Microscale assay for screening of inhibitory activity of Lactobacillus.

The inhibitory activity of Lactobacillus species has been well documented (1–5,7,11–13,15) and is usually a result of the secretion of secondary metabolites including hydrogen peroxide, organic acids, and peptides (2–5, 11–13,15). Peptides that exhibit an inhibitory effect on any other microorganisms are classified as bacteriocins (5). Bacteriocins have been shown to inhibit a wide range of microorganisms including Gram-positive bacteria (2,4,5, 7,11–15), Gram-negative bacteria (2,5, 9,10), and even different bacteria within the same species (2,5,11). The traditional method for screening of bacteriocins is the agar diffusion method (7,11). The traditional agar diffusion assay is used to screen for direct antagonism of bacteriocin-producing microorganisms against other microorganisms. The assay was first described by Tagg et al. (11) and involves the use of a cell-free supernatant from a Lactobacillus test culture placed into wells on seeded agar plates. The corresponding zones of inhibition are measured in millimeters and are used to quantify the antimicrobial activity. However, the agar diffusion assay depends directly on zone of inhibition to quantify inhibitory activity, and the activity can be influenced by the rate of diffusion of the inhibitory compound through the agar medium (2,7,9,14,15). The agar diffusion assay is not cost effective; each assay requires the use of a large amount of specialized agar plates and sterilized well-forming rings. In addition, the agar diffusion assay requires incubation times of 24, 48, and 72 h to produce accurate analysis of the inhibitory effect of Lactobacillus (11). To facilitate the inhibitory screening of Lactobacillus, we developed a microscale optical density assay (MODA) and compared the effectiveness to the agar diffusion method. MODA uses optical density to correlate the secretion of inhibitory compounds with a decrease in indicator cell density. MODA compares the relative decrease in absorbance between the cultures inoculated with Lactobacillus plantarum (8014; ATCC, Manassas, VA, USA) supernatant and cultures that are inoculated with media. The MODA examines the effectiveness of secreted inhibitory products from bacterial species based on direct antagonism between these species and target microorganisms in a liquid medium. This removes any effect that the rate of diffusion may have on the results, while only requiring a 24-h incubation time. The MODA also allows for the rapid examination of the effects of proteolytic enzymes, temperature, and pH on the bacteriocin. These are all common tests used in the screening for the inhibitory activity of Lactobacillus and are important tools to evaluate the antimicrobial activity of Lactobacillus (1–5,7,9,11–13). To test the characteristics of the bacteriocin mentioned above, Lactobacillus supernatant can be treated with several proteases and catalase to rule out hydrogen peroxide. The supernatant can also be adjusted with 1 M HCl or 1 M NaOH to examine the effect of pH and/or dialyzed against a standard buffer (pH 7.8) to rule out the effect of organic acids present only in Modified Rogosa Broth (MRS) (BD Biosciences, San Jose, CA, USA) used to grow L. plantarum that might not be present in normal MRS. Finally, the supernatant can be heated to various temperatures to examine the effect of heat. This versatility allows the MODA to be used as a rapid method to distinguish between bacteriocins and bacteriocinlike factors, which may not be proteins. The result is a rapid and quantifiable analysis of the antimicrobial action of the bacteriocin-producing species, which is helpful when screening either a large number of target microorganisms or a large number of bacteriocinproducing species, or both. To perform either the agar diffusion assay or MODA, the following culture conditions were used. L. plantarum cultures are grown overnight in MRS at 25°C with shaking. Sterile cell-free supernatant was achieved by growing overnight cultures followed by centrifugation at 6500× g for 5 min. The supernatant is decanted and filtered through a 0.2-μm low-protein binding syringe filter (Millipore, Bedford, MA, USA). The inhibitory test cultures (Table 1) are all grown overnight in Nutrient Broth (BD Biosciences) at 37°C with shaking. The cultures are then diluted 1:1000 in Nutrient Broth. This dilution was selected because it was the first dilution in which an OD600 < 2.00 was measured before running the protocols below. The well diffusion assay is carried out following the protocol described by Tagg et al. (11). To 25 mL liquid Nutrient agar (BD Biosciences), tempered to 45°C–48°C, 1 mL diluted test culture (1:1000) is added. The inoculated agar is then poured onto petri dishes and allowed to solidify. Using a sterilized 10mm cork borer, wells are cut into the center of the plates, to which 250 μL Lactobacillus supernatant are added; to a second plate, 250 μL MRS (pH 4.8) are added to serve as a negative control. The plates are then incubated for 24, 48, and 72 h, with the relative zones of inhibition measured at each time point. The MODA involves the addition of 100 μL diluted test cultures to a well of a 96-well cell culture plate (Corning, Acton, MA, USA). This is done in four separate wells, one to serve as a negative control (no supernatant or media), two to serve as test wells (supernatant, duplicate), and one additional negative control (media). To the test wells, 15 μL cell-free L. plantarum supernatant are added; to the media test well, 15 μL MRS adjusted to pH 4.8 (the pH of the cell-free supernatant) are added; to the final well only, diluted test culture is added (no supernatant). The 96-well plate is then covered with a sterile lid and incubated at 37°C for 24 h. After incubation, the plate is read on a Multiskan MCC/340 microplate reader (Labsystems, Fisher Scientific, Pittsburgh, PA, USA) at a wavelength of 600 nm to measure optical density, which can be correlated to cell density. Table 1 shows the measured inhibitory activity of L. plantarum using the MODA. The L. plantarum supernatant showed a strong ability to inhibit all test cultures examined (Table 1). The observations that show that the L. plantarum supernatant inhibited both Gram-positive and Gram-negative microorganisms are interesting. Most studies of antimicrobial properties of Lactobacillus show them to be more inhibitory to closely related species (i.e., Gram-positives) Benchmarks