Hydrogen peroxide production

Hydrogen peroxide production by the four LAB strains grown in MRS and TJ broth was determined in sodium phosphate buffer. Each of the media was inoculated with a suspension of lactobacilli and incubated for 24 h at 30 or 37 °C, depending on the strains. The cells were then harvested by centrifugation at 6000 ´ g at 4 °C for 15 min. The cells were washed twice with 10 mL of cold, sterile phosphate buffer solution (1 M, pH=6.5). A mass of 1 g of the cell pellet was resuspended in 10 mL of cold, sterile sodium phosphate buffer (1 M, pH=6.5) and then for the preparation of the final buffer solutions, the initial dilution was further diluted (1:10) in phosphate buffer. In the final buffer solutions approx. 108 CFU/mL of Lactobacillus population were present. The cells were then incubated at 5 °C for a period of 3 days. After 0, 2, 24, 48 and 72 h of incubation, the cells were removed by centrifugation at 6000 ´ g at 4 °C for 15 min and the supernatants were assayed for hydrogen peroxide according to the methods of Villegas and Gilliland (19). The supernatant was also used for evaluation of inhibitory activity against pathogens by agar diffusion method. Each value presented here represents the mean value of two determinations.

Production of crude bacteriocin suspensions

For the production of bacteriocin suspension LAB strains were grown in the two broths at 30 °C for 24 h. Supernatants were harvested by centrifugation at 6000 ´ g at 5 °C for 20 min, adjusted to pH=6.5, filtered through a 0.22-m pore size filter and heat-treated at 80 °C for 10 min to inactivate proteases and kill the bacteria that might be present in the filtrates. We had observed earlier that the bacteriocin suspension did not lose its inhibitory effect after the heat treatment and other researchers also came to the same conclusion that the most bacteriocins from lactobacilli are heat stable (20).  

Production of concentrated bacteriocin suspension

Crude bacteriocin suspensions were made and then freeze-dried and redissolved in sterile water to give three-fold concentration in comparison with crude bacteriocin suspension.

Agar diffusion method

To test whether the concentration of hydrogen peroxide and bacteriocin produced by LAB can inhibit the growth of test microorganism, wells were made in the nutrient agar plate. The wells contained 450 L (in three portions of 150 L) of the culture supernatant with different concentrations of hydrogen peroxide, the crude and concentrated bacteriocin suspension and the plates were incubated at 50 °C for 30 min to accelerate the diffusion of the solutions into the agar. A volume of 7 mL of soft nutrient, BHI, MRS or TGE agar (agar 0.7 %) containing the test microorganism (700 L added from the appropriately diluted bacterial suspension to soft agar and cooled down at 40 °C to obtain the bacterial concentration of 104 CFU/mL) was poured onto the agar plate. The plates were incubated at 30 or 37 °C for 18 h. The width of the inhibition zones was expressed in mm as

difference in radius. 

Microtiter assay method

In the microplate assay, 125 L of the crude or concentrated bacteriocin in MRS or TGE broth (125 L) were prepared in a 96-well microtiter plate, respectively. A volume of 25 L of overnight culture of the indicator strains appropriately diluted (about 105 CFU/mL) was added to each well or the same quantity of water to the control. We used as control of cell growth the appropriate broth (MRS, TGE) with sterile, distilled water instead of bacteriocin solution, and it was inoculated with the test organisms, which showed the growth of L. sakei or C. glabrata when nothing influenced their growth 

(Control 1). We used broth-control: 125 L of broth (usual or three-fold concentrated MRS and TJ broth) were added to 125-L media of test organisms and it was inoculated with 25 L of diluted test microorganisms. Thus we obtained the effect of different broths on test organisms and in this way we could predict the possibility of inhibitory effect of broths (mainly the concentrated broths) (Control

2). The plates were incubated at 30 °C for 24 or 40 h and the absorbance, beginning the 12th hour of incubation, was measured at 630 nm using a microtiter plate reader (Dynatech MR7000, Dynatech Laboratories Inc., Chantilly, USA). The absorbance was measured every hour. The absorbance of wells containing only broth and bacteriocin suspension without the indicator strain was subtracted from the absorbance of the wells that contained the indicator strain. Thus we obtained the absorbance of the cells. This absorbance is in direct proportion with the amount of cells, within certain limits.