Of Inactivation of and Colony Formation by Heated Spores and Vegetative Cells of Molds L
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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. candi- dum, 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 concen- tration, 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 suspen- sions 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.