Effect of Citrate on Production of Diacetyl and Acetoin
by Lactococcus lactis ssp. lactis CNRZ 483
Cultivated in the Presence of Oxygen
H. BOUMERDASSI,* C. MONNET,* M. DESMAZEAUD,†
*Institut National de la Recherche Agronomique, Laboratoire de Génie
et Microbiologie des Procédés Alimentaires, 78850 Thiverval-Grignon, France
ABSTRACT
The effect was studied of trisodium citrate addition
on the growth and formation of diacetyl and acetoin
by Lactococcus lactis ssp. lactis CNRZ 483 in a whey-
based medium under an oxygen pressure of 2 atm.
Growth increased, as shown by the final absorbance
of the culture, which was 1.2 in the absence of citrate
and 2.8 in the presence of 26 mM citrate. In the
absence of citrate, maximal concentrations of diacetyl
and acetoin were 0.09 and 0.29 mM, respectively.
When citrate was added at 6.2, 11.5, and 26 mM, the
maximal diacetyl concentrations were 0.45, 0.53, and
0.85 mM, and those of acetoin were 3.6, 4.5, and 8.5
mM, respectively. However, under these conditions,
the yield of citrate bioconversion to diacetyl and ace-
toin decreased as the initial concentration of citrate
increased. Yields were 121, 83, and 70% in the
presence of 6.2, 11.5, and 26 mM citrate, respectively.
( Key words: Lactococcus lactis ssp. lactis CNRZ 483,
citrate, diacetyl, acetoin)
INTRODUCTION
Citrate is considered to be the principal precursor
of diacetyl (butter flavor) in fermented dairy
products. The concentration of citrate in milk is about
1.7 g/L, and citrate can be metabolized by lactic acid
bacteria of the genus Leuconostoc or by citrate-
utilizing Lactococcus lactis ssp. lactis. Citrate is
transported inside cells via citrate permease. Citrate
is then cleaved to acetate and oxaloacetate by citrate
lyase, and oxaloacetate is decarboxylated to pyruvate
by oxaloacetate decarboxylase. a-Acetolactate syn-
thase then condenses two pyruvate molecules to form
CO2 and a-acetolactate. The latter compound is un-
stable, and its spontaneous decarboxylation may be
Received March 11, 1996.
Accepted August 21, 1996.
1997 J Dairy Sci 80:634–639 634
and G. CORRIEU*
†Institut National de la Recherche Agronomique,
Unité de Recherches Laitières, 78350 Jouy-en-Josas, France
oxidative, yielding diacetyl ( 8 ) , or nonoxidative,
forming acetoin. Acetoin is also produced from a-
acetolactate by a-acetolactate decarboxylase or by the
reduction of diacetyl by acetoin dehydrogenase (22,
25).
Diacetyl is important for the organoleptic quality of
dairy products, such as cottage cheese, butter, and
fermented cream. For this reason, considerable work
has been devoted to improving the production of this
aromatic compound. In the absence of citrate, L. lactis
ssp. lactis 3022 grown in MRS broth produced 0.02
mM diacetyl, but 0.29 mM was synthesized when 2
g/L of citrate were added (13). Similarly, Petit et al.
( 1 9 ) reported that diacetyl production by nonprolifer-
ating cultures of L. lactis ssp. lactis CNRZ 124 in-
creased from 0.2 to 1.4 mM when the citrate concen-
tration was increased from 8.8 to 59 mM. Those
researchers ( 1 9 ) showed that diacetyl production in-
creased twofold when citrate was batch-fed to cells at
the end of growth compared with citrate addition at
the beginning of growth. Citrate stimulated growth,
increased the rate of lactose utilization, and increased
the production of diacetyl, acetoin, and 2,3-butanediol
by Leuconostoc mesenteroides ssp. cremoris (21).
Oxygen also had a positive effect on production of
diacetyl and acetoin (1, 14, 15, 18) because of its
stimulation of a-acetolactate synthase ( 1 ) and NADH
oxidase (1, 3 ) activities and its enhancement of the
oxidative chemical decarboxylation of a-acetolactate
to diacetyl (17, 27, 28). We ( 2 ) recently studied
different oxygenation conditions of a culture of L.
lactis ssp. lactis CNRZ 483 in a whey-based medium
containing 6.2 mM citrate. When fermentation was
conducted under a nitrogen pressure of 1 atm, di-
acetyl production reached 0.015 mM; under an oxygen
pressure of 2 atm, diacetyl production increased to
0.45 mM. The objective of the present work was to
determine the effect of citrate addition on the produc-
tion of diacetyl and acetoin by L. lactis ssp. lactis
CNRZ 483 grown under conditions of oxygenation
that were favorable for diacetyl production.
 DIACETYL PRODUCTION BY LACTOCOCCUS SPECIES
MATERIALS AND METHODS
Strain
Citrate-utilizing L. lactis ssp. lactis CNRZ 483
(from the collection of Institut National de la
Recherche Agronomique, Jouy-en-Josas, France) was
preserved in reconstituted NDM (100 g·L–1; Elle &
Vire, Union Laitière Normande, Condé-sur-Vire,
France) that had been sterilized for 15 min at 110°C.
Stock cultures were obtained after incubation of the
strain in sterilized reconstituted NDM for 8 h at 30°C
and inoculation of 200 ml of the culture in 2 ml of
sterilized litmus milk. Cultures were stored at –20°C
without previous incubation.
Culture Conditions
The growth medium contained dried whey (60
g·L–1; Besnier, Bourgbarré, France), bactopeptone ( 5
g·L–1; Difco Laboratories, Detroit, MI), and yeast ex-
tract ( 3 g·L–1; OSI, Maurepas, France); the pH was
adjusted to 6.8 with HCl. The whey did not contain
citrate; trisodium citrate dihydrate (Prolabo, Paris,
France) was added to the medium in the range of 0 to
26 mM. After saturation of the medium with pure
oxygen introduced in the headspace, cultures were
grown in a 7-L fermenter (Inceltech, Toulouse,
France) that was pressurized at 2 atm of oxygen ( 2 ) .
Temperature and agitation were maintained at 30°C
and 400 rpm, respectively.
Analyses
Growth was monitored at 575 nm with a spec-
trophotometer (UV-160; Shimadzu, Kyoto, Japan).
Cultures were diluted in fresh medium when the
absorbance values exceeded 0.6. For chemical ana-
lyses, cell-free supernatant fluids that were obtained
by centrifugation of media at 14,000 × g for 10 min
were used for the assay of substrates and products.
Diacetyl and acetoin were determined using the
colorimetric method described by Walsh and Cogan
(29). The concentrations of lactic acid, acetic acid,
2,3-butanediol, and citrate were determined by HPLC
as previously described by Bassit et al. ( 1 ) . Ethanol
and formate were determined by an enzymatic
method (Boehringer Mannheim, Mannheim, Germa-
ny).
Determination of the Maximum
Rate of Acidification
Maximum rates of acidification were determined by
using a Weibull function as previously described ( 2 ) .
635
Growth rate was determined by the slope of the linear
portion of a plot relating logarithms of absorbances to
time.
RESULTS AND DISCUSSION
Effect of Citrate on Growth
and Acidification
Lactococcus lactis ssp. lactis CNRZ 483 was grown
in a whey-based medium under an oxygen pressure of
2 atm as described by Boumerdassi et al. ( 2 ) . The
changes in absorbance, pH, and the concentrations of
lactate and acetate as a function of time are shown in
Figure 1. The maximal growth rate was 0.72/h in the
absence of citrate and 0.70/h, 0.75/h, and 0.74/h with
6.2, 11.5, and 26 mM of citrate, respectively, showing
that citrate had no effect on the growth rate of the
strain. However, when citrate was added to the
medium, the maximal absorbance increased from 1.2
in the absence of the substrate to 2.8 with 26 mM
citrate. This absorbance increase could be due to the
buffering effect of citrate ( 9 ) , modulating the
decrease of pH in the medium, which favored growth.
Indeed, the rates of acidification in the absence and
presence of 26 mM citrate were 0.7 and 0.2 pH unit/h,
respectively. After 9 h of incubation, the pH of a
citrate-free medium was 4.5 but was 5.5 when the
initial citrate concentration was 26 mM. The pH
curves of cultures with 6.2 and 11.5 mM citrate were
quite similar, which was probably due to lower initial
cell concentration in the culture with 11.5 mM citrate.
Effect of Citrate on Lactate
and Acetate Production
Lactate production increased as initial concentra-
tions of citrate (Figure 1 ) increased. The maximal
lactate concentration was 16.2 mM in the absence of
citrate and reached 44.8 mM in the presence of 26
mM citrate. As a result of the buffering effect of
citrate, strain CNRZ 483 probably grew in less acidic
conditions and thus produced more lactate from lac-
tose. These findings differ from the results of Libud-
zisz and Galewska (16), who observed no increase in
lactate production by citrate-utilizing L. lactis ssp.
lactis in milk that had been augmented with citrate.
According to Cogan ( 4 ) , the relationship between the
growth of the strain CNRZ 483 and lactate production
was linear, as is shown in Figure 2. At each level of
initial citrate concentration, the plots obtained were
almost identical, and the corresponding slopes varied
from 0.071 to 0.076. Also, lactate production related
to growth was not modified by citrate addition.
Journal of Dairy Science Vol. 80, No. 4, 1997
636 BOUMERDASSI ET AL.

In the absence of citrate, the maximal acetate
production of 4.2 mM (Figure 1 ) was equivalent to
almost 26% of the lactate concentration. This hetero-
lactic fermentation was due to the presence of oxygen
because the same culture in anaerobic conditions
yielded primarily lactate (results not shown). For all
cultures, no significant production of formate and
ethanol occurred. Acetate production in aerobic condi-
tions has often been observed in lactococci (5, 7, 10,
23). The acetate that was produced by L. lactis ssp.
lactis CNRZ 483 in the absence of citrate probably
arose from pyruvate via pyruvate dehydrogenase.
This enzyme converts pyruvate to acetyl-coenzyme A
with the simultaneous production of NADH and CO2.
Acetyl-coenzyme A is transformed to acetyl phosphate
by phosphoacetyl transferase; then, acetate kinase
produces acetate and ATP. Pyruvate dehydrogenase
expression was highest in aerobic conditions and
when the ratio of NADH to NAD was low (24). In
addition, the presence of oxygen increased NADH
oxidase activity in lactococci (1, 3). This enzyme
reoxidizes the NADH produced during glycolysis and
thus could replace lactate dehydrogenase in the
presence of oxygen for the regeneration of NAD. Thus,
starting from pyruvate, a portion of the carbon flux
could be diverted toward acetate, leading to an energy
gain.
For cultures in the presence of citrate, acetate
production increased as initial citrate concentration
increased (Figure 1). When lactococci utilize citrate,
acetate is formed by citrate lyase. In all fermenta-
tions, acetate production and citrate consumption had

Figure 1. Effect of initial citrate concentration on changes in absorbance, pH, lactate concentrations, and acetate concentrations in
cultures of Lactococcus lactis ssp. lactis CNRZ 483. Cultures were incubated at 30°C in a whey-based medium under 2 atm of oxygen
pressure. Initial citrate concentration: 0 mM ( ÿ) , 6.2 mM ( ♦) , 11.5 mM ( ⁄) , or 26 mM ( π) .

Journal of Dairy Science Vol. 80, No. 4, 1997

 DIACETYL PRODUCTION BY LACTOCOCCUS SPECIES 637

conditions employed, L. lactis ssp. lactis CNRZ 483

did not reduce diacetyl or acetoin because acetoin and
diacetyl reduction occurred only when an excess of
NADH2 occurred. In presence of oxygen, the NADH2
was reoxidized by lactate dehydrogenase and by
NADH oxidase ( 1 ) ; thus, less NADH2 was available

Figure 2. Absorbance as a function of lactate production by

Lactococcus lactis ssp. lactis CNRZ 483. Cultures were incubated at
30°C in a whey-based medium under 2 atm of oxygen pressure.
Initial citrate concentration: 0 mM ( ÿ) , 6.2 mM ( ♦) , 11.5 mM ( ⁄) ,
or 26 mM ( π) .

a linear relationship. The molar ratio of acetate

produced to citrate consumed, however, was higher
than unity for all cultures, because acetate was also
produced from lactose in the presence of oxygen. This
ratio was 1.9 and 1.6 in the presence of 6.2 and 11.5
mM citrate and decreased to 1.1 when the initial
substrate concentration was 26 mM. This reduction
probably occurred because acetate synthesis from ci-
trate became predominant over that from lactose,
when the initial citrate concentration increased.

Effect of Citrate on Diacetyl

and Acetoin Production
Figure 3 shows the time course of changes in ci-
trate, diacetyl, and acetoin concentrations in cultures
of L. lactis ssp. lactis CNRZ 483. In cultures with 6.2
and 11.5 mM citrate, citrate was consumed in 5 h,
and, with 26 mM citrate, citrate was exhausted in 9
h. In the absence of citrate, diacetyl and acetoin were
produced at 0.09 and 0.29 mM, respectively. After
addition of 6.2, 11.5, and 26 mM citrate to the
medium, the maximal diacetyl concentration in-
creased to 0.45, 0.53, and 0.85 mM and that of acetoin
reached 3.6, 4.5, and 8.5 mM, respectively. Maximal Figure 3. Effect of initial citrate concentration on changes in the
production of diacetyl and acetoin corresponded to concentrations of citrate, diacetyl, and acetoin during incubation of
Lactococcus lactis ssp. lactis CNRZ 483 at 30°C in a whey-based
citrate depletion. Furthermore, no 2,3-butanediol was medium under 2 atm of oxygen pressure. Initial citrate concentra-
produced (data not shown). Thus, with the culture tion: 0 mM ( ÿ) , 6.2 mM ( ♦) , 11.5 mM ( ⁄) , or 26 mM ( π) .

Journal of Dairy Science Vol. 80, No. 4, 1997

638 BOUMERDASSI ET AL.
for the reduction of diacetyl and acetoin. Many strains
of lactococci do not reduce acetoin or diacetyl (1, 17).
The amounts of diacetyl and acetoin produced and
the citrate consumed in cultures incubated with 6.2,
11.5, and 26 mM citrate are depicted in Figure 4. The
coefficient of determination for the different lines
varied from 0.97 to 0.98 at various initial citrate
concentrations, showing that the production of di-
acetyl and acetoin was proportional to citrate con-
sumption. However, the slopes of the different lines
Figure 4. Diacetyl and acetoin production as a function of citrate
consumption in cultures of Lactococcus lactis ssp. lactis CNRZ 483.
Cultures were incubated at 30°C in a whey-based medium under 2
atm of oxygen pressure. Initial citrate concentration: 6.2 mM ( ♦) ,
11.5 mM ( ⁄) , or 26 mM ( π) .
Journal of Dairy Science Vol. 80, No. 4, 1997
decreased considerably as the initial citrate concen-
tration increased. The slopes for diacetyl were 0.074,
0.045, and 0.031 with initial citrate concentrations of
6.2, 11.5, and 26 mM. This decrease was less impor-
tant for acetoin because the slopes were 0.53, 0.37,
and 0.34 in the presence of 6.2, 11.5, and 26 mM of
citrate, which suggested that the molar ratio of di-
acetyl to acetoin decreased slightly when initial ci-
trate concentration increased. Thus, at the end of
fermentation, this ratio was 0.13, 0.12, and 0.10 in
the presence of 6.2, 11.5, and 26 mM citrate, respec-
tively. Theoretically, 2 mol of citrate were required to
form 1 mol of diacetyl or acetoin. The molar yield of
citrate bioconversion ( Y C) into diacetyl and acetoin
can be calculated by the following equation: YC = 2 ×
[(diacetyl/citrate) + (acetoin/citrate)]; slopes of these
ratios are shown in Figure 4. Molar yield decreased
considerably as the initial citrate concentration in-
creased: 121, 83, and 70% in the presence of 6.2, 11.5,
and 26 mM citrate, respectively. The yield obtained
with 6.2 mM indicated that diacetyl and acetoin
production was higher than citrate consumption; di-
acetyl and acetoin production apparently occurred
even in the absence of citrate. The decrease of the
yield of citrate bioconversion was probably due to an
effect on pH, which increased as initial citrate concen-
trations increased. Similarly, the formation of
a-acetolactate subsequently transformed to diacetyl
and acetoin is favored by acidic pH. When citrate is
utilized at low pH, the intracellular pyruvate concen-
tration is very high (26), favoring the activity of
a-acetolactate synthase, which has a very low affinity
for pyruvate (12, 24). Collins ( 6 ) has suggested that
the formation of diacetyl and acetoin is a means for
the cell to eliminate excess toxic pyruvate.
CONCLUSIONS
In the presence of oxygen, the addition of citrate to
the culture medium increased the production of di-
acetyl and acetoin. However, the increase of initial
citrate concentration required a longer fermentation
time for total citrate consumption. In addition, the
yield of bioconversion of citrate to diacetyl and acetoin
decreased considerably when the initial concentration
of citrate (as trisodium citrate) increased. This
decrease was probably due to the increased buffering
effect of higher citrate concentrations. This study
showed the limit of a citrate addition strategy to
improve diacetyl production. Interesting studies of
metabolic engineering in Lactococcus lactis have been
recently published (11, 20); the resulting strains ob-
tained produced diacetyl in the absence of citrate.
 DIACETYL PRODUCTION BY LACTOCOCCUS SPECIES
ACKNOWLEDGMENTS
The financial support of the European Union under
the agro-industrial research (contract number AIR3-
CT94-2010) program is greatly appreciated. H. Bou-
merdassi was financed by the Algerian Higher Educa-
tion Ministry and the French Foreign Affairs Minis-
try.
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