detection in the gel. Strains G50 and H61 grew well when using glucose, lactose, or galactose as a carbohydrate source. Cell extracts from strains G50 and H61 grown with these three carbohydrates were analyzed by SDS-PAGE; a 24-kDa protein band indicative of lactococcal SOD (30) was seen (data not shown). The SOD activity of these two strains was also detected on a nondenaturing polyacrylamide gel as a single band (data not shown).

Effects of carbon sources on SOD and NADH oxidase activities. The SOD activity of L. lactis subsp. lactis G50 was significantly (P < 0.05) higher in lactosecontaining and galactose-containing media than in glucosecontaining medium (Fig. 1A). The difference between the activity on lactose and galactose was a trend (P = 0.0624). The final pH values of the broths in strain G50 were 4.46 (glucose), 4.70 (lactose), and 4.41 (galactose). The pH value was lowest on galactose-containing medium, higher on glucose-containing medium, and highest on lactose-con­ taining medium; these values were significantly (P < 0.001) different. In contrast, the SOD activity of L. lactis subsp. cremoris H61 did not differ significantly among the glucose-, lactose-, and galactose-containing media (Fig. 1A). The NADH oxidase activity of strain G50 cells was highest in lactose-containing medium, lower in galactosecontaining medium, and lowest in lactose-containing medium (Fig. IB); these differences were significant (P < 0.05). The NADH oxidase activity of strain H61 cells on these three carbohydrates, however, did not differ signifi­ cantly, although the activity on galactose-containing medium tended to be higher (P = 0.0870) than that on lactose-containing medium. Metabolites produced by different carbon sources. Metabolite production depended on the bacterial strain and the carbon source (Table 1). Culture of strain G50 in glucose-containing medium produced mainly lactate (more than 96% of the products formed) and low amounts of acetate, formate, and ethanol. In lactose- and galactosecontaining media, lactate, acetate, formate, and ethanol were also produced. The percentages of lactate produced by cells grown on lactose and galactose were significantly (P < 0.01) lower than those produced by cells grown on glucose. Strain H61, on the other hand, produced mainly lactate (more than 94%) with small amounts of acetate, formate, and ethanol when cultured in glucose-, lactose-, and galactose-containing media, although the percentages of

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TABLE 1. Lactate formation by Lactococcus lactis strains G50 and H61 in batch cultures grown on different carbon sources Strain

Carbon source

G50

Glucose Lactose Galactose Glucose Lactose Galactose

H61

% of lactate" 96.5 37.7 81.9 94.9 96.5 94.3

± + ± ± ± +

1.1 a 1.1c 1.1 b 0.1 b 0.1 a 0.1 c

a The percentage of lactate was calculated by using equation 1. Values are means + standard errors of triplicate samples. Mean values with different letters within each strain are significantly different (P < 0.01).

lactate produced under the three growth conditions differed significantly (P < 0.01). Effects of metabolic inhibition on SOD activity, growth efficiency, and metabolite production. Because the SOD activity of strain G50 was influenced by the carbon source used, we further evaluated the carbohydrate utilization of strain G50. Strain G50 was cultured for 24 h, with and without agents that affected metabolic rate, to determine the effect of these agents on growth rates (Fig. 2). In cultures with CCCP and without CCCP (i.e., with methanol only), the maximum growth rates were 0.079/h and 0.199/h, respectively. These values were significantly (P < 0.001) different. After culture for 24 h, the cell yields of the cultures containing CCCP (OD620 = 0.63) were also significantly (P < 0.001) lower than those of the controls (OD620 = 1.04). In contrast, cultures containing DCCD showed a lag in their cell growth. The maximum growth rates of cultures with DCCD and without (i.e., with ethanol only) were 0.078/h and 0.199/h, respectively. These values were significantly (P < 0.01) different. However, the cell yields from DCCD-containing cultures after culture for 24 h (OD620 = 1.23) were similar to those from controls (OD62o = 1.15). There were differences in SOD activity between cells treated with CCCP or DCCD and their respective controls (Table 2). The SOD activity of strain G50 exposed to DCCD was similar to that of its control. In contrast, the SOD activity of strain G50 exposed to CCCP was significantly (P < 0.001) higher than that of the control. There were also differences in growth efficiency between CCCP and DCCD relative to their controls (Table 2). The Yg (i.e,, growth efficiency) of cells cultured with DCCD was similar to that of the control, whereas the Yg of cells cultured with CCCP was significantly lower than that of the control (P < 0.001). The main fermentation product of strain G50 cultured with or without growth inhibitor was lactate (more than 96%), with a small amount of acetate and ethanol (Table 2). The percentage of lactate produced by strain G50 cultured with CCCP was significantly (P < 0.001) lower than that of the control (methanol). The percentage of lactate produced by strain G50 cultured with DCCD was significantly (P < 0.05) higher than that produced by the control (ethanol).

FIGURE 2. Growth curves of Lactococcus lactis strain G50 cultured with agents that affect metabolic rate (O, methanol; • , CCCP in methanol; □ , ethanol; ■, DCCD in ethanol). Values are triplicate means. Effects of aerobic and glucose-limited conditions on SOD activity, growth efficiency, and metabolite produc­ tion. The SOD activity of strain G50 cultured aerobically was significantly greater (P < 0.05) than that of cells cultured in batch (control) conditions (Table 3). However, the SOD activity of bacteria cultured under glucose-limited conditions (0.1% glucose) was similar to that of bacteria cultured under batch glucose-rich control conditions (1.0% glucose). The Yg of bacteria cultured under aerobic conditions was similar to that of the batch control, whereas the Yg of cells cultured in glucose-limited conditions was significant­ ly higher (P < 0.05) than that of the control (Table 3). The end product of glucose fermentation under batch conditions was lactate (Table 3); however, under aerobic conditions, strain G50 produced acetate and acetoin, besides lactate. In addition to calculating the grams of acetoin produced, we calculated the percentage of lactate produced under aerobic conditions, by using equation 1, and found it to be less than 90%, which was significantly (P < 0.05) lower than that of the control. Under glucose-limited conditions, strain G50 produced acetate, formate, and ethanol in addition to lactate, and the percentage of lactate was significantly (P < 0.05) lower than that in the control. DISCUSSION We confirmed here that strains G50 and H61 have SOD, and we showed that the SOD activity of L. lactis strain G50 varied significantly with the carbon source used. Sanders et al. (30) reported that growth of L. lactis MG1363 lowered the pH of the growth medium and that the lowered pH enhanced SOD activity. Sanders et al. (30) compared SOD activities between cells grown on buffered medium at pH 6.8 and those grown on unbuffered medium. Under the latter conditions, the pH of the culture was 4.8. BrunoBarcena et al. (7) suggested that acid stress in Streptococcus thermophilus triggers an iron-mediated oxidative stress that can be reduced by manganese superoxide dismutase and

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TABLE 2. SOD activity, molar growth yield (Yg), and lactate formation in the presence of metabolic inhibitors (CCCP and DCCD) in the case of Lactococcus lactis strain G50 cultured in glucose-containing medium in batch culturea Growth condition Methanol (control: CCCP) CCCP Ethanol (control: DCCD) DCCD

SOD activity (U/mg) 11.9 46.2 9.8 12.2

± ± + ±

2.1 2.0*** 1.4 2.0

u 24.7 14.2 24.7 26.0

± + + +

0.5 0.1*** 0.2 0.8

% of lactate 97.9 96.6 98.3 98.9

± + ± +

0.1 0.1*** 0.1 0.1*

° Values are means + standard errors of triplicate samples. Asterisks denote statistically significant differences between values and controls within each column: * P < 0.05; *** P < 0.001.

iron chelators. Our pH values for G50 grown on the three carbohydrates ranged from 4.4 to 4.7—i.e., they were low. The effect of low pH on SOD activity reported by Sanders et al. (30) would likely apply to all cells grown on different carbons; therefore, the dependence of SOD activity on the carbon source in the strain G50 cultures cannot be attributed to low pH effects. Metabolism in homolactic acid bacteria results in more than 90% conversion of sugars to lactate. Under certain conditions, other metabolites, such as acetate, formate, ethanol, and carbon dioxide, are produced in addition to lactate; this is generally referred to as mixed-acid fermen­ tation. A shift from homolactic to mixed-acid fermentation has been observed in lactic acid bacteria in certain circumstances, such as with galactose utilization (8, 34) and under aerobic conditions (32). Catabolite control protein A can control the balance between homolactic and mixedacid fermentation (9). The fermentation of lactose by typical lactococci in batch culture results in simultaneous conver­ sion of both glucose and galactose moieties almost entirely to lactate. Lactococci cultured on galactose have the enzymatic potential to metabolize galactose via two initially separate pathways: the D-tagatose-6-phosphate (Tag6P) pathway and the Leloir pathway (6). In the present study, strain G50 fermented galactose to lactate via the Leloir pathway, and less than 90% of the products were formed via this pathway; metabolism was shifted to mixed-acid fermentation (28). Strain H61 fermented galactose to lactate and more than 90% of the products were formed via the Tag6P pathway, i.e., homolactic fermentation. Our study showed that the effect of sugar on SOD activity depends on the L. lactis strain; differences are likely related to utilization of the Leloir or Tag6P pathways for galactose utilization. Hansson and Haggstrom (12) also reported that TABLE 3. SOD activity, molar growth yield (Yg), and lactate formation of Lactococcus lactis strain G50 cultured in 1.0% glucose-containing broth under batch (control) and aerobic conditions and in 0.1% glucose-containing (glucose-limited) broth in batch conditiona Growth condition SOD activity (U/mg) Control Aerobic Glucose limited

10.1 ± 2.7 54.7 ± 2.7* 7.0 + 2.7

U

% of lactate

20.1 + 0.9 98.2 + 1.2 17.3 ± 0.9 85.2 -1- 1.2* 70.5 ± 0.9* 88.3 ± 1.2*

a Values are means ± standard errors of triplicate samples. Asterisks denote statistically significant differences between values and controls within each column: * P < 0.05.

SOD activity in L. lactis ATCC 19435 depends on the carbon source. However, in contrast to our findings, they found that the SOD activity of cells grown on galactose was higher than that of cells grown on glucose. This difference may be attributable to differences in culture conditions: their culture conditions involved pH-controlled culture at 6.9 and supplementation with N2 and C 0 2 (95:5), whereas our culture was done under batch conditions, and no gas was supplied. A constant NAD+ to NADH ratio is generally maintained in glycolysis, because the NAD+ cofactor reduced during glycolysis is regenerated during pyruvate reduction by L-lactate dehydrogenase. Numerous lactic acid bacteria, including L. lactis, use molecular oxygen or hydrogen peroxide to regenerate NAD+ via NADH oxidase and NADH peroxidase (2). Overproduction of NADH oxidase (2NADH + 2H+ + 0 2 -» 2NAD+ + 2H20 , or NADH + H + 0 2 —* NAD+ + H20 2) causes mixedacid fermentation because the availability of NADH is low, resulting in inhibition of NADH-dependent enzymes, such as L-lactate dehydrogenase (13). When L-lactate dehydro­ genase is inhibited, pyruvate metabolism changes from homolactic to mixed-acid fermentation. We found here that the NADH oxidase activity of strain G50 was enhanced in mixed-acid fermentation. Oxygen is reduced by the increased activation of NADH oxidase and reactive oxygen species, such as superoxide radicals, are produced by the reduction of molecular oxygen in biological systems. We, therefore, suggest that the SOD activity of lactococci in mixed-acid fermentation was affected by the NADH oxidase activity. In fact, in strain G50, the NADH oxidase and SOD activities were linked by the type of sugar (lactose, galactose, or glucose) available for use. Conversely, it has been suggested that, when SOD activity is enhanced and oxygen is generated, NADH oxidase activity is enhanced. It is not clear which enzyme’s activity (SOD or NADH oxidase) is enhanced first in strain G50. In contrast, Hansson and Haggstrom (12) reported no clear correlation between SOD and NADH oxidase—NADH peroxidase levels in L. lactis ATCC 19435. We, too, found that the activities of SOD and NADH oxidase of strain H61 were not linked. The correlation between SOD and NADH oxidase activities thus appears to depend on the strain. Next, we investigated the effects of different growth conditions (growth rate, carbohydrate concentration, and aerobic conditions) on SOD activity with glucose as the carbon source. Garrigues et al. (8) have reported that, in L. lactis, homolactic fermentation is observed in glucose-containing

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medium, supporting high growth rates, whereas substrates that support the lowest growth rates (e.g., lactose and galactose) show mixed-acid fermentation. In our previous study, we found that the growth rates of strain G50 were highest when the bacteria were cultured on glucose, were significantly lower on galactose, and lower still on lactose (20). We found here that strain G50 showed homolactic fermentation on glucose and mixed-acid fermentation on galactose and lactose and that there may be a correlation between growth rate and SOD activity in the presence of different carbon sources. However, the low growth efficiency and low growth rate of strain G50 in the presence of CCCP affected SOD activity, but the low growth rate of strain G50 in the presence of DCCD did not. In addition, metabolite analysis revealed that culturing the bacteria in the presence of CCCP and DCCD led to homolactic fermentation. Thus, a low growth rate, in the absence of any change in carbohydrate metabolic pathway, does not necessarily affect SOD activity. On the other hand, the effect of low growth efficiency on SOD activity should be considered more carefully. Yields of cells per mole of energy source, y(substrate) and TAtp, have been widely used in analyses of the molecular energetics of biochemical pathways. A Tatp value of 10.5 has been generally accepted as a biological constant (4). Accordingly, the Yg of an organism that ferments glucose via the hexose diphosphate pathway should be 21 g (dry weight) of cells per mole of glucose. The Yg of strain G50 grown in the presence of CCCP was considerably lower. Low-growth efficiency of bacteria can be caused by stressors such as low pH (26) or exposure to bile (18), and low pH induces SOD activity in L. lactis (30). Thus, the stress of reduced growth efficiency may affect SOD activity. A shift from homolactic to mixed-acid fermentation in strain G50 was observed under both aerobic and glucoselimited conditions. The SOD activity of strain G50 was greater under aerobic conditions than under batch condi­ tions. Aerobic conditions provide more oxygen, and in response, G50 cells increase their SOD activity. This finding is in agreement with previous studies (12, 32). In the present study, under aerobic conditions, the growth efficiency, Yg, was similar to that for the batch control, indicating that stress was not induced. Under glucoselimited conditions, SOD activity was not altered. In this case, the Yg of strain G50 was higher than that of the control (i.e., no treatment), suggesting that the high growth efficiency does not influence SOD activity. Lactococci are widely used as starter bacteria in manufacturing cheese and other fennented dairy products, and some strains are candidates for probiotics (25, 33). Recently, in many countries, consumers have begun to favor the many types of organoleptic properties and food textures of dairy foods (14, 35), including probiotic activity. Accordingly, there is much interest in the development of new starter cultures for fermented products. Establishing the effective probiotic properties (i.e., SOD activity) of lactococci by altering culture condition such as carbon source could lead to development of enhanced probiotic dairy foods. In the dairy industry, lactose, galactose, and glucose are the main sugars. We found that the SOD activity

of strain H61 was high and was not influenced by the carbohydrate source available; this would be a useful trait for probiotic dairy starters grown on lactose in milk. The SOD activity of strain G50 was highest in lactosecontaining broth; this is also a desirable characteristic for starters for making milk products. ACKNOWLEDGMENTS The authors thank Ms. C. Koitabashi, Ms. X. Y. Yu, Ms. M. Nishimura, and Ms. K. Yamasaki for their technical assistance.

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