Organisms used. The routine test organism was a putrefactive anaerobe, Company, and Bacilus stearothermophilus, strain NCA 1518.

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1 THE EFFECT OF OXIDATIVE RANCIDITY IN UNSATURATED FATTY ACIDS ON THE GERMINATION OF BACTERIAL SPORES NORMAN G. ROTH2 AND H. 0. HALVORSON Department of Bacteriology, University of Illinois, Urbana, Illinois Received for publication September 7, 1951 It has been reported that some lots of commercial media contain substances that inhibit germination of anaerobic spores which can be overcome by adding starch (Foster and Wynne, 1948a). Foster and Wynne ascribed this activity to the unsaturated C18 fatty acids. Halvorson (1950) suggested that this effect may be due to the formation of peroxides from the fatty acids. This investigation was undertaken to test the latter hypothesis. MATERIALS AND METHODS Organisms used. The routine test organism was a putrefactive anaerobe, strain P.A Later tests were also run on Clostridium botulinum, strain 7948, Clostridium pasteurianum, strain 7040, Bacillus subtilis, strain of Upjohn Company, and Bacilus stearothermophilus, strain NCA Preparation of spore suspensions. Anaerobic spores were collected after inoculating Blake bottles containing Yesair's pork infusion agar (Wynne and Foster, 1948) with a suspension of cells containing both spores and vegetative cells. The inoculum for each bottle consisted of 1.5 ml of a 72 hour egg-meat culture (Difco). The bottles were incubated for one week at 37 C in a desiccator which had been evacuated and the air replaced with natural gas. Cells were harvested by washing off the agar surface with sterile distilled water. They were spun down in a centrifuge, washed twice, and suspended in sterile distilled water. This suspension was heated to 80 C for 25 minutes to kill the vegetative cells and stored in a refrigerator. There was no apparent loss in viability of these spores over a four month period as shown by plate counts. The approximate number of viable spores in the suspensions was determined by serial dilution and plating in Yesair's pork agar. After the agar had solidified, all plates were layered with 2 per cent agar plus 5 per cent sodium thioglycolate and incubated at 37 C for 72 hours in an evacuated and gassed desiccator. Spore suspensions were diluted with distilled water to a final count of 100 to 300 viable spores per ml. The two suspensions of aerobic spores were laboratory stock preparations. B. msbtilis spores had been prepared by growth in a glucose-glutamate synthetic medium, washed twice with distilled water, and dried in vacuo over calciuim chloride. B. stearothermophilus had been grown on tryptone glucose agar, washed twice, heated to 80 C for 20 minutes, and stored in phosphate buffer at ph 7.0. Method of testing for inhiwtory action. All inhibitors of anaerobic spores were 1 This work was aided by a research grant from Swift & Co., Chicago, Illinois. 2 Present address: Camp Detrick, Frederick, Maryland. 429

2 430 NORMAN G. ROTH AND H. 0. HALVORSON [VOL. 63 tested by plating one ml of the diluted spore suspension in Yesair's pork infusion agar to which the various agents to be tested had been added before plating. It was found that a uniform emulsion of the fats or fatty acids could be obtained by adding them directly to the melted agar and shaking vigorously. The air added by this technique was not sufficient to affect germination of the spores. After inoculation, plates were layered with 2 per cent agar plus 0.5 per cent sodium thioglycolate. Control plates were run with the same diluted spore suspension and the same desiccator in all experiments. All plates were incubated at 37 C in an evacuated and gassed anaerobic desiccator. The desiccators were evacuated for 10 minutes with an ordinary laboratory water pump, and the air was replaced with natural gas. A slight vacuum was left in the jars for gas expansion in the incubator. After 72 hours, the plates were removed from the desiccator and the colonies counted. All results were recorded as number of colonies per plate. Statistical analyses were run on the resultant counts; the confidence interval was taken to be the mean plus or minus 2 times the standard error (standard deviation of the mean). A significant decrease in the number of colonies as compared with the controls was considered to be evidence for inhibition. EXPERIMENTAL RESULTS Effect of fats on spores of a putrefactive anaerobe. Corn oil (Mazola) and lard were tested in a fresh and a rancid state for their effect on germination of spores of a putrefactive anaerobe. Fresh fat was made rancid by bubbling oxygen through it and subsequently heating to boiling for a few minutes. Vigorous treatment was necessary in the case of corn oil to overcome the action of the tocopherol which is naturally present as an antioxidant. Ten plates of each sample were set up for statistical analysis. Kreis test (Jamieson, 1943) were run preliminary to this and all subsequent experiments to determine whether the samples were rancid. The results shown in table 1 indicate that oxidative rancidity in lard is a definite inhibitory factor for spore germination in this organism. Similar results were obtained with the corn oil. Effect of methyl esters of oleic, linoleic, and linolenic acids on spores of a putrefactive anaerobe. Pure, unoxidized (Kreis test negative) methyl esters of the Cis unsaturated fatty acids were obtained from the Hormel Foundation, Austin, Minnesota. Portions of these fatty esters were made rancid in the same manner as the fats. Fresh and rancid samples of all three esters were compared by the plate count technique. In all three cases, the fresh, unoxidized methyl esters were not inhibitory at 0.01 per cent concentration. Upon oxidation, all three samples caused significant inhibition at the same concentration. In these cases of methyl linoleate and methyl linolenate, inhibition was about 95 per cent; with methyl oleate, about 50 per cent. The difference between the degree of inhibition produced by the oleate and by the linoleate and linolenate is probably not due to any difference in the type of ester, but rather in the state of oxidation. Methyl oleate contains only one double bond and does not oxidize as readily as linoleate and linolenate which have two and three double bonds, respectively.

3 1952] OXIDATIVE RANCIDITY IN FATTY ACIDS TABLE 1 Effect of oxidative rancidity of lard on the germination of spores of a putrefactive anaerobe 1 ~~~~23 LARD (1:100) RNCID LAR (1:100) NOT OOL (Ernxis +) R.I4NCT (muis - OTO Plate counts Mean Standard error Confidence, interval Effect of pure oleic, linoleic, and linolenic acids on spores of a putrefactive anaerobe. Pure oleic, linoleic, and linolenic acids also were obtained from the Hormel Foundation, Kreis tested, and immediately plated. The oleic acid was negative to the Kreis test, the linoleic acid was slightly positive, and the linolenic acid very strongly positive in the initial test. All three acids were inhibitory in the rancid state at 1:1,000 against spores of the putrefactive anaerobe. Fresh oleic acid (1: 1,000) showed no inhibition (table 2). Rancid linolenic acid was shown to be inhibitory through 1:1,000,000. As these fatty acids were prepared anaerobically and received in vials which had been evacuated and the air replaced with nitrogen, they probably were not rancid upon arrival. However, they were opened in the presence of air. The linoleic and linolenic acids are highly unsaturated and readily subject to auto- oxidation. Therefore, it seems likely that a method of opening the vials and testing the inhibitory action in the complete absence of oxygen will be necessary to adequately test the more highly unsaturated fatty acids. This effect on the germination of spores is probably only an inhibitory one and not a direct lethal effect on the spores. A spore suspension, 10 times the strength normally used for plating, was set up in 0.25 per cent sterile, "nonnutrient" agar. Rancid linolenic acid was added at a concentration of 1:100, The suspension was shaken thoroughly; the small amount of agar produced a uniform emulsion of the fatty acid, yet left the solution entirely liquid. This spore suspension containing the oxidized linolenic acid was placed in a shaker at room temperature. At the end of 1 and 2 hours, 0.1 ml samples were plated in 10 ml of Yesair's agar. In this way, the linolenic acid was diluted out to 1:10,000,000, a nontoxic concentration, during the plating. Controls were 431

4 432 NORMAN G. ROTH AND H. 0. HALVORSON [VOL. (53 made by plating 0.1 ml aliquots from the spore suspension (before the addition of linolenic acid) into Yesair's pork infusion agar with no added fatty acids, and into Yesair's agar containing 1:100,000 linolenic and 1:10,000,000 ilnolenic acid. It was found that the oxidized linolenic acid had no lethal effect on the spores in 2 hours. In the controls, 1:100,000 linolenic was inhibitory in the plating medium, but 1:10,000,000 was not. Effect of benzoyl peroxide on spores of a putrefactive anaerobe. Benzoyl peroxide was chosen as a test agent because of its structural similarity to the fatty acid peroxides and its ready availability. It was found, by the same technique as that used with the fatty acids, that benzoyl peroxide gave an inhibition against spores of the same order of magnitude as that of the rancid fatty acids. TABLE 2 Effect of oxidative rancidity of oleic acid on the germination of spores of a putrefactive anaerobe OLzIC (1:1,00) RANc OrzIC (1:1,000) NOT (mi +) i]ucid (KR - CONThOL Plate counts Mean Standard error Confidence interval Reversal of the inhibition. If the action of the rancid fatty acids actually was due to peroxide, it seemed likely that catalase might overcome the inhibition. Consequently, the linolenic acid inhibition at 1:10,000 was tested with catalase added to the medium. Beef liver catalase (250 U per ml) was obtained from Worthington Biochemical Laboratory, Freehold, New Jersey. It was found that 0.01 per cent of this catalase preparation was able to overcome partially the inhibition of germination of spores (table 3). Other organism. Additional tests of the effect of rancid unsaturated fatty esters were run on spores of two other anaerobes, C. botulinum and C. pasteurianum, using Yesair's agar as the germination medium, and, two aerobes, B. aubtilis plated in nutrient agar and B. stearothermophilus in tryptone glucose agar (table 5). In all four cases, the rancid esters inhibited at 0.01 per cent while the nonrancid esters did not inhibit at the same concentration.

5 1952] 2OXIDATIVE RANCIDITY IN FATTY ACIDS 433 B. subtilis was completely inhibited by rancid methyl oleate (0.01 per cent) after 24 hours' incubation at 37 C; there was no inhibition with the nonrancid methyl ester at the same concentration after 24 hours. After 5 days' incubation TABLE 3 Effect of catalase on reversal of inhibition caused by rancid linolenic acid (1:10,000) on spores of a putrefactive anaerobe x.xoznmc (1:10 000) LINOLEMC (1: 10,000) CO3OL CATALASE (0.025 i/pll) NO CATALAS Plate counts Mean Standard error Confidence interval TABLE 4 Summary of effect of oxidative rancidity in fatty acids and fatty acid eaters on the germination of spores of a putrefactive anaerobe (All numbers are averages of ten plate counts) INHIBITOR NO. OF COLONIZS PER PLATE.- Rancid sample Fresh sample Controls Lard (1:100) Corn oil (1:100) Methyl oleate (1:100) Methyl oleate (1:100) Methyl linoleate (1:100) Methyl linoleate (1:100) Methyl linolenate (1:100) Methyl linolenate (1:100) Oleic acid (1: 1,000) Linoleic acid (1:1,000) Linolenic acid (1:1,000) of the 10 plates containing rancid methyl oleate showed 1 to 4 colonies. (Control plates averaged 50 colonies.) The suspension of spores used was shown to be catalase positive. It seems probable that a few of these spores contained enough

6 434 NORMAN G. ROTH AND H. 0. HALVORSON [vol. 63 catalase to overcome the inhibition. None of the other spore suspensions showed any spontaneous ability to overcome the inhibition first noted, and none of them gave catalase positive reactions. TABLE 5 Effect of oxidative rancidity in fatty acid esters on the germination of spores of several organisms (All numbers are averages of ten plate counts) ORGANSM ESTER PLATE COUNTS Rancid Fresh sample Control Bacillus subtilis Methyl oleate (1:100) Bacillus stearothermophilus Methyl linoleate 0 1,000 1,000 (1:100) (approximate) (approximate) Clostridium botulinum Methyl linoleate (1:100) Clostridium pasteurianum Methyl linoleate (1:100) DISCUSSION It has been shown that unsaturated fatty acids and methyl esters do not inhibit spore germination unless they are rancid. Thus, it can be concluded that fatty acids themselves are not inhibitory but that some product formed during the course of oxidation is the cause of the inhibition. The most likely inhibitory agent involved is a peroxide. It was shown that benzoyl peroxide, which is structurally very similar to the fatty acid peroxides, gave an inhibition of the same order of magnitude as the rancid fatty acids. The action of catalase in partially reversing the inhibition clearly indicates a peroxide effect. However, it is suggested that this effect may not be due to the organic peroxides themselves but to hydrogen peroxide which may be formed as a temporary breakdown product of the organic peroxides. It was found that the spore suspension of B. subtilis caused a violent bubbling when brought in contact with hydrogen peroxide, but did not react with benzoyl peroxide or any of the rancid fatty acids or fatty acid esters. Similarly, the commercial catalase preparation was very active against hydrogen peroxide but not against the organic peroxides. It is possible that a peroxide effect may be involved in the phenomenon of dormancy shown by bacterial spores. Foster and Wynne (1948b) reported that oleic, linoleic, and linolenic acids inhibited germination in a manner similar to the dormancy phenomenon. Halvorson (1950) postulated that this might be due to the presence of peroxides rather than the unsaturated fatty acids themselves. The evidence cited in this paper supports the latter view.

7 1952] OXIDATIVE RANCIDITY IN FATrY ACIDS 435 SUMMARY Rancid corn oil and lard inhibited the germination of spores of a putrefactive anaerobe, whereas nonrancid samples at the same concentration had no effect. Oleic, linoleic, and linolenic acids and their methyl esters were inhibitory only when rancid. The experiments were repeated and the results were confirmed with Clostridium botulinum, Clostridium pasteurianum, Bacillus subtilis, and Bacillus stearothermophilus. Benzoyl peroxide gave an inhibition of the same order of magnitude as rancid, unsaturated fatty acids. Catalase partially reversed the inhibition produced by the rancid fatty acids. The possible relationship of this inhibition to the phenomenon of dormancy is discussed. REFERENCES FOSTER, J. W., AND WYNNE, E. S. 1948a Physiological studies on spore germination with special reference to Clostridium botulinum. IV. Inhibition of germination by unsaturated C08 fatty acids. J. Bact., 55, FOSTER, J. W., AND WYNNE, E. S. 1948b The problem of "dormancy" in bacterial spores. J. Bact., 55, HALVORSON, H Factors affecting bacterial spore germination. Proc. Second Conf. Res. Am. Meat Inst., JAMIESON, G. S Vegetable Fats and Oils. Reinhold Publishing Corporation, New York, New York. WYNNE, E. S., AND FOSTER, J. W Physiological studies on spore germination with special reference to Clostridium botulinum. I. Development of a quantitative method. J. Bact., 55, Downloaded from on July 13, 2018 by guest