Vol. 56, No. 8APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1990, p. 2319-23260099-2240/90/082319-08$02.00/0Copyright © 1990, American Society for Microbiology

Colony Formation by Sublethally Heat-Injured Zygosaccharomycesrouxii as Affected by Solutes in the Recovery Medium

and Procedure for Sterilizing MediumDAVID A. GOLDEN AND LARRY R. BEUCHAT*

Department ofFood Science and Technology, The University of GeorgiaAgricultural Experiment Station, Griffin, Georgia 30223-1797

Received 15 March 1990/Accepted 21 May 1990

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as

influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, andoatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose,

glycerol, or sorbitol and sterilized by autoclaving (110°C, 15 min) and by repeated treatment with steam

(100°C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplementedYMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated andheated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days ofincubation at 25°C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm indiameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter whichwere formed by heat-injured cells on YMA at an a, of 0.880 was 2 to 3 logs less than the total number ofcolonies detected, i.e., on YMA at an av of 0.933 and using no limits of exclusion based on colony diameter.A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increasedsensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by mediumsterilization procedure when glucose or sorbitol was used as the a,-suppressing solute. In 7 of 80 paircomparisons (autoclaving versus steaming), significant differences were detected in populations recovered on

media supplemented with sucrose and glycerol. In six of these seven pairs, significantly higher populations weredetected on steamed versus autoclaved media. Significant differences in recovery of unheated cells were due tosolute type in 16 of 80 comparisons; for heated cells, significant differences were noted for 34 comparisons.

When differences did occur, the enhanced effect of solute on recovery of unheated and heated cells was typicallyin the order of glucose 2 sucrose 2 glycerol 2 sorbitol and glucose 2 glycerol 2 sucrose 2 sorbitol.

Osmophilic yeasts are commonly encountered as organ-isms responsible for spoilage of concentrated fruit juices,honey, molasses, syrups, soft-centered candies, jams andjellies, confectionery products, and a variety of other high-sugar and salted products (2, 16, 19, 22, 27, 29). Among themost common of the osmophilic spoilage yeasts is Zygosac-charomyces rouxii (= Saccharomyces rouxii), which cantolerate concentrations of up to 22% NaCI, 80% glucose, and80% sucrose (19). Some strains can grown in the presence of0.1% potassium sorbate (22; L. M. Lenovich, Ph.D. thesis,Drexel University, Philadelphia, Pa., 1986).

Traditionally, enumeration procedures used to detect os-mophilic yeasts are similar to procedures used to enumerateother yeasts (30). However, populations of osmophilicyeasts may be substantially underestimated or not detectedunless special provisions are made for their recovery. De-tection of osmophilic yeasts in high-sugar products can beimproved by plating on media formulated to resemble moreclosely the natural environment of the organism. Severalinvestigations have revealed that increasing the sugar con-tent, and thus reducing the water activity (aJ), of recoverymedia results in enhanced recovery of osmophilic fungi (11,14, 18, 24).

Increasing the sugar content of recovery media can,however, create problems. Nonenzymatic browning result-ing from Maillard reactions occurs when organic materialscontaining reducing sugars and amines (e.g., from amino

* Corresponding author.

acids) are subjected to elevated temperatures (12). Maillardreactions can lead to the formation of high-molecular-weightnitrogenous polymers such as premelanoidins and melanoi-dins (10). The rate of browning as a result of Maillardreactions in foods can double or triple for each 10°C rise intemperature (9).Many of the products formed by Maillard reactions disrupt

cellular metabolism in microorganisms (1, 7, 15, 26). Conse-quently, when media containing high concentrations of sugarare sterilized by using routine autoclaving procedures(121°C, 15 min), a reduction in recovery of osmophilic yeastsmay occur due to inhibition by products of Maillard reac-tions. Heat-stressed cells may be particularly sensitive to thetoxic effects of these products.

Early microbiologists sterilized media by a process ofintermittent sterilization (Tyndallization) which involvedsuccessive boiling treatments over a period of a few days(28). Similarly, a process in use by some mycologists work-ing with osmophilic and xerophilic fungi involves dissolvingmedium ingredients in water and then steaming for 30 min,after which the medium is allowed to stand at room temper-ature for 1 day. The medium is again steamed for 30 min (oruntil all ingredients are in solution), allowed to stand at roomtemperature for a second day, and finally steamed a thirdtime before use. This process reduces the occurrence ofbrowning reactions while inactivating vegetative microor-ganisms and spores that germinate during the periods inter-mittent to the three steam treatments. Other researcherscontinue to use traditional or modified autoclaving proce-

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2320 GOLDEN AND BEUCHAT

dures to sterilize high-sugar media, despite the potentialproblems associated with toxic browning products.The investigation reported here was undertaken to deter-

mine the suitability of media sterilized by autoclaving(110°C, 15 min) and by repeated treatment with steam(100°C) for recovering healthy and heat-injured cells of Z.rouxii. The rate of colony development of Z. rouxii on mediacontaining various a,-suppressing solutes was examined.

MATERIALS AND METHODS

Strains tested and culturing conditions. Five strains of Z.rouxii were investigated. Strains NRRL Y-12691 (diploid),NRRL Y-2547 (+ mating type), and NRRL Y-2548 (-mating type) were obtained from C. P. Kurtzman, NorthernRegional Research Center, Agricultural Research Service,U.S. Department of Agriculture, Peoria, Ill.; strains ATCC2623 (type strain) and UGA 36, a laboratory stock strain,were also investigated. All cultures were maintained on YMagar (YMA; YM broth [Difco Laboratories, Detroit, Mich.]plus 1.5% agar) supplemented with 33% glucose (YMA-33;a, of 0.933; pH 5.0) at 5°C and activated in YM brothsupplemented with 33% glucose (YMB-33; a, of 0.933; pH5.0) at 25°C. YMA-33 was prepared by dissolving glucose(493 g/liter) and other dry ingredients in deionized water ona hot plate/stirrer followed by autoclaving at 110°C for 15min. YMB-33 was prepared by dissolving 21 g of the YMBformulation in 100 ml of deionized water and autoclaving at121°C for 15 min; the sterile YMB was then mixed with afilter-sterilized (0.22-pLm) solution of glucose (493 g/900 ml ofdeionized water) to obtain a final concentration of 33%glucose, i.e., 493 g/liter. The pH of sterile YMA-33 andYMB-33 was adjusted to 5.0 with sterile 1.0 N HCl. Yeastswere activated from stock cultures by inoculating 50 ml ofYMB-33 in 125-ml Erlenmeyer flasks and incubating at 25°Cin a gyratory incubator shaker (150 rpm; New BrunswickScientific Co., Inc., Edison, N.J.). A minimum of twosuccessive transfers (10 pil) at 48-h intervals were madebefore subjecting cells to heat treatment and recovery ex-periments.

Procedure for inducing heat injury. One milliliter of a 48- to50-h culture of each strain was inoculated into 100 ml ofYMB-33 (pH 5.0) in a 250-ml Erlenmeyer flask immersed ina recycling oil bath (B. Braun Instruments, Burlingame,Calif.). Flasks were immersed up to the neck in the oil bath,such that the surface of the YMB-33 was ca. 7.0 cm belowthe surface of the oil. The YMB-33 heating menstruum wasadjusted to 47, 48, 50, 52, 55, or 58 + 0.1°C prior toinoculation with yeast cultures. Magnetic stirring bars wereused to mix the YMB-33 during heat treatment. Samples (3.0ml) were withdrawn from the heated suspensions at 6-minintervals over a 36-min period of heating and dispensed intosterile test tubes (20 by 125 mm). Samples were eithersurface plated (0.1 ml) without dilution or serially diluted in0.1 M potassium phosphate buffer (pH 7.0) before beingsurface plated (0.1 ml) in duplicate onto agar recoverymedia. Two replicate experiments were done for each strainat each temperature.

Procedure for determining heat injury. To determinewhether that injury occurred upon exposing cells to elevatedtemperature, heated and unheated (control) cells were sur-face plated (0.1 ml) onto YMA supplemented with 0 (a,,0.998), 33 (a,, 0.933), or 49% (a,, 0.880) glucose (corre-sponds to 0, 493, or 961 g of glucose per 1,000 ml ofdeionized water, respectively). Recovery media were pre-pared by dissolving dry ingredients in the appropriate

TABLE 1. a,s of recovery media supplemented with 33%glucose, 45% sucrose, 21% glycerol, or 33% sorbitol

aw

Basal medium Solute SteamAutoclaving sterilization

YMA Glucose 0.936 0.936Sucrose 0.938 0.935Glycerol 0.936 0.937Sorbitol 0.937 0.936

Wort agar Glucose 0.938 0.933Sucrose 0.938 0.936Glycerol 0.934 0.937Sorbitol 0.938 0.937

Cornmeal agar Glucose 0.936 0.936Sucrose 0.934 0.937Glycerol 0.934 0.937Sorbitol 0.938 0.938

Oatmeal agar Glucose 0.934 0.936Sucrose 0.935 0.935Glycerol 0.933 0.937Sorbitol 0.938 0.937

amount of deionized water on a hot plate/stirrer, adjusting topH 7.0 ± 0.2 with 1.0 N NaOH, and autoclaving at 110°C for15 min. After autoclaving, the pH of sterile media wasreadjusted to 7.0 + 0.2, using sterile 1.0 N NaOH. Mediawere dispensed into 90-mm diameter petri dishes and al-lowed to stand at room temperature (ca. 22°C) for 24 h priorto use. Media on which heat-stressed and unheated cellswere surface spread were incubated at 25°C for 7 days.Sublethal heat injury was determined by assessing the rela-tive sensitivity, i.e., number of colonies formed by heat-stressed cells on various YMA media compared with thenumber of colonies formed by unheated cells on the samemedia. In addition, colonies were enumerated at 12- to 24-hintervals throughout a 7-day incubation period with aBiotran III automatic count/area totalizer (New BrunswickScientific Co.). Rates of increase in colony size (diameter)and number were determined by examining four platesinoculated with selected dilutions of each strain plated oneach YMA medium. The same plates were examined severaltimes throughout the 7-day incubation period. The meannumber of colonies which exceeded 0.2 and 0.5 mm indiameter was recorded at each time of analysis. In thoseinstances when colonies did not reach 0.2 mm in diameter,no limit of exclusion based on diameter was used to deter-mine the total number of colonies on each plate on day 7 ofincubation.

Procedure for evaluating method of sterilization of recoverymedia. Four basal media were evaluated for their suitabilityto recover unheated and heat-stressed cells of Z. rouxii.Media evaluated were YMA, wort agar (Oxoid U.S.A., Inc.,Columbia, Md.), cornmeal agar (Oxoid), and oatmeal agar,which consisted of 15.0 g of oatmeal (Quaker Oats Co.,Chicago, Ill.), 15.0 g of wheat germ (Quaker Oats Co.), 20.0g of agar, and 1,000 ml of deionized water.Each medium was adjusted to an a, of 0.933 to 0.938

(Table 1) by adding various solutes. Concentrations of 33%glucose, 45% sucrose, 21% glycerol, and 33% sorbitol (cor-responds to 493, 818, 266, and 493 g of solute per 1,000 ml ofdeionized water, respectively) resulted in an a¾ in this range.YMA, wort agar, and cornmeal agar recovery media were

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COLONY FORMATION BY HEAT-INJURED Z. ROUXII

NRRL Y-254a (47C)

,~~~0r~6 12 18 24 0

UGA 36 (48rC)

|~~~~~~~i

NRRL Y-1 2691 (48C)

IE^

6 12 1' 24

Heating time (min)FIG. 1. Recovery of five strains of Z. rouxii heated at 47 or 48°C in YMB-33 and plated on YMA at aws of 0.998, 0.933, and 0.880.

prepared by combining preformulated dry ingredients, sol-ute, and deionized water and then heating the mixture on a

hot plate/stirrer to dissolve all ingredients. Each mediumwas divided into 500-ml aliquots in 1,000-ml flasks, and thepH was adjusted to 7.0 + 0.2 with 1.0 N NaOH. Oatmealagar was prepared by combining dry ingredients and supple-mental solutes in deionized water and then slowly bringingthe mixture to a boil on a hot plate/stirrer. After boiling forabout 30 min, the mixture was filtered through cheeseclothand the filtrate was divided into 500-ml aliquots. The pH wasadjusted to 7.0 + 0.2 with 1.0 N NaOH.Two procedures designed to render media "sterile" were

evaluated. While neither procedure can be described as a

sterilization process in the real sense, the terms sterile andsterilized will be used in the following text to describe mediasubjected to heat treatment sufficient to render them essen-tially free of microbial contaminants which might eventuallygrow and interfere with colony development of Z. rouxii.Each freshly prepared molten medium (500 ml) was steril-ized by autoclaving at 110°C for 15 min. Likewise, 500-mlportions of each medium were sterilized in an autoclaveunder free-flowing steam at 100 to 102°C (hereafter referredto as steaming) for 45 min for the first treatment, 60 min forthe second treatment, and 75 min for the final treatment.Media were allowed to solidify and stand at room tempera-ture for 24 h between each steam treatment. Media contain-ing sucrose had a stronger gel strength than media containingthe other solutes and required a final steam treatment of 100min to melt the agar. After sterilization, the pH of auto-

claved and steamed media was readjusted to 7.0 0.2 withsterile 1.0 N NaOH. All sterile media were poured into90-mm-diameter petri dishes and allowed to stand at room

temperature for 24 h prior to use.

Based on observations from heat stress studies, 48- to 50-hcultures of strains NRRL Y-12691 and UGA 36 were heatedin YMB-33 for 12 min at 48°C, as described above, to inducesublethal injury of a substantial portion of surviving cells; 48-to 50-h cultures of strains NRRL Y-2547, NRRL Y-2548, andATCC 2623 were heated in YMB-33 for 12 min at 47°C.Heat-injured and unheated cells were serially diluted in 0.1M potassium phosphate buffer (pH 7.0) as needed andsurface plated (0.1 ml) in duplicate onto autoclaved andsteamed media. Plates were incubated at 25°C for 7 daysbefore colonies were enumerated. Two replicate experi-ments were conducted.Measurements of a,. The a,s of media were measured at

25°C with a Hygroscop DT hygrometer (Rotronic InstrumentInc., Huntington, N.Y.) equipped with two a, measuringstations. The hygrometer was standardized at 25°C with a

saturated aqueous solution of potassium chloride (a, 0.86)(23).

Statistical analyses. Analysis of variance and Duncan'smultiple-range tests were used to determine statistical differ-ences (P s 0.05) between values obtained from experimentswith autoclaved and steamed media and to determine differ-ences in the number of colonies formed as influenced by thetype of solute used in basal media. Data representing actual

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4 A-A OW Oaw32..

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NRRL Y-2547 (4rC)

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2322 GOLDEN AND BEUCHAT

Unheated ATCC 2623 Unheated4 A U.m NRRL Y-2547

2 3 4 5 6 7 2 3 4 5 6 7

UnheatedNRRL Y-2548

Al.

-~~~~

Incubation time (days)FIG. 2. Recovery of three strains of uninjured and heat-injured (47°C, 12 min) cells of Z. rouxii on YMA incubated at 25°C. Colonies were

enumerated on YMA at a,s of 0.998 (0), 0.933 (O), and 0.880 (A), using a 0.2-mm-diameter exclusion limit, and on YMA at a,s of 0.998 (0),0.933 (U), andO.880 (A), using a 0.5-mm exclusion limit. No exclusion limit based on diameter was used to determine the total number ofcolonies formed after 7 days on YMA at a,s 0.998 (V), 0.933 (V), and 0.880 (K).

populations were transformed to log1o values prior to statis-tical analysis.

RESULTS AND DISCUSSION

Heat stress. Results from heat stress studies indicated thatall five strains of Z. rouxii examined were quite sensitive toinactivation at 50, 52, 55, and 58°C. When heated at 52°C,viable populations (105 to 106 cells per ml) were essentiallynondetectable within 6 min; heating at 55 and 58°C for 6 minresulted in undetectable populations. At 47 and 48°C, inac-tivation rates were slower, although strains UGA 36 andNRRL Y-12691 exhibited substantially greater resistance at47°C than strains ATCC 2623, NRRL Y-2547, and NRRLY-2548 (Fig. 1). The rates of thermal inactivation for strainsATCC 2623, NRRL Y-2547, and NRRL Y-2548 at 47°C weresimilar. The viable populations of strains ATCC 2623,NRRL Y-2547, and NRRL Y-2548 were reduced by 2 to 2.5logs when held for 24 min at 47°C. Strain UGA 36 was themost heat-resistant strain investigated. No reduction in theviable population of strain UGA 36 occurred until after 6 minof heating at 48°C, and only about a 1-log decrease wasobserved after 24 min. Strain NRRL Y-12691 was rapidlyinactivated during the first 6 min of heating at 48°C (about 3.5logs); however, subsequent heating for up to 18 min wasconsiderably less effective in further reducing the viablepopulation.

It is difficult to compare the relative heat sensitivity ofstrains examined in this study with that reported by others

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UnheatedNRRL Y-1 2691

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HeatedNRRL Y-1 2691

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Incubation time (days)FIG. 3. Recovery of two strains of uninjured and heat-injured

(48°C, 12 min) cells of Z. rouxii on YMA incubated at 25°C. Seelegend to Fig. 2 for explanation of symbols.

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COLONY FORMATION BY HEAT-INJURED Z. ROUXII

due to the great variability associated with aw, solute, pH,and chemical composition of media in which Z. rouxii hasbeen suspended during heat treatment. Lowering the a, ofthe heating medium generally results in an increase intolerance of osmophilic yeasts to elevated temperatures (6).Corry (5) reported that the heat resistance of Z. rouxii wasincreased by solutes in the heating medium in the order ofsucrose > sorbitol > glucose > glycerol. Whether the sameorder of tolerance of heat-injured cells to these solutes inrecovery media exists is not known. Certainly, the order ofsolute tolerance of the five unheated and heat-injured Z.rouxii strains examined in the present study was somewhatdifferent than that reported by Corry for relative toleranceduring the actual time cells were being exposed to heat.

Recovery of all test strains on YMA at an a, of 0.933 wasas good as or slightly better than recovery on YMA at an a,of 0.998. Heat-stressed cells of all strains had increasedsensitivity to an a, of 0.880; strains NRRL Y-2547 andNRRL Y-2548 were the least tolerant to a low a. Thisindicates that substantial portions of the heated cells con-sisted of injured cells. Since unheated cells of strains NRRLY-2547 and NRRL Y-2548 had the least tolerance to a lowa., it is more difficult to assess the degree of heat injurysustained by these strains with this criterion alone. Theoptimum a, for growth of Z. rouxii, as well as othermicroorganisms, is dependent on several environmentalfactors. Nevertheless, data presented in Fig. 1 suggest anoptimum a, in the intermediate range, i.e., 0.933. Thisobservation is particularly noteworthy when recovery ofinjured cells is a consideration.

Rate of colony development. The presence of injured cellswas also confirmed by a decrease in the number of coloniesexceeding 0.2 or 0.5 mm in diameter which were formed byheated cells after any given incubation time compared withthe number of colonies exceeding 0.2 and 0.5 mm, respec-tively, formed by unheated cells after the same incubationtime. Relative differences in the total number of coloniesformed by unheated and heated cells on YMA at various awsafter 7 days of incubation, regardless of diameter, would alsodemonstrate that sublethal heat injury occurred. The magni-tude of differences in populations detected on YMA atvarious aws would reveal the relative sensitivity of injuredcells to suboptimal aws.

Rates of appearance and increase in diameter of coloniesdeveloped by unheated and heated (12 min at 47°C) cells ofstrains ATCC 2623, NRRL Y-2547, and NRRL Y-2548 areillustrated in Fig. 2. Colonies which developed from un-heated cells on YMA at aws of 0.998 and 0.933 generallyexceeded 0.5 mm in diameter within 3.5 to 4 days ofincubation, whereas colonies formed from unheated cells onYMA at an aw of 0.880 typically did not exceed 0.5 mm indiameter until after 5.5 to 6.5 days. Colony developmentfrom heat-stressed cells occurred at a slower rate comparedwith that by unheated cells. The number of colonies exceed-ing 0.5 mm in diameter which were formed by heated cells onYMA at an aw of 0.880 was 2 to 3 logs less than the totalnumber of colonies detected, i.e., on YMA at an a& of 0.933and using no limits of exclusion based on colony diameter.The rates of appearance and increase in diameter of

colonies formed by unheated cells to strains UGA 36 andNRRL Y-12691 (Fig. 3) generally followed the same patternsas those observed for strains ATCC 2623, NRRL Y-2547,and NRRL Y-2548. Somewhat smaller differences were

observed in the number of colonies enumerated on YMA atan a, of 0.880 with 0.2- and 0.5-mm diameter exclusionlimits for unheated and heated cells of strain NRRL Y-12691

compared with the other four strains examined. In addition,smaller differences in the number of colonies exceeding 0.5mm in diameter on YMA at an awof 0.880 and in the numberof colonies enumerated with no diameter exclusion limitwere detected for strains UGA 36 and NRRL Y-12691compared with respective differences for strains ATCC2623, NRRL Y-2547, and NRRL Y-2548.Slow rates of colony development by heated cells of all

strains on media at an a, of 0.880 were made apparent,especially during the initial days of incubation, by differ-ences detected in the number of colonies with diameters of.0.2 and 0.5 mm. This observation further supports theconclusion that a portion of the heat-stressed cells was

sublethally injured. While colony development from un-

heated cells was also delayed on media at an aw of 0.880, thedelayed associated with colonies which developed fromheated cells was generally longer. Additional evidence indi-cating sublethal injury lies with the reduced number ofcolonies exceeding 0.5 mm in diameter which were formedon YMA at an a, of 0.880 compared with the total number ofcolonies formed at the end of the 7-day incubation period.Comparing these observations with those from colony

formation on YMA at an a, of 0.933, it is apparent that thelarge percentage of colonies that never reached 0.5 mm indiameter on YMA at an a, of 0.880 resulted from sublethallyinjured cells. The difference between the number of coloniesenumerated by using 0.5-mm-diameter exclusion limits andthe total number of colonies indicates that at least 90% of thecells were injured by treatment at 47 or 48°C for 12 min.Therefore, these time-temperature combinations were cho-sen for evaluating the recovery of heat-injured cells on

autoclaved and steamed media.Method of sterilization. Data obtained for recovery of

uninjured (control) and heat-injured cells of Z. rouxii on

media containing glucose, sucrose, glycerol, or sorbitolwhich had been sterilized by autoclaving or steaming are

presented in Table 2. In no instance was recovery of Z.rouxii significantly affected by sterilization procedure whenglucose or sorbitol was used as the a,-suppressing solute. In7 of 80 pair comparisons (four basal media, two solutes, fivestrains, unheated and heat-injured cells), significant differ-ences in populations recovered on autoclaved and steamedmedia supplemented with sucrose and glycerol were de-tected. In six of these seven pairs, significantly higherpopulations were detected on steamed versus autoclavedmedia. It is therefore concluded that autoclaving media at

110°C for 15 min does not result in browning reactionproducts that are toxic to Z. rouxii.

Within strain, basal medium, culture treatment, and me-

dium sterilization procedure, some significant differences inpopulations resulted from the use of various solutes to

suppress the a, (Table 2). When considering unheated cells,significant differences due to solute type were observed in 16of 80 comparisons; for heated cells, significant differenceswere noted for 34 of the 80 comparisons. When differencesdid occur, the enhanced effect of solute on recovery ofunheated cells was typically in the order of glucose 2sucrose 2 glycerol 2 sorbitol, whereas recovery of heatedcells as affected by solute was generally in the order ofglucose 2 glycerol 2 sucrose 2 sorbitol. The observeddifferences in recovery of Z. rouxii as influenced by solutewere not surprising. It is commonly accepted that xerophilicfungi exhibit variable growth responses at a given a, whendifferent solutes are used to adjust the aw of growth media(20, 21). Pitt and Hocking (21) reported that several xero-

philic molds grew better on media containing a glucose-

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2324 GOLDEN AND BEUCHAT

TABLE 2. Populations of uninjured (control) and heat-injured cells of Z. rouxii recovered on media (am, 0.933 to 0.938)sterilized by autoclaving and steaming

Culture Sterilization Log1o CFU/mla recovered on media supplemented with:Strain Basal medium tramnpocdeStrainBasalmediumtreatment procedure Glucose Sucrose Glycerol Sorbitol

ATCC 2623 YMA Control Autoclaved 5.64 5.63 5.51 5.58

Heated

Wort agar Control

Heated

Cornmeal agar

Oatmeal agar

NRRL Y-2547 YMA

Wort agar

Cornmeal agar

Oatmeal agar

NRRL Y-2548 YMA

Control

Heated

Control

Heated

Control

Heated

Control

Heated

Control

Heated

Control

Heated

Control

Heated

Wort agar

Cornmeal agar

Oatmeal agar

YMAUGA 36

Control

Heated

Control

Heated

Control

Heated

Control

Heated

SteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

AutoclavedSteamedAutoclavedSteamed

5.573.76 a3.64

5.75 a5.663.48 a3.51

5.68 a5.65 ab3.30 a3.41 a

5.635.68 a3.45 a3.47 a

5.675.674.985.05

5.655.604.95 a5.03 a

5.71 a5.65 a4.87 a5.04 a

5.69 a5.65 a5.05 a5.06 a

5.905.865.114.92 a

5.905.914.95 a4.77 a

5.94 a5.88 a4.88 a4.91 a

5.89 a5.89 a5.01 a5.01 a

5.925.905.21 a5.19

5.703.42 ab3.39

5.59 ab5.643.09 ab3.10

5.54 ab5.68 a2.48 b

*3.18 a

5.705.68 a2.75 ab2.99 ab

5.565.504.914.96

5.475.554.57 a4.61 ab

5.32 ab5.46 ab3.35 ab4.15 a

5.34 a5.45 a3.88 b4.96 a

5.815.834.774.81 ab

5.755.874.54 a4.58 a

5.43 a5.77 a2.00 c3.69 a

5.76 a5.67 a2.30 c

*2.69 bc

5.915.865.16 ab5.10

5.642.92 c3.14

5.68 ab5.602.78 ab2.40

5.49 ab5.60 ab2.49 b2.56 b

5.605.47 a2.48 bc2.44 b

5.525.624.674.75

5.335.233.90 ab

*4.07 ab

5.32 ab4.81 c4.41 a4.55 a

4.69 b5.28 a4.38 ab4.38 a

5.755.894.684.71 ab

5.835.893.49 b3.41 b

5.83 a5.80 a3.87 b3.56 a

5.70 a5.77 a3.67 b3.69 b

5.885.935.18 a5.17

5.543.02 bc3.15

5.57 b5.602.39 b2.65

5.41 b5.53 b

b

2.30 b

5.244.92 b2.00 c

5.545.554.694.67

5.205.322.80 b3.35 b

4.21 b4.92 bc2.00 b2.54 b

3.77 c3.65 b2.49 c2.00 b

5.855.804.584.47 b

5.475.682.15 c2.39 c

3.83 b3.97 b2.00 c2.15 b

3.69 b3.82 b

2.00 c

5.885.815.07 b5.06

Autoclaved 5.85Steamed 5.86

5.82*5.93

5.86 5.825.86 a 5.87

Continued on following page

Wort agar Control

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COLONY FORMATION BY HEAT-INJURED Z. ROUXI 2

TABLE 2-Continued

Culture Sterilization Log1o CFU/mla recovered on media supplemented with:treatment procedure Glucose Sucrose Glycerol Sorbitol

Heated Autoclaved 5.17 a 5.04 a 4.71 b 2.71 cSteamed 5.27 a 5.15 ab 4.77 b 3.26 c

Cornmeal agar Control Autoclaved 5.91 a 5.73 ab 5.85 ab 5.34 bSteamed 5.96 a *5.96 a 5.89 a 5.29 b

Heated Autoclaved 5.18 a 2.65 b 5.04 a 2.30 bSteamed 5.28 a 4.13 b 5.12 ab 2.48 c

Oatmeal agar Control Autoclaved 5.88 a 5.69 a 5.81 a 4.92 bSteamed 5.92 a 5.90 a 5.88 a 5.23 b

Heated Autoclaved 5.18 a 4.41 b 4.67 b 3.90 cSteamed 5.31 a *4.99 b 4.90 b 2.60 c

NRRL Y-12691 YMA Control Autoclaved 5.93 5.81 5.89 5.95Steamed 5.88 5.89 5.85 5.81

Heated Autoclaved 3.37 a 2.99 b 3.22 ab 2.27 cSteamed 3.41 a 3.01 b 3.27 ab 2.60 c

Wort agar Control Autoclaved 5.85 5.76 5.87 5.85Steamed 5.83 5.90 5.86 5.89

Heated Autoclaved 3.18 a 2.65 b *2.42 bSteamed 3.12 a 2.76 a 2.00 b 2.85 a

Cornmeal agar Control Autoclaved 5.84 5.82 5.88 5.75Steamed 5.90 5.88 5.81 5.72

Heated Autoclaved 3.29 a 2.62 b 2.90 abSteamed 3.31 a 2.36 b 2.81 ab

Oatmeal agar Control Autoclaved 5.86 5.89 5.73 5.43Steamed 5.87 a 5.87 a 5.82 a 5.43 b

Heated Autoclaved 3.27 a 2.69 ab 2.73 ab 2.00 bSteamed 3.42 a 2.78 ab 2.96 a 2.15

a Values in columns within the same strain, basal medium, and culture treatment which are marked by an asterisk are significantly higher (P < 0.05). Valuesin the same row which are not followed by the same letter are significantly different (P - 0.05).

b -, Colonies were too small and/or too numerous to count.

fructose mixture than on media containing sucrose. Simi-larly, Homer and Anagnostopoulos (13) reported thatglycerol was less inhibitory to yeasts than sucrose.

While variable growth responses to solutes are common,the mechanism involved in solute tolerance has not beenfully elucidated. Brown (3) suggested that there was nocorrelation between the inhibitory activity of sugars andtheir molecular weights but proposed that stereochemicalfactors play a major role in solute tolerance. Also, theinfluence of various organic substances on compatible soluteproduction and accumulation should be considered. Com-patible solutes, particularly glycerol and arabitol, affordprotection of osmophilic yeasts against the inhibitory effectsof low a, by serving as osmoregulators (3, 4). Moran andWitter (17) reported that D-arabitol production by Saccha-romyces rouxii was favored by high concentrations of glu-cose but not by sucrose, which suggests that certain solutesmay act as enzyme inhibitors.

In some instances, data were not obtained when heat-injured cells were plated on sorbitol-supplemented mediabecause populations on plates on which 0.1 ml of theundiluted suspension was applied were too numerous tocount but no colonies were detected at the next highest10-fold dilution. The phenomenon causing our inability tocount colonies on media supplemented with sorbitol remainspuzzling. Since the heated cells were already sublethallystressed, it is possible that an additional stress imposed byosmotic shock sustained from exposure to high a, in the

potassium phosphate buffer diluent was sufficient to cause anincrease in sensitivity to sorbitol in recovery media. Incontrast to these results, Graumlich and Stevenson (8)reported enhanced recovery of thermally injured S. cerevi-siae cells after prolonged storage in water prior to plating onrecovery media. However, since S. cerevisiae is not anosmophilic yeast, susceptibility to osmotic shock at high a,may not have been as great compared with that of Z. rouxii.Several reports have indicated that when foods with low a,are examined, the a, of the diluent should also be reduced toavoid osmotic shock of osmophilic yeasts (6, 22, 24, 25).The varying inhibitory activity of glucose, sucrose, glyc-

erol, and sorbitol observed in this investigation can befurther assessed by comparing the maximum colony diame-ters reached by Z. rouxii on media containing these solutes(Table 3). Recovery of unheated and heated cells wasfavored on media containing glucose. All strains producedcolonies with approximately the same maximum diameter ona particular medium, regardless of the heat treatment appliedto cultures or procedure used to sterilize media. However,the number of colonies reaching the maximum diameterwhich developed from heated cells was substantially lessthan those formed from unheated cells. The maximumdiameter of colonies on YMA and cornmeal agar was ob-served to be in the order of glucose > glycerol > sorbitol >sucrose, while the maximum diameter for growth on wortagar and oatmeal agar was determined to be in the order ofglucose > glycerol > sucrose > sorbitol.

VOL. 56, 1990 2325

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2326 GOLDEN AND BEUCHAT

TABLE 3. Maximum diameter of typical colonies of five strainsof Z. rouxii recovered on media (a,, 0.933 to 0.938)

containing various solutesa

Maximum colony diam (mm) on basal mediaBasal medium supplemented with:

Glucose Sucrose Glycerol Sorbitol

YMA 4.0 2.5 3.8 3.2Wort agar 2.2 1.5 2.0 1.2Cornmeal agar 1.0 0.5 0.9 0.8Oatmeal agar 2.2 1.2 1.5 1.0

a After 7 days at 25°C, all strains produced colonies with approximately thesame maximum diameter regardless of the heat treatment applied to culturesor procedure used to sterilize media.

In summary, time-consuming and tedious steaming pro-cesses used to sterilize media at a^, values as low as 0.880can be replaced by autoclaving at 110°C for 15 min withoutadversely affecting the total number of colonies formed byunheated and heat-injured Z. rouxii. We recommend thatglucose be the solute used to reduce the aws of media used toenumerate Z. rouxii, since it is less inhibitory than glycerol,sucrose, and sorbitol. Finally, consideration should be givento reducing the aws of diluent as well as recovery media toenumerate Z. rouxii, especially when metabolically or struc-turally injured cells may be present in the food beinganalyzed.

ACKNOWLEDGMENTS

We express our gratitude to Lynn M. Perkins for assistance withtechnical work. Appreciation is also extended to Jih-Shiang Chernfor assistance with statistical analyses.

LITERATURE CITED1. Adrian, J. 1974. Nutritional and physiological consequences of

the Maillard reaction. World Rev. Nutr. Diet. 19:71-122.2. Barnett, J. A., R. W. Payne, and D. Yarrow. 1983. Yeasts:

characteristics and identification. Cambridge University Press,Cambridge.

3. Brown, A. D. 1976. Microbial water stress. Bacteriol. Rev.40:803-846.

4. Brown, A. D., and J. R. Simpson. 1972. Water relations ofsugar-tolerant yeasts: the role of intracellular polyols. J. Gen.Microbiol. 72:589-591.

5. Corry, J. E. L. 1976. The effect of sugars and polyols on the heatresistance and morphology of osmophilic yeasts. J. Appl. Bac-teriol. 40:269-276.

6. Corry, J. E. L. 1987. Relationships of water activity to fungalgrowth, p. 51-99. In L. R. Beuchat (ed.), Food and beveragemycology. Van Nostrand Reinhold, New York.

7. Cuzzoni, M. T., G. Stoppine, G. Gazzani, and P. Mazza. 1988.Influence of water activity and reaction temperature of ribose-lysine and glucose-lysine Maillard systems on mutagenicity,absorbance, and content of furfurals. Food Chem. Toxicol.26:815-822.

8. Graumlich, T. R., and K. E. Stevenson. 1978. Recovery ofthermally injured Saccharomyces cerevisiae: effects of mediaand storage conditions. J. Food Sci. 43:1865-1870.

9. Greenwood, C. I., and D. N. Munro. 1979. Carbohydrates, p.35-76. In R. J. Priestly (ed.), Effects of heating on foodstuffs.Applied Science Publishers, Ltd., London.

10. Hawthorn, J. 1981. Foundations of food science, p. 156-162.W. H. Freeman, Oxford.

11. Hocking, A. D., and J. I. Pitt. 1980. Dichloran-glycerol mediumfor enumeration of xerophilic fungi from low-moisture foods.Appl. Environ. Microbiol. 39:488-492.

12. Hodge, J. E., and E. M. Osman. 1976. Carbohydrates, p.41-138. In 0. R. Fennema (ed.), Principles of food science, partI. Food chemistry. Marcel Dekker, Inc., New York.

13. Horner, K. J., and G. D. Anagnostopoulos. 1973. Combinedeffects of water activity, pH and temperature on the growth andspoilage potential of fungi. J. Appl. Bacteriol. 36:427-436.

14. Ingram, M. 1959. Comparisons of different media for countingsugar tolerant yeasts in concentrated orange juice. J. Appl.Bacteriol. 22:234-247.

15. Ingram, M., D. A. A. Mossel, and P. de Lange. 1955. Factors,produced in sugar-acid browning reactions, which inhibit fer-mentation. Chem. Ind. 1:63-64.

16. Kurtzman, C. P., R. Rogers, and C. W. Hesseltine. 1971.Microbiological spoilage of mayonnaise and salad dressings.Appl. Microbiol. 21:870-874.

17. Moran, J. W., and L. D. Witter. 1979. Effect of sugars onD-arabitol production and glucose metabolism in Saccharomy-ces rouxii. J. Bacteriol. 138:823-831.

18. Mossel, D. A. A., and F. E. M. J. Sand. 1968. Occurrence andprevention of microbial deterioration of confectionery products.Onserva 17:23-32.

19. Onishi, H. 1963. Osmophilic yeasts. Adv. Food Res. 12:53-94.20. Pitt, J. I. 1975. Xerophilic fungi and the spoilage of foods of

plant origin, p. 273-307. In R. B. Duckworth (ed.), Waterrelations of foods. Academic Press, Inc. (London), Ltd., Lon-don.

21. Pitt, J. I., and A. D. Hocking. 1977. Influence of solute andhydrogen ion concentration on the water relations of somexerophilic fungi. J. Gen. Microbiol. 101:35-40.

22. Restaino, L., S. Bills, K. Tscherneff, and L. M. Lenovich. 1983.Growth characteristics of Saccharomyces rouxii isolated fromchocolate syrup. Appl. Environ. Microbiol. 45:1614-1621.

23. Rockland, L. B. 1960. Saturated salt solutions for the staticcontrol of relative humidity between 5° and 40°. Anal. Chem.32:1375-1376.

24. Seiler, D. A. L. 1980. Yeast spoilage of bakery products, p. 135-152. In F. A. Skinner, S. M. Passmore, and R. R. Davenport(ed.), Biology and activities of yeasts. Academic Press, Inc.(London), Ltd., London.

25. Seiler, D. A. L. 1986. Effect of diluent and medium wateractivity on recovery of yeasts from high sugar coatings andfillings, p. 162-163. In A. D. King, Jr., J. I. Pitt, L. R. Beuchat,and J. E. L. Corry (ed.), Methods for the mycological exami-nation of food. Plenum Publishing Corp., New York.

26. Shinohara, K., R.-T. Wu, N. Jahan, M. Tanaka, N. Morinaga,H. Murakami, and H. Omura. 1980. Mutagenecity of the brown-ing mixtures by amino-carbonyl reactions on Salmonella typhi-murium TA 100. Agric. Biol. Chem. 44:671-672.

27. Tilbury, R. H. 1980. Xerotolerant yeasts at high sugar concen-trations. Tech. Ser. Soc. Appl. Bacteriol. 15:103-128.

28. VanDemark, P. J., and B. L. Batzing. 1987. The microbes: anintroduction to their nature and importance, p. 11. Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif.

29. Walker, H. W., and J. C. Ayres. 1970. Yeasts as spoilageorganisms, p. 463-527. In A. H. Rose and J. S. Harrison (ed.),The yeasts, vol. 3. Academic Press, Inc., New York.

30. Zotolla, E., and H. W. Walker. 1984. Osmophilic microorgan-isms, p. 170-175. In M. L. Speck (ed.), Compendium ofmethods for the microbiological examination of foods, 2nd ed.American Public Health Association, Washington, D.C.

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