Wodd Journal of Microbiology and Biotechnology 9, 587-589

Protease production by Leuconostoc Oenos

strains isolated from wine

G.C. Roll&v, M.E. Farias and M.C. Manta de Nadra*

Two separate, extracellular proteolytic activities were demonstrated in four strains of Leuconostoc oenos isolated
from Argentinian wines. The first took place in the early growth phase and the other had its maximum at the end
of growth. The two proteolytic enzymes had different pH and temperature optima. Divalent metal ions had different
effects not only on each L. oenos strain but also on each of the two proteases from each strain.

Key words: Leuconostoc oenos, proteases, wine.

Wine is not a good substrate for the growth of lactic acid Medium and Culture Condifions
bacteria because few nutrients are available (Guilloux- The basal semi-synthetic medium contained IO g yeast extract, 5 g
glucose, 1.0 ml Tween 80, and 170 ml grape juice per 1. The pH
Benatier et al. 1985). Leuconosloc oenos strains have complex
was adjusted to 4.5 with 0.1 M HCl before sterilization by
nutritional requirements (Kunkee 1967; Radler 1975); amino autoclaving for 15 min at 121°C. The effects of pH and
acids are important for growth, both as nitrogen (Bidan temperature on the growth of the strains were determined in the
1967; Garvie 1967) and carbon sources (Kuensch et al. 1974). ranges of 3.0 to 6.5 and 20 to 5O”C, respectively.
The proteolytic system of L. oenos is important for its
growth because this microorganism has an absolute Dehrnination of Proteolytic Activity
The method of Hull (1943, modified by Citti et al. (1963), was
requirement for several amino acids (Garvie 1967). Only used to evaluate the proteolytic activity of the cultures. The effects
arginine and glutarnic acid appear to be available in high of pH and temperature on the activity were determined in the
enough concentrations at the onset of the alcoholic ranges of 3.0 to 7.0 and 20 to 6O”C, respectively. The results were
fermentation (Feuillat et al. 1985) that rearranges and expressed as mg tyrosine released per 100 ml of trichloracetic acid
filtrate per h.
completes the amino acid composition of the must. Feuillat
et al. (1977) have shown that wine contains very small
concentrations of some oligopeptides that function as
growth factors for lactic acid bacteria. As there was no Results and Discussion
information about bacterial proteolytic activity in wine, it
was the aim of this work to investigate the ability of L. The optimum growth temperature was 35°C for L. oenos
oenos strains from wine to degrade proteins. strains X,L, L, and m and 30°C for strain ST. The optimum
pH was 5.5 for L, and ST, 6.5 for X,L, and 5.0 for m.
All four strains produced two proteolytic activities each
Materials and Methods during growth (Figure 1). The first protease synthesis
occured during the early growth phase, starting after 10 h
Microorganisms
and increasing for the next 16 h. The other protease
Le~onostoc oenos X,L, L,, m and ST strains were isolated from red
wines under commercial conditions during the 1986 vintage in production period was maximal at the end of growth, when
Cafayate, Argentina (Manta de Nadra & Strasser de Saad 1987). the medium was nutritionally poor, i.e. after 77 h at 30°C
for strains X,L (Figure la) and L, (Figure lb) and after 100 h
at the same temperature for strains m (Figure lc) and ST
G.C. Roll&n is with the Centro de Referencia para Lactobacilos (CERELA),
Chacabuco 145. 40000 TucumBn, Argentina. M.E. Farias and M.C. Manca
(Figure Id).
de Nadra are with the Facultad de Bioqulmica, Quimica y Farmacia, The optimum pH values and temperatures for proteolytic
Universidad National de Tucuman and Centro de Referencia para
Lactobacilos (CERELA). Chacabuco 145, 40000 TucumBn, Argentina; fax: enzymes production in each strain are shown in Table 1.
54 81 311462 or 54 81 310465. ‘Corresponding author. The optimum pH for production of the first enzyme was
@I 1993 Rapid Communications of Oxford Ltd

World ~ownal of Muoblology and Biotechnology, Vol 9, 1993 587

 (a) I I
(W

4- 0.8.

Time (h) Time (h)

0.8-

Time (h) Time (h)

Figure 1. Growth at 30°C (a), measured turbidometrically at 560 nm. and proteolytic activity (O), measured as release
of tyrosine, in Leuconosfoc oenos strains (a) X,L. (b) L,, (c) m, and (d) ST.

4.5 for each of the four strains; there was no activity at pH In Lacfobacillus murims, maximum protease production
4.0 or 5.0. The second enzyme was produced optimally at occurred at 45’C and pH 6.6 (Strasserde Saad et al. 1987).
pH 5.5 for strains X,L, L, and m but at pH 4.5 for strain ST. Clostridim bifemenfuns proteolysis was optimal at pH 7.0
Proteolytic enzyme production was very high at the (Macfarlane & Macfarlane 1992).
optimum temperature but was much lower at all other Of various divalent cations (Table 2), Cal + and Mg’ ’
assayed temperatures. The pH and temperature optima for stimulated production of the first enzyme in all the strains.
growth are not always the same as those for enzyme The proteolytic activity of hf. mtrrinttsalso increased in the
production. presence of MgZ + (Strasserde Saad et al. 1988). Mn’ +
increased activity of the first enzyme in Leuc. oenus ST and
decreased it in the other strains. Mr? + has been found to
stimulate proteolytic activity in Sfrepfomycesclavuligerus
Table 1. Medium pH and temperature optima for Leuconosfoc
(BascarAnet al. 1990) and lacf. mttrin~ (Strasserde Saad et
oenos protease production.*
al. 1988). Zn’+ inhibited production of the first protease in
Strain PH Temperature (“C) all four strains.
In terms of production of the second protease, Zn’ + and
I II I II Mnz + were strongly inhibitory in all strains, Ca2+ was
w- 4.5 5.5 30 40 strongly inhibitory in X,L, L, and ST and Mg’ + at 1 mM
l-2 4.5 5.5 40 30 was slightly stimulatory only in strain m. Mg’ + at 10 mM
m 4.5 5.5 40 30 also stimulated proteolysis in X,L but only I mM was needed
ST 4.5 4.5 30 40
with Lewonosfoc oenos m whereas this cation completely
* Protease production was measured in a basal semi-synthetic inhibited the activity in L, and ST.
medium. I-Protease produced during the early growth phase; To our knowledge, this is the first report of proteolytic
Il-Protease produced at the end of the growth phase. activity in Leuconosfocoenosisolated from wine.
 Protease production by Leuconostoc oenos

Table 2. Effect of inclusion of divalent cations in growth medium on proteases productlon.*

Leuconosfoc Enzyme I (%) Enzyme II (%)

oenos straln
Ca*+ (mM) Mg*+ (mM) Mn2+ (mm) Zn*+ (mm) Ca2+ (mm) IMg*+ (mm) Mn*+ (mM) Zn*+ (mM)

1 10 1 10 1 10 1 10 1 10 1 10 1 10 1 10

w- 125a 169 100 190 16 0 0 0 22 12 100 194 20 0 0 0

L2 170 190 100 154 33 0 0 0 37 0 0 0 0 0 0 0
m 138 117 112 129 93 53 53 41 120 100 130 100 0 0 0 0
ST 115 180 104 133 110 136 25 0 40 0 80 63 18 0 0 0

l Protease production was measured in a basal semi-synthetic medium. Values are percentages of control assays which
contained no divalent cations and are the means of duplicate experiments. Enzyme I was produced during the early growth
phase and Enzyme II at the end of the growth phase.

Acknowledgements lactiques isolees du vin: effet stimulant des autolysats de

levures. Vitis 24, 59-74.
Hull, M.E. 1947 Studies on milk proteins, II. Calorimetric
This work was partially supported by grants from the
determination on the partial hydrolysis of the proteins in milk.
Consejo National de Investigaciones Cientificas y Tecnicas
journal of Dairy Science 30, 881-884.
(CONICET), Argentina, and from the Consejo de Kuensch, U., Termperli, A. & Mayer, K. 1974 Conversion of
Investigaciones de la Universidad National de Tucum6n arginine to omithine during malo-lactic fermentation in red
(CIUNT). Swiss wine. American journal of Enology and Vitictrlhrre 25,
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Kunkee, R.E. 1967 Malo-lactic fermentation. Advances in Applied
Microbiology 9, 235-279.
Macfarlane, G.T. & Macfarlane, S. 1992 Physiological and
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