Arginine metabolism in malolactic wine lactic acid bacteria and its oenological implications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
L-Arginine is a major amino acid found in grapes and wine which is
degraded by some wine lactic acid bacteria (LAB). The mechanism of this
degradation and its oenological implications were examined in this research.
It was found that wine LAB able to degrade arginine do so by means of the
arginine deiminase pathway, demonstrated by measuring the enzyme
activitie since l l-free extracts: arginine deiminase, ornithinetrans carbamylase
and carbamate kinase. These enzymes we represent in most
heterofermentative lactobacilli and leuconostocs, but were absent in
homofermentative lactobacilli and pediococci.
The presence of arginine increased the activities of arginine deiminase
pathway enzymes in heterofermenters, but failed to induce these enzymes in
homofermenters even under conditions of low glucose concentration (1g/L).
Glucose did not repress arginine utilisatio n but fructose appeared to do so,
as fructose and arginine were metabolised sequentially, with arginine being
metabolised mainly after utilisation of the fructose.
D etailed studies o n Leuconostoc oenos OENO, Lactobacillus b uchneri
CUC-3 and Lactobacillus brevis 250 showed that arginine was converted
stoichiometrically to ammonia and ornithine as the major end-products and
that arginine catabolism could supply energy (ATP) to support growth. lt was
also demonstrated that citrulline was excreted during arginine catabolism by
both the lactobacilli and the leuconostoc. Some of the excreted citrulline was
reassimilated and catabolised after arginine depletion by the lactobacilli, but
not by the leuconostoc.
The implication of citrulline excretion for the wine industry was explored by
studying the formation of the carcinogen ethyl carbamate (urethane) in a
synthetic wine and a white wine, since citrulline is a known precursor of ethyl
carbamate. During growth of Le. oenos OENO and Lb. buchneri CUC-3 in
the synthetic wine and wine, significant amounts of ethyl carbamate were
found i n the two wine types upon heat treatment of samples. The formatio n
of ethyl carbamate correlated well with arginine degradation and citrulline
excretion. Citrulline excretion during arginine degradation is of concern to
the winemaker, since the reaction of citrulline and ethanol to form ethyl
carbamate has been shown by other workers to occur even at normal wine
storage temperatures. Winemakers, therefore , should avoid using argininedegrading
LAB starter cultures for inducing malolactic fermentation (MLF). In
addition, spontaneous MLF in wine by undefined LAB strains should be
discouraged, as this may lead to formation of ethyl carbamate precursors.
Ammonia detection with Nessler's reagent provides a simple, rapid test to
assess arginine degradation by wine LAB in a complex medium, but is useful
only for strains showing strong ammonia formation . The more sensitive
enzymatic determination of ammonia is required for strains showing weak