Influence of Water Activity and Temperature on Survival of and Colony Formation by Heat-Stressed Chrysosporium farinicola Aleuriospores

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Oct. 199, p t9/1291-6$2./ Copyright ( 199, American Society for Microbiology Vol. 6, No. 1 Influence of Water Activity and Temperature on Survival of and Colony Formation by Heat-Stressed Chrysosporium farinicola Aleuriospores L. R. BUCHATt* AND J. I. PITT Division of Food Processing, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales 2113, Australia Received 12 March 199/Accepted 26 May 199 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.88 to.9 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 2 C were less resistant to heat inactivation compared with aleuriospores from 2-day-cultures. Increased populations of heat-stressed aleuriospores were recovered as the aw of YGA was decreased from.9 (glucose and glycerol) and.94 (sorbitol) to.89 and.88, respectively. In NaCl-supplemented YGA, populations recovered at an aw of.94 were greatly reduced compared with populations detected at an a, of.92; no colonies were formed on NaCI-supplemented YGA at an aw of.88. Tolerance to aw values above.88 to.89 as influenced by solute type was in the order of glucose > sorbitol > glycerol > NaCl. Incubation at 2 C generally resulted in an increase in recoverable aleuriospores compared with incubation at 2 C or at 3 C for 14 days followed by 2 C for 1 days. The lethal effect of NaCl on heat-stressed aleuriospores was enhanced at 3 C. The retention of viability of aleuriospores held in sucrose-peptone water diluent (aw,.936) for 2 min was essentially the same as that observed when aleuriospores were held in peptone water (a,,.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 foodstuffs, although spoilage by this group of microorganisms tolerant of low water activity (aj) is most commonly associated with intermediate-moisture fruits, fish and meats, confectionery products, jams and jellies, syrups, nuts, and cereal grains (13). Standard methods for analyzing foods for yeasts and molds are inadequate for detecting or enumerating xerophiles because some genera, such as Chrysosporium, will not grow at the high a, values of the media employed. The species studies here, Chyrsosporium farinicola, has been found on dried prunes (14), honey (16), and desiccated coconut (1). It can grow at a, values as low as.69 (14) and does not grow well at a, values above.9. Clearly, the a, values of media such as acidified potato dextrose agar, antibiotic-supplemented plate count agar, and dichloran rose bengal chloramphenicol agar are too high to support the growth of this or other xerophilic species (13). The susceptibility of nonxerophilic fungal spores and vegetative cells to sublethal injury upon exposure to physical and chemical stress is well documented (4). However, the behavior of xerophilic mold spores exposed to such conditions is essentially unknown. New media and methods for enumerating xerophilic molds in foods are still needed, and such media and methods must be formulated to resuscitate injured cells. * Corresponding author. t Present address: Department of Food Science and Technology, University of Georgia, Agricultural xperiment Station, Griffin, GA Reported here are the results of studies to determine whether aleuriospores of C. farinicola undergo sublethal injury upon exposure to heat treatment and to determine the response of heat-stressed aleuriospores to various solutes used to adjust the a, of enumeration media. The effects of aw and holding time in diluent as well as the temperature used to incubate enumeration media were also investigated. MATRIALS AND MTHODS Mold. C. farinicola Burnside (Skou) FRR 377, obtained from the culture collection of the Commonwealth Scientific and Industrial Research Organisation, Division of Food Processing, North Ryde, New South Wales, Australia, was used in all experiments. The fungus was isolated from unspoiled prunes by J. I. Pitt in Young, New South Wales, Australia, in Preparation of aleuriospores used as test inocula. C. farinicola was grown on a modification of yeast extract-glucose agar (YGA, ph 6.2) (6). The modified medium (aw,.92) consisted of 1. g of yeast extract, 3 g of glucose monohydrate, 4. g of K2PO4, 1. g of agar, and 64 ml of distilled water. The ingredients were dissolved in water by steaming for 3 min. After 24 h at 23 C, the medium was again steamed for 3 min; this was followed by allowing the medium to again set at 23 C for 24 h and steaming it for 3 min before cooling it to 48 to C and pouring it (2 ml) into 9-mm-diameter petri dishes. An aleuriospore suspension was prepared by flooding 14- to 2-day-old cultures of C. farinicola grown at 2 C on modified YGA with a sterile.1% peptone solution. Aleuri-

2 292 BUCHAT AND PITT ospores were dislodged by gently rubbing the surface of the 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 peptone water. Procedure for determining sensitivity to heat. The survival of C. farinicola aleuriospores in.1% peptone when heated at 48,, 2, 4, and 6 C for 1 min was determined. The aleuriospore inoculum (. ml) harvested from 14- and 2-day-old cultures was mixed with 4. ml of peptone water adjusted to the desired heating temperature in a water bath. The suspension, contained in 13- by 1-mm test tubes, was positioned in the water bath such that its surface was at least 2 cm below the level of the constantly circulating water. Samples were withdrawn after 1 min of heat treatment, immediately diluted in.1% peptone water, and surface plated (.1 ml) in duplicate on glucose-supplemented YGA (aw,.92). Unheated aleuriospores were likewise plated on glucose-supplemented YGA. Colonies were counted after 12 days of incubation at 2 C. Recovery of heat-stressed aleuriospores on YGA containing various solutes. Peptone water suspensions of aleuriospores from 2-day-old cultures were heated at 2 C for, 1, 2, 3, and 4 min as described above. Serial dilutions (.1 ml) were surface plated in duplicate on YGA supplemented with glucose, sorbitol, glycerol, or NaCl to yield a, values ranging from.88 to.9 (ph 6.2 to 6.9). Colonies were counted after 12 to 14 days of incubation at 2 C. The diameters of colonies formed on YGA containing various concentrations of solutes were measured after 12 days of incubation at 2 C. Influence of incubation temperature on recovery of heatstressed aleuriospores. Aleuriospores from a 2-day-old culture were heated in.1% peptone water at 2 C for 3 min and surface plated on YGA containing various concentrations of glucose, sorbitol, glycerol, or NaCl (aw,.88 to.9). Plates were incubated at 2, 2, and 3 C, and colonies were counted after 12 days. ffect of diluent on heat-stressed aleuriospores. Suspensions of aleuriospores harvested from 14-day-old cultures were heated at 2 C for 3 min as described above before transferring.1 ml of the suspension to 9.9 ml of sterile.1% peptone water supplemented with, 2, and 4% sucrose to yield a, values of.997,.964, and.936, respectively. Suspensions were then either diluted immediately in.1% peptone or held for 2 min at 23 C before being diluted and surface plated on various YGA media containing glucose (a, values of.89,.92, and.9) and NaCl (a, values of.88,.92, and.94). Colonies were counted after 14 days of incubation at 2 C. Survival of aleuriospores in cornstarch. Commercial cornstarch was equilibrated to aw values of.43 and.71 at 2 C by placing 1 g in sealed desiccators containing vessels of saturated potassium carbonate and strontium chloride, respectively, for 17 days. Inoculation with C. farinicola aleuriospores was achieved by inverting plates containing 16- day-old cultures grown on glucose-supplemented YGA (aw,.9; 2 C) directly over the starch and tapping the bottoms gently. After thorough mixing, portions of starch at each a¾ were hermetically sealed in glass jars and stored at 1 and 2 C. Viable aleuriospores were enumerated on the day of inoculation after 9, 2, and 4 days of storage. Duplicate samples (. g) were combined with 4 ml of.1% peptone water in 2-ml pharmaceutical bottles, shaken vigorously, :D Ui- \ 4 3 -{}- 2 days days Temperature ( C) FIG. 1. Survival of C. farinicola aleuriospores heated in.1% peptone for 1 min. Aleuriospores were harvested from 14- and 2-day-old cultures. serially diluted, and surface plated on glucose-supplemented YGA (aw values of.89,.92, and.9). Colonies were counted after 12 to 14 days of incubation at 2 C. Measurement of aw. The aw values of all media and diluents were determined with a Sina-scope instrument (Sina, Zurich, Switzerland) at 2 C. All data reported represent the means of values from at least two replicate experiments performed in duplicate. RSULTS AND DISCUSSION Sensitivity of heat-stressed aleuriospores to solutes. The thermal sensitivity of C. farinicola aleuriospores harvested from 14- and 2-day-old cultures is illustrated in Fig. 1. Initial viable populations of aleuriospores before exposure to 1-min heat treatments were 1. x 1 CFU/ml (14-day culture) and 9.6 x 14 CFU/ml (2-day culture). Aleuriospores from the older culture were clearly more resistant to heat inactivation. This difference was magnified as the treatment temperature increased from 48 to 6 C. Changes in the resistance of fungal spores to heat inactivation as affected by age have been reported by others. Conidia of Aspergillus flavus and Aspergillus parasiticus appear to reach a maximum tolerance to heat and then revert to a more sensitive state as aging progresses (8). Ascospores of Neosartorya fischeri (7) and Talaromyces flavus (), on the other hand, were shown to develop increased heat resistance over 114- and 8-day test periods, respectively. The degree of maturity of various types of fungal spores, then, influences their sensitivity to heat, and aleuriospores of C. farinicola are no exception. The recovery of heat-treated C. farinicola aleuriospores as influenced by a, and type of solute is shown in Fig. 2. Recovery increased as the a, APPL. NVIRON. MICROBIOL. of YGA was decreased from.9 (glucose and glycerol) and.94 (sorbitol) to.89 and.88, respectively. In NaCI-supplemented YGA, populations recovered at an aw of.94 were greatly reduced compared with populations detected at an aw of.92; no colonies were formed on YGA at an aw of.88. Tolerance to aw values above.88 to.89 as influenced by solute type was in the order of glucose > sorbitol > glycerol > NaCl. The response of heat-stressed aleuriospores to glucose, sorbitol, and glycerol in recovery media at a¾ values less than.88 was not determined. Thus, the optimum

3 VOL. 6, 199 INFLUNCS ON SURVIVAL OF C. FARINICOLA ALURIOSPORS 293 SORBITOL D U- 6 -j 4 I I o GLYCROL -A O.9 \~~~~~~~~~~~~~~~~~P NaCI Time (min) FIG. 2. Recovery of C. farinicola aleuriospores heated at 2 C and plated on YGA supplemented with glucose, sorbitol, glycerol, and NaCl. a, for recovery and colony formation on YGA containing these solutes cannot be concluded from this study. Increased sensitivity of heat-stressed fungal spores to a, values departing from the optimum for germination and growth has been demonstrated by other researchers. Adams and Ordal (1) observed that when sucrose, glycerol, and NaCl were used to reduce the a, of media for recovering heat-stressed A. parasiticus conidia, viability was reduced by NaCl and least by sucrose. Likewise, heat-stressed A. flavus conidia are more sensitive to sucrose than to NaCl (2). The sensitivity of xerophilic molds, including other Chrysosporium species, to NaCl has been reported. Unstressed spores of Chrysosporiumfastidium were markedly intolerant to NaCl (1) and glycerol (9). The use of NaCl to reduce the a, of media used to detect or enumerate Chrysosporium species in foods, particularly if the spores are sublethally impaired by heat treatment, should not be considered. ffect of solute on colony development. Illustrated in Fig. 3 are the mean diameters of C. farinicola colonies formed on YGA supplemented with glucose, sorbitol, glycerol, and NaCl. An increase in colony size as influenced by a, was generally correlated with the increased number of colonies formed by heat-stressed aleuriospores on the same medium (Fig. 2). Maximum colony size on NaCl- and sorbitolsupplemented YGA was observed between aw values of.882 and.93 and aw values of.88 and.942, respectively (Fig. 3); maximum size in the presence of glucose and glycerol was at or below aw values of.886 and.89, respectively. The growth rate of C. farinicola has been reported to be more rapid in glucose-fructose-supplemented media compared with that in media containing glycerol (1) and to be more adversely affected by KCl than by sucrose or glucose (12). Observations of C. farinicola colony development on media containing glucose, sorbitol, glycerol, and NaCl tend to confirm the general sensitivity of Chrysosporium species to low-molecular-weight polyols and ionic solutes. The ability of a mycological enumeration medium to support the development of large colonies is not necessarily desirable. Rather, the formation of easily detected small colonies is preferred so as to not interfere with the counting of colonies formed by other mycoflora. Considering the sensitivity of heat-stressed and healthy aleuriospores to various solutes, especially at reduced a, values at which nonxerophiles cannot grow, as well as colony size as affected by solute and a, sorbitol may be the solute of choice to incorporate into media to detect and enumerate C. farinicola in foods. a) Q) O Le Water Activity FIG. 3. Mean diameters of C. farinicola colonies formed on YGA supplemented with glucose, sorbitol, glycerol, and NaCl.

4 294 BUCHAT AND PITT APPL. NVIRON. MICROBIOL. 6 [ Glu 2C Sorb Gly NaCI 2C 3/2C k LL - 4F 3F 2 L Water Activity FIG. 4. Recovery of C. fririnicola aleuriospores heated at 2 C for min (1) and 3 min (U) and plated on YGA supplemented with glucose, sorbitol, glycerol, and NaCl. Plates were incubated at 2 and 2 C for 14 days or at 3 C for 14 days followed by 2 C for 1 days. Interacting effect of solutes and incubation temperature. The results from experiments to determine the interacting effects of the type of solute in recovery medium and incubation temperature on colony formation by unheated and heat-stressed (3 min, 2 C) C. ffarinicola aleuriospores are shown in Fig. 4. The data obtained when plates inoculated with heat-stressed aleuriospores were incubated at 2 C are similar to those shown in Fig. 2. Within the range of a, tested (Fig. 4), increased numbers of colonies were correlated with decreased aw, regardless of heat treatment or the type of solute. Incubation at 2 C generally resulted in an increase in recoverable aleuriospores compared with incubation at 2 C or at 3 C for 14 days followed by 2 C for 1 days, particularly in media containing glycerol and NaCl. Colonies formed on YGA containing glucose, sorbitol, and glycerol were barely visible after 14 days at 3 C and failed to reach the size of colonies which developed on the same media incubated at only 2 or 2 C, even after being subjected to additional incubation at 2 C for 1 days. Fewer than 12 CFU/ml were detected when unheated and heated aleuriospores plated on NaCl-supplemented media were initially incubated at 3 C. Apparently the combined stress induced by NaCl and high incubation temperature has a lethal effect on C. farinicola aleuriospores. These observations are in agreement with those of conidia of another xerophilic mold, Wallemia sebi, which exhibit less tolerance to incubation temperatures and a, values departing from the optima after exposure to sublethal heat stress (6). The optimum temperature for recovering C. farinicola is in the range from 2 to 2 C, which is slightly lower than the optimal growth temperatures of halophilic xerophiles (17). Survival of aleuriospores in diluent. The effects of holding unheated and heated C. farinicola aleuriospores for 2 min in diluent containing sucrose before plating them oln YGA containing various solutes were determined (Fig. ). The data plotted in this figure also confirm the increased heat resistance of mature (2-day-old) aleuriospores compared with that of 14-day-old cultures (Fig. 1, 2, and 4). Lessmature aleuriospores, regardless of heat treatment, were also more sensitive to the lethal effects of glycerol at a high a, (-9) and NaCl at aw values of.88 and.94, i.e., at aw values further from the growth optima for each solute. Considering each solute at a given aw, aleuriospore viability appears little affected by holding time ( or 2 min) and the concentration of sucrose in the diluent. There were slight reductions in viable aleuriospores recovered from sucrosesupplemented diluent held for 2 min compared with those recovered from diluent with no holding time before plating on YGA (aw,.92) containing added glycerol or NaCl. As observed in experiments to determine the effects of solutes at various aw values on colony development by unheated and heated aleuriospores (Fig. 2), glucose and sorbitol are clearly superior to glycerol and NaCl at a given aw. The concentration of intracellular solutes in vegetative cells of C. fastidium, a species closely related to C. farinicola, has been shown to be affected by hypoosmotic and hyperosmotic conditions in the suspending medium (11), and this phenomenon may also occur in aleuriospores; however, the stress imposed by sucrose diluents at aw values of.936,.964, and.997 over a 2-min holding period apparently is not severe enough to result in death or irreversible repair of aleuriospores upon plating on nonstressing recovery media. Survival of aleuriospores in cornstarch. The aw values (.43 and.71) of cornstarch and temperatures (1 and 2 C) of storage for 4 days had no effect on the viability of C. farinicola aleuriospores. Likewise, the a, values (.89,.92, and.9) of glucose-supplemented YGA recovery media had no effect on the recovery of aleuriospores. Populations of aleuriospores ranged from 1.1 x 13 to 8.3 x 13 CFU/g of starch over the 4-day storage period, and no population decrease was observed in starch subjected to various aw, temperature, or recovery medium conditions. The observation that the aw of recovery media did not influence colony formation by aleuriospores suggests that the storage conditions did not cause detectable injury or stress. At similar aw values (.44 and.66) and storage temperatures (4 and 21 C), conidia of A. flavus have also been observed to retain high viability in cowpea flour for 2 months (3). In summary, media traditionally used to enumerate yeasts and molds in foods are not suitable for enumerating C. farinicola aleuriospores. Supplementation of the media with sorbitol to give a, values from.88 to.89 would undoubt-

5 VOL. 6, 199 Glucose aw.92 Held 2 mh before pking INFLUNCS ON SURVIVAL OF C. FARINICOLA ALURIOSPORS 29 aw.89 Held 2 mh before plating Sorbitol QW.94 a.92 aw.88 D LL J) -J a W aw NaCI 6- CW Concentration (%) of Sucrose in Diluent a aw FIG.. Recovery of unheated () and heated (2 C for 3 min) (k) C. farinicola aleuriospores on YGA supplemented with glucose, sorbitol, glycerol, and NaCl as affected by holding time ( and 2 min) in.1% peptone containing % (aw,.997), 2%o (a,.964), and 4o (aw,.936) sucrose. edly enhance the recovery and ease of counting of C. farinicola colonies. Incubation at 2 C rather than at 2 C should be considered. The optimum formulation of media and temperature of incubation for recovering C. farinicola aleuriospores injured by other stress environments such as freezing or chemicals may be different. Additional experiments are needed to determine the most suitable conditions for enumerating aleuriospores which have been subjected to these stress environments. ACKNOWLDGMNTS L.R.B. is grateful to the Sir Frederick McMaster Fellowship program, Commonwealth Scientific and Industrial Research Organization, which provided financial support to enable this investigation to be conducted. Appreciation is also expressed to Sonya Dyer for her technical assistance. LITRATUR CITD 1. Adams, G. H., and Z. J. Ordal ffects of thermal stress and reduced water activity on conidia of Aspergillus parasiticus. J. Food Sci. 41: Beuchat, L. R Combined effects of solutes and food preservatives on rates of inactivation of and colony formation by heated spores of vegetative cells of molds. Appl. nviron. Microbiol. 41: Beuchat, L. R Survival of Aspergillus flavus conidia and other fungi in cowpeas during long-term storage under various environmental conditions. J. Stored Prod. Res. 2: Beuchat, L. R Injury and repair of yeasts and moulds. Soc. Appl. Bacteriol. Symp. Ser. 12: Beuchat, L. R Thermal tolerance of Talaromyces flavus ascospores as affected by growth medium, temperature and age, and sugar content in the inactivation medium. Trans. Br. Mycol. Soc. 9: Beuchat, L. R., and J. I. Pitt Influence of solute, ph, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia. Appl. nviron. Microbiol. 6: Conner, D.., and L. R. Beuchat fficacy of media for promoting ascospore formation by Neosartorya fischeri, and influence of age and culture temperature on heat resistance of ascospores. Food Microbiol. 4: Doyle, M. P., and. H. Marth Thermal inactivation of conidia from Aspergillus flavus and Aspergillus parasiticus. I. ffects of moist heat, age of conidia, and sporulation medium. J. Milk Food Technol. 38: Hocking, A. D., and J. I. Pitt Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods. Appl. nviron. Microbiol. 39: Kinderlerer, J. L Fungi in desiccated coconut. Food Microbiol. 1: Luard,. J ffect of osmotic shock on some intracellular solutes in two filamentous fungi. J. Gen. Microbiol. 128: Luard,. J., and D. M. Grffin ffect of water potential on fungal growth and turgor. Trans. Br. Mycol. Soc. 76: Pift, J. I Xerophilic fungi and the spoilage of foods of plant origin, p In R. B. Duckworth (ed.), Water relations of foods, Academic Press, Inc. (London), Ltd., London.

6 296 BUCHAT AND PITT 14. Pitt, J. I., and J. H. B. Christian Water relations of xerophilic fungi isolated from prunes. AppI. Microbiol. 16: Pitt, J. I., and A. D. Hocking Influence of solute and hydrogen ion concentration on the water relations of some xerophilic fungi. J. Gen. Microbiol. 11:3-4. APPL. NVIRON. MICROBIOL. 16. van Oorschot, C. A. N A revision of Chrysosporium and allied genera. Stud. Mycol. 2: Wheeler, K. A., A. D. Hocking, and J. I. Pitt Influence of temperature on the water relations of Polypaecilum pisce and Basipetospora halophila, two halophilic fungi. J. Gen. Microbiol. 134: