Vol. 35 – 2009 - Ecologia Mediterranea - Université d'Avignon et des ...

KACEM MOURAD, KAZOUZ HAFIDA
Figure 1 – Agar well
diffusion assay showing
the antibacterial activity of
FI83 from Rhizobium sp.
ORN83 strain (A) and FI83
from Rhizobium sp. ORN24
(B) strain.
Sinorhizobium sp. ORN16
was used as the indicator
strain.
94
were used (Table 1) with the agar diffusion
test, using the appropriate agar media and
incubation conditions for their growth: lactobacilli
(Lactobacillus plantarum) were tested
in MRS agar at 30 o C for 18 h, Propionibacterium
strains in YGL agar medium at 37 o C
for 48 h, Pseudomonas in Brain Heart Infusion
agar at 32 o C for 48 h, and E. coli and
Erwinia in Nutrient agar at 37 o C for 3 days
(Kacem 2007).
Results
Figure 1 show the typical antagonism produced
by cell free supernatants (FI) from Rhizobium
sp. ORN24 (designated SI24) and
ORN83 (designated SI83) among the six
strains screened for their antagonistic activity.
These two strains caused respectively, an inhibition
zone of 10 mm and 25 mm in diameter
Table 1 – Concentration and partial purification of FI24 produced by Rhizobium sp. ORN24 strain.
on indicator (Sinorhizobium sp. ORN16)
strain. We can observe that the zone of inhibition
produced by FI83 on indicator plates is
extremely clear and large compared to zone
of inhibition produced by FI24. Based on the
quality and size of the zones of inhibition,
Rhizobium sp. ORN83 and ORN24 were
therefore considered in this study as antibacterial
agent-producing strains. On the other
hand, Sinorhizobium sp. ORN16 was selected
as an indicator strain.
Concerning the result of the concentration
procedure, in the case of Rhizobium sp.
ORN83 strain, no activity was detected in precipitate
phase after treatment of the fraction
FI83 (until 60%) with solid ammonium sulphate,
while activity was only found in the
supernatant (designated FII83), suggesting
that the inhibitory agents present in FI83 have
not ability to precipitate by “salting-out”. In
addition, the inhibitory activity was not
retained when FII83 was ultrafiltered through
filtron membranes (10,000-molecular weight
cut-off).
In the case of Rhizobium sp. ORN24 strain,
Table 1 and Figure 2 shows that the activity
recovery was achieved by including ammonium
sulphate (55%) and dialysis. Each step
resulted in a considerable loss of protein concentration
while, specific activity increases.
Antibacterial agent in this fraction (designated
FII24) was able to pass through cellulose
membranes with 10,000-molecular weight
cut-off (FIII24).
The antibacterial activity of FII24 or FII83
was estimated directly from the first dilution
where inhibition of indicator strain is not
Purification Stages Volume Activity 1 Total 2 Total protein 3 Specific activity 4 (5) Purification
(ml) (AU/ml) activity (µg/ml) (AU/µg) factor
Faction FI24
Ammonium sulphate 100 400 40000 210 1.9 1
precipitation and dialysis (FII24) 10 1600 16000 140 5.7 3
Membrane molecular weight cut-off ( * ) AU (% Initial FIII24 activity)
Retentate (%) Eluted fraction (%)
1,00,000 200 (12.5) 800 (50.0)
10,000 800 (50.0) 400 (25.0)
1,000 1600 (100.0) (FIII24) 0 (0.0)
1. Antimicrobial activity of the bacteriocin solution against Sinorhizobium sp ORN16 strain (AU/ml).
2. Multiplication of total volume (ml) by activity (AU/ml).
3. Determined by the Bradford method.
4. Activity (AU/ml) (column 2) divided by the protein concentration (µg/ml) (column 4).
5. Fold increase in the initial specific activity, when compared to previous step.
* Initial bacteriocin activity was 1600 AU/ml.
ecologia mediterranea – Vol. 35 – 2009