Antimicrobial properties of extracts regions of Tunisia.pdf


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18

A.-M. ZOUHIR ET AL.

The FIC index of agents A and B in combination is the sum of their respective
FIC indexes: FICA + B = FICA + FICB. The interaction between two agents was
considered synergistic if FICA + B was ⱕ0.5, additive if it was from 0.5 to 1.0,
indifferent if it was between 1.0 and 4.0, and antagonistic if it was >4.0
(Barchiesi et al. 2001).
Death Time Study
The effects of divergicin M35/garlic extract combinations on the viability
of L. monocytogenes LSD338, LSD525 and LSD535 were determined at two
stages of growth. Cells grown for 6 and 18 h (stationary phase) in TSBYE
broth (25 mL, inoculated at 1% v/v) were harvested by centrifugation at
7,000 ¥ g for 15 min, washed twice with 0.01 M phosphate-buffered saline
(PBS) at pH 6.5 and resuspended to a final concentration of approximately
107 cfu/mL. The divergicin/garlic combination in PBS was then added to
obtain concentrations twice the MBC of each inhibitor, as determined by
microdilution assay. Tubes containing 10 mL of bacterial suspension were
incubated aerobically at 30C for 3 h or at 10C for 24 h. Samples (100 mL) were
withdrawn in duplicate at 0, 1, 2 and 3 h for incubation at 30C, and at 0, 3, 6,
9 and 24 h for incubation at 10C, and were serially diluted 10-fold in peptone
water (0.1% w/v). Appropriate dilutions were plated in duplicate on TSBYE
agar and were incubated aerobically at 30C for 48 h. Each experiment was
repeated three times.
Validation of Inhibition of L. monocytogenes LSD535 in
Cold-smoked Salmon
L. monocytogenes-free cold-smoked pacific salmon (Sockeye salmon)
fillets (25.0 ⫾ 1.0 g each) were obtained from Grizzly Smoke House
Company (St. Augustin, Province of Quebec, Canada). Sixty-three fillets were
spiked with fresh culture of L. monocytogenes LSD535 at a final concentration
of 5 ¥ 104 cfu/g and were subjected to the following treatments:
(A) Twenty-one fillets: considered as control.
(B) Twenty-one fillets: freeze-dried divergicin M35 was reconstituted in distilled water, filter sterilized through 0.45-mm pore size membrane and
spread on each fillet to obtain a final concentration of 0.125 mg/g.
(C) Twenty-one fillets: freeze-dried divergicin M35 and garlic aqueous extract
were reconstituted in distilled water, filter sterilized through 0.45-mm pore
size membrane and then spread on each fillet to final concentrations of
0.125 and 1.25 mg/g, respectively.
All fillets were kept in a laminar-flow biological safety cabinet for
approximately 10 min in order to dry off excessive liquid. Fillets were vacuum