Freezing, and Ultraviolet

Transcription

1 APPLIED MICROBIOLOGY, May, 1966 Vol. 14, No. 3 Copyright ( 1966 American Society for Microbiology Printed in U.S.A. Effect of Pimaricin on the Resistance of Saccharomyces cerevisiae to Heat, Freezing, and Ultraviolet Irradiation G. K. YORK Department of Food Science and Technology, UIniversity of California, Davis, California Received for publication 4 February 1966 ABSTRACT YORK, G. K. (University of California, Davis). Effect of pimaricin on the resistance of Saccharomyces cerevisiae to heat, freezing, and ultraviolet irradiation. Appl. Microbiol. 14: Exposure of washed cells of Saccharomyces cerevisiae C-299 to inhibitory concentrations of pimaricin decreased resistance to lethal temperatures and to freezing and thawing. Varying the ph of the recovery medium had little effect on the decrease in heat resistance, but addition of peptone or yeast extract resulted in a partial recovery of apparent heat resistance. Addition of peptone or calcium ion to the heating menstruum did not affect the reduction in resistance caused by pimaricin. Varying the composition of the recovery medium similarly affected the resistance to freezing and thawing of pimaricin-exposed and unexposed cells. Sensitivity to ultraviolet light was apparently not affected by pimaricin either by preliminary exposure or when irradiated in the presence of pimaricin. It is suggested that pimaricin lowers the heat resistance of S. cerevisiae by depleting essential nutrients of the cells through a disruption of permeability, and that heat-damaged cells cannot resynthesize these nutrients. It is also suggested that spoilage of acid foods bys. cerevisiae may be retarded by a combination of mild heat treatment and the addition of pimaricin. 451 Pimaricin, a polyene antifungal antibiotic, is active against a wide range of fungi in concentrations of 5 to 50 mg per liter (9). It absorbs light in the ultraviolet region, with peaks at 289, 303, and 317 m,u (Gunetileke and York, submitted for publication). Pimaricin increases the rate of efflux of amino acids, nucleotides, inorganic phosphate, and potassium ions from washed cells of Saccharomyces cerevisiae, and the mechanism of inhibition is believed to be the alteration of the cytoplasmic membrane (Gunetileke and York, submitted for publication). Several antibiotics, for example, subtilin, nisin, and tylosin, have been reported to decrease the heat resistance of certain bacterial endospores (1, 2, 4-7, 13). Pimaricin has been suggested as a chemical preservative to retard spoilage in both fresh and processed foods (3, 10, 11). Because of its advocacy for use in foods and because S. cerevisiae can spoil certain acid foods, the effect of pimaricin on the resistance of this yeast to heat, freezing and thawing, and ultraviolet light was investigated. MATERIALS AND METHODS Measurement of pimaricin. The optical density of acidified solutions of pimaricin was measured at 317 mua and compared with standard solutions in the range of 1 to 10 ppm of pimaricin by the method of Gunetileke and York (submitted for publication). Exposure of cells to pimaricin. Cells of S. cerevisiae no. C-299 were harvested from 48-hr cultures in Yeast Nitrogen Base (Difco) plus 1% glucose, washed twice with sterile water, and suspended in a solution containing 10 ;umoles of glucose in 0.06 M phosphate buffer (ph 6.0) and various concentrations of pimaricin. The suspensions were incubated aerobically for 3 hr at 20 C with constant agitation. The cells were removed by centrifugation and washed twice with sterile 0.89% sodium chloride. Determination of thermal death rate. The heating menstruum used was 0.06M phosphate buffer (ph 6.0), with or without added peptone, yeast extract, or calcium chloride; suspensions of approximately 107

2 452 YORK APPL. MICROBIOL. (I) -J LLu 00 L] 5 z TIME, MIN. FIG. 1. Effect of exposure to pimaricin on the rate ofheat destruction at 55 C of washed cells of Saccharomyces cerevisiae C-299. Heating menstruum: 0.06 M phosphate buffer. Recovery medium: Yeast Nitrogen Base-agar (ph 6.0) with 1% glucose. Symbols: * = no pimaricin; 0 = 10 ppm of pimaricin; A = 20 ppm of pimaricin. cells per milliliter were heated at 55 i 1 C in a threenecked flask under constant agitation (14). Samples were withdrawn aseptically, cooled in an ice-water bath, and appropriate dilutions were made in sterile physiological saline. The spread plate procedure was used, and colony counts were made after 48 hr of incubation at 30 C. The time necessary to reduce the viable count by 90% (D value) was calculated from the straight-line portion of a plot of the logarithms of number of cells versus time. Determination of death by freezing and thawing. A 20-ml amount of 0.06 M phosphate buffer (ph 6.0), containing approximately 107 cells per milliliter, was frozen in 125-ml Erlenmeyer flasks at -20 C. The suspensions were stored for 24 hr at -20 C and then thawed for 2 hr at 30 C. The concentration of viable cells was determined after 48 hr at 30 C by spread plate procedures. Freezing and thawing of the suspensions was repeated twice, and viable counts were determined after each thawing. Determination of death by ultraviolet irradiation. A suspension containing approximately 108 cells per milliliter in phosphate buffer (ph 6.0) was placed in the bottom of a sterile petri dish (20 by 150 mm) at a depth of 6 mm. The suspension was gently agitated with a Teflon-coated magnetic stiffer bar (3 by 10 mm) and irradiated with a mercury arc ultraviolet lamp placed 15 cm from the surface of the suspension. Samples of 0.1 ml were withdrawn with a steiile 1.0- ml syringe and diluted, and viable counts were made by the spread plate method. Incubation was done in the dark for 48 hr at 30 C. Recovery media. Double-strength Yeast Nitrogen Base (Difco) containing 2% glucose was adjusted to ph 4.0 or 6.0 and sterilized by filtration. Peptone or yeast extract was added, when desired, before filtersterilization to a concentration of 0.2%. Just prior to pouring plates, the double-strength broth medium was mixed with an equal volume of melted and cooled (45 C) 3% agar. The final concentrations of ingredients were single-strength Yeast Nitrogen Base, 1% glucose, 0.1% peptone or 0.1% yeast extract, and 1.5% agar. RESULTS Effect on heat resistance. Figure 1 shows the rate curves obtained when cells of S. cerevisiae were heated in 0.06 M phosphate buffer (ph 6.0) at 55 C and viable counts were made on Yeast Nitrogen Base-agar (ph 6.0). Cells which had been exposed to 10 and 20 ppm of pimaricin for 3 hr and then washed were more rapidly destroyed than unexposed, washed cells (Fig. 1). In addition to an increase in thermal death rate, there was a change in the shape of the curves after exposure to pimaricin as well as a decrease in the initial number of cells (Fig. 1). Table 1 shows the thermal death rates at 55 C when the ph of the recovery medium was lowered from 6.0 to 4.0. Although there was a significant decrease in the D values at ph 4.0, proportional decrease was essentially the same for cells exposed to pimaricin and for unexposed cells. When the recovery medium at ph 6.0 was supplemented with 0.1% peptone or yeast extract, there was less decrease in apparent heat resistance after exposure to pimaricin (Table 2). Peptone was apparently more effective than yeast extract TABLE 1. Effect of ph of recovery medium* on apparent heat resistance of Saccharomyces cerevisiae C-299 after exposure to pimaricin D value (min at 55 C) Pimaricin Ratio, D4,/D6 :0 ph 4.0 ph 6.0 ppm * Recovery medium: Yeast Nitrogen Base-agar with 1% glucose.

3 VOL. 14, 1966 EFFECT OF PIMARICIN ON S. CEREVISIAE 453 TABLE 2. Effect ofpeptone and yeast extract added to the basal recovery medium (ph 6.0) on the apparent heat resistance of Saccharomyces cerevisiae C-299 exposed to pimaricin Apparent heat resistance* TABLE 3. Effect of peptone and calcium chloride added to heating menstruum on the apparent heat resistance of Saccharomyces cerevisiae C-299 exposed to pimaricin* Apparent heat resistancet Recovery medium Pimaricin (ppm) D Per D Per D Per D Per cent cent cent cent Basal Basal + 0.1% peptone Basal + 0.1% yeastextract * D in min in 0.6 M phosphate buffer (ph 6.0) at 55 C. Per cent = (D control/d pimaricin) X 100. in counteracting the effect of pimaricin on heat resistance of S. cerevisiae (Table 2). When the heating menstruum was supplemented with 0.1 % peptone, there was no apparent effect on the reduction of heat resistance caused by exposure to 20 ppm of pimaricin (Table 3). Increasing the concentration of peptone to 1% resulted in only a slight counteraction of the reduction of heat resistance (Table 3). The addition TABLE 4. Freeze and thaw Effect of exposure Pimaricin Heating menstruumt ppm) D Per D Per cent cent Buffer Buffer + 0.1% peptone Buffer + 1.0% peptone Buffer ppm of Ca * Recovery medium: Yeast Nitrogen Base-agar (ph 6.0) with 1% glucose. t Buffer: 0.06 M phosphate, ph D in min at 55 C. Per cent = (D control/d pimaricin) X 100. of 100 ppm of calcium ion (as the chloride) to the heating menstruum had no effect on the apparent heat resistance (Table 3). Exposure to pimaricin for 3 hr markedly reduced the resistance of washed cells of S. cerevisiae to freezing and thawing (Table 4). When to pimaricin on the viability of cells of Saccharomyces cerevisiae C-299 subjected to freezing and thawing* Cells/ml in control Cells/ml with pimaricin (20 ppm) Initial Final Per cent Initial Final recent reduction ntilfal reduction 1st 5.3 X 10' 3.8 X X X nd 3.8 X X X X rd 2.6 X X X X * Recovery medium: Yeast Nitrogen Base-agar (ph 6.0) with 1% glucose. TABLE 5. Effect of added peptone and ph of the recovery medium on resistance ofpimaricin-treated cells of Saccharomyces cerevisiae C-299 to freezing and thawing Pimaricin (20 ppm) Recovery medium* No. of cells/ml No. of cells/ml Pei cent ALogt no. Per cent ALogt no. reduction of cells reduction of cells Initial Final Initial Final ph X X X X ph X X X X ph % peptone X X X X * Yeast Nitrogen Base-agar with 1% glucose. t ALog = (log initial no. - log final no.).

4 454 YORK APPL. MICROBIOL. Cl) -LJ [Luj ~ 2 3 TIME7MIN. FIG. 2. Effiect of pimaricin on sensitivity to ultraviolet light of washed cells ofsaccharomyces cerevisiae C-299. Suspending menstruum: 0.06 M phosphate buffer. Recovery medium: Yeast Nitrogen Base-agar (ph 6.0) with 1% glucose. Symbols: * = no pimaricin; O = exposure to 20 ppm ofpimaricin for 3 hr at 30 C; A = irradiated in the presence of 20 ppm of pimaricin. the ph of the recovery medium was lowered from 6.0 to 4.0, there were fewer surviving cells after freezing and thawing both with pimaricin-exposed and unexposed cells (Table 5). Comparison of the differences in the logarithms of the initial and final numbers of cells indicates that the ph of the recovery medium similarly affected the pimaricin-exposed cells and unexposed cells (Table 5). The addition of 0.1I% peptone to the recovery medium resulted in an increased resistance to freezing and thawing in unexposed cells and a partial reversal of the effect of pimaricin in treated cezs (Table 5). Cells of S. cerevisiae exposed to 20 ppm of pimaricin had essentially the same resistance to ultraviolet radiation as did unexposed cells (Fig. 2). Irradiation in the presence of 20 ppm of pimaricin also did not affect resistance to ultraviolet lighit (Fig. 2). DISCUSSION Pimaricin in concentrations which inhibit growth apparently lowers the heat resistance of cells of S. cerevisiae. Exposure to pimaricin, followed by washing before heating, and diluting before plating precludes carry-over of inhibitory amounts of pimaricin to the recovery medium. One of the effects of pimaricin on cells of S. cerevisiae is an increase of efflux of certain cellular components, for example, amino acids and nucleic acids (Gunetileke and York, submitted for publication). Because exposure to heat may destroy the ability of the cells to synthesize a particular component or components, the increased loss of such material caused by exposure to pimaricin may result in the inability of the heated cells to reproduce when placed on a recovery medium devoid of such compounds. This is suggested by the partial reversal of the effect of pimaricin on heat resistance when the recovery medium is supplemented with a complex nutrient source, i.e., peptone or yeast extract. It is also possible, however, that the disruption of the normal function of the cytoplasmic membrane by pimaricin may account for the increased sensitivity of the cells to heat if one assumes that the membrane is a site of heat damage. Therefore, addition of peptone or yeast extract to the recovery medium would partially counteract the leakage of essential nutrients of the pimaricinexposed, heated cells. The former hypothesis appears more likely in view of the observation that peptone added to the heating menstruum had less effect on reversing the decrease in heat resistance than did the addition of peptone to the recovery medium. Experiments are necessary to determine whether individual nutrients, e.g., amino acids or vitamins, cause a similar reversal when added to the recovery medium. The failure of a lower ph of the recovery medium (ph 4.0) to give a decrease in heat resistance in pimaricin-treated cells proportionately larger than a higher ph (6.0) is difficult to explain in light of the increased permeability of these cells. Perhaps the ability of S. cerevisiae to grow at lower ph levels reflects a tolerance of higher internal concentrations of hydrogen ion, or indicates that the cell wall, per se, rather than the cytoplasmic membrane is involved in acid tolerance. Since changes in osmotic pressure are, at least partly, responsible for death of microbial cells during freezing and thawing (8), it appears that damage to the cytoplasmic membrane by pimaricin results in a marked reduction of resistance to freezing and thawing. The similar effects on pimaricin-exposed and unexposed cells of the ph of the recovery medium and supplementation with peptone suggests that pimaricin damaged the cytoplasmic membrane but did not appreciably alter the synthetic metabolism of the cell.

5 VOL. 14, 1966 EFFECT OF PIMARICIN ON S. CEREVISIAE 455 Failure of pimaricin to influence the sensitivity of S. cerevisiae to ultraviolet light when irradiated in the presence of pimaricin may be attributed to ultraviolet absorption by the antibiotic (Gunetileke and York, submitted for publication). It is difficult to explain, however, the failure of previously exposed cells to react differently to ultraviolet light than do unexposed cells. Perhaps resistance to ultraviolet irradiation does not primarily involve the cytoplasmic membrane, but involves some other structure, for example, the cell wall or the nuclear apparatus. Since pimaricin apparently lowers the heat resistance of S. cerevisiae and loses activity upon storage in acid foods (12), it may be advantageous to combine mild heat and pimaricin to extend the storage life of such foods. LITERATURE CITED 1. ADAMS, A. T., J. C. AYERS, AND R. B. TISCHER The effect of subtilin and heat in preventing spoilage of comminuted meat. Food Technol. 5: ANDERSEN, A. A., AND H. D. MICHENER Preservation of foods with antibiotics. I. The complementary action of subtilin and mild heat. Food Technol. 4: AYERS, J. C., AND E. L. DENISEN Maintaining the freshness of berries using selected packing materials and antifungal agents. Food Technol. 12: CAMPBELL, L. L., AND R. T. O'BRIEN Antibiotics in food preservation. Food Technol. 9: CAMPBELL, L. L., E. E. SNIFF, AND R. T. O'BRIEN Subtilin and pisin as additives that lower the heat process requirements of canned foods. Food Technol. 13: DENNY, C. B., L. E. SHARPE, AND C. W. BOHRER Effects of tylosin and nisin on canned food spoilage bacteria. Appl. Microbiol. 9: EL-BisI, H. M., Z. J. ORDAL, AND A. I. NEKON The effect of certain fungicides on the thermal death rate of spores of Bacillus coagulans var. thermoacidurans. Food Res. 20: G. INGRAHAM, J. L Temperature relationships, p In I. C. Gunsalus and R. Y. Stanier [ed.], The bacteria, vol. 4. Academic Press, Inc., New York. 9. KLIS, J. S., L. D. WITTER, AND Z. J. ORDAL The effect of several antifungal antibiotics on the growth of common food spoilage fungi. Food Technol. 13: SHAHANI, K. M., K. M. NILSON, AND P. A. DAvIs Effect of antifungal antibiotics upon the keeping quality of cottage cheese. Intern. Dairy Congr. 15th, vol. 2, p SHIRK, R. L., AND W. L. CLARK The effect of pimaricin in retarding the spoilage of fresh orange juice. Food Technol. 17: SHIRK, R. J., E. F. KLINE, AND W. L. CLARK The effect of pimaricin in the control of yeast growth in orange juice concentrates. Food Technol. 20: WHEATON, E., AND G. L. HAYS Antibiotics and the control of spoilage in canned foods. Food Technol. 18: YOKOYA, F., AND G. K. YORK Effect of several environmental conditions on the "thermal death rate" of endospores of aerobic, thermophilic bacteria. Appl. Microbiol. 13: