Of Inactivation of and Colony Formation by Heated Spores and Vegetative Cells of Molds L

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1981, p. 472-477 Vol. 41, No. 2 0099-2240/81/020472-06$02.00/0

Combined Effects of Solutes and Food Preservatives on Rates of Inactivation of and Colony Formation by Heated Spores and Vegetative Cells of Molds L. R. BEUCHAT Department of Food Science, University of Georgia Agricultural Experiment Station, Experiment, Georgia 30212

The combined and independent effects of sucrose, sodium chloride, potassium sorbate, and sodium benzoate on heat inactivation of conidia ofAspergillus flavus and Penicillium puberulum, ascospores of Byssochlamys nivea, and vegetative cells of Geotrichum candidum were studied. In addition, the effects of solutes and preservatives on colony formation by unheated and heated conidia of A. flavus were evaluated. Increased concentrations of sucrose were accompanied by increased tolerance to heat by A. flavus, B. nivea, and G. candidum. Low concentrations (3 and 6%) of sodium chloride protected A. flavus and G. candidum, whereas up to 12% sodium chloride protected B. nivea, but had little effect on the heat stability of P. puberulum. Potassium sorbate and sodium benzoate acted synergistically with heat to inactivate all four molds. At the same concentration, the two preservatives had varied degrees of effectiveness on molds and were influenced by the type of solute in the heating menstrua. Heated conidia of A. flavus had increased sensitivity to preservatives and reduced water activity, whether achieved by the presence of sucrose or sodium chloride, thus demonstrat- ing heat-induced injury. At the same concentration, potassium sorbate was clearly more inhibitory than was sodium benzoate to colony formation by A. flavus, and the presence of sucrose and sodium chloride enhanced this inhibition. The rates of death of mold propagules differ imal a, values vary, depending upon the species at various water activities (a.) and temperatures in question, the pH, redox potential and availa- unfavorable for growth, depending upon the spe- bility of nutrients in recovery media, and tem- cies, type of cell, and factors associated with the perature of incubation (13). Acott and Labuza environment in which cells are suspended. Rates (1) stated that the method of addition of water of death of conidia ofAspergillus flavus at non- to a food as well as the final a, are important in lethal temperatures are more rapid as the a, of determining survival and rates of growth of mi- foods is increased from 0.32 to 0.78 (5). Doyle croorganisms. Chicken cubes (a., 0.79) prepared and Marth (11) reported that an increase in the by a desorption system supported growth of A. amount of sodium chloride, sucrose, or glucose niger, whereas cubes at the same a, prepared in heating media was accompanied by a decrease by an adsorption system did not. The influence in the rate at which conidia of A. flavus and of three solutes, sodium chloride, glycerol, and Aspergillusparasiticus were inactivated. At the a glucose-fructose mixture, on germination and same aw, the three solutes failed to protect co- growth of six xerophilic molds was studied by nidia to an equivalent degree. The rate of ther- Pitt and Hocking (14). Germination times and mal inactivation of ascospores of Byssochlamys growth rates were affected by solute type, thus nivea in grape juice is significantly decreased supporting earlier observations that solute type when sucrose concentration is increased (7). At plays a subordinate but important role to a,, in any given a,, characteristics of the heating men- the growth of molds (13). The type of solute struum, such as pH and type of acid present, can used to achieve a reduced a,, can also affect the influence survival of molds. Shibasaki and rate of growth of nonxerophilic molds (2, 12). Tsuchido (16) reported that the addition of sor- The study reported here was designed to com- bic acid to a heating medium reduced the D- pare the effects of sucrose and sodium chloride value of conidia ofAspergillus niger by 21%, but on rates of heat inactivation of four molds of had no effect on the D-value of conidia of Peni- spoilage and public health concern. A second cillium thomii. objective was to determine whether potassium Germination and outgrowth ofspores ofmolds sorbate and sodium benzoate acted synergisti- are inhibited at reduced a,, values (10, 17). Min- cally with heat to kill various types of mold 472 VOL. 41, 1981 EFFECTS OF SOLUTES AND PRESERVATIVES ON MOLDS 473 propagules and, further, to determine what ef- of 500 and 1,000 ug/ml. Finally, all heating menstrua fects solutes might have on this synergism. were adjusted to pH 4.5 by adding 1 M HCI. Third, experiments were conducted to deter- Just before the thermal inactivation experiments, mine the extent of colony formation by heated supplemented buffer and broth were tempered in a water bath shaker at suitable elevated temperatures, and unheated conidia when plated on recovery depending upon the organisms to be tested. Relative agar supplemented with solutes and food pre- sensitivities to heat had been determined in prelimi- servatives. nary experiments. Inocula (1 ml) of spore suspensions of A. flavus, P. puberulum, and B. nivea were trans- MATERIALS AND METHODS ferred to heated buffers, whereas diluted cell suspensions of G. candidum were transferred to heated Cultural conditions for production oftest cells. YMPG broth. After periods of time ranging to 80 min, Four molds were examined: A. flavus, Penicillium samples of heated cells were withdrawn, serially di- puberulum, B. nivea, and Geotrichum candidum. The luted in phosphate-Tween buffer (A. flavus, P. pub- basal medium (YMPG) used for culturing consisted of erulum, and B. nivea) or 0.1 M phosphate buffer 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, lacking Tween 80 (G. candidum) at 22°C, and plated 10 g of glucose, and 1 liter of deionized water (pH 5.5). in YMPG agar (pH 5.5) by a pour-plate technique. Agar (2%) was added to YMPG broth before sterilizing Colonies were counted after a 3- to 5-day incubation at 121°C for 15 min, pouring into petri dishes, and period at 30°C. Tests were replicated three times allowing to dry at 22°C overnight. Plates were inocu- before calculating decimal reduction times (D-value is lated by spreading with 0.2-ml suspensions of conidia the time at a constant temperature required to reduce of A. flavus or P. puberulum in 0.1 M potassium viable populations by 90%) from portions of inactiva- phosphate buffer (pH 7.0) containing 0.01% Tween 80 tion curves which showed exponential decreases in (phosphate-Tween buffer). Similarly, heat-shocked (1 viable populations over three or more log cycles. h, 70°C) ascospores of B. nivea were inoculated onto Effects of solutes and preservatives on colony YMPG agar. A. flavus and P. puberulum were incu- formation. Conidial suspensions (1 ml) of A. flavus bated for 20 days at 25°C (80% relative humidity), and were inoculated into heat-tempered phosphate-Tween B. nivea was incubated for 35 days at 30°C (60% buffer (pH 4.5). After 20 min, suspensions were serially relative humidity). G. candidum was cultured in diluted in phosphate-Tween buffer, and 0.1-ml quan- YMPG broth; 100 ml of broth was dispensed into 250- on solidified YMPG ml Erlenmeyer flasks, sterilized, cooled, and inocu- tities were spread agar (pH 4.5) lated. The organism was prepared for testing by suc- supplemented with 0, 10, 30, or 60% sucrose, 5 or 10% cessively transferring 2-day-old cultures which had sodium chloride, and 500 or 1,000 ug of potassium been incubated at 30°C while being continuously agi- sorbate or sodium benzoate per ml. Plates were incu- tated (150 rpm) on a rotary shaker. bated at 25°C, and visual observations for colony Procedure for harvesting cells. Conidia of A. formation were made at 1- to 3-day intervals for a flavus and P. puberulum and ascospores of B. nivea period of 10 days. were harvested by a procedure which minimized the RESULTS AND DISCUSSION amount of mycelium removed from the cultures. The on plates were flooded with phosphate-Tween buffer and Effects of solutes and preservatives gently rubbed with a sterile bent glass rod. Spore thermal inactivation. The D-values for four suspensions were then removed and filtered through test molds as influenced by independent and sterile glass wool. The washing and filtering proce- combined effects of sucrose, potassium sorbate, dures were repeated a minimum of three times. Stock and sodium benzoate are presented in Fig. 1. suspensions of spores were held at 4°C for a maximum Increased sucrose concentrations to 10, 30, 45, of 12 (P. puberulum), 27 (A. flavus), and 38 (B. nivea) and 60% were accompanied by decreased a. days before subjecting to test conditions. values to 0.99, 0.98, 0.95, and 0.89 (+0.005), re- Suspensions (46 to 48 h old) of vegetative cells of G. spectively. As a result of this reduction in a., candidum cultured in YMPG broth were diluted 100- conidia of A. flavus and ascospores of B. nivea fold in 0.1 M potassium phosphate buffer (pH 7.0) to before being used as inocula for testing tolerance to had increased tolerance heat. The resistance heat. of vegetative cells of G. candidum to heat was Effects of solutes and preservatives on ther- increased as the sucrose concentration ofheating mal inactivation. Sucrose and sodium chloride were media was increased from 0 to 45%. The effects added separately to 0.1 M phosphate-Tween buffer of heat on rates of inactivation of P. puberulum, (pH 7.0) at concentrations of 10, 30, 45, and 60% (wt/ however, did not appear to be influenced by wt) and 3, 6, 9, and 12% (wt/wt), respectively, before sucrose at any of the concentrations tested. The sterilization of 100-ml quantities in 250-ml Erlenmeyer data for A. flavus confnrm those reported by flasks. Similarly, solutes were added to YMPG broth Doyle and Marth (11). These researchers sug- (pH 7.0) before sterilizing. For tests involving preserv- that increased thermal resistance of co- atives, 1 ml of sterile 5 or 10% solutions of potassium gested sorbate (Monsanto Industrial Chemicals Co., St. nidia in the presence of sucrose may be largely Louis, Mo.) and sodium benzoate (Pfizer Inc., New a result of dehydration, which confers greater York, N.Y.) were added to cooled, solute-supple- stability on protein and possibly reduces loss of mented buffer and broth to give final concentrations spore components. 474 BEUCHAT APPL. ENVIRON. MICROBIOL.

60 A. flovus, 52° - P. puberulum, 490

C 45 E 30 0) E *I - " ._ 15 _

c I I I I 0 4- U B. niveo,80 10 0 _H E 0 0

I I I I I 0 15 30 45 60 0 15 30 45 60 Concentration of Sucrose (, wt/wt) FIG. 1. Effects of sucrose, potassium sorbate, and sodium benzoate on rates of heat inactivation of molds. Symbols: 0, no preservative in heating medium; A, 500 pg ofpotassium sorbateper ml; [, 1,000 iLg ofpotassium sorbate per ml; A, 500 jig of sodium benzoate per ml; , 1,000 jig of sodium benzoate per ml.

Regardless of the sucrose concentration of tivation of ascospores of B. nivea was increased heating media, the presence of potassium sor- with each incremental increase of sodium chlo- bate or sodium benzoate reduced the D-values ride. Survival of conidia of A. flavus was en- of all molds tested. Preservatives were more hanced in buffer containing up to 6% sodium effective at 1,000 than at 500 jig/ml; however, chloride, whereas only a 3% concentration fa- one preservative was not superior to the other vored G. candidum. The solute imparted no for reducing D-values of all molds. At the same protection to conidia of P. puberulum. Elevated concentration, sodium benzoate was more det- concentrations of sodium chloride were clearly rimental to P. puberulum and G. candidum than detrimental to the survival of A. flavus and G. was potassium sorbate, whereas little if any dif- candidum. ference was noted between the effects of the two As noted in sucrose studies, potassium sorbate preservatives on the extent of reduction of D- and sodium benzoate acted synergistically with values forA. flavus and B. nivea. The synergistic heat to increase the rate of thermal inactivation effects of preservatives with heat on rates of in control (no solute) menstrua. Data from ex- inactivation of A. flavus and perhaps B. nivea periments not reported here indicate that colony appeared to be retarded as sucrose concentration formation by A. flavus and P. puberulum in agar was increased, whereas effects on reducing D- containing up to 1,000 jig of these preservatives values of P. puberulum were enhanced as su- per ml is not retarded. Thus, reductions in D- crose concentration was increased. values of these molds, at least, are truly due to Results from experiments to determine the the combined effects of preservatives and heat. effects of sodium chloride and preservatives on At the same concentration, sodium benzoate rates of heat inactivation of molds are presented had a more pronounced effect than did potas- in Fig. 2. Concentrations of 3, 6, 9, and 12% sium sorbate on inactivating propagules of A. sodium chloride in heating media resulted in a. flavus, B. nivea, and G. candidum at elevated values of 0.99, 0.97, 0.95, and 0.93 (+0.005), re- temperatures. Potassium sorbate, on the other spectively. Responses of molds were somewhat hand, was more effective than sodium benzoate different than those observed in studies of the in reducing D-values of P. puberulum. Contrary effects of sucrose. Protection against heat inac- to the retarding effects of increased sucrose in VOL. 41, 1981 EFFECTS OF SOLUTES AND PRESERVATIVES ON MOLDS 475 the action of preservatives against A. flavus, Effects of solutes and preservatives on elevated amounts of sodium chloride appeared colony formation. Results from experiments to enhance the effects of sodium benzoate. The to determine the rate and extent of colony for- effects of increased sodium chloride on the effec- mation by unheated and heated conidia of A. tiveness of preservatives against B. nivea are flavus on recovery agar supplemented with su- comparable to those noted for sucrose. crose and preservatives are presented in Fig. 3. Observations from studies on A. flavus heated The time required for colony formation was in the presence of sodium chloride differ from increased as the concentration of sucrose was those reported by Doyle and Marth (11). They increased, regardless of pretreatment of conidia. noted that decreased a, values to 0.90, achieved In YMPG agar containing 30 and 45% sucrose, by adding sodium chloride to media, were ac- the rates of colony formation by heated conidia companied by increased heat stability of conidia were retarded to a greater degree compared with of A. flavus and A. parasiticus. In the present rates of colony formation by unheated conidia. study, a. values of c0.95 had a detrimental The number of colonies formed by unheated effect on the viability of conidia of A. flavus. G. conidia at the end of the 10-day observation candidum was adversely affected in salt-supple- period was essentially the same on all recovery mented broth with an a. of c0.97. Thus, an media not containing preservatives. The same overall evaluation ofdata reveals that sensitivity statement can be made concerning heated co- to heat at reduced a. depends upon the type of nidia, although the total population was initially propagule as well as the genus of mold. Further- reduced during heat treatment. more, although reduced a. can act to protect The decreased rate at which colonies were cells against heat inactivation, solutes used to formed by heated conidia indicates that a por- achieve any given a. may differ with regard to tion of these cells had undergone injury or stress the extent of protection afforded. Above some as a result of heat treatment. Demonstration of optimum concentration of solute, i.e., below injury of conidia has been made by other re- some optimum a,,,, solutes may actually ad- searchers (8, 9). Adams and Ordal (3) reported versely affect the survival of molds at elevated that colonies from heated (injured) conidia ofA. temperatures. parasiticus developed more slowly than did col- 60 -A . f1 v us,, 520 P. puberulum, 49°

C -- 45 0"-- E :.We .0 30 _ 0 30 0 E _

Il I I I ._0 0 J O 60 G. candidum,52° nivea, 800 0 W. 451 _ ! 0 30 - 0o E \~ 0,l | | I I I I 0 0

0 15

I a 1 I I 0 0 3 6 9 12 0 3 6 9 12 Concentration of NaCI (%, wt/wt) FIG. 2. Effects of sodium chloride, potassium sorbate, and sodium benzoate on rates of heat inactivation of molds. Symbols: 0, no preservative in heating medium; A, 500 ,ug ofpotassium sorbate per ml; a1 1,000 jg ofpotassium sorbate per ml; A, 500 pug of sodium benzoate per ml; l, 1,000 pug of sodium benzoate per ml. 476 BEUCHAT APPL. ENVIRON. MICROBIOL. Unheated (520,20 min) flavus were subject to reversible injury upon IHeated exposure to heat. No sucrose added Potassium sorbate, at the same concentration 6 in sucrose-supplemented agar, was clearly more 0-1 m 5 inhibitory than was sodium benzoate to colony )9 formation by A. flavus, regardless of treatment 4 L 0_o_o - of conidia. Inhibition was more pronounced as .O ...... concentration was increased, e.g., no 3 _ the sucrose colonies were formed by unheated conidia on / / 2 /0_ .l' YMPG agar containing 30 or 45% sucrose and I I I 1 I I I I I 1,000 ,ug of potassium sorbate per ml. Heated conidia failed to form colonies on all recovery 6 10% Sticrose agars containing 1,000 jLg of potassium sorbate per ml. Thus, the inhibitory action of potassium 5 sorbate is enhanced both by high sucrose concentration and the stressed disposition ofconidia as induced by heat. This is in agreement with previous observations on mold spores indicating 0. B. are more 2 that heated ascospores of nivea J sensitive to potassium sorbate than to sodium I I I I1I 1I I 1I 0oi 4 benzoate (4) and that heated conidia ofA. flavus had increased sensitivity to potassium sorbate 30% S ucrose (25 ,ug/ml) compared with sodium benzoate (150 0 ,tg/ml) (6). Przybylski and Bullerman (15) re- in 0 ported that a decrease adenosine triphosphate C- 4 level in conidia of A. parasiticus was related to 0 decreased viability after exposure to sorbic acid. -J 3 Shown in Fig. 4 are the effects of sodium 2 chloride on colony formation by unheated and I I I I I heated conidia of A. flavus. Data from the control recovery agar are plotted in Fig. 3 (no su- 45% Stucrose crose added). Compared with sucrose studies at equivalent a, values, sodium chloride more se- verely retarded colony development by A. flavus. The effects of preservatives were also mag- nified in salt-supplemented agar. Colonies were not formed on 5 or 10% salt-supplemented agar containing potassium sorbate, regardless of pretreatment of conidia, and sodium benzoate com- pletely inhibited colony formation in 10% salt 0 2 4 6 10 0 2 4 6 10 agar. Considering the 5% salt agar and compar- Time of Incubation (doys) ing data with those obtained by using nonsup- FIG. 3. Effects of sucrose potassium sorbate, and plemented agar, it can be concluded that conidia sodium benzoate on colony formation by unheated underwent heat injury within 20 min at 52°C and heated conidia of A. flavus. Symbols: 0, no which resulted in increased sensitivity to sodium preservative in recovery medium; A, 500 pg ofpotas- chloride, potassium sorbate, and sodium ben- sium sorbate per ml; [:, 1,000X g ofpotassium sorbate zoate. per ml; A, 500 Mg of sodium benzoate per ml; 1,000 of sodium benzoate per ml. Observations on relative sensitivities of molds jig heated at reduced a, in the presence of preservatives are pertinent to the design of thermal onies from unheated conidia recovered on a me- processing schemes for foods and food ingredi- dium supplemented with 10% sodium chloride. ents. Depending upon the nature of the solute Cytoplasmic membrane damage was shown to and a,, the presence of preservatives during be heat induced, allowing the leakage of nucleic processing could contribute to the lethality of acid and proteinaceous materials into the heat- molds to an extent which might permit a reducing menstruum. Beuchat and Jones (6) used tion in the time-temperature treatment without sodium chloride-supplemented recovery agar to sacrificing any degree of sterility. Pilot plant demonstrate that conidia of another strain ofA. studies involving specific food items to evaluate VOL. 41, 1981 EFFECTS OF SOLUTES AND PRESERVATIVES ON MOLDS 477 Unheated min) Sci. 41:541-546. IHeated(520,20 3. Adams, G. H., and Z. J. Ordal. 1976. Effects of thermal stress and reduced water activity on conidia of Asper- 6 5% NaCl gillusparasiticus. J. Food Sci. 41:547-550. 4. Beuchat, L. R. 1976. Effectiveness of various food pre- 5 servatives in controlling the outgrowth ofByssochlamys nivea ascospores. Mycopathologia 59:175-178. E 4 -8 5. Beuchat, L. R. 1979. Survival of conidia of Aspergillus flavus in dried foods. J. Stored Prod. Res. 15:25-31. s1053 6. Beuchat, L. R., and W. K. Jones. 1978. Effects of food a- preservatives and antioxidants on colony formation by c 2 O- Q1 1 heated conidia of Aspergillus flavus. Acta Aliment. 0 Ia--I--II L.Zi_iZZ_ Acad. Sci. Hung. 7:373-384. 0. 1JI *./1I I I I I I I 7. Beuchat, L. R., and R. T. Toledo. 1977. Behaviour of Byssochlamys nivea ascospores in fruit syrups. Trans. o6I 10% NoCI Brit. Mycol. Soc. 68:65-71. O 6 8. Charlang, G., and N. H. Horowitz. 1974. a- -0 Membrane o7o- permeability and the loss of germination factor from 5 0 Neurospora crassa at low water activities. J. Bacteriol. C04 117:261-264. 0 9. Charlang, G. W., and N. H. Horowitz. 1971. Germina- -J tion and growth of Neurospora at low water activities. 3 _ /0__O o Proc. Natl. Acad. Sci. U.S.A. 68:260-262. 10. Corry, J. E. L. 1978. Relationships of water activity to 2 fungal growth, p. 45-83. In L. R. Beuchat (ed.), Food and beverage mycology. Avi Publishing Co., Inc., West- 0 2 4 6 8 lo o 2 4 6 8 l0 port, Conn. Time of Incubation 11. Doyle, M. P., and E. H. Marth. 1975. Thernal inacti- (days) vation of conidia from Aspergillus flavus and Aspergil- FIG. 4. Effects ofsodium chloride and sodium ben- lus parasiticus. II. Effects of pH and buffers, glucose, zoate on colony formation by unheated and heated sucrose, and sodium chloride. J. Milk Food Technol. conidia ofA. flavus. Symbols: 0, no sodium benzoate 38:750-758. 12. Horner, K. J., and G. D. Anagnostopoulos. 1973. Com- in recovery medium; A, 500 ofsodium benzoate per pg bined effects of water activity, pH and temperature on ml; 1,00X pg of sodium benzoate per ml. the growth and spoilage potential of fungi. J. Appl. Bacteriol. 36:427-436. 13. Pitt, J. I. 1975. Xerophilic fungi and the spoilage of foods the combined effects ofsolutes and preservatives of plant origin, p. 273-307. In R. B. Duckworth (ed.), Water relations of foods. Academic Press, Inc., New on heat inactivation of molds should be con- York. ducted before recommendations to the food in- 14. Pitt, J. I., and A. D. Hocking. 1977. Influence of solute dustry can be made. and hydrogen ion concentration on the water relations of some xerophilic fungi. J. Gen. Microbiol. 101:35-40. 15. Przybylski, K. S., and L. B. Bullerman. 1980. Influence LITERATURE CITED of sorbic acid on viability and ATP content of conidia 1. Acott, K. M., and T. P. Labuza. 1975. Microbial growth ofAspergillusparasiticus. J. Food Sci. 45:375-376,385. response to water sorption preparation. J. Food Tech- 16. Shibasaki, I., and T. Tsuchido. 1973. Enhancing effect nol. 10:603-611. of chemicals on the thermal injury of microorganisms. 2. Acott, K., A. E. Sloan, and T. P. Labuza. 1976. Evalu- Acta Aliment. Acad. Sci. Hung. 2:327-349. ation of antimicrobial agents in a microbial challenge 17. Snow, D. 1949. The germination of mould spores at study for an intermediate moisture dog food. J. Food controlled humidities. Ann. Appl. Biol. 36:1-13.