Effects of Potassium Sorbate and Sodium Benzoate on Inactivating Yeasts Heated in Broths Containing Sodium Chloride and Sucrose

765

L. R. BEUCHAT

Department ofFood Science, University of Georgia Agricultural Experiment Station, Experiment, Georgia 30212

(Received for publication November 21, 1980) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/10/765/1653722/0362-028x-44_10_765.pdf by guest on 27 September 2021

ABSTRACT potassium sorbate acted synergistically with heat to Six genera of yeasts possessing a wide range of physiological inactivate cells of yeasts and conidia of molds (10). characteristics were tested for their sensitivities to heat when The present study was designed to determine the suspended in media (pH 4.5) with reduced water activities (aw). effects of sodium chloride and sucrose on thermal Five of six strains had increased tolerance to heat, compared to inactivation of several species of previously untested controls, when cells were suspended in broth containing 3% yeasts. A second objective was to evaluate the effects of sodium chloride. Further protection was afforded to three solutes together with potassium sorbate and sodium strains in broth containing 6o/o salt, whereas one strain showed benzoate on thermal destruction of yeasts. increased tolerance to heat when sodium chloride was present in broth at a concentration of 12% (aw = 0.926). Sucrose, at levels up to 60% (aw = 0.892), protected five of six strains MATERIALS AND METHODS against heat inactivation. Addition of potassium sorbate or sodium benzoate at 500 or 1000 ppm to heating menstrua Yeasts resulted in significantly decreased D values for all yeasts. At the Three ascomycetous strains IDebaryomyces hansenii NRRL Y-7268, same concentration, the extent to which the two preservatives Pichia membranaefaciens 67-272 (obtained from Dr. M. W. Miller, acted synergistically with heat was dependent upon the nature University of California, Davis) and Saccharomyces cerevisiae of the solute used to lower the aw of the heating media. UGA-102] and three asporogenous strains [Candida krusei NRRL Y-7179, Kloeckera apiculata NRRL Y-1382 and Rhodotoru/a rubra C-46A (obtained from Dr. M. W. Miller)] of yeasts were examined. The microorganisms responsible for spoilage of acid Stock cultures were maintained at 4 Con potato dextrose agar (PDA, pH 5.5) slants. foods or foods with reduced water activities (aw) are often Growth and heating media yeasts. Environmental conditions characteristic of such Yeast extract - malt extract - peptone - glucose (YMPG) broth was foods retard growth of bacteria and thus provide a used for culturing the yeasts. The formula consisted of (grams per liter non-competitive situation which promotes growth of of deionized water): yeast extract, 3; malt extract, 3; peptone, 5 and yeasts. Pasteurization of foods having aw values in a glucose, 10. Sodium chloride [3. 6, 9, and 12% (wtlwt)] and sucrose 10, 30, 45, and 60% (wt/wt)] were added to YMPG broth to result in range in which yeasts can grow is complicated by the fact decreased aw values as shown in Table 1. The YMPG broth used for that tolerance of microorganisms to heat at these values culturing yeasts in preparation for tests was adjusted to pH 5.5 with 6 is generally higher compared to tolerance in foods with M HCI and dispensed in 100-ml quantities into 250-ml Erlenmeyer aw values approaching 1.0. Corry (2, 3) investigated the flasks before autoclaving at 121 C for 15 min. Broth to which sodium relative tolerance of Saccharomyces rouxii and Schizo chloride or sucrose was added was adjusted to pH 7 .0, autoclaved, saccharomyces pombe to heat when suspended in various cooled to 45 C and then adjusted to pH 4.5 with sterile HCl. For experiments designed to evaluate the combined effects of preservatives solutions of sugars and polyols. Resistance to inactiva and solutes on the rate of inactivation of yeasts, 1 ml of a tion was maximum in solutions of sucrose. Shibasaki and filter-sterilized water solution of 5.0% or 10% potassium sorbate Tsuchido (10) reported that sodium chloride had a (Monsanto Industrial Chemicals Co., St. Louis, MO) and sodium pronounced protective effect toward heated cells of benzoate (Ptizer Inc., New York, NY) was added to 100 ml of cooled YMPG to give tina! concentrations of 500 or 1000 ppm, respectively. Candida utilis. Addition of preservatives was made before adjustment of pH to 4.5. Some chemical preservatives act synergistically with Preparation of cultures for testing heat to inactivate microorganisms. Pederson and The YMPG broth was inoculated with stock cultures of yeasts and Tressler (8) demonstrated that sodium benzoate and incubated at 30 C while being constantly agitated (150 rpm) on a rotary sulfur dioxide lowered the effective temperature for shaker. Loop inocula were transferred to sterile YMPG broth at 2- to 3-day intervals. The age of the cultures used for various tests ranged pasteurization of apple juice. A combination of sodium from 46 to 48 h. sorb ate and mild heat greatly increased the storage life of Inactivation at elevated temperatures apple cider, peach slices and fruit salad (9). whereas Suspensions (1 ml) of vegetative yeast cells were transferred to 100 ml

JOURNAL OF FOOD PROTECTION, VOL 44, OCTOBER 1981 766 BEUCHAT

TABLE 1. Water activities of YMPG breth containing sodium medium had a protective effect against heat inactivation. chloride and sucrose. Further protection was noted for P. membranaefaciens, Concentration of Water C. krusei and K. apiculata when a concentration of 6o/o solute (o/o, wtlwt) Molality activity was present in the heating medium, whereas concentra Control 0.996 tions ranging to 12o/o enhanced the heat stability of D. Sodium chloride hansenii. With the exception of D. hansenii, the 3 0.53 0.988 maximum sodium chloride concentration affording 6 1.09 0.974 protection against heat inactivation of yeasts was less 9 1.69 0.949 than 12o/o. The presence of sodium chloride at all levels 12 2.80 0.926 tested enhanced the rate of inactivation of R. rubra, Sucrose compared to rates observed in YMPG broth containing 10 0.32 0.992 no sodium chloride. 30 1.25 0.975 45 2.39 0.946 40 D. hansen// ,48° C. krusei,54° Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/10/765/1653722/0362-028x-44_10_765.pdf by guest on 27 September 2021 60 4.39 0.892 30 ofYMPG broth supplemented with 0, 500 or 1000 ppm of preservatives and various amounts of sodium chloride or sucrose. Before initiation of 20 heat treatment, all heating media were adjusted to selected temperatures, depending upon the relative heat resistance of test yeasts (1). Temperatures maintained during periods of up to 80 min of 10 treatment were: K. apiculata, 47 C; D. hansenii, 48 C; S. cerevisiae, 51 C; P. membranaefaciens and R. rubra, 52 C; and C. krusei, 56 C. The 0 initial population of viable cells ranged from 105 to 2 x 106 per ml of c YMPG heating broth. E 40 P membranaefaciens, 51° K. apiculala ,46° Enumeration ofyeasts Q) E Following exposure to elevated temperature for periods ranging to 80 30 1- min. samples (1 ml) were withdrawn from the heating flask, dispensed c:: in 9- or 99-ml quantities of 0.1 M potassium phosphate buffer (pH 7.0) 0 u 20 at room temperature, serially diluted and plated on YMPG broth ::J "0 containing 2% agar (pH 5.5). Colonies were counted after 5 days of (lJ incubation at 30 C. Cl: 10 Decimal reduction times (D value = time required at a given 0 E temperature to inactivate 90%ofthe viable population) were calculated u 0 (lJ for all test yeasts. Tailing was occassionally observed, but D values were 0 calculated from straight-lined portions of inactivation curves which 40 5 cerevisiae, 51° transcended at least three log cycles. Inactivation at 22 C 30 Using the procedure described for determining rates of inactivation at elevated temperatures, it was possible that cells may have undergone 20 osmotic shock as a result of transfer from YMPG culturing broth to YMPG containing added solutes, then back to phosphate buffer before plating on YMPG agar. If shock was severe enough to be lethal or cause 10 injury to cells, correspondingly reduced numbers of colonies may have appeared on YMPG agar. It was desirable to design a test to determine the effect of changes in osmotic pressure of suspending media on the 0 3 6 9 12 0 3 6 viability of cells separate from the lethal effects of elevated Concentrat,on of NaCI (%, wt/wt) temperature. This was accomplished by repeating the experiments outlined in the two preceding subsections, except that treatment for up Figure 1: The effects of sodium chloride, potassium sorbate, to 80 min was carried out at 22 C rather than at temperatures ranging and sodium benzoate on decimal reduction times (JJ values) of from 47 to 56° C. six yeasts exposed to elevated temperatures (C). Symbols: 0, no Measurement of water activity potassium sorbate or sodium benzoate added to YMPG heating The aw of YMPG broths was measured at 21 C with a Sina-scope medium; /:;., 500 ppm of potassium sorbate; D, 1000 ppm of hygrometer (Beckman Instruments Inc., Cedar Grove. NJ). potassium sorbate; "'· 500 ppm of sodium benzoate; •· I 000 Statistical ana(vses ppm of sodium benzoate. An absence of data points signifies Data presented represent means of a minimum of two replications that D values were< 3.5 min. done in duplicate. The multiple range test as described by Duncan (5) was used throughout the study to test for statistically significant differences (P ..;; 0.05). Without exception, addition of potassium sorbate and sodium benzoate at 500 and 1000 ppm to heating media RESULTS cau~ed more rapid inactivation of yeasts, compared to rates of inactivation observed in respective controls Results of studies concerning the combined effects of containing the same level of sodium chloride. Trends sodium chloride and preservatives on heat inactivation of showing increases and decreases in D values were similar yeasts are shown in Fig. 1. With the exception of R. to those attributed to chaP~es in the level of sodium rubra, addition of 3o/o sodium chloride to the heating chloride in heating media.

JOURNAL OF FOOD PROTECTION. VOL. 44. OCTOBER 1981 HEAT INACTIVATION OF YEASTS 767

A comparison of the lethal effects of the two test 60 D. hansenil, 4 8 o C. kruse!, 56° preservatives reveals that in any given formulation of YMPG broth, sodium benzoate exerted a more lethal 45 effect on C. krnsei and K. apiculata than did potassium 0--:z: sorbate. However, examination of data from experiments 30 ,/ jY" involving the other four test yeasts does not clearly 15 I .f indicate a superiority of one of the preservatives with 0 .~~ regard to enhancement of heat inactivation. This is .~~ particularly relevant when comparing D values of cells 0 '2 heated in salt-supplemented broth. 60 .s P membranae foe/ens, 52 o K. apiculata ,4 7° Data from experiments designed to evaluate the Q) E combined and independent effects of sucrose, potassium 45 1- sorbate and sodium benzoate on heat inactivation of c: 0

yeasts are presented in Fig. 2. With the exception of P. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/10/765/1653722/0362-028x-44_10_765.pdf by guest on 27 September 2021 u 30 ::> membranaefaciens heated in YMPG broth containing ""0 Q) --0'--...... Iii 60% sucrose, addition of up to 60% of this solute 0: 0 15 enhanced the tolerance of yeasts to heat. Data for D. 0 ~ A~• E i===='~~8~D-D hansenii and S. cerevisiae heated in nonsupplemented u Q) 0 YMPG compare favorably to those shown in Fig. 1 for 0 cells heated in the same broth. Since z values for test 60 5 cerevts1ae 51° ~~---6 yeasts differ (1), and the temperature of treatment was 45 ~~:;:::..- 0---=~ increased for sucrose studies involving P. membranaefa ciens, C. krnsei, K. apiculata and R. rnbra, it is difficult to compare the effects of solutes at any given aw. 30 /~?·~· However, it appears that sodium chloride at 9 and 12% 15 ~# may exert a detrimental ionic effect on test yeasts which Q?;:-· is greater than the protective effect of reduced aw. In 0~----L---~--~--~ sucrose media at lower aw values than those containing 0 15 30 4 5 60 0 15 30 45 60 high levels of sodium chloride, a continuous trend toward Concentration of Sucrose (%, wt/wt) increased tolerance to heat is observed. Figure 2: The effects of sucrose, potassium sorbate, and sodium Both potassium sorbate and sodium benzoate reduced benzoate on decimal reduction times (/] values) of six yeasts the tolerance of yeasts to heat when present in exposed to elevated temperatures (C). Symbols: 0, no sucrose-supplemented broth. The effects of preservatives potassium sorbate or sodium benzoate added to YMPG heating on C. krnsei were reversed from those noted in medium; !J., 500 ppm of potassium sorbate; D, 1000 ppm of salt-supplemented broth, i.e., in any given sucrose potassium sorbate; •· 500 ppm of sodium benzoate; •· 1000 formulation containing the same concentration of ppm of sodium benzoate. An absence of data points signifies preservatives, potassium sorbate was more lethal than that D values were< 3.5 min. was sodium benzoate. The two preservatives differed little with respect to their effects on D. hansenii, P. aw values at 22 C are presented in Tables 2 and 3. With membranaefaciens, K. apiculata and R. rnbra, whereas the exception of R. rnbra, transfer of cells from one sodium benzoate appeared to be more lethal toward S. medium to the other had no significant effect on viable cerevisiae than was potassium sorbate. populations (P ~ 0.05). A significant reduction in Results from experiments to determine the extent of population of R. rnbra occurred when cells were inactivation of yeasts suspended in media with various suspended in YMPG broth containing 9 and 12%sodium

TABLE 2. Viable populations ofyeasts stored at 22 C in YMPG broth containing sodium chloride.

Mean log10 viable population per ml

After 80 min in YMPG broth Yeast Initial containing NaCl (o/o, wt/wt) 0 3 6 9 12 D. hansenii 5.881 5.79 5.76 5.76 5.78 5.75 P. membranaefaciens 5.96 6.04 6.02 5.92 5.92 5.82 S. cerevisiae 5.82 5.73 5.78 5.77 5.85 5.69 C. krusei 5.99 6.03 6.03 6.06 6.04 5.91 K. apiculata 6.35 6.49 6.49 6.48 6.48 6.43 R. rubra 5.62a 5.66a 5.70a 5.67a 4.50b 3.78c

'Values in the same row not followed by the same letter are significantly different (P ~0.05).

JOURNAL OF FOOD PROTECTION, VOL. 44, OCTOBER 1981 768 BEUCHAT

TABLE 3. Viable populations ofyeasts stored at 22 C in YMPG broth containing sucrose.

Mean log10 viable population per ml

After 80 min in YMPG broth Yeast Initial containing sucrose (o/o, wt/wt) 0 10 30 45 60 D. hansenii 5.71 1 5.71 5.77 5.71 5.68 5.67 P. membranaefaciens 6.08 6.10 6.09 6.10 6.23 5.94 S. cerevisiae 5.81 5.77 5.77 5.77 5.80 5.74 C. krusei 6.09 6.04 6.06 6.05 6.05 6.05 K. apiculata 6.04 6.07 6.07 6.10 6.08 6.08 R. rubra 5.84a 5.72ab 5.70ab 5.59b 4.34c 4.33c

1Values in the same row not followed by the same letter are significantly different (P ~0.05). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/10/765/1653722/0362-028x-44_10_765.pdf by guest on 27 September 2021 chloride (fable 2). Transfer of 46- to 48-h-old cells of R. tion and other constituents in the heating medium. rubra to YMPG broth containing 30o/o sucrose resulted It is noteworthy that P. membranaefaciens, a yeast in a significant reduction in viable population; further with exceptionally high tolerance to sodium chloride in reductions were significant when cells were held in growth media, was, compared to other yeasts examined, YMPG broth containing 45 and 60% sucrose (fable 3). also afforded the greatest protection against inactivation Thus, of the six yeasts examined, R. rubra was most when sodium chloride was present in the heating sensitive to the detrimental effects of osmotic shock and medium. The D 51 value increased from 10.8 min in should be evaluated with respect to heat inactivation in control YMPG broth to 32.2 min in broth containing 6% media with reduced aw with this fact in mind. sodium chloride. D. hansenii is also tolerant to salt in growth media, but was not affected by the same order of magnitude as was P. membranaefaciens with respect to DISCUSSION increased protection by sodium chloride against heat inactivation. However, D. hansenii was the only yeast Observations presented here showing correlations tested which had increased D values with each between decreased aw and increased tolerance of yeasts incremental increase of sodium chloride in the heating to heat confirm reports on other fungal species. Gibson medium. One is tempted to suggest that tolerance of (6) studied the osmophilic yeast, S. rouxii, and found that yeasts to sodium chloride in growth media may be increased sucrose concentration of suspending media positively correlated to the relative response of cells to the resulted in decreased cell volume. She attributed an protective effects of salt against heat inactivation. increased heat resistance exhibited by cells in sucrose As reported in an early publication from our solutions of low aw to dehydration together with a laboratory (1), potassium sorbate and sodium benzoate reduction in the pore size of the cell wall. Corry (2) also act synergistically with heat to inactivate several yeasts noted that sucrose enhanced the heat resistance of S. over a pH range of 2.5 to 5.0 and in some instances at rouxii and S. pombfi . The increased heat resistance of higher pH. Shibasaki and Tsuchido (10) indicated that yeasts in orange concentrates as compared to single· the enhancing effect of sorbic acid on thermal strength juices has been attributed to both sugars and inactivation of C. uti/is was not influenced significantly citric acid (7). Doyle and Marth (4) demonstrated that by factors such as composition of the suspending increased concentrations of sodium chloride up to 16% menstruum. Studies reported herein using sodium (w/v) and sucrose up to 60% (w/w) were correlated with chloride and sucrose as solutes in heating media tend to decreased rates of heat inactivation of conidiospores of confirm this observation. The six yeasts examined Aspergillus .flavus and Aspergillus parasiticus. They showed the same general patterns of rates of inactivation stated that the mechanism of protection afforded by in broth containing potassium sorbate compared to sucrose may be similar to that of sodium chloride. respective sodium chloride and sucrose controls. Similar Shibasaki and Tsuchido (10) demonstrated that sodium observations were made with respect to sodium benzoate. chloride (up to 10%) protected cells of C. uti/is against As noted above, however, we did observe that relative thermal destruction. Data reported here with respect to effectiveness of the two chemical preservatives differed, protection of cells of yeasts against heat inactivation are depending upon the nature of the solute. not in agreement with the latter findings, at least when A mechanism of resistance of Saccharomyces bailii to one compares the effects of high levels of sodium chloride sorbic and benzoic acids has been offered by Warth (11). (9 and 12 %) with those elicited by sucrose at similar aw It was suggested that resistance apparently results values. Perhaps the differences in response of conidio primarily from an inducible, energy-requiring system spores and various strains of yeasts to sodium chloride which transports preservatives from the cells. It is and sucrose at elevated temperatures are in part due to doubtful, however, that uninduced cells like those used differences in cell structure, mechanisms for osmoregula- in the present study possess such a mechanism to afford

JOURNAL OF FOOD PROTECTION, VOL. 44, OCTOBER 1981 HEAT INACTIVATION OF YEASTS 769 protection against the lethal affects of sorbic and benzoic Bacteriol. 40:269-276. acids during heat treatment. 3. Corry, J. E. L. 1976. Sugar and polyol permeability of Salmonella and osmophilic yeast cell membranes measured by turbidimetry, In summary, studies of yeasts possessing a wide range and its relation to heat resistance. J. Appl. Bacteriol. 40:277-284. of physiological and growth characteristics have shown 4. Doyle, M. P., and E. H. Marth. 1975. Thermal inactivation of that all are protected against heat inactivation by sucrose conidia from Aspergillus flavus and Aspergillus parasiticus. II. and, to a lesser extent, by sodium chloride. Sensitivity of Effects of pH and buffers, glucose, sucrose, and sodium chloride. cells to heat can be increased by adding potassium J. Milk Food Techno!. 38:750-758. 5. Duncan, D. B. 1955. Multiple range and multiple F tests. sorbate or sodium benzoate to broth during treatment. Biometrics 11:1-42. Food processors could readily benefit by this practice, 6. Gibson, B. 1973. The effect of high sugar concentrations on the since the time/temperature requirements for pasteuriza heat resistance of vegetative microorganisms. J. Appl. Bacteriol. tion of certain acid food products containing relatively 36:365-376. high levels of sodium chloride or sucrose could be 7. Juven, B. J., J. Kanner, and H. Weisslowicz. 1978. Influence of orange juice composition on the thermal resistance of spoilage reduced, thus reducing the amount of energy required yeasts. J. Food Sci. 43:1074-1076, 1080.

and possibly maintaining more desirable sensory and 8. Pederson, C. S., and D. K. Tressler. 1938. Flash pasteurization of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/10/765/1653722/0362-028x-44_10_765.pdf by guest on 27 September 2021 nutritional qualities. apple juice. Ind. Engineer. Chern. 30:954-959. 9. Robinson, J. F., and C. H. Hills. 1959. Preservation of fruit products by sodium sorbate and mild heat. Food Techno!. REFERENCES 13:251-253. 10. Shibasaki. I., and T. Tsuchido. 1973. Enhancing effect of 1. Beuchat, L. R. 1981. Synergistic effects of potassium sorbate and chemicals on thermal injury of microorganisms. Acta Alimentaria sodium benzoate on thermal inactivation of yeasts. J. Food Sci. 2:327-349. 46:771-777. 11. Warth, A. D.1977. Mechanism of resistance of Saccharomyces 2. Corry, J. E. L. 1976. The effect of sugars and polyols on the heat bailii to benzoic, sorbic, and other weak acids used as food resistance and morphology of osmophilic yeasts. J. Appl. preservatives. J. Appl. Bacteriol. 43:215-230.

JOURNAL OF FOOD PROTECTION. VOL. 44, OCTOBER 1981