**2.2 Culture conditions**

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 incubated at 35 ± 1.0°C for 24 h.

### **2.3 Preparation of cell-free supernatant**

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 25 cm Hg.

## **2.4 Antimicrobial activity**

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 bacteria culture (105 CFU/ml) on TSA plates previously solidified, and four wells (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.

## **2.5 Storage stability**

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.

**93**

*Stability of the Antimicrobial Activity of Lactobacillus plantarum NRRL B-4496 Supernatants…*

Statistical software Minitab (v.17, LEAD Technologies Inc., USA) was used to

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

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 Gram-

The use of the supernatants of *L. plantarum* is a good alternative in the industry

of foods to be used as biopreservatives, since these are able to inhibit *S. aureus* during a period of time of 20 weeks when stored at 25 ± 1.0°C; although there is a significant difference between the antimicrobial activity at time 0 and time 20, the

perform an analysis of variance (ANOVA) with the 95% level of confidence.

*DOI: http://dx.doi.org/10.5772/intechopen.83691*

be corroborated in further experiments.

**2.6 Statistical analysis**

**3. Results**

negative bacteria.

**4. Conclusions**

**Figure 1.**

antimicrobial activity remains effective.

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

*Stability of the Antimicrobial Activity of Lactobacillus plantarum NRRL B-4496 Supernatants… DOI: http://dx.doi.org/10.5772/intechopen.83691*

### **2.6 Statistical analysis**

Statistical software Minitab (v.17, LEAD Technologies Inc., USA) was used to perform an analysis of variance (ANOVA) with the 95% level of confidence.
