APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1990, p. 2761-2763 Vol. 56, No.

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0099-2240/90/092761-03$02.00/0
Copyright ( 1990, American Society for Microbiology

Enzyme Activities Affecting End Product Distribution by

Lactobacillus plantarum in Response to Changes in pH and O2t
CHING-PING TSENG' AND THOMAS J. MONTVILLE2*
Department of Food Science2 and Graduate Program in Microbiology,' New Jersey Agricultural Experiment Station,
Cook College, Rutgers-The State University, New Brunswick, New Jersey 08903
Received 26 January 1990/Accepted 2 July 1990

Lactobacillus plantarum catabolic end products changed in response to environmental conditions. While
lactate was always the major end product, acetate was produced in alkaline and aerobic environments. Acetoin

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levels decreased under alkaline conditions. Changes in acetoin dehydrogenase, acetate kinase, NADH oxidase,
pyruvate oxidase, and acetate kinase activities correlated with changes in end product distribution.

Homofermentative and facultatively heterofermentative
lactobacilli oxidize glucose to pyruvate through the Embden-
Meyerhof-Parnas pathway (14). The pyruvate can be con-
verted into lactate, acetate, acetoin, ethanol, and 2,3-butane-
diol (12, 13). The reduction of pyruvate to lactate by lactate
dehydrogenase (LDH) allows for NAD regeneration, which
is obligatory for continued glycolysis (9). The LDH of some
organisms (such as Lactobacillus casei and most lactococci)
has an absolute and specific requirement for fructose 1,6-
diphosphate (FDP) (8, 34); in its absence, LDH has low
activity and catabolism is shifted to other end products.
Lactobacillus plantarum and other homofermentative lacto-
bacilli which lack an FDP-activated LDH also form diverse
catabolic products, but the mechanisms are unknown (12,
30). Even though some L. plantarum catabolic enzymes
have been characterized (11, 25, 26), little is known about
how these lactobacilli regulate catabolite distribution. In
alkaline (19) and aerobic (24) environments, L. plantarum
produces acetate in addition to lactate, but acetoin levels
decrease with increasing pH. The objective of this study was
to measure the enzyme activities of L. plantarum grown
under a combination of acid, alkaline, aerobic, and anaero-
bic conditions so that changes in enzyme activities could be
correlated with the shift of glucose catabolism which occurs
in response to environmental conditions.
MATERIALS AND METHODS
L. plantarum ATCC 8014 was maintained as previously
described (24) and cultured in a modification (22) of the
medium of Craig and Snell (6), designated CS-T, which was
prepared in 400 mM phosphate buffer at pH 5.5 or 7.7.
Glucose was sterilized separately and added to a final
concentration of 25 mM. Cultures were grown for 20 h at
37°C under anaerobic (GasPak jars; BBL Microbiology
Systems, Cockeysville, Md.) or aerobic (baffle flasks with
shaking at 200 rpm) conditions.
End products were quantified by high-pressure liquid
chromatography (23). In some instances, lactate concentra-
tions were also determined enzymatically by monitoring the
increase in NAD A340 (Sigma Chemical Co., St. Louis, Mo.).
Cells were harvested and washed with 100 mM potassium
phosphate buffer (pH 7.0). Cell extracts were prepared with
*
Corresponding author.
t This is manuscript D-10112-1-90 of the New Jersey State Agri-
cultural Experiment Station.
2761
a Sonifier cell disruptor (Branson Sonic Power Co., Dan-
bury, Conn.) for six cycles of 50 s of sonication and 10 s at
rest with cells on ice. Unbroken cells and debris were
removed by refrigerated (4°C) centrifugation at 14,000 x g
for 5 min.
Pyruvate formate lyase, ethanol dehydrogenase, and 2,3-
butanediol dehydrogenase activities in the cell extracts were
assayed by methods described in references 31, 15, and 18,
respectively. Acetaldehyde dehydrogenase (18), acetoin de-
hydrogenase (18), and NADH oxidase (28) activities were
determined in a potassium phosphate buffer (pH 7.0) by the
change in A340. The assays for lactate racemase (20) and
pyruvate dehydrogenase (3) were modified slightly by using
potassium phosphate buffer at pH 7.0. oa-Acetolactate syn-
thase was measured as described previously (4), and aceto-
lactate was then decarboxylated to acetoin, which was
quantified colorimetrically (35). Pyruvate oxidase (16, 29),
acetate kinase (27), and phosphotransacetylase (2, 16) were
assayed spectrophotometrically. The assay for NAD-depen-
dent LDH (nLDH) (20) was modified by slight changes in
cofactor concentrations; 0.12 mM NADH and 2.3 mM
sodium pyruvate were used in 100 mM phosphate buffer at
pH 7.5. Activation of LDH by FDP was determined by the
method of Gotz and Schleifer (10). Negative control assays,
in which the cell extract was replaced with buffer and the
reaction-specific substrate was omitted, confirmed the spec-
ificity of each assay. The enzyme activity levels presented
(see Table 2) are averages of triplicate determinations which
differed by <6% and are expressed as international units per
milligram of protein. Protein was measured by the method of
Bradford (1).
RESULTS AND DISCUSSION
When L. plantarum was cultured at pH 5.5 or pH 7.7 and
grown aerobically or anaerobically in separate cultures,
differences in end products and cell mass were observed
(Table 1). The terminal pH differed from the initial medium
pH by <0.1 (data not shown). Cultures grew to higher cell
densities in aerobic than in anaerobic environments; pH 5.5
cultures produced more mass than pH 7.7 cultures did.
Lactate was always the major fermentation product, ac-
counting for >60% of the carbon catabolized. Lactate con-
centrations were highest under anaerobic and alkaline con-
ditions. L. plantarum contains two separate nLDHs which
form L-lactate and D-lactate (7, 9, 21). In this study, no
lactate racemase was detected. nLDH activities were higher
in pH 5.5 aerobic cultures than in pH 5.5 anaerobic cultures
2762 TSENG AND MONTVILLE APPL. ENVIRON. MICROBIOL.

TABLE 1. Cell dry weights and catabolite levels of cultures (Table 2), but again the requirement for NAD
L. plantarum cultures regeneration limited acetate production to the pH 5.5 cul-
Catabolite level (mmol/mg of tures (Table 1). Acetate production was accompanied by the
Culture conditions Cell dry wt cell dry wt) formation of one extra ATP molecule, a 50% bonus over the
(mg/liter) normal fermentative yield of two ATP molecules per mole of
Lactate Acetate Acetoin glucose.
Aerobic
pH 5.5 215.3 0.64 0.33 0.09 ACKNOWLEDGMENTS
pH 7.7 83.2 1.89 NDa 0.07
This work was supported by New Jersey state appropriations and
Anaerobic U.S. Hatch Act funds.
pH 5.5 160.2 0.93 ND 0.14
pH 7.7 45.9 1.97 ND 0.07 LITERATURE CITED
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VOL. 56, 1990
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