Hydrogen peroxide formation by lactobacilli and its effect on Staphylococcus aureus.

Culture filtrates of Lactobacillus lactis and Lactobacillus bulgaricus were observed to contain material inhibitory against Staphylococcus aureus. The concentration of inhibitor increased on storage of the lactobacilli at 5 C, and the maximum was attained af ter five days. A carbohydrate source was necessary for the formation of this inhibitory activity by resting cell suspensions. The inhibitory factor was identified as hydrogen peroxide. Storage of lactobacilli in dextrose at neutral p H and at 5 C resulted in maximum accumulation of hydrogen peroxide. Inhibi tory activity associated with cultures of lactobacllli has been shown previously by several workers (1, 3, 6, 8, 9, 14, 15, 17). In addition to lactic acid, a heat labile compound(s) has been eommonly implicated as an inhibitory product (10, 16). Wheater et al. (18) observed inhibition of Staphylococcus aurcus by a strain of Lactobacillus lavt~s. They proposed that inhibition of S. aureus resulted from the formation of hydrogen peroxide by the L. lactis. However, they were unable to show the presence of hydrogen peroxide in the lactobacillus growth medium and indicated that viable lactobacilli were necessary for the inhibition of the staphylococcus. During our studies we also observed that inhibition of S. aureus by certain strains of L. lactis and L. bulgaricus was caused by the formation of H~O~ by lactobacilli. However, we were able to show the accumulation of H20~ in the lactobacillus growth medium. This observation prompted further work to obtain information on the factors influencing the hydrogen peroxide formation by the lactobacilli and to clarify the influence of this compound on the growth of S. aureus. Materials and Methods Organ,~ms. Single strains of laetobacflE were isolated from yogurt s tarter cultures obReceived for publication October 20, 1967. 1 Paper number 2503 of the Journal Series of the :North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. tained from commercial supply houses by plating on lactic agar (2). Al l isolates were transferred in sterile litmus milk and subcultures were made with 1% inoculum; incubation was at 35 C for approximately 16 hr. F o r inoculation of lactic broth or trypticase soy broth, cultures were transferred in the same medium for at least three transfers before