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Heat Resistance of Vegetative Cells and Asci of Two Zygosac- Charomyces Yeasts in Broths at Different Water Activity Values

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Heat Resistance of Vegetative Cells and Asci of Two Zygosac- Charomyces Yeasts in Broths at Different Water Activity Values 835 Journal of Food Protection, Vol. 50, No. 10, Pages 835-841 (October 1987) Copyright1 International Association of Milk, Food and Environmental Sanitarians Heat Resistance of Vegetative Cells and Asci of Two Zygosac- charomyces Yeasts in Broths at Different Water Activity Values MARCO F. G. JERMINI1 and WILHELM SCHMIDT-LORENZ* Food Microbiology Laboratory, Department of Food Science, Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/835/1651027/0362-028x-50_10_835.pdf by guest on 01 October 2021 (Received for publication February 2, 1987) ABSTRACT solutions of sucrose or in sucrose-glucose mixtures at re­ duced a (19). Since the influence of lyophilization on The heat resistance of vegetative cells and asci of two os- w motolerant yeasts (Zygosaccharomyces rouxii and Z. bailii) was heat resistance has not yet been investigated, great cau­ tion is needed in evaluating those results. investigated in two different broths of aw 0.963 and 0.858, re­ spectively. The highest heat resistance was observed with asci Corry (12) demonstrated that the heat resistance of of Z. bailii LMZ 108, showing a decimal reduction time CD- Saccharomyces rouxii at 65°C, pH 6.5 and aw 0.95 was value) at 60°C and aw 0.858 of 14.9 min. Asci of Z. rouxii at the highest levels in solutions of sucrose, less in sor­ v LMZ 100 were less heat resistant (D60=c- alue at aw 0.858 = 3.5 bitol and least in solutions of glucose, fructose and min). The heat resistance (D-values) of asci at aw 0.963 proved glycerol. She, however, investigated heat resistance of to be 20- to 50-fold and 5- to 8-fold higher than the D-valucs vegetative cells only. Comparative studies of the heat re­ of the corresponding vegetative cells of Z. rouxii and Z. bailii, sistance of vegetative cells and spores of four Sac­ respectively. However, the lower the a of the heating broth, w charomyces spp. and of a strain of S. cerevisiae were the smaller the differences between heat resistance of asci and carried out by Put and de Jong (33) and Splittstoesser that of vegetative cells. Morcver, different preparations of the same cell material were found to lead to different heat resis­ et al. (40), respectively. In both researches cells and as- tances. cospores were, however, prepared in different media, al­ though the composition of the culture medium in which the organism is grown before heating as well as the age of the culture were reported to influence the thermal re­ Processing of foods by heat remains the most important sistance of microorganisms (8,22). method in modern food technology, and the most effec­ tive one when safety is the main aim of processing. Unfortunately other experimental approaches to heat re­ Hence the heat resistance of microorganisms is one of sistance of osmotolerant yeasts were carried out without their most important attributes. any consideration to other parameters influencing destruc­ According to Schmidt (38) and Hansen and Riemann tion of yeasts by heat (e.g. number and conditions of (22) the heat resistance of microoganisms is affected by cells involved, aw and other parameters of the heating a large number of factors including the inherent resistance medium). The heat resistance was expressed loosely in (e.g. differences among species of a certain genus, strains term of time and temperature combinations for cell inacti- of a certain species and cells versus spores), environmen­ vation (15,35,37). tal factors which are operative during growth and during Special note must be made of the work of Beuchat cell or spore formation and environmental factors which who investigated the influence of preservatives on the are operative during the time of heating. In addition, sev­ heat resistance of various osmotolerant yeasts eral authors pointed out that recovery conditions after (4,5,6,7,9,16). heat treatment also play an important role (6,20, The results of the investigations of Gibson (19), Corry 23,29,30,31,41,42). (12) and Put and de Jong (33) are fundamental for further It is difficult to make an accurate appraisal of the fac­ studies on the heat resistance of osmotolerant yeasts; tors affecting the heat resistance of osmotolerant yeasts however, they need to be confirmed, since the involve­ from a survey of the literature. Moreover, data regarding ment of unrecognized experimental factors make an inter­ the thermal destruction of osmotolerant yeasts, especially pretation of the reported data difficut. Zygosaccharomyces bailii, are limited. Vegetative cells of Therefore, it was the purpose of the present trials to Z. rouxii and Torulopsis globosa obtained from investigate the influence of aw on the heat resistance of lyophilized cultures, showed enhanced heat resistance in osmotolerant yeasts. Vegetative cells (prepared in two different ways) and asci of two strains representing the Present address: Fermentation Microbiology Laboratory, Department of Applied Biological Sciences, Massachusetts Institute of Technology, most important species, Z. rouxii and A. bailii, were Cambridge, MA 02139. used. Glucose was adopted as the solute in the heating JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 836 JERMINI AND SCHMIDT-LORENZ broth, because of its widespread use as a humectant in centrifugation and resuspended in diluent DS30. Total counts foods. by the counting chamber method (Helber chamber) were carried out after which the suspensions were diluted with DS30 to at­ MATERIALS AND METHODS tain a count of approximately 108 vegetative cells/ml. Preparation of vegetative cells and asci on solid medium. Preparation of media and solutions When asci were desired, yeast cells preenriched and suspended All the media: malt extract glucose 30 (MEG30) broth [aw in presporulation broth MYG30 were used to inoculate the 0.964; pH 5.0, 30% (w/w) D( + )-glucose; 3% (w/w) malt ex- sporulation agar MEG30. In accordance with Fowell (17), tractl, malt extract glucose 30 (MEG30) agar [aw 0.961; pH Adams and Miller (7) and Bettelheim and Gay (3), to stimulate 5.5; MEG30 broth supplemented with 1.5% (w/w) agar], sporulation and therefore increase the ratio of asci/vegetative mycophil glucose 30 (MYG30) broth [aw 0.966; pH 6.6; 30% cells, cultures were incubated at slightly reduced 02 and in­ (w/w) glucose; 1% (w/w) polypeptone peptone], yeast extract creased C02 level. These conditions were attained by placing glucose 30 (YEG30) broth [aw 0.963; pH 4.5; 30% (w/w) glu­ the cultures for 10 d at 32°C in a Gas Pack jar (model BBL) cose; 0.5% (w/w) yeast extract], yeast extract glucose 60 with 2 Gas Pack disposable H2 + C02 generator envelopes (YEG60) broth [aw 0.858; pH 4.5; 60% (w/w) glucose; 0.5% (BBL No. 70304). The initial concentrations of 02 and C02 (w/w) yeast extract], yeast extract glucose 10 (YEG10) agar [aw in the jar, measured by means of gas analyser tubes (DragerDownloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/835/1651027/0362-028x-50_10_835.pdf by guest on 01 October 2021 0.990; pH 7.0; 10% (w/w) glucose; 0.5% (w/w) yeast extract; GmbH, Liibeck, West Germany), were 14.0% (v/v) and 4.0% 1.5% (w/w) agar], yeast extract glucose 30 (YEG30) agar [aw (v/v), respectively. The yeasts (a mixture of vegetative cells and 0.960; pH 4.6; YEG30 broth supplemented with 1.5% (w/w) asci) grown on the agar were then brought into suspension by agar], yeast extract glucose 50 (YEG50) agar [aw 0.906; pH adding 10 ml of DS30 and shaking. Any cells remaining on 4.05; 50% (w/w) glucose; 0.5% (w/w) yeast extract; 1.5% (w/ the agar surface were scratched off with a glass rod. Total w) agar] as well as the diluent DS30 (aw 0.958; pH 6.5; 30% microscopic counts of vegetative cells and asci were carried out (w/w) glucose, 0.1% (w/w) polypeptone peptone] were made with a Helber counting chamber after which the suspensions by dissolving the chemicals in distilled water in a steam-boiler were diluted with DS30 to attain a count of approximately 108 at 90°C and by autoclaving at 110°C for 15 min. vegetative cells/ml. The percent of asci present in the suspension was between Yeast taxa 2.4 and 3.3. Asci contained 1 to 3 ascospores, but most fre­ Z. rouxii strain LMZ 100 and Z. bailii strain LMZ 108, pre­ quently 2 ascospores per ascus were observed. viously isolated from spoiled fitness drink and candied pineap­ ples, respectively, and maintained for 3 years on agar slants Thermal treatment (YEG50 agar) at 4°C, with subculturing at 5-month intervals, The method consisted of transferring 2.5 ml of a suspension as described by Jermini et al. (24), were used in this investiga­ (approx. 108 cells/ml) of (a) vegetative cells only or (b) a mix­ tion. ture of vegetative cells and asci into 247.5 ml of sterile heating broth (YEG30 or YEG60 of aw 0.963 and 0.858, respectively), Physical methods in 3-necked round bottomed 500-ml flasks, previously adjusted pH Adjustment. After sterilization, YEG30 and YEG60 to the selected temperature. The heating broth was maintained broths were adjusted to pH 4.5, with a few drops of 85% or- by immersing all but the necks of the flasks in a temperature thophosphoric acid, whilst YEG10 agar was adjusted to pH 7.0 regulated water bath. The water temperature was controlled with a few drops of a 1 M NaOH solution.
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