APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1990, p. 2951-2956 Vol. 56, No. 10 0099-2240t90/102951-06$02.00/0 Copyright (© 1990, American Society for Microbiology Influence of Water Activity and Temperature on Survival of and Colony Formation by Heat-Stressed Chrysosporium farinicola Aleuriospores L. R. BEUCHATt* AND J. I. PITT Division of Food Processing, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales 2113, Australia Received 12 March 1990/Accepted 26 May 1990 The ability of sublethally heat-stressed aleuriospores of Chrysosporium farinicola to form colonies on yeast extract-glucose agar (YGA) supplemented with sufficient glucose, sorbitol, glycerol, and NaCl to achieve reduced water activity (a,) in the range of 0.88 to 0.95 was determined. The effects of the aw of diluent and incubation temperature during recovery and colony formation were also investigated. Aleuriospores harvested from 14-day-old cultures grown at 25°C were less resistant to heat inactivation compared with aleuriospores from 20-day-cultures. Increased populations of heat-stressed aleuriospores were recovered as the aw of YGA was decreased from 0.95 (glucose and glycerol) and 0.94 (sorbitol) to 0.89 and 0.88, respectively. In NaCl-supplemented YGA, populations recovered at an aw of 0.94 were greatly reduced compared with populations detected at an a, of 0.92; no colonies were formed on NaCI-supplemented YGA at an aw of 0.88. Tolerance to aw values above 0.88 to 0.89 as influenced by solute type was in the order of glucose > sorbitol > glycerol > NaCl. Incubation at 20°C generally resulted in an increase in recoverable aleuriospores compared with incubation at 25°C or at 30°C for 14 days followed by 20°C for 10 days. The lethal effect of NaCl on heat-stressed aleuriospores was enhanced at 30°C. The retention of viability of aleuriospores held in sucrose-peptone water diluent (aw, 0.936) for 20 min was essentially the same as that observed when aleuriospores were held in peptone water (a,, 0.997). Considering the sensitivity of heat-stressed and healthy aleuriospores to various solutes, especially at reduced aw values at which nonxerophiles cannot grow, as well as colony size as affected by the type of solute and a,, sorbitol may be the solute of choice for incorporating into media to detect and enumerate C. farinicola in foods. Xerophilic fungi are present on a wide variety of food- Reported here are the results of studies to determine stuffs, although spoilage by this group of microorganisms whether aleuriospores of C. farinicola undergo sublethal tolerant of low water activity (aJ) is most commonly asso- injury upon exposure to heat treatment and to determine the ciated with intermediate-moisture fruits, fish and meats, response of heat-stressed aleuriospores to various solutes confectionery products, jams and jellies, syrups, nuts, and used to adjust the a, of enumeration media. The effects of aw cereal grains (13). Standard methods for analyzing foods for and holding time in diluent as well as the temperature used to yeasts and molds are inadequate for detecting or enumerat- incubate enumeration media were also investigated. ing xerophiles because some genera, such as Chrysos- porium, will not grow at the high a, values of the media MATERIALS AND METHODS employed. The species studies here, Chyrsosporium farini- Mold. C. farinicola Burnside (Skou) FRR 377, obtained been on dried prunes (14), honey (16), and cola, has found from the culture collection of the Commonwealth Scientific It can at values as low as desiccated coconut (10). grow a, and Industrial Research Organisation, Division of Food 0.69 (14) and does not grow well at values above 0.95. a, Processing, North Ryde, New South Wales, Australia, was Clearly, the a, values of media such as acidified potato used in all experiments. The fungus was isolated from dextrose agar, antibiotic-supplemented plate count agar, and unspoiled prunes by J. I. Pitt in Young, New South Wales, rose agar are too high to dichloran bengal chloramphenicol Australia, in 1969. support the growth of this or other xerophilic species (13). Preparation of aleuriospores used as test inocula. C. farin- The susceptibility of nonxerophilic fungal spores and icola was grown on a modification of yeast extract-glucose upon exposure to physical vegetative cells to sublethal injury The modified medium 0.92) and chemical stress is well documented (4). However, the agar (YGA, pH 6.2) (6). (aw, condi- consisted of 10.0 g of yeast extract, 350 g of glucose behavior of xerophilic mold spores exposed to such 15.0 of and 640 ml of tions is essentially unknown. New media and methods for monohydrate, 4.0 g of K2PO4, g agar, distilled water. The ingredients were dissolved in water by enumerating xerophilic molds in foods are still needed, and steaming for 30 min. After 24 h at 23°C, the medium was such media and methods must be formulated to resuscitate the cells. again steamed for 30 min; this was followed by allowing injured medium to again set at 23°C for 24 h and steaming it for 30 min before cooling it to 48 to 50°C and pouring it (20 ml) into * 90-mm-diameter petri dishes. Corresponding author. 14- t Present address: Department of Food Science and Technology, An aleuriospore suspension was prepared by flooding University of Georgia, Agricultural Experiment Station, Griffin, GA to 20-day-old cultures of C. farinicola grown at 25°C on 30223-1797. modified YGA with a sterile 0.1% peptone solution. Aleuri- 2951 2952 BEUCHAT AND PITT APPL. ENVIRON. MICROBIOL. ospores were dislodged by gently rubbing the surface of the 5 culture with a sterile, bent glass rod. These suspensions served as inocula for all studies involving heat treatment. For viability and injury studies using corn starch as a storage medium, aleuriospores were transferred directly from a 16-day-old culture surface to the starch without the use of E :D peptone water. Ui- Procedure for determining sensitivity to heat. The survival 4 of C. farinicola aleuriospores in 0.1% peptone when heated 0 at 48, 50, 52, 54, and 56°C for 10 min was determined. The 0 aleuriospore inoculum (0.5 ml) harvested from 14- and \ -{}- 20 days 20-day-old cultures was mixed with 4.5 ml of peptone water ---- 14 days adjusted to the desired heating temperature in a water bath. The suspension, contained in 13- by 100-mm test tubes, was positioned in the water bath such that its surface was at least 3 2 cm below the level of the constantly circulating water. 48 50 52 54 56 Samples were withdrawn after 10 min of heat treatment, immediately diluted in 0.1% peptone water, and surface Temperature (°C) plated (0.1 ml) in duplicate on glucose-supplemented YGA FIG. 1. Survival of C. farinicola aleuriospores heated in 0.1% (aw, 0.92). Unheated aleuriospores were likewise plated on peptone for 10 min. Aleuriospores were harvested from 14- and glucose-supplemented YGA. Colonies were counted after 12 20-day-old cultures. days of incubation at 25°C. Recovery of heat-stressed aleuriospores on YGA containing serially diluted, and surface plated on glucose-supplemented various solutes. Peptone water suspensions of aleuriospores YGA values and were were heated at (aw of 0.89, 0.92, 0.95). Colonies from 20-day-old cultures 52°C for 0, 10, 20, counted after 12 to 14 days of incubation at 25°C. 30, and 40 min as described above. Serial dilutions (0.1 ml) Measurement of aw. The aw values of all media and were surface plated in duplicate on YGA supplemented with diluents were determined with a Sina-scope instrument glucose, sorbitol, glycerol, or NaCl to yield a, values (Sina, at All to 0.95 6.2 to were Zurich, Switzerland) 25°C. data reported repre- ranging from 0.88 (pH 6.9). Colonies sent the means of values from at least two replicate experi- counted after 12 to 14 days of incubation at 25°C. ments performed in The diameters of colonies formed on YGA containing duplicate. various concentrations of solutes were measured after 12 days of incubation at 25°C. RESULTS AND DISCUSSION Influence of incubation temperature on recovery of heat- Sensitivity of heat-stressed aleuriospores to solutes. The stressed aleuriospores. Aleuriospores from a 20-day-old cul- thermal sensitivity of C. farinicola aleuriospores harvested ture were heated in 0.1% peptone water at 52°C for 30 min from 14- and 20-day-old cultures is illustrated in Fig. 1. and surface plated on YGA containing various concentra- Initial viable populations of aleuriospores before exposure to tions of glucose, sorbitol, glycerol, or NaCl (aw, 0.88 to 10-min heat treatments were 1.05 x 105 CFU/ml (14-day 0.95). Plates were incubated at 20, 25, and 30°C, and colonies culture) and 9.60 x 104 CFU/ml (20-day culture). Aleurios- were counted after 12 days. pores from the older culture were clearly more resistant to Effect of diluent on heat-stressed aleuriospores. Suspen- heat inactivation. This difference was magnified as the sions of aleuriospores harvested from 14-day-old cultures treatment temperature increased from 48 to 56°C. Changes in were heated at 52°C for 30 min as described above before the resistance of fungal spores to heat inactivation as af- transferring 0.1 ml of the suspension to 9.9 ml of sterile 0.1% fected by age have been reported by others. Conidia of peptone water supplemented with 0, 20, and 40% sucrose to Aspergillus flavus and Aspergillus parasiticus appear to yield a, values of 0.997, 0.964, and 0.936, respectively.