NADH Oxidase of Streptococcus thermophilus 1131 is Required for the Effective Yogurt Fermentation with Lactobacillus delbrueckii subsp. bulgaricus 2038.

We previously reported that dissolved oxygen (DO) suppresses yogurt fermentation with an industrial starter culture composed of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) 2038 and Streptococcus thermophilus 1131, and also found that reducing the DO in the medium prior to fermentation (deoxygenated fermentation) shortens the fermentation time. In this study, we found that deoxygenated fermentation primarily increased the cell number of S. thermophilus 1131 rather than that of L. bulgaricus 2038, resulting in earlier l-lactate and formate accumulation. Measurement of the DO concentration and hydrogen peroxide generation in the milk medium suggested that DO is mainly removed by S. thermophilus 1131. The results using an H2O-forming NADH oxidase (Nox)-defective mutant of S. thermophilus 1131 revealed that Nox is the major oxygen-consuming enzyme of the bacterium. Yogurt fermentation with the S. thermophilus Δnox mutant and L. bulgaricus 2038 was significantly slower than with S. thermophilus 1131 and L. bulgaricus 2038, and the DO concentrations of the mixed culture did not decrease to less than 2 mg/kg within 3 hr. These observations suggest that Nox of S. thermophilus 1131 contributes greatly to yogurt fermentation, presumably by removing the DO in milk.

INTRODUCTION

Yogurt, a very important dairy product, is fermented by a mixed culture of two thermophilic lactic acid bacteria: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Protocooperation (or symbiosis) exists between these two bacteria via an exchange of metabolites that are necessary for the growth of each bacterium in milk. For example, L. bulgaricus provides amino acids and peptides for S. thermophilus, and S. thermophilus provides formate and carbon dioxide for L. bulgaricus [1,2,3]. Several reviews on protocooperation have been published [4,5,6,7] and the mutual benefits often result in higher acidification rates, which are important for yogurt fermentation.

Many new aspects of protocooperation in yogurt fermentation have been revealed using the current post-genomic techniques. The complete genomes of five S. thermophilus strains [8,9,10,11,12] and four L. bulgaricus strains have been characterized [13,14,15,16] and post-genomic studies to analyze the protocooperation have recently been reported. Microarray and proteome analyses of S. thermophilus LMG18311 and a qRT-PCR analysis of L. bulgaricus ATCC 11842 have shown that the coculture results in not only nutritional exchanges but also dramatic physiological changes in these two bacteria. Transcriptional changes for genes related to nitrogen, nucleotide bases and iron metabolism were observed in S. thermophilus LMG18311 [17]. Another study [18] showed, using microarray of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365, that the interactions between the purine, amino acid, exopolysaccharide and long-chain fatty acid metabolisms are affected by coculture. The genome of S. thermophilus LMD-9 encodes eight two-component systems, and qRT-PCR analysis data indicated that coculture with L. bulgaricus ATCC11842 induced expression of two response regulators among them [19].

Recently, we reported that dissolved oxygen (DO) greatly affects yogurt fermentation with an industrial starter culture composed of L. bulgaricus 2038 and S. thermophilus 1131 [20]. The starter began to produce acid actively only after the DO concentration in the yogurt mix was reduced to almost 0 mg/kg. Fermentation was suppressed in the presence of more than 1 mg/kg of DO, and this suppression was compensated partially by the addition of formate to the medium [21]. We have also found that the fermentation time was shortened by 30 min if DO in the yogurt mix was removed in advance (deoxygenated fermentation). These observations clearly demonstrated that DO in milk would greatly affect yogurt fermentation, and at the same time, they indicated that these bacteria have a DO-consuming enzyme(s) presumably required for fermentation.

Like other lactic acid bacteria, S. thermophilus and L. bulgaricus do not produce cytochrome oxidases required for energy-linked respiratory metabolism due to the inability to synthesize heme, an essential cofactor for cytochrome oxidase. Instead of heme cofactor oxidases, lactic acid bacteria consume molecular oxygen through the action of flavoprotein oxidases, including NADH oxidase, pyruvate oxidase, α-glycerophosphate oxidase, L-amino acid oxidase, and lactate oxidase. Although these oxidases have not been directly linked to energy metabolism, many functions of these oxidases in the physiology of lactic acid bacteria have been reported [22,23,24,25,26,27,28,29,30]. Teraguchi et al. characterized the NADH oxidase activity of S. thermophilus and suggested that high NADH oxidase activity might shift metabolism from homo lactic acid to mixed acid fermentation by affecting the cellular NAD+/NADH ratio [31]. NADH oxidase activity of L. bulgaricus has been reported to be a source of hydrogen peroxide (H2O2) when the bacterium was cultured under aerobic conditions [32]. However, the role of these oxidases in yogurt fermentation has not been elucidated.

In the present study, the beneficial effects of deoxygenated fermentation on yogurt fermentation were investigated from the viewpoint of protocooperation. We also analyzed the contributions of these bacterial oxidases in yogurt fermentation, and identified the NADH oxidase of S. thermophilus 1131 as the major enzyme required for the DO reduction and fermentation of milk.

MATERIALS AND METHODS

Strains and culture conditions

Yogurt starter strains of L. bulgaricus 2038 and S. thermophilus 1131 were obtained from the stock cultures of the Food Science Institute of Meiji Co., Ltd., which are used in the commercial production of Meiji Bulgaria Yogurt LB81 in Japan. For the coculture medium, 10% (wt/wt) skim milk was prepared by heating it to 95°C for 2 min and immediately cooling it to 43°C. For monoculture experiments of L. bulgaricus 2038 or S. thermophilus 1131, either 1 mM sodium formate (Wako Pure Chemical Industries, Ltd., Japan) or 0.1% (wt/wt) casein peptides (CE90GMM, Nippon Shinyaku Co., Ltd., Japan) was added, respectively, to the skim milk medium prepared above. Yogurt fermentation with or without deoxygenated fermentation was performed as described previously [20, 21]. Since the Δnox mutant (described below) did not grow well on skim milk medium, the preculture of this strain was prepared on M17L medium [M17 (Becton, Dickinson and Company) supplemented with 0.5% lactose] in monoculture. The precultures of S. thermophilus 1131 and the Δnox mutant were grown for 16.5 hours at 37°C on M17L using an Anaero Pack system (anaerobic cultivation system; Mitsubishi Gas Chemical Company Inc.) and then inoculated to 10% skim milk supplemented with 0.1% casein peptides to give the final OD660 of 0.05. The growths of S. thermophilus 1131 and the Δnox mutant were monitored at 43°C under static conditions. For coculture experiments, the preculture obtained above containing S. thermophilus 1131 or Δnox was inoculated to 10% skim milk together with a culture of L. bulgaricus 2038 grown on MRS (Becton, Dickinson and Company) at 37°C. S. thermophilus 1131 or the Δnox mutant and L. bulgaricus 2038 were inoculated to 10% skim milk medium at a 1:1 ratio, corresponding to a final OD660 of 0.025.

Measurements of CFU, DO, lactate and formate

In coculture experiments, a portion of skim milk medium was withdrawn at predetermined time intervals and spread on M17G agar plates and on Rogosa agar plates. The plates were incubated at 43°C for 48 hours under anaerobic conditions using an Anaero Pack system. The numbers of colony-forming units (CFU) for S. thermophilus and L. bulgaricus were determined by counting colony numbers on M17G plates and on Rogosa plates, respectively.

Concentration of DO in the skim milk medium was measured using optical oxygen sensors (VisiFerm DO optical sensors, Hamilton Company, Reno, NV, USA).

l-lactate, D-lactate and formate in the skim milk medium was measured using an F-kit (Roche Diagnostics K.K.) suitable for each material as described previously [20, 21].

Measurement of hydrogen peroxide in skim milk medium

In order to measure small quantities of H2O2 in the milk medium, the following improved method was used in this study. An aliquot of culture medium (0.5 g) was harvested at predetermined time intervals and diluted with 0.5 mL of distilled water, and 30 µL of Carrez I and Carrez II solutions [33] were added. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was diluted by three-fold with 100 mM PIPES (pH 6.5). One hundred microliters of this diluted sample received 100 µL of 1 mM DA64 (Wako Pure Chemical Industries Ltd., Osaka, Japan), a sensitive chromophore N-(carboxymethylaminocarbonyl)-4,4’-bis(dimethyamino)-diphenylamine sodium salt [34], and 2 µL of peroxidase (Wako Pure Chemical Industries Ltd., Osaka, Japan). After 10 min of incubation at 16°C, the absorbance at 727 nm was determined.

Construction of a nox knockout mutant of S. thermophilus 1131

Plasmids containing the nox gene, inactivated by insertion of a spectinomycin resistance cassette, were constructed as follows. A 1369-bp internal fragment of the nox gene was obtained by PCR using the primer pairs PYY0034 (5′-CTCGAGTCAAAAATCGTAGTTGTCGG-3′) and PYY0037 (5′-CTCGAGTATTCAGCGGAGATAGCTGC- 3′), genomic DNA from S. thermophilus 1131, and Takara Ex Taq DNA polymerase (Takara Bio Inc., Otsu, Japan). The amplified fragment was cloned into pGEM-T (Promega K.K., Tokyo, Japan) to obtain pGEM-T-nox. The primer pairs PYY0035 (5′-GCACCAACGACTGCTACAC-3′) and PYY0036 (5′-TGCGTACGATGTAGATATGG-3′) were used to introduce blunt ends in the middle of the nox gene of pGEM-T-nox by PCR. pSPC1 [25] was digested with BamHI to obtain a DNA fragment containing a terminator-less spectinomycin resistance cassette. Then the BamHI fragment was ligated with the blunt ends of the PCR product of pGEM-T-nox after generating blunt ends by treatment with the Klenow fragment of E. coli DNA polymerase I (Takara Bio Inc., Otsu, Japan). pGEM-T-nox carrying the spectinomycin resistance cassette in the middle of the nox gene in the same direction was selected and designed as pGEM-T-nox::spcr. The nox gene carrying the spectinomycin resistance cassette was then transferred into the XhoI site of pG+host6 (Appligene, Pleasanton, CA, USA), a temperature-sensitive cloning vector for gram-positive bacteria. The resulting plasmid was designated as pG+host6-nox::spcr and introduced into S. thermophilus 1131 by electroporation. Double-cross-over events leading to the expected gene replacements were obtained as previously described [35]. Correct insertion of the spectinomycin resistance cassette into genomic DNA was confirmed by PCR analysis using primer pairs PYY0034 and PYY0037.

Determination of NADH oxidase and whole-cell oxygen consumption activities of S. thermophilus

To determine NADH oxidase activity, 1 mL of overnight culture of each strain was transferred into a 300 mL flask containing 50 mL of fresh M17G medium, and incubated at 37°C with vigorous shaking (180 cycles/min) under aerobic conditions until the mid-log phase (OD600 = ~1.0). Then the bacterial cells were collected by centrifugation at 6,300 g for 5 min at 4°C, and washed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 mM EDTA, and suspended in 0.8 mL of the same buffer. The cell suspensions were transferred into a screw cap tube containing 0.3 g zirconium beads and disrupted by a Mini-Beadbeater (BioSpec Products, Inc., Bartlesville, OK, USA) at 4,600 rpm for 120 sec. The cell debris was removed by centrifugation at 16,000 g for 15 min at 4°C, and the resulting supernatant was used for NADH oxidase activity measurement as described previously [25]. One unit of enzyme was defined as the amount that catalyzed oxidation of 1 µmol of NADH to NAD per min at 25°C. Protein concentrations were determined according to the Bradford method [36] using bovine serum albumin as a standard.

To monitor oxygen consumption activity of S. thermophilus cells, each strain was grown up to an early log phase (OD600 = ~0.5) in M17G medium with vigorous shaking (180 cycles/min) as described above. The bacterial cells were collected by centrifugation at 6,300 g for 5 min at 4°C, and washed with phosphate buffered saline (PBS) and suspended in 0.8 mL of the same buffer. Then an aliquot of the suspension was injected into a rubber-capped vial filled with 3.5 mL of oxygen-saturated PBS to give an OD600 of 2.0. After the vial containing the bacterial suspension was incubated for 5 min at 37°C with stirring, 20% glucose solution was injected into the vial at a final concentration of 0.1%, and the rate of oxygen consumption of the bacterial suspension was monitored using an by the oxygen meter (Fibox 3, PreSens, Regensburg, Germany) through a sensor tip fixed to the bottom of the vial.

RESULTS

Growth acceleration by deoxygenated fermentation was more prominent in S. thermophilus 1131 than L. bulgaricus 2038

The viable cell numbers and lactate productions of S. thermophilus 1131 and L. bulgaricus 2038 in yogurt fermentation were compared under deoxygenated and normal fermentation conditions [20, 21]. Deoxygenated fermentation in coculture significantly increased the number of colony-forming units of S. thermophilus 1131 and production of both l-lactate and formate at 90 min (Fig. 1). However, the number of colony-forming units and l-lactate concentrations of the cultures with or without deoxygenated fermentation were almost the same at the end of fermentation. Although the number of colony-forming units of L. bulgaricus 2038 and D-lactate production were also slightly affected by deoxygenated fermentation, the changes were small compared with those of S. thermophilus 1131. These results indicate that l-lactate, produced by S. thermophilus 1131, mainly contributed to the acceleration of acidification in coculture, especially between 60 and 120 min of fermentation in the deoxygenated milk medium.

Fig. 1.

Influence of deoxygenated fermentation on viable cell numbers and lactate and formate concentrations in coculture of S. thermophilus 1131 and L. bulgaricus 2038. For deoxygenated fermentation (DF), skim milk medium was deoxygenated with nitrogen gas before fermentation. Normal fermentation (NF) was performed without this treatment. Incubation was at 43°C. In (A), CFU of S. thermophilus 1131 in DF (■) and NF (□) and CFU of L. bulgaricus 2038 in DF (♦) and NF (◊) are shown. In (B), l-lactate concentrations in DF (■) and NF (□) and D-Lactate concentrations in DF (●) and NF (○) are shown. In (C), formate concentrations in DF (●) and NF (○) are shown. The error bars represent the standard deviations of three independent experiments.

DO consumption and H2O2 generation in monoculture of S. thermophilus 1131 and L. bulgaricus 2038

DO concentrations and pH changes in the skim milk medium in monoculture of each lactic acid bacterium were examined in relation to H2O2 generation. The milk medium received either 0.1% of casein peptides or 1 mM sodium formate to promote growth of S. thermophilus 1131 or L. bulgaricus 2038, respectively. H2O2 concentrations in the culture medium were determined because L. bulgaricus produces H2O2 in the presence of oxygen [32, 37, 38] and S. thermophilus changes the expression of genes involved in iron metabolism during the coculture with L. bulgaricus presumably to avoid the harmful effects of H2O2 [17, 18].

Although it is reportedly difficult to measure H2O2 concentrations in milk due to the presence of several milk components including casein [17], we successfully determined H2O2 concentrations using a sensitive chromophore, DA-64. Linear standard curves of H2O2 in the milk were obtained repeatedly in a range between 18 and 300 μM (data not shown). This new method was used to measure H2O2 concentrations in the skim milk medium.

As shown in Fig. 2, both lactic acid bacteria in monoculture reduced DO in the medium to less than 1 mg/kg within 120 min. Although L. bulgaricus 2038 produced up to 120 μM of H2O2 in accordance with the decrease of DO, no H2O2 (less than 18 μM, the quantitative determination limit) was detected in the monoculture of S. thermophilus 1131. These results indicate that both lactic acid bacteria are able to consume DO in the skim milk medium and that L. bulgaricus 2038 does not completely reduce molecular oxygen to H2O, resulting in production of H2O2.

Fig. 2.

DO consumption and H2O2 generation in monoculture of S. thermophilus 1131 and L. bulgaricus 2038. DO (□) and H2O2 (■) concentrations and pH (○) in monoculture of S. thermophilus 1131 (A) and L. bulgaricus 2038 (B) at 43°C are shown. The error bars represent the standard deviations of three independent experiments.

DO consumption and H2O2 generation in coculture

The concentrations of DO and H2O2 in the culture medium together with the pH values were measured in coculture of S. thermophilus 1131 and L. bulgaricus 2038. As shown in Fig. 3, DO in the medium decreased to less than 1 mg/kg within 90 min, and H2O2 was not detected until 60 min of fermentation. The maximum concentration of H2O2 was around 40 μM, which was approximately 3 times lower than that of the L. bulgaricus 2038 monoculture (Fig. 2B).

Fig. 3.

DO consumption and H2O2 generation in coculture.

DO (■) and H2O2 (□) concentrations and pH (○) in coculture of S. thermophilus 1131 and L. bulgaricus 2038 at 43°C are shown. The error bars represent the standard deviations of three independent experiments.

The result showing that H2O2 was not detected in the first 60 min of fermentation, when the rapid decrease of DO occurred, strongly suggests that the DO decrease in coculture can be attributed to S. thermophilus 1131. This result together with the increased cell number and production of l-lactate in yogurt fermentation (Fig. 1A and 1B) indicates that the metabolic activity of S. thermophilus 1131 is the main contributor to DO reduction during yogurt fermentation.

Construction of an NADH oxidase knockout mutant of S. thermophilus 1131

The results obtained above strongly suggest the importance of the DO reduction activity of S. thermophilus 1131 during yogurt fermentation. To identify the oxidase(s) that contributes to DO reduction during yogurt fermentation, we focused on an H2O-forming NADH oxidase (Nox) homologue of S. thermophilus 1131.

An S. thermophilus 1131 nox-inactivated strain was constructed by insertion into nox of a terminator-less spectinomycin resistance gene as described in Materials and Methods. Insertion of the spectinomycin resistance gene into the chromosomal nox gene was confirmed by PCR analysis (data not shown). Log phase cultures of the resulting Δnox mutant and S. thermophilus 1131 were prepared under aerobic conditions and used for NADH oxidase and rate of whole-cell oxygen consumption determinations. As shown in Fig. 4, NADH oxidase activity was diminished in the Δnox mutant to less than 4% of that in wild-type strain. Inactivation of the nox gene also reduced the rate of whole-cell oxygen consumption by 65%. These results indicate that NADH oxidase encoded by the nox gene is the major oxygen-consuming enzyme in S. thermophilus 1131.

Fig. 4.

NADH oxidase and whole-cell oxygen consumption activities of S. thermophilus 1131 and the Δnox mutant. Log-phase cultures of S. thermophilus 1131 and Δnox were used for NADH oxidase activity (A) and whole-cell oxygen consumption determinations (B). The error bars represent the standard deviations of three independent experiments.

Comparison of growth and DO consumption in monoculture of S. thermophilus 1131 and the Δnox mutant

Growth properties of S. thermophilus 1131 and the Δnox mutant were compared in a monoculture on skim milk medium supplemented with 0.1% casein peptides. As shown in Fig. 5, S. thermophilus 1131 effectively reduced the DO concentrations of the medium, and fermentation seemed to be accelerated after the DO concentrations were below 1 mg/kg. By contrast, the Δnox mutant did not rapidly reduce the DO concentrations, and the pH value of the medium was kept high even after 4 hours of fermentation. We also compared the growth of both strains on M17L medium under the same static conditions and found no significant differences between the two strains (data not shown). These results demonstrated that NADH oxidase of S. thermophilus 1131 was required for effective fermentation in the skim milk medium but not in M17L medium.

Fig. 5.

Growth, DO concentration, and formate production in monoculture of S. thermophilus 1131 and Δnox mutant. Growth of S. thermophilus 1131 and Δnox mutant were monitored at 43°C in skim milk medium supplemented with 0.1% casein peptides. In (A), pH (■) and DO (●) of S. thermophilus 1131, and pH (□) and DO (○) of the Δnox mutant are shown. In (B), formate concentrations in monoculture of S. thermophilus 1131 (■) and the Δnox mutant (□) are shown. The error bars represent the standard deviations of three independent experiments.

Formate concentrations of the skim milk medium were determined (Fig. 5B), because NADH oxidase has a possible role in activating formate production in S. thermophilus. Under anaerobic conditions, formate is generally produced by pyruvate formate lyase (Pfl), an oxygen-sensitive enzyme whose activity is easily inactivated by molecular oxygen [40]. NADH oxidase may be able to activate Pfl by removing DO in the medium. As shown in Fig. 5B, formate was detected in the culture medium of S. thermophilus 1131 after 2 hours of fermentation, but not in that of the Δnox mutant, supporting this hypothesis.

Growth and DO removal of S. thermophilus 1131 and the Δnox mutant in coculture with L. bulgaricus 2038

The behavior of the S. thermophilus Δnox mutant in coculture with L. bulgaricus was examined. The coculture conditions were essentially the same as standard coculture conditions except that M17L was used as a preculture of S. thermophilus 1131 and the Δnox mutant. The consumption of DO and decrease in pH caused by coculture of the Δnox mutant were by far slower than those caused by coculture of the wild type (WT) strain, resulting in a strong retardation of yogurt fermentation (Fig. 6). The H2O2 concentrations in the coculture medium of the Δnox mutant were in the range of 40 to 70 µM, a little higher than the concentration observed in Fig. 3, indicating that NADH oxidase activity might contribute to the reduction of H2O2 generation during the coculture.

Fig. 6.

Growth and DO concentration in coculture with L. bulgaricus 2038. pH (■) and DO (●) in coculture of S. thermophilus 1131 and L. bulgaricus 2038, and pH (□) and DO (○) in coculture of the S. thermophilus Δnox mutant and L. bulgaricus 2038 at 43°C are shown. The error bars represent the standard deviations of three independent experiments.

DISCUSSION

We have previously reported that deoxygenated fermentation shortened the yogurt fermentation time by 30 min, and the details of this preferable effect were investigated in the present study. Monitoring the growth of S. thermophilus 1131 and L. bulgaricus 2038 in skim milk demonstrated that DO removal mainly accelerates the growth of S. thermophilus 1131. Formate production was also increased by pre-fermentation removal of DO from the medium. Accumulation of formate would favor to the growth of L. bulgaricus 2038, because formate is a well-known growth factor for L. bulgaricus in milk [41]. However, DO removal stimulated the growth of S. thermophilus 1131. This result implies that formate accumulation may also facilitate the growth of S. thermophilus in milk. Using proteome analysis, Derzelle et al. identified Pfl of S. thermophilus lMG18311 as a protein strongly induced during growth in skim milk medium [42]. They also demonstrated that the addition of formate or purine bases diminishes the overexpression of Pfl and increases the growth yield of S. thermophilus in skim milk medium [42]. Their observations suggest that formate produced by Pfl may be utilized in an essential biosynthetic pathway such as purine biosynthesis of S. thermophilus in milk. It would therefore not be surprising if the deoxygenated milk activated the Pfl, thereby promoting the growth of S. thermophilus 1131 by the addition of formate.

In this study, we identified Nox of S. thermophilus 1131 as the major oxygen-consuming enzyme that promotes the fermentation of milk. The S. thermophilus Δnox mutant could not effectively reduce the DO concentrations and pH values of the skim milk medium in either monoculture or coculture. Nox is a flavoprotein conserved in most Streptococcaceae and has been reported to be a major part of the oxidase machinery in several lactic acid bacteria [25, 28, 39]. Although most flavoprotein oxidases of lactic acid bacteria generate H2O2 as an end product of the reaction, Nox is able to reduce molecular oxygen to H2O without producing H2O2.

As mentioned above, a possible role for Nox in milk fermentation is activation of Pfl, an oxygen-sensitive enzyme, by the removal of DO from the medium. Consistent with this hypothesis, formate was detected in the culture medium of S. thermophilus 1131 but not that of the Δnox mutant. However, we need to also pay attention to other possibilities concerning the function of Nox in S. thermophilus 1131 because various roles have been proposed as physiological functions of Nox in streptococci and lactococci. One major role of Nox is regulation of the intercellular ratios of NAD+/NADH by oxidizing NADH with molecular oxygen. Overexpression or a defect of Nox has been shown to affect the intercellular NAD+/NADH ratio and change the fate of the metabolic end products in Lactococcus lactis, Streptococcus mutans, and Streptococcus agalactiae [25, 28, 43]. In Streptococcus thermophilus, the difference in NADH oxidase activities between strains has been proposed to affect the extent of mixed acid fermentation [31]. Regeneration of NAD+ by Nox has been shown to be necessary for the assimilation of sugar alcohols in S. mutans and for fatty acid synthesis in S. agalactiae, under aerobic conditions [25, 28]. Although the mechanism is not clearly understood, Nox activity is involved in the development of natural competence in Streptococcus pneumoniae [43, 44]. Nox has also been shown to contribute to the oxidative stress resistance of S. pyogenes, S. mutans, and S. pneumoniae [45,46,47]. In these bacteria, Nox has been proposed to reduce the generation of reactive oxygen species (ROS) by affecting cellular metabolism or removing DO, a precursor of ROS, from the medium [45,46,47]. Although we propose the possibility that Nox can promote formate production by establishing anaerobic conditions, further study is necessary to understand the precise role(s) of Nox in milk fermentation and protocooperation with L. bulgaricus.

Recently, Techon et al. characterized the noxE (or nox, nox2) deficient mutant of L. lactis and demonstrated that NoxE was required for the reduction of DO and redox potential of milk [39]. However, in the case of L. lactis, NoxE deficiency only slightly affected the growth of the bacterium in milk. They also found that the L. lactis strains isolated from cheese did not exhibit NADH oxidase activity [32], indicating that Nox is not important for the growth of L. lactis in milk. Although the knowledge about NADH oxidase activities of S. thermophilus isolated from dairy products is limited, it is useful to assess whether the results obtained in the present study for S. thermophilus and L. bulgaricus are also relevant to other strains and strain combinations.

In this study, we demonstrated the H2O2-producing activity of L. bulgaricus 2038 in milk, while S. thermophilus 1131 did not produce detectable H2O2 under the same conditions (Fig. 2). During the coculture of these bacteria in milk, H2O2 was transitionally detected at 90 and 120 min of fermentation (Fig. 3). This suggested that L. bulgaricus 2038 might also contribute to DO reduction and that either S. thermophilus 1131 or the skim milk medium may scavenge H2O2. To assess the fate of H2O2 during coculture, we measured the whole-cell H2O2-scavenging activity of S. thermophilus 1131. H2O2 degradation was not promoted by the addition of S. thermophilus 1131 cells to the PBS or skim milk medium to a final concentration of 109 CFU/ml (data not shown). Instead, this experiment indicated that the medium itself has the ability to decrease H2O2, i.e., 200 μM H2O2 added to the skim milk medium at 43°C decreased to about 90 μM in one hour. The presence of H2O2 during milk fermentation has been suggested in several studies [17, 18]. Further research will clarify the effects of H2O2 on yogurt fermentation.