Production by Lactococcus lactis subsp. lactis

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p. 1893-1897 99-224/93/61893-5$2./ Copyright 1993, American Society for Microbiology Vol. 59, No. 6 Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis NAIMA BASSIT, CLAIR-YVES BOQUIEN,* DANIEL PICQUE, AND GEORGES CORRIEU Laboratoire de Genie des Procedegs Biotechnologiques Agro-alimentaires, Institut National de la Recherche Agronomique, 7885 Thiverval-Grignon, France Received 3 November 1992/Accepted 27 March 1993 The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 3 C at different initial oxygen concentrations (, 21, 5, and 1% of the medium saturation by oxygen). Regardless of the initial 2 concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was.1 and 2.4 mm, respectively, at % oxygen. Maximum acetoin concentration reached 5.4 mm at 1% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 5, and 1,o respectively. The diacetylacetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 1% (3.3%) than at O%o oxygen (.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 1% oxygen, specific activity of a-acetolactate synthetase (.15 U/mg) and NADH oxidase (.4 U/mg) was 3.6 and 5.4 times lower, respectively, than at 1o oxygen. The increasing cv-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds. Many lactic acid bacteria, especially Lactococcus lactis subsp. lactis biovar diacetylactis, use citrate to produce aroma compounds such as acetoin and diacetyl. The latter is the most desired aroma compound in fresh, unripened cheeses. Citrate fermentation pathways (Fig. 1) have been studied by several authors (9, 1, 16). The substrate enters the cells via a citrate permease and is split to oxaloacetate and acetate by a citrate lyase (9). The decarboxylation of oxaloacetate yields pyruvate, which is transformed to acetaldehyde-thiamine pyrophosphate and a-acetolactate. It is established that diacetyl arises from the oxidative decarboxylation of a-acetolactate (11, 23, 25). Acetoin is produced by the enzymatic decarboxylation of ot-acetolactate by acetolactate decarboxylase or by reduction of diacetyl by diacetyl reductase. The extent of formation of these aroma compounds depends on the L. lactis strain (7), the citrate concentration in the medium (8, 18, 19), the presence of hemin and/or metal ions such as Cu2+ (13), temperature and ph (2), and cell immobilization (21, 22). Prior studies have shown the importance of oxygen for diacetyl and acetoin production, which increases by a factor of 3 to 9 when the culture medium is agitated (4, 14) or when air is introduced during growth (3). To complete these studies, we have tested the effect of atmospheres containing different initial oxygen concentrations on the production of diacetyl and acetoin, as well as on growth and acidification. Experiments were carried out in the culture conditions of fresh, unripened cheese production (nonagitated culture). Results were obtained initially with L. lactis subsp. lactis * Corresponding author. biovar diacetylactis CNRZ 483 and subsequently generalized to other lactococci. MATERIALS AND METHODS Microorganisms. Nine strains were used: L. lactis subsp. lactis NCDO 763 was provided by the National Collection of Dairy Organisms (Shinfield, Reading, United Kingdom). L. lactis subsp. lactis biovar diacetylactis SD 13 and CDI 1 were isolated from two different industrial mixed-strain starters. L. lactis subsp. lactis biovar diacetylactis CNRZ 483, DRC1 (also called CNRZ 124), DRC2 (CNRZ 125), DRC3 (CNRZ 126), and CNRZ 365 and L. lactis subsp. cremoris AM2 (CNRZ 38) were obtained from the Institut National de la Recherche Agronomique collection of lactic acid bacteria (Jouy-en-Josas, France). They were stored in litmus milk at -2 C. Culture medium. The culture medium contained skimn powdered milk (Elle & Vire; ULN, Conde-sur-Vire, France) reconstituted at 1% (wt/vol) and pasteurized at 92 C for 5 min. It was supplemented with.1% of calf rennet at 52 mg of active chymosin (Boll, Arpajon, France) per liter. Batch cultures. The culture medium was inoculated at 3% (vol/vol) with a culture grown in skim milk for 8 h at 3 C. Medium (4 ml) was poured into sterile conical flasks (15-ml total volume). Four initial concentrations of dissolved oxygen were tested; they were determined with an oxygen probe (Ingold, Urdorf, Switzerland) previously calibrated in the same medium at % 2 with nitrogen and then at 1% 2 with 2. Next, 1, 5, 21, and % of the saturation by oxygen were obtained by flushing the medium with oxygen, a mixture of oxygen and air controlled at 5% 2 with a gas analyzer (Servomex, Crowborough, England), air, or nitrogen, respectively. The flasks were hermetically closed and 1893

1894 BASSIT ET AL. NAD(P)H NAD(P) 2 CITRATE citrate lyase ;_ 2 ACETATE] 2 oxaloacetate 2 C2 oxaloacetate 2 decarboxylase 2 PYRUVATE co 2 ACETALDEHYDE-TPP acetolactate synthetase ACETOLACTATE acetolactate 2 decarboxylase diacetyl ACETOIN reductase NAD(P) acetoin reductase 2,3-BUTANE DIOL NAD(P)H 2 DIACETYL FIG. 1. Citrate metabolism in L. lactis subsp. lactis biovar diacetylactis (6, 11). incubated at 3C without agitation. The dissolved oxygen concentration was monitored on-line with the probe in one of the flasks. Samples (one flask per sample) were taken hourly until 14 h of culture and then after 24 h. Bacterial population, ph, and concentrations of citrate, lactic acid, diacetyl, acetoin, and 2,3-butanediol were determined in every sample Ėxperiments with L. lactis subsp. lactis biovar diacetylactis CNRZ 483 were run in duplicate. Only the and 1% initial oxygen concentrations were tested with L. lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AM2. Bacterial enumeration. A culture sample was treated with a TURRAX disperser for 3 s. The population was determined by plating on M17 agar (Biokar, Beauvais, France). The ability to metabolize citrate was assayed with the medium of Kempler and McKay (15). Analyses. The concentrations of citrate, lactic acid, acetoin, and 2,3-butanediol were determined by high-performance liquid chromatography (HPLC). The HPLC system (Waters) was composed of an automatic injector, two serial detectors (a UV detector [214 nm for citrate] and a differential refractometer [for acetoin and lactic acid]), and two integrators. The cation-exchange column (Aminex HPX- 87H) was maintained at 6 C. Sulfuric acid (.1 M) was used as mobile phase at a flow rate of.6 ml/min. Samples were precipitated with trichloroacetic acid at 4% and centrifuged at 11, x g for 3 min. Butyric acid (.1%) was used as an internal standard. Diacetyl was determined with the colorimetric method of Walsh and Cogan (26). A 2-g sample was steam distilled, APPL. ENVIRON. MICROBIOL. and diacetyl was assayed in the first 1 ml of the distillate. Each value is the mean of two determinations. Determination of the maximum growth rate (Um,.) and of the maximum rates of lactic acid production (VL,,) and acidification (VPH). Data for bacterial population, ph, and lactic acid concentration were fitted with the following Weibull function (17): X = XO ± a[1 - exp (-bt)], where a, b, and c are constants, t is time (hours), X is ph, lactic acid concentration, or the logarithm of bacterial population at time t, and XO is the corresponding value at t =. The function is an increasing one for bacterial population and lactic acid and decreasing for ph. The first derivative of this equation was determined. Its maximum (UJmax for bacterial population, VLA for lactic acid, and V H for ph) was reached when the derivative was cancelfed with reference to time. Enzyme assays. Cells of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 were grown in M17 medium for 8 h at 3 C in conical flasks. The medium was initially saturated with oxygen (1% 2), the mixture of air and oxygen (5% 2), air (21% 2), or nitrogen (% 2). Cells were harvested by centrifugation at 3, x g for 15 min at 4 C, washed twice, and resuspended in an appropriate volume of 5 mm Na phosphate buffer (ph 7). Cells were lysed after incubation for 1 h at 37 C with lysozyme (15, U/ml) and mutanolysin (15 U/ml). The supernatant was recovered after centrifugation at 2, x g for 4 min. Acetoin reductase (6), diacetyl reductase (6), lactate dehydrogenase (24), and NADH oxidase (1) were assayed in the cell extract by monitoring the decrease ina34 due to NADH oxidation. No NADH oxidase activity was found in the assays of acetoin reductase, diacetyl reductase, and lactate dehydrogenase. One unit of activity oxidized 1,umol of NADH per min. a-acetolactate synthetase was determined by measuring the conversion of pyruvate into acetoin (6). One unit of activity formed 1 pzmol of acetoin per min. Protein assay. The quantity of protein was assayed by the method of Bradford (2) with bovine serum albumin as the standard. RESULTS Citrate consumption and production of aroma compounds by L. lactis subsp. lactis biovar diacetylactis CNRZ 483. Figure 2 shows concentration changes of citrate, diacetyl, and acetoin versus time during the culture of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 at different initial oxygen concentrations. The initial citrate concentration was 11 mm. Citrate was consumed after 5 h when the initial atmosphere was N2 (% 2) and after 6 h when the initial oxygen concentration was 21 or 5% (Fig. 2B). Oxygen was consumed during the fermentation, and the dissolved 2 concentration was nearly % when citrate was exhausted (Fig. 3). At an initial 2 content of 1%, citrate exhaustion was observed after 6 h of growth (Fig. 2B) while the dissolved 2 concentration was still 4% (Fig. 3). Diacetyl production stopped after 5 h of culture in all atmospheres tested (Fig. 2C) regardless of the dissolved 2 concentration in the medium at that time (Fig. 3). There was no decrease in the diacetyl concentration at the end of culture resulting from reduction to acetoin. The maximum concentration of diacetyl varied significantly with initial oxygen concentration. It was.1 mm in the presence of nitrogen and increased by factors of 2, 6, and 18 at 21, 5, and 1% 2, respectively. After 6 h of culture, acetoin production stopped or in-

VOL. 59, 1993 OXYGEN AND AROMA PRODUCTION 1895 _~ 2 2 m1 b - TABLE 1. Acidification characteristicsa of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 at different initial oxygen concentrations xeb ph24 VPH (ph U/h) LA24 (mm) VLA (mm/h) 1 4.58 ±.5.193 ±.6 89.9 ±.1 7.41 ±.3 5 4.56 ±.6.211 ±.7 93.1 ±.8 7.98 ±.25 21 4.55 ±.4.218 ±.8 94.4 ± 3.1 8.49 ±.47 4.49 ±.4.234 ±.13 95.9 + 2.5 9.69 ±.13 a ph24 and LA2, ph and lactic acid concentration reached at 24 h of culture, respectively; VPH, maximum acidification rate; VA, maximum rate of lactic acid production. All values are mean values + standard deviations. Expressed as percentage of the medium saturation by oxygen..) 5 1 15 2 25 FIG. 2. Bacterial population (A), citrate utilization (B), and diacetyl (C) and acetoin (D) production at different initial oxygen concentrations by L. lactis subsp. lactis biovar diacetylactis CNRZ 483 at 3 C. Oxygen concentration is expressed as a percentage of the medium saturation by oxygen. Symbols: *, 1% 2;, 5% 2; C, 21% 2; *, % 2- creased only slightly (Fig. 2D). The maximum concentration was reached when all the citrate had disappeared. Acetoin production was 2.4 mm at % 2 and increased with initial oxygen concentration up to 5.4 mm at 1% 2. There was no formation of 2,3-butanediol at 1% initial 2. At, 21, and 5% 2, 2,3-butanediol was produced only at the end of the fermentation (results not shown). After 24 h of culture, the production reached.21,.21, and.3 mm, respectively. At 1% initial 2, the sum of diacetyl, acetoin, and 2,3-butanediol concentration was 5.6 mm, while it was only 2.6 mm in a nitrogen atmosphere. The ratio of diacetyl to acetoin concentration (DIA) char- 1-1 C. C ) 1 8-6 - 4-2 - 44 44 44 "I, 4 49 411 4 *# t1 ~~~~1 ~~~~~1 4# 4 4 1 46 1% I~~~ 4 ~~~~~~~~~~~~~~~ 2 4 6 8 1 12 FIG. 3. On-line determination of oxygen concentration (expressed as a percentage of the medium saturation by oxygen) during growth of L. lactis subsp. lactis biovar diacetylactis CNRZ 483. Three different initial oxygen concentrations were assayed: 1% (---), 5% ( - -), and21%( ). acterizes curd flavor. The higher this ratio, the better the curd quality. Acetoin, the major compound produced by citrate metabolism, is less desirable than diacetyl aroma. This ratio increased with initial oxygen concentration in the atmosphere: it was eight times higher at 1% 2(3.3%) than at % 2 (.4%). Growth of and acidification by L. lactis subsp. lactis biovar diacetylactis CNRZ 483. The initial bacterial population was 7 x 17 CFU/g (wet weight) (Fig. 2A). Growth stopped after 5 h, and the population reached 2.1 x 19, 1.8 x 19, 2.4 x 19, and 1.8 x 19 CFU/g at, 21, 5, and 1% 2, respectively. Thus, the final population was not significantly affected by the presence of oxygen. Furthermore, the maximum growth rate (Imax) was.65.2 h-1 regardless of the initial 2 concentration. Table 1 lists the acidification characteristics of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 expressed as a function of initial oxygen concentration. Lactic acid concentration reached after 24 h of culture (LA24) increased from 89.9 to 95.9 mm between 1 and % 2, while ph at 24 h (ph2a) decreased from 4.58 to 4.49. The maximum rate of acidification (VPH) and of lactic acid production (VL) was inversely proportional to initial oxygen concentration. At 1% 2, V H and V_A were 18 and 24% lower than at %, respectivef;. These data show that the acidifying ability of the strain was maximal in the presence of nitrogen and decreased as the initial oxygen concentration increased. Effect of oxygen on enzyme activity. Regardless of the initial 2 concentration in the culture medium, specific activity of diacetyl reductase, acetoin reductase, and lactate dehydrogenase in L. lactis subsp. lactis biovar diacetylactis CNRZ 483 was 2.83.8, 1.2.14, and 25. + 2.9 U/mg, respectively (Table 2). At % oxygen, specific activity of NADH oxidase was.4 U/mg and that of a-acetolactate synthetase was.15 U/mg. Both activities were increased TABLE 2. Enzyme activity determined in cell lysates of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 grown for 8 h under different gas atmospheres Sp act (U/mg of total protein) Oxygena Diacetyl Acetoin Acetolactate NADH Lactate reductase reductase synthetase oxidase dehydrogenase 1 2.83 1.1.55.21 24.1 5 2.74 1.26.3.15 22.2 21 2.93 1.34.26.9 29. 2.83 1.21.15.4 25.2 a Initial 2 concentration expressed as a percentage of medium saturation by oxygen.

1896 BASSIT ET AL. 3 4-3- 2- n w 2 4 6 8 Oxygen concentration (%) FIG. 4. DIA ratio determined for the seven strains of L. lactis subsp. lactis biovar diacetylactis used (CNRZ 483 [El], CNRZ 365 [], DRC1 [X], DRC2 [A], DRC3 [-], SD 13 [-], and CDI 1 [A]) versus initial oxygen concentration. with initial oxygen concentration; they were 3.6 and 5.4 times higher at 1% oxygen, respectively. Diacetyl and acetoin production by other lactococci. The effect of initial oxygen concentration in the atmosphere on diacetyl and acetoin production by other lactococci was determined. The results were compared with those obtained with strain CNRZ 483. Apart from strain SD 13 (only.9 mm diacetyl and 2.8 mm acetoin produced at 1% oxygen), production by other L. lactis subsp. lactis biovar diacetylactis strains (CNRZ 365, DRC1, DRC2, DRC3, and CDI 1) was comparable to that of strain CNRZ 483. Thus, changes versus initial 2 concentration were similar (.17 +.2 mm diacetyl, 5.5 +.8 mm acetoin, and ratio of diacetyl to acetoin [DIA] of 3.1% +.4% at 1% 2). The DIA ratio increased around 1 times as initial oxygen varied from to 1% (Fig. 4). A linear relationship was established for the seven strains of L. lactis subsp. lactis biovar diacetylactis tested between the DIA concentration ratio and the initial oxygen concentration (Fig. 4): DIA (%) =.28 (% 2) +.187. A high determination coefficient (r2 =.94) and a significant slope value (standard deviation =.1) were determined. L. lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AM2 do not metabolize citrate. They were tested only at and 1% 2. At % 2, the two compounds assayed were absent, while at 1% initial oxygen, some acetoin (1 to 1.5 mm) and diacetyl (.3 mm) were produced by the strains tested. Diacetyl production at 1% oxygen was nearly the same as that of strain CNRZ 483 in the presence of 21% oxygen. Acetoin production was significantly lower than that by L. lactis subsp. lactis biovar diacetylactis, while the DIA ratio was similar (2. to 3.%). DISCUSSION The results show that diacetyl and acetoin production by all the L. lactis strains was clearly improved when the initial oxygen concentration increased. Two hypotheses can explain this effect. (i) The specific activity of ot-acetolactate synthetase was 3.6 times higher in cells of L. lactis subsp. lactis biovar diacetylactis CNRZ 483 grown in the presence of oxygen in comparison to cells grown in the presence of nitrogen. Cogan et al. (5) have found that this activity in L. lactis is 2 to 2.5 times higher in aerobically grown cells than in anaerobically grown cells. This could lead to a higher production of acetoin and probably diacetyl under aerobic 1 APPL. ENvIRON. MICROBIOL. conditions. (ii) NADH oxidase activity in L. lactis subsp. lactis biovar diacetylactis was 5.4 times higher at 1% than at % oxygen. Bruhn and Collins (3) have shown that this activity in L. lactis is high in the presence of air. This enzyme ensures the reoxidation of NADH generated during glycolysis. Its activity would explain 2 consumption observed during growth of the strain CNRZ 483. It would replace the role of lactate dehydrogenase, acetoin reductase, and diacetyl reductase in the regeneration of NAD (Fig. 1). This hypothesis is consistent with the fact that the quantity of lactic acid produced was slightly lower in oxygen (9 mm) than in nitrogen (96 mm) and the maximal rates of acidification and lactic acid production decreased in the presence of oxygen (by about 2%). In these conditions, diacetyl formation is a means of eliminating accumulated pyruvate that may be toxic (1). It is to be noted that some pyruvate arising from lactose is also used by L. lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AM2 to produce diacetyl and acetoin (only in the presence of oxygen), since neither strain metabolizes citrate. The hypothesis is also consistent with the fact that no notable reduction of diacetyl and acetoin was observed with L. lactis subsp. lactis biovar diacetylactis CNRZ 483 at the end of culture. Cogan (7) and Petit et al. (19) have shown that the diacetyl concentration decreases after all the citrate is consumed, but Kaneko et al. (14) reported that this is preferentially observed in nonagitated culture. A chemical oxidation of acetoin under oxidative conditions might be responsible for diacetyl production. However, no diacetyl formation was observed when acetoin (5 mm) was incubated at 3 C for 15 h in a milk solution under an oxygen atmosphere, regardless of the presence of end products such as acetic acid (1 mm) or lactic acid (9 mm). These results indicate that diacetyl was only formed by oxidative decarboxylation of acetolactate. The results obtained in this study at a 1% initial 2 content show that the presence of oxygen was not sufficient to produce diacetyl after citrate exhaustion. However, diacetyl production was limited at low initial 2 concentration ( or 21%). Diacetyl production by L. lactis subsp. lactis biovar diacetylactis CNRZ 483, CNRZ 365, DRC1, DRC2, DRC3, and CDI 1 was around.17 mm at 3 C under oxygen. This production is relatively high in comparison to usual observations (less than.1 mm) for L. lactis strains (8, 18). Only one strain, described by Jordan and Cogan (12), produces quantities of diacetyl almost six times higher (1 mm). But this strain is unusual, since it accumulates a-acetolactate which could be broken down into diacetyl during sample distillation, leading to false values for diacetyl (12). The present results show that in the presence of oxygen, the quantity of diacetyl and acetoin produced by L. lactis increases. The DIA ratio is a linear function of initial oxygen concentration. This finding can thus be used to improve the aroma qualities of a fresh, unripened cheese curd during its manufacture. ACKNOWLEDGMENTS We thank G. Yonnet and E. Ferreira for their technical assistance and T. Cogan for his advice concerning the methods used for the determination of diacetyl and enzyme activity. REFERENCES 1. Anders, R. F., D. M. Hogg, and G. R. Jago. 197. Formation of hydrogen peroxide by group N Streptococci and its effect on their growth and metabolism. Appl. Microbiol. 19:68-612.

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