Antimicrobial Activity of Selected Antioxidants

Abstract

Tripticase soy broth containing 100, 150, 200 and 400 ppm of butylated hydroxyanisole (BHA), propyl gallate, nordihydroguaiaretic acid (NDGA), or combined BHA and propyl gallate was inoculated with Escherichia coli or Staphylococcus aureus to determine the antimicrobial effect of these antioxidants. Both propyl gallate and NDGA at 400 ppm had a strong lethal effect against E. coli. BHA and the BHA-propyl gallate combination were more effective against S. aureus than against E. coli. NDGA had the strongest antimicrobial activity. Only SO ppm of NDGA were highly inhibitory to S. aureus. Most antioxidants in use today are phenolic compounds which are added to numerous food products to inhibit development of oxidative rancidity. Several workers have investigated the possiblity that certain phenolic antioxidants might exhibit and antimictobial effect. Kaufmann and Ahmad (5) reported that 140 ppm of nordihydroguaiaretic acid inhibited growth of Saccharomyces cerevisiae and Epstein et al. (2) reported that the LD 50 for Tetrahymena pyriformis was only 5 ppm for NDGA. Growth of Salmonella senftenberg was inhibited slightly by l.Oo/o of butylated hydroxy-toluene (BRT) (10). Recently, Chang and Branen (1) found that 250 ppm of butylated hydroxyanisole (BRA) could inhibit growth of Aspergillus parasiticus and 150 ppm could inactivate Staphylococcus aureus. In this study BRA, NDGA, and propyl gallate were tested against Escherichia coli and S. aureus to determine their anitmicrobial effect. Combined BHA and propyl gallate were also tested since this combination has been widely used in fat-containing products. :ItA TERIALS A:>ID METHODS Organisms S. aureus. A TCC 6538P, and E. col~ A TCC 9723 were used as test organisms. Twelve-hour-old cultures of each organism were prepared by transferring one loop of S. au reus or E. coli into SO ml of Trypticase Soy Broth (fSB, BBL). Cultures were then incubated at 35 C for 12 h. Inhibition studies BHA and propyl gallate were obtained from Sigma Chemical Co. and NDGA was obtained from Aldrich Chemical Co. The above antioxidants and combined BHA-propyl gallate (50/50) each were dissolved in ethanol to achieve a 1.0% concentration of antioxidant. Ethanol was removed by evaporation in a drying oven at 120 C for 2 h. Each antioxidant was then combined with 100 ml ofTSB to make stock solutions. Media containing 100, 150, 200, and 400 ppm of antioxidant were prepared by adding appropriate amounts of each antioxidant stock solution to SO ml of TSB. Flasks of media containing antioxidants were autoclaved. After cooling, media containing antioxidants were inoculated with 1 ml of a 12-hold culture. After 0, 4, 8, 12 and 24 h of incubation at 35 C, 1 ml from each TSB culture was removed and appropriate dilutions were made with phosphate buffer solution. Cells were plated in Trypticase Soy Agar (BBL) and incubated for 48 h at 35 C. The anitmicrobial activity was shown by the curve of log 10 cell numbers/ml vs. hours of incubation with different concentrations of antioxidants. Only one set of data is reported since it would not be valid to report mean number of cells when the intial cell numbers varied. RESULTS AND DISCUSSION Propyl gallate and NDGA had a lethal effect against E. coli at the 400 ppm level, (Fig. 1 and 2) but BRA and combined propyl gallate-BRA were not as effective in killing E. coli (Fig. 3 and 4). When tested against S. aureus BRA and combined propyl gallate-BRA had strong antimicrobial activity at the 400 ppm level (Fig. 5 and 6), but propyl gallate alone had little effect agianst S. aureus (Fig. 7). NDGA was the most effective inhibitor of S. aureus growth. Levels as low as SO ppm had a strong lethal effect (Fig.8). Apparently NDGA, BRA, and the propyl gallate-BRA combination were more effective against S. aureus than E. coli. These results are in accord with the concept that phenols have more antimicrobial activity against gram-positive than against gram-negative cells (4). An exception to the above was seen when propyl gallate showed greater inhibitory effects against E. coli than against S. aureus. More study is needed to explain propyl gallate's mechanism oflethality. In the present study, 400 ppm of BHA were required to inhibit effectively growth of E. coli and S. sureus Chang and Branen (1) found that 150 ppm of BHA inactivated S. aureus but 400 ppm were required for