Tomoaki Okitsu

Studies on the Bactericidal Efficacy of Furylfuramide

The bactericidal effect of furylfuramide [2-2(furyl)-3-(5-nitro-2-furyl) acrylamide; FF], a newly permitted food preservative in Japan has been examined. And results obtained may be summarized as follows: 1. Bactericidal effects of FF and nitrofurazone (NFS) were compared each other by the official phenol coefficient method4). S. typhi was killed off with FF at a dilution equivalent 1:10, 000 in 5 minutes which corresponded to 120 in terms of phenol coefficient number. To the contrary, NFS with 1:5, 000 dilution exhibited almost no killing effect on the same test organism in 120 minutes (Tables 1 to 3). 2. Bactericidal activities of FF and NFS against E. coli and Staph. aureus were measured at their growing stage in the nutrient broth containing varying concentrations of each of the drugs and exposed for different period of time. As shown in Fig. 1, E. coli was killed off with more than 0.2 ppm of FF, and Staph. aureus with more than 1.6 ppm of the drug within 6 hours of incubation. To the contrary, no marked killing effect could be seen with NFS even at 200 ppm (Fig. 2). 3. Similar bactericidal effects on the growing stage of E. coli and Staph. aureus were noted with FF which had been added to the tuna flesh infusion, however, the efficacy was slightly lowered as compared with that observed in the nutrient broth (Fig. 3).

STUDIES ON THE FOOD PRESERVATIVES-IV

Acquisition of resistance against chlortetracycline (CTC) by Pseudomonas fluorescens, which is closely associated with putrefaction of fish and shellfish, was studied. The results may be summarized as follows: 1. Three starins of P8. fluorescens were exposed to CTC of either a constant dose at the maximal tolerance level (M. T. L.) of each strain or 0.1 ppm, or an increasing dose at M. T. L. at each stage. After 60 successive subculturings in nutrient broth (Difco) containing a constant dose of CTC, the organisms acquired 4 to 16-fold higher resistance at the M. T. L. and 2 to 8-fold at 0.1 ppm, while the corresponding values obtained at the increasing dose were 16 to 64-fold that of the original culture (Figs. 1 and 2). The resistance acquired in vitro seemed to be comparatively stable since 25 successive subculturings of the in vitro resistant organisms in plain nutrient broth held almost the same level of their resistance. 2. A complete cross-resistance against CTC and oxytetracycline was observed. A slight cross-resistance against chloramphenycol was recorded with the CTC-resistant organisms, while that against streptomycine was not appreciable (Table 1). 3. No marked difference in the biochemical characters, especially those relating to putrefaction, was observed between the resistant and sensitive strains (Fig. 3). 4. The distribution curve of resistance of individual cells of a sensitive strain obtained by plotting the numbers of the colonies counted in agar plates added with various levels of CTC appears to be a S shape (Fig. 4), which indicates heterologous sensitivity to CTC by individual cells. While, the curves for the resistant descendants are composed of two linear lines crossing at nearly right angle, which means the majority of the individuals has the same level of resistance. Neither the sensitive nor resistant strain showed CTC-dependence because the largest number of colonies was counted in the plates without CTC.

Effect of Mixed Preservatives on the Retardation of Bacterial Growth

It is a well accepted fact that the effect of a certain preservative can be attributed to its specific action against spoilage bacteria. And many efforts have been paid for finding out non-toxic compounds capable of inhibiting the growth of microorganisms responsible for food decomposition. However, it may be also important to fortify the action of known preservatives by improving the method of application. The purpose of the present study is to ascertain whether or not the mixing. of two prerervatives can develop more effective inhibition than a single application of each preservative used for mixing.In the present test, formula of inhibition coefficient is defined as follows: H=1-Tc/T where H=coefficient of growth inhibition. Tc=time required for bacteria to reach a definite growth showing 0.1 of the turbidity of a cultural medium (viable counts, 4×lO7/c. c, see Fig. 1). T=time required for bacteria to reach the same turbidity of the medium, in which the test preservative is added. Examining the inhibiting effect of some preservatives (Table 1) on the growth of bacteria, it has been ascertained that there are two types of growth-inhibition. An obvious example of type I is sodium nitrite, though most of other preservatives tested were found to belong to this type as well. The only exceptions are two sulphonamides, which indicate type II of the growth-inhibition (Fig. 2). To clarify the quantitative relationship existing between the growth-inhibition and concentration of preservatives, the concentration-inhibition curves have been sought from the growth curves of bacteria in the presence of preservatives (Fig. 3). The result reavled that there is a tendency to increase in effect of inhibition against bacterial growth by a proper combination of two preservatives (Fig. 5).

Inhibition of Bacterial Deamination by Nitrofuran Derivatives

Inhibition of dehydrogenation systems involved in carbohydrate metabolism of certain bacterial cells is considered as one of the causes of bacteriostasis provoked by 5-nitro-2-furfuraldehyde semicarbazone (NFS). In connection with metabolism of nitrogenous compound, few information have been made available for the action of nitrofuran derivatives against bacterial dearnination except a fact that NFS has no remarkable effect upon protease, amino-acid decarboxylase, an tryptophanase of bacterial origin. However, as specific dehydrogenase systems are known to participate in some bacterial deamination, we have made an assum ?? tion that NFS might be effective in inhibiting bacterial deamination. With this view in mind we have carried out a series of experiments to ascertain the effects of nitrofuran derivatives, NFS and NFA (5-nitro-2-furylacrylic amide), on the oxydative deamination of glycine, alanine, and glutamic acid in resting cell suspensions of E. col ?? , The results obtained are as follows; 1) In resting cell suspensions of E. col ?? , nitrofuran derivatives (NFS and NFA) and sodium nitrite inhibit oxydative deamination of glycine and alanine. 2) Amoung these compounds, NFA showed inhibition more active than NFS, while sodium nitrite was the least effective of them in inhibition. This sequence of the agents in the inhibition seems to correspond with that of their effects in bacteriostatic action. 3) Furfural semicarbazone, one of the non-nitrated furan derivatives, gives little inhibitory action. 4) From these results, it may be said that the inhibitory action of nitrofuran derivatives and sodium nitrite on the bacterial deamination is connected with the bacteriostatic action of these agent to some extent, and that incidence of the inhibitory action of nitrofuran derivatives depends not only on nitro-group but also on some structural relation of the compounds. 5) Deamination of glycine is more strongly inhibited by the agents than that of alanine.