**3. Results**

*Technology, Science and Culture - A Global Vision*

**2. Methodology**

**2.1 Bacterial strains**

**2.2 Culture conditions**

25 cm Hg.

incubated at 35 ± 1.0°C for 24 h.

**2.4 Antimicrobial activity**

bacteria culture (105

**2.5 Storage stability**

**2.3 Preparation of cell-free supernatant**

species of *Lactobacillus* produce a variety of antimicrobial compounds that differ in their inhibitory spectrum, mode of action, structure and biochemical properties. *Lactobacillus plantarum* has an antagonistic effect on Gram-positive organisms and, in some cases, on Gram-negative organisms, such as *Listeria monocytogenes,* 

*Staphylococcus aureus, Streptococcus, Salmonella*, and *Pseudomonas* [3].

The objective of this work was to evaluate the stability of the antimicrobial activity of *Lactobacillus plantarum* NRRL B-4496 supernatants against *Staphylococcus aureus* ATCC 29413 during 20 weeks of storage at 25 ± 1.0°C.

The *L. plantarum* NRRL B-4496 used in this study and the indicator strain *S. aureus* ATCC 29413 were obtained from the Food Microbiology Laboratory strain collection of the Universidad de las Americas Puebla (Puebla, Mexico). *L. plantarum* NRRL B-4496 was maintained on MRS Agar (de Man, Rogosa and Sharpe) (Difco™ BD, Sparks, Maryland) and *Staphylococcus aureus* ATCC 29413 on Trypticase soy agar (TSA, Bioxon® BD, Edo. de Mexico, Mexico), at 5 ± 1.0°C.

The cultures were prepared by inoculating *L. plantarum* NRRL B-4496 into 30 mL of MRS broth (Difco™ BD, Sparks, Maryland) and incubated at 35 ± 1.0°C for 48 h under anaerobic conditions, whereas *S. aureus* ATCC 29413 was inoculated into 10 mL of Trypticase soy broth (Bioxon® BD, Edo. de Mexico, Mexico) and

The cell-free supernatant was collected by centrifugation at 12000× g for 10 min (Marathon 21 K/R, Fisher Scientific, Germany), filtrated through 0.45-μm Millipore membrane filter and supernatants concentrated 10-fold by vacuum evaporation on a Buchi R-210/215 rotary evaporator (Buchi, Flawil, Switzerland) at 70 ± 1.0°C and

The antimicrobial activity was evaluated by the agar-well diffusion method [4], performed in duplicate, which consists on spreading 0.1 mL of the indicator

To evaluate the stability of *L. plantarum* supernatant through time, supernatant was stored at 25 ± 1.0°C. The antimicrobial activity with the agar-well diffusion method (previously described) was determined every 7 days for 5 months.

(8 mm diameter) were punched in the plate. Three of the wells were filled with 100 μL of the concentrated *L. plantarum* supernatant; the fourth well was filled with 100 μL concentrated MRS broth as a negative control. The plates were incubated at 37 ± 1.0°C for 24 h. The bacterial growth was observed, and the diameter of the inhibition zones (mm) around the wells including the well was measured with a

digital Vernier caliper (Metax, Mexico), by triplicate.

CFU/ml) on TSA plates previously solidified, and four wells

**92**

The zone of inhibition generated from the supernatant of *L. plantarum* against *S. aureus,* during storage at 25 ± 1.0°C, obtained at time zero was 20.05 mm and after 20 weeks of storage was 16.24 mm. As it can be observed in **Figure 1**, there is a significant difference (P < 0.05) in the antimicrobial activity of *L. plantarum* supernatants between time 0 and after 20 weeks, although the ability to inhibit *S. aureus* was still observed after storage time. The observed antimicrobial activity can be attributed to the presence of secondary metabolites such as organic acids, hydrogen peroxide, carbon dioxide, diacetyl, pyroglutamic acid, among others, what should be corroborated in further experiments.

Other researchers such as Anas et al. [5] and Kareem et al. [6] observed that the supernatants of *L. plantarum* have the ability to inhibit the growth of pathogenic microorganism Gram-positive such as *S. aureus* ATCC 25923 and also Gramnegative bacteria.

**Figure 1.** *Plot of intervals of inhibition zones (mm) vs. storage time (week).*
