The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.

SummaryA method is described for estimating the growth of Group N streptococci in milk and other media by measuring the 260 nm-absorbing material extracted from the cells with 0·1 M-NaOH at 100°C. A good correlation between the absorbance at 260 nm of the hot alkali extracts and the dry weight of the cells was obtained for cultures of Streptococcus lactis during the logarithmic and early stationary phases of growth.

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Lactoperoxidase (EC 1.11.1.7) catalysed the oxidation of NADH by hydrogen peroxide in the presence of either thiocyanate, iodide or bromide. In the presence of thiocyanate, net oxidation of thiocyanate occurred simultaneously with the oxidation of NADH, but in the presence of iodide or bromide, only the oxidation of NADH occurred to a significant extent. In the presence of thiocyanate or bromide, NADH was oxidized to NAD+ but in the presence of iodide, an oxidation product with spectral and chemical properties distinct from NAD+ was formed. Thiocyanate, iodide and bromide appeared to function in the oxidation of NADH by themselves being oxidized to products which in turn oxidized NADH, rather than by activating the enzyme. Iodine, which oxidized NADH non-enzymically, appeared to be an intermediate in the oxidation of NADH in the presence of iodide. NADPH was oxidized similarly under the same conditions. An assessment was made of the rates of these oxidation reactions, together with the rates of other lactoperoxidasecatalysed reactions, at physiological concentrations of thiocyanate, iodide and bromide. The results indicated that in milk and saliva the oxidation of thiocyanate to a bacterial inhibitor was likely to predominate over the oxidation of NADH.It is well established that certain peroxidases (e.g. horseradish peroxidase and uterine peroxidase) can catalyse the oxidation of NADH by hydrogen peroxide provided that a suitable cofactor (e.g., certain phenols) is present (Akazawa & Conn, 1958;Hollander & Stephens, 1959). Oram & Reiter (1966b) Bacteria. Streptococcus cremori8 972 was maintained and grown as described previously (Hogg & Jago, 1970 NADase treatment of oxidation product8. To 2.0ml of the above reaction mixtures was added 0.1 ml of NADase solution (1.8mg of protein/ml). The solutions were incubated at 37°C for 30min, and the presence of NAD+ was tested for by the addition of 2.Oml of m-KCN as described above.Other method8. The amperometric methoai for studying inhibition of oxygen uptake by bacteria, the polarographic method for detecting the antibacterial oxidation product of SCN-and the method for estimating cyanide, thiocyanate and ammonia after acidification of the reaction mixture have been described elsewhere (Hogg & Jago, 1970).