number or vitality. Spores from strain 62A were used for the major part of this EFFECT OF SUBTILIN ON SPORES OF CLOSTRIDIUM

Transcription

1 EFFECT OF SUBTILIN ON SPORES OF CLOSTRIDIUM BOTULINUM A. A. ANDERSEN Western Regional Research Laboratory,' Albany, California Received for publication January 9, 1952 Within the last few years considerable interest has developed in the possible use of antibiotics in food preservation (Curran and Evans, 1946; Andersen and Michener, 1950). Before any new food process can be seriously considered commercially, it must be established that it involves no hazard to public health. In the processing of low-acid foods, in particular, the effect of the process on CMstridium botulinum is of vital importance. For this reason studies of the action of antibiotics on this organism have been undertaken. Subtilin is considered in this paper; studies on other antibiotics will be reported later. Eight cultures of C. botulinum and a putrefactive anaerobe, N.C.A (listed by the American Type Culture Collection as Clostridium sporogenes, ATCC 7955), were used in this work. METHODS AND MATERIALS Two spore suspensions of C. botulinum, strains 62A and 213B, were obtained from the National Canners Association Laboratory in 1946 for use in a previous study (Perry et al., 1948). These suspensions were concentrations of spores in part of the original culture liquor. There appeared to have been no decrease in number or vitality. Spores from strain 62A were used for the major part of this work because this culture is the most widely used botulinum culture in food research and represents the most important food poisoning type. A suspension of spores of putrefactive anaerobe, strain 3679, was also obtained from the National Canners Association, and 6 cultures of C. botulinum (62A, 109A, 457A, 115B, 169B, and 1267B) were obtained from the University of Chicago. These cultures contained many spores and were used, after heating, as spore suspensions. These preparations were stored in the refrigerator, and appropriate dilutions were used in the various experiments described hereafter. The medium2 and counting method employed were described in a recent publication (Andersen, 1951). In those experiments where subtilin was used and it was necessary to prevent subtilin from being carried over into the counting plates in inhibitory concentrations, a sufficient number of spores (34,000 to 65,000 per ml) was 1 Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 2 The medium consisted of the following: 800 ml pork infusion, 200 ml pea infusion, 5 g peptone, 1.6 g tryptone, 1.25 g K2HPO4, 1 g soluble starch, 0.5 g Na thioglycolate, NaOH to ph 7.2, and 16 g agar. At time of pouring 0.4 ml NaHCOs 5 g per 100 ml was added. Agar was omitted when liquid medium was used. 145

2 146 A. A. ANDERSEN [VOL. 64 used so that dilutions could be made to bring the subtilin concentration in the plates to 0.01 ppm or lower. As is shown later, this concentration does not inhibit colony development. The subtilin was produced (Garibaldi and Feeney, 1949) and purified (Fevold et al., 1948) in this laboratory and was 70 per cent as active as the most potent material prepared by counter-current distribution (Brink et at., 1951). Concentrations are expressed in terms of the impure material. EXPERIMENTAL RESULTS Sensitivity of spores of C. botulinum to subtilin in agar medium. The spore suspension of C. botulinum, strain 62A, was diluted 1: 5,000, and 3 ml of the diluted suspension were heated in a tube for 1 minute in steam, cooled, and diluted 1:100. One ml aliquots were plated in anaerobic petri dishes with agar medium2 containing various amounts of subtilin. Counts made at 40 hours were as follows: no subtilin, 340; 0.01 ppm subtilin, 339; 0.1 ppm, 177; 0.3 ppm, 18; 0.4 TABLE 1 The fate of spores of Clostridium botulinum heated and incubated in a liquid medium2 with and without subtilin TIM O COUNTS COUNTS ON TE MEDIM CONTAINIG 20 ppm subtilin No subtilin Initial 65,000 65,000 After 2 min heat at 100 C 42,500 56,400 4 hr, 28 C 10,500 45, hr, 28 C 4,200 50, hr, 28 C 200 Millions 36 hr, 28 C 0* Millions 66 hr, 28 C 0* Millions * No colonies from the 1:100 dilution. ppm, 0; and 1.0 ppm, 0. Recounts at 30 days were the same. Similar results were obtained when the experiment was twice repeated. Action of subtilin and mild heat. A spore suspension of C. botulinum, strain 62A, containing 65,000 spores per ml was heated at 100 C for 2 minutes in 12.5 ml liquid HCO--medium, containing 20 ppm subtilin in one case and no subtilin in another. At the end of the heat treatment the tubes were cooled and incubated at 28([1) C. Anaerobic plate counts were made after the 2 minute heat treatment and at intervals during incubation as shown in table 1. The results indicate that destruction takes place as the spores begin to germinate. Further work will be required in order to 'determine whether the greater destruction observed when the spores were heated in the presence of subtilin is significant. Immediately following the heat treatment in the previous experiment, 1 ml quantities of the spore suspensions were diluted to 100 ml, and 1 ml was transferred to plates containing 0, 0.1, 0.4, and 1 ppm subtilin. The resulting counts

3 1952] SUBTILIN ON SPORES OF C. BOTULINUM 147 showed that only 7 per cent of the spores surviving subtilin-heat treatment grew in plates containing 0.1 ppm subtilin, whereas 20 per cent of the spores surviving heat alone were able to grow in plates containing 0.1 ppm subtilin. Again 0.4 ppm subtilin prevented all colony development. Subtilin activity in rapid and slow germination media. The following experiment was designed to determine the effect of subtilin in rapid and slow germination media. It has been shown (Andersen, 1951) that germination of C. botulinum spores in pork infusion media is slow if HCO (or CO2) is absent from the medium but very rapid and uniform if HCO0 is present. Unheated spores were placed in liquid medium2 containing: (1) HCOj without subtilin, (2) HCO and subtilin, and (3) subtilin without HCO. These were plated after varying intervals as indicated in table 2. This table shows: (1) 20 ppm subtilin is effective in lowering the viable count in a medium which supports rapid and uniform germination and growth; (2) if germination is delayed by omission of HCO, the spores TABLE 2 Effect of subtilin (S0 ppm) on Clostridium botulinum in rapid and 810w germination media at 28 C TDM PLATED COLONY COUNT PROM 1 ML OF 1:100 DILUTION Medium containing HCOT Medium containing HC0_ Medium containing subtilin (no subtilin) and subtilin (no HCO) hours , >10, * * A small part of this count was due to a contaminant which showed as atypical colonies and proved to be a gram negative rod. are not rapidly destroyed. In water or buffer containing subtilin the spore count likewise was reduced only slowly, or not at all. Sensitivity to subtilin of other cultures of C. botulinum and a putrefactive anaerobe. The subtilin sensitivity of 8 cultures of C. botulinum and a putrefactive anaerobe was determined in anaerobic petri dishes. After each suspension was diluted 1:100, 3 ml were heated in a test tube in boiling water for 1 minute and further diluted so that 1 ml, the inoculum for each plate, would contain an appropriate number of spores. A solution of 10 ppm subtilin in sterile water was adjusted to ph 4.0 with HCl, and the solution was steamed 5 minutes to destroy nonspore formers which might be resistant to subtilin. To each plate was added enough of this solution to give, below the glass plate, the concentrations indicated in table 3. Subtilin was not added to the thioglycolate agar above the glass plate. Diffusion of subtilin from the medium (around edge of glass plate) to the thioglycolate agar above undoubtedly lowered the concentration somewhat below that indicated. Table 3 shows that, while the cultures varied some-

4 148 A. A. ANDERSEN [vol. 64 what in their response, all were sensitive to low concentrations of subtilin under the conditions of the experiment. Effect of size of inoculum. A series of plates containing 20 ppm subtilin was set up with inocula ranging from 160 to 800,000 spores per plate. No colonies. developed in 30 days of incubation. In another experiment with inocula as described before but with only 1 ppm subtilin, a number of colonies developed as shown in table 4. TABLE 3 Sensitivity of several cultures of anaerobes to subtilin in agar medium2 at 28 C PPM SUBTILI CULTURE j 0.5 J 1.0 Number of colonies Clostridium botulinum, strain 1267B Clostridium botulinum, strain 115B Clostridium botulinum, strain 457A Clostridium botulinum, strain 169B Clostridium botulinum, strain 109A Clostridium botulinum, strain 62A Clostridium botulinum, strain 62A, N.C.A Clostridium botulinum, strain 213B Putrefactive anaerobe, strain Ot * Although practically all colonies were detectable in 1 day and all had appeared in 2 days, final counts were not made until 48 days. t No colonies appeared in plates containing 2, 5, and 10 ppm subtilin. TABLE 4 Effect of inocula on the number of colonies of Clostridium botulinum developing in anaerobic plates containing 1 ppm subtilin INOCULUM NO. OP COLONXES AT 5 DAYS 800, , , , * Average of duplicate plates. No additional colonies appeared in 23 days. Working with spores of a "flat-sour" organism, no. 1518, Cameron and Bohrer (1951) suggested that the action of subtilin is bacteriostatic rather than bactericidal, because of the observation that the count goes down in the presence of subtilin but partly recovers if the spores are given a heat treatment prior to plating. The following experiments suggest that C. botulinum does not respond in a similar manner. Suspensions of spores of C. botulinum, strain 62A (34,000 per ml), were incubated 25 hours at 30 C in a liquid medium2 containing 20 ppm subtilin. At 0, 6, 18, and 25 hours, samples were taken, diluted 1 to 100, and

5 19521 SUBTILIN ON SPORES OF C. BOTULINUM 149 plated. The subtilin level in the plate was thus about ppm. At 25 hours the count indicated that the number of viable spores had been reduced to about 100 per ml. Attempts to revive the "killed" spores by heating samples at 100 C for 1 and 5 minutes and at 80 C for 10 minutes prior to plating did not give higher counts. SUMMARY AND CONCLUSIONS Under the conditions used, 0.4 ppm subtilin prevented as many as 517 spores of Clostridium botulinum, strain 62A, from producing colonies in pork infusion agar. The colony count was reduced to approximately 50 per cent by 0.1 ppm subtilin. Subtilin reduced the count rapidly in a medium which supports rapid germination. In a medium where germination is delayed, subtilin reduced the count very slowly. This suggests that subtilin is not effective against many of the spores until the germination proces is started. All 8 cultures of C. botulinum tested and a putrefactive anaerobe were sensitive to low subtilin concentrations in a pork infusion medium containing HCOa. Inocula as high as 800,000 spores per plate were stopped by 20 ppm subtilin. One ppm subtilin permitted 253 out of 800,000 spores to produce colonies. REFERENCES ANDERSEN, A. A A rapid plate method of counting spores of Clostridium botulinum. J. Bact., 62, ANDERSEN, A. A., AND MICHENER, H. D Preservation of foods with antibiotics. I. The complementary action of subtilin and mild heat. Food Technol., 4, BFANK, N. G., MAYFIELD, J., AND FOLKERS, K The purification of subtilin concentrates by counter-current distribution. J. Am. Chem. Soc., 73, CAMERON, E. J., AND BOHRER, C. W Food preservation with antibiotics: the problem of proof. Food Technol., 5, CURRAN, H. R., AND EVANS, F. R The activity of penicillin in relation to bacterial spores and the preservation of milk. J. Bact., 52, FEVOLD, H. L., DIMIcK, K. P., AND KLOsE, A. A Isolation of subtilin from submerged cultures. Arch. Biochem., 18, GARIBALDI, J. A., AND FEENEY, R. E Subtilin production. Ind. Eng. Chem., 41, PERRY, H., TOWNSEND, C. T., ANDERSEN, A. A., AND BERRY, J. A Studies on Clostridium botulinum in frozen pack vegetables. Food Technol., 2,