Preservative Resistance in Yeast Species

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1989, p Vol. 55, No /89/ $2./ Copyright 1989, Americn Society for Microbiology Reltionships mong Cell Size, Membrne Permebility, nd Preservtive Resistnce in Yest Species ALAN D. WARTH Commonwelth Scientific nd Industril Reserch Orgnistion Division of Food Processing, P.O. Box 52, North Ryde, New South Wles 2113, Austrli Received 6 Mrch 1989/Accepted 3 July 1989 The rte of uptke of propnoic cid nd the cell dimensions were mesured for 23 yests differing in their resistnce to wek-cid-type preservtives. Reltionships between reciprocl uptke rte, reciprocl permebility, cell volume, cell re, volume/re, nd the MICs of benzoic cid nd propnoic cid for the yests were tested by correltion nlysis on pirs of prmeters. The MIC of methylprben, which is not wekcid-type preservtive, ws included. The most significnt reltionships found were between both reciprocl uptke rte nd reciprocl permebility nd the MICs of propnoic nd benzoic cids Cell volume, re, nd volume/re were ech individully correlted with propnoic nd benzoic cid MICs, but less strongly. In multiple regression nlyses, inclusion of terms for volume, re, or volume/re did not mrkedly increse the significnce. The MIC of methylprben ws unrelted to the uptke nd permebility prmeters, but did show correltion with cell volume/re. Schizoscchromyces pombe ws nomlous in hving very low permebility. Exclusion of these outlying dt reveled prticulrly strong reltionships (P <.1) between both reciprocl uptke rte nd reciprocl permebility nd the benzoic cid MIC. MICs for Zygoscchromyces bilii isoltes were substntilly higher thn for the other species, nd therefore Z. billii isoltes hd lrge influence on the regressions. However, the reltionships observed remined significnt even fter removl of the Z. bilii dt. In showing correltion between the rte t which propnoic cid enters yest cells nd the bility of the cells to tolerte this nd other wek-cid-type preservtives, but not methylprben, the results suggest tht the resistnce mechnism, in which preservtive is continuously removed from the cell, is common nd mjor determinnt of the preservtive tolernce of yest species. Yest species differ in their tolernce to the wek-cidtype preservtives (1, 7, 1). Under cid conditions, preservtive enters cells in its undissocited form. It ws shown for Zygoscchromyces billii (6, 9) tht in the presence of n energy source, preservtive is continuously eliminted from the cell, thus mintining both low intrcellulr concentrtion of preservtive nion nd high intrcellulr ph, but t the expense of continuous expenditure of energy. The system ppers to be inducible in mny yest species, nd its induction corresponds to n increse in preservtive tolernce in them (8), suggesting tht it is importnt in determining preservtive resistnce. Differences in resistnce mong species could be due to differences in the rte of penetrtion of the cell by preservtive, the cpcity of the system to remove it, or n intrinsic sensitivity to preservtive or its nion. A study of the effect of benzoic cid on the growth yields of smll number of yests suggested tht resistnt species my be less permeble thn sensitive ones to undissocited benzoic cid (8). In the present study, the rte of uptke of propnoic cid nd the cell dimensions were determined for lrger number of yests, nd severl prmeters relevnt to the bove mechnism were clculted nd tested for sttisticl correltions with yest resistnce to wek-cid preservtives. The response of these prmeters to preservtive tht cnnot be hndled by this mechnism, methylprben, ws lso investigted. MATERIALS AND METHODS Sttistics. Multiple correltion coefficients were clculted by using the Minitb computer progrm. Growth of cells. Yests were grown t 25 C in yest extrct 2995 medium (ph 3.5) contining 5% glucose (7), without the ddition of preservtive. Exponentil-phse cells (.1 to.5 mg/ml; 3 x 1' to 3 x 16 cells per ml) were used for the determintion of MICs, cell dimensions, nd propnoic cid uptke rtes. Uptke rte nd permebility. Propnoic cid uptke rtes were mesured t 25 C;.1 mm [1-14C]propnoic cid ws used (9). Permebility ws clculted by using the uptke rte nd membrne re (9). Cell dimensions. The cell re nd volume were determined by using phse-contrst microscopy, tking the outer edge of the phse-drk zone s the cell surfce. This corresponded to the point of contct between cells in most species nd to the mximum zone of stining when direct microscopy nd severl different stins were used. However, some species, including Z. bilii, ppered to hve less substntil structure outside the principl phse-drk boundry, which ws ignored for the purpose of the clcultions. The membrne re (A), the cytoplsmic volume (V), nd the volume/re rtio (V/A) were clculted for individul cells by ssuming wll thickness of.125 Ixm. In most cses, cells pproximted closely to prolte ellipsoids nd were clculted s such. Other cells were mesured nd clculted s hemisphericlly cpped cylinders. Buds were treted s individul cells nd weighted for verging ccording to their volume. Cross-wlls were not included in the clcultion of membrne re. At lest two seprte cultures were used, nd 1 cells from ech were mesured. MIC. The MIC dt were obtined principlly from previous publiction (1). The MICs re for cells grown in the bsence of dded preservtive (undpted cells) nd for cells grown in the presence of benzoic cid. Downloded from on October 26, 218 by guest

2 2996 WARTH APPL. ENVIRON. MICROBIOL. TABLE 1. Propnoic cid permebility nd uptke rte, cytoplsmic volume, membrne re, nd MIC of benzoic cid for yests Orgnism P (mis, 18) R V (m3) A (Lm2) Benzoic cid MIC Kluveromycesfrgilis FRR Kloecker picult FRR Pichi ohmeri FRR Hnsenul noml FRR Scchromyces cerevisie FRR Scchromyces cerevisie FRR Scchromyces cerevisie FRR Zygoscchromyces rouxii FRR Zygoscchromyces rouxii FRR Zygoscchromyces bisporus FRR Zygoscchromyces bisporus FRR Cndid krusei FRR Scchromycodes ludwigii FRR Scchromycodes ludwigii FRR Schizoscchromyces pombe FRR Schizoscchromyces pombe FRR Zygoscchromyces bilii FRR Zygoscchromyces bilii FRR ,1 Zygoscchromyces bilii FRR ,2 Zygoscchromyces bilii FRR ,2 Zygoscchromyces bilii FRR ,25 Zygoscchromyces bilii FRR ,25 Zygoscchromyces bilii FRR ,3 R is expressed in nnomoles per milligrm (dry weight) per micromolr concentrtion of propnoic cid in superntnt per minute. RESULTS Yests. Twenty-three yest isoltes covering wide rnge of preservtive resistnce were exmined (Tble 1). Most were isolted from spoiled foods which hd contined preservtive. Severl isoltes of Z. billii were included, reflecting the prevlence nd importnce of this species. Six cultures were from different products in Austrli, nd ll were very resistnt. Strin FRR2476 ws isolted from cider in the United Kingdom nd ws less tolernt of benzoic cid. Z. bisporus nd Z. rouxii were included s representtives of the sme genus s Z. billii nd were isolted from spoiled foods not contining preservtive. Scchromyces cerevisie FRR1298 is wine isolte. Four cultures were unselected common lbortory strins nd hd low preservtive tolernce. Prmeters. A number of prmeters relting to permebility, size, nd preservtive resistnce were considered in the regression nlyses, but they re not ll independent. The membrne re (A) is n importnt prmeter in nonspecific trnsport processes such s permetion of undissocited preservtives. The cytoplsmic volume (V) ws clculted to give n indiction of the metbolic cpcity of the cell. The rtio of cytoplsmic volume to re (V/A) my determine the bility of the cytoplsm to cope with the flux of preservtive through the membrne. These three prmeters were ech clculted from the cell dimensions nd re not independent. The uptke rte (R) is n independently determined prmeter. Propnoic cid ws used in preference to benzoic cid becuse its generlly slower uptke llowed more ccurte determintion of rtes. The permebility (P) ws clculted from R nd A nd is therefore not independent of these prmeters. Reciprocls of rte (1IR) nd permebility (1P) were included becuse of the negtive correltions nd hyperbolic distributions observed between the direct prmeters nd preservtive resistnce nd becuse under the preservtive cycling model, liner reltionships between the reciprocl vlues nd the MICs of preservtive were expected. Resistnce to wek-cid-type preservtives is highly correlted mong species (7, 1). The MIC of benzoic cid for preservtive-dpted cultures ws tken s being the prmeter most relevnt to preservtive resistnce, but correltions were lso clculted for propnoic cid nd for undpted cells. The MIC of methylprben ws included s control, since methylprben cnnot function in the wek-cid-cycling mechnism, but its MIC my give mesure of generl direct sensitivity of yests to otherwise similr compounds. The dt re shown in Tble 1. Correltion mtrix. Correltions between the prmeters re shown in Tble 2. 1/R nd 1/P were significntly correlted with the benzoic cid MIC. Similr but negtive correltion coefficients were found for R nd P, showing tht the reltionship ws evident not only mong the more resistnt species, which re effectively more weighted in the reciprocl reltionship, but lso mong the sensitive ones, which hve greter influence on the direct comprison. V, A, nd V/A were lso correlted with the benzoic cid MIC, but less strongly. V/A gve the best reltionship. It ws very highly correlted with the cell dimeter (R =.97), which, in prctice, would be much simpler to mesure. In generl, the reltionships with the benzoic cid MIC pplied similrly to the MICs of undpted benzoic cid nd propnoic cid. On the other hnd, there ws only slight reltionship between the MIC of methylprben nd R or P, lthough reltionship with V/A ws pprent. Distribution of dt. The plot of 1/P ginst the benzoic cid MIC (Fig. 1) showed most of the orgnisms forming single cluster but with two outlying clusters. A group of six points with exceptionlly high preservtive resistnce consisted of strins of Z. billii, nd two points t exceptionlly high 11P were strins of Schizoscchromyces pombe. In the plot of V/A (lso V nd A seprtely [dt not shown]) ginst the benozic cid MIC (Fig. 2), the six Z. billii points were gin distnt from the min cluster of dt. The presence of these outlying points could severely distort the Downloded from on October 26, 218 by guest

3 1 wz...-''--''a''t 1~~ VOL. 55, 1989 PERMEABILITY AND PRESERVATIVE RESISTANCE OF YEASTS 2997 TABLE 2. Multiple correltion coefficients for physicl prmeters nd preservtive resistnces of yests Correltion coefficient for: Prmeter MIC of: P 1/P R 1/R V A V/A Methyl- Propnoic Benzoic prben cid cidb 1IP -.68 R IR V A V/A MICs Methylprben Propnoic cid Benzoic cidb Benzoic cid n = 23. b Benzoic cid MIC for undpted cells. sttisticl nlysis. Therefore, the dt were nlyzed seprtely fter exclusion of ech group. between the permebility of yest cells nd preservtive respectively, indicting very close reltionship (P <.1) Removl of both the Z. billii nd the Schizoscchromyces pombe dt incresed the correltion coefficients for ll to resistnce, wheres volume nd re becme less signif- resistnce. V/A retined significnt (P <.1) reltionship the permebility, uptke rte, nd size prmeters (Tble 3), icnt. showing tht the reltionships found by using the full dt set 1/P hd no cler reltionship with resistnce to methylprben. Schizoscchromyces pombe hd low resistnce nd remined fully pplicble to the remining species. Inclusion of the Schizoscchromyces pombe dt reduced the correltion coefficient for 1/P only slightly. However, the slope low permebility, nd no trend ws evident mong other low permebility (Fig. 3). Z. billii hd high resistnce nd (.25) ws different from tht of the min cluster (1.16) nd species. the significnce ws reduced, suggesting tht this species is Multiple regression nlysis. When the Schizoscchromyces pombe dt were excluded, both 1/R nd 11P gve very typicl in the reltionship between permebility nd benzoic cid resistnce. On the other hnd, it did not pper to good predictions of the benzoic cid MIC (Tble 4). V/A, be unusul with respect to volume nd re. A very low when included, contributed to the sums of squres of the coefficient ws shown with respect to volume nd re. The regression but gve only slight overll increse. Significnce decresed s consequence of the loss of one degree very low coefficient shown with respect to resistnce to methylprben shows tht for most species there is very of freedom (Tble 4). Methylprben mde prcticlly no little reltionship between resistnce to methylprben nd further contribution to the sum of squres. All the regressions were highly significnt nd ccounted for nerly 9% of resistnce to benzoic cid nd tht the correltion found depends hevily on the presence of Z. billii. Exmintion of the totl vribility. the effect of excluding only the Schizoscchromyces pombe The sme conclusions pplied even when the Z. billii dt confirmed its typicl permebility properties. Regression coefficients for 1/P nd 1/R incresed to.935 nd.951, cnce of the 1/P regression slightly but hd little effect on dt were excluded. Inclusion of V/A incresed the signifi- 11R. Anlysis of the full dt, including the Schizoscchromyces pombe dt, lso yielded highly significnt (P < 151.1) predictions bsed on 1/R or 1/P nd V/A. Inclusion of E %-1 c6oo E ~~~ N 1i z w 51 m U...-- A- C-) Downloded from on October 26, 218 by guest /16 x PERMEABILITY TO PROPANOIC ACID (sec/m) FIG. 1. Reltionship between the reciprocl of the permebility to propnoic cid of the cell membrne nd benzoic cid MIC. Symbols:, 15 yests;, 6 isoltes of Z. billii; A, 2 isoltes of Schizoscchromyces pombe. Regression lines:, 15 yests; ---, 15 yests plus Z. billii;..., 15 yests plus Schizoscchromyces pombe. N z w m C- 5. ". A.**, CELL VOLUME/MEMBRANE AREA (,sm) FIG. 2. Reltionship between the V/A rtio of yest cells nd the benzoic cid MIC. Symbols re s in Fig. 1. *.1~~~~S..

4 2998 WARTH APPL. ENVIRON. MICROBIOL. TABLE 3. Correltion coefficients on the benzoic cid MIC' Correltion coefficient for: Dt excluded n MIC of: P 1/P R 1/R V A VIA Propnoic Benzoic Methylprben cid cid' None S. pombe Z. bilii S. pombe nd Z. bilii Outlying dt excluded. b Benzoic cid MIC for undpted cells. methylprben dt further improved the predictions, but this ws due only to its bility to reconcile the nomlous Schizoscchromyces pombe dt. Reltionships within species. In three species, S. cerevisie, Z. rouxii, nd Scchromycodes ludwigii, differences in benzoic cid resistnce were ssocited with corresponding differences in permebility. In ddition, the resistnt strins of S. cerevisie were much lrger nd hd greter V/A thn the sensitive bkers' yest strin. The two strins of Z. bisporus differed significntly in benzoic cid resistnce but hd similr permebilities. However, the more sensitive strin ws smller (with lower V/A rtio) nd ws less tolernt of methylprben. Both strins hd higher permebilities nd were considerbly smller thn Z. billii. Both strins of Schizoscchromyces pombe hd very low permebilities, which distinguished them from other species. Becuse of the mjor difference in morphology nd mode of cell division between this genus nd most of the other yests, it is not surprising tht it did not closely fit the pttern shown by them. Schizoscchromyces pombe ws sensitive to methylprben, nd it is possible tht its lower-thn-expected tolernce of benzoic cid ws due to n intrinsic sensitivity to both substnces. Among the six resistnt strins of Z. billii there ws only slight reltionship between differences in size, permebility, nd resistnce. However, strin FRR2476 ws less resistnt nd correspondingly hd higher permebility nd slightly smller size thn the other strins. DISCUSSION The sttisticl reltionships between the uptke rte nd permebility prmeters nd preservtive resistnce suggest 1- z Li m -I I~- LL *e * * S ~~~~~~A /16 x PERMEABILITY TO PROPANOIC ACID (sec/m) FIG. 3. Reltionship between the reciprocl of the permebility to propnoic cid of the cell membrne nd the methylprben MIC. Symbols re s in Fig. 1. A6 strongly tht these re involved in physiologicl mechnisms which determine the level of tolernce to preservtives in mny yest species. The involvement of uptke rte supports mechnisms in which the flux of preservtive into the cell is importnt. This implies tht form of preservtive must be eliminted t comprble rte. Were this not the cse, given the high rte t which preservtive enters the cell (9), cytoplsmic preservtive concentrtions would rpidly rech levels which re determined by the electrochemicl equilibrium nd re independent of the rte of uptke. If the MIC depended solely upon the mximum rte t which the cytoplsm could hndle preservtive, the MIC would be expected to be proportionl to 11R. Tht this ws not precisely the cse indictes tht other fctors, including the level of ctivity of the elimintion system, the energyproducing cpcity of the cytoplsm, nd direct sensitivity to preservtive, vry significntly mong species. In prticulr, Schizoscchromyces pombe, lthough notbly resistnt to benzoic cid, ws not s tolernt s its very low permebility would indicte. It ws, however, reltively more tolernt of propnoic nd cetic cids (1), suggesting tht it my hve prticulr sensitivity to benzoic cid. Schizoscchromyces species hve number of differences from most of the other yests, including the composition of the cell wll (4) nd the mode of cell division. In the present study, ll the yests were grown under the sme conditions nd hrvested during exponentil growth, nd they ppered to hve very reproducible morphology nd dimensions. The vrince in the verge cell length nd width between replicte crops of number of species ws 7%. The men cell volume of S. cerevisie depended on the growth rte (5); however, most of the chnge ws in cell length rther thn in dimeter. The protoplst volume in Z. billii NCYC 563 vried mrkedly with the preservtive concentrtion (2), but TABLE 4. Multiple liner regression" on the MIC of benzoic cid for dt excluding Schizoscchromyces pombeb 16 Sequentil sums of squres 11R 11P ~~Methylprben V/A MIC Totl < < < < <.1 Benzoic cid MIC 119 = x 11R = x 11P x IIR x V/A x 11P x V/A. bn = 21. Downloded from on October 26, 218 by guest

5 VOL. 55, 1989 PERMEABILITY AND PRESERVATIVE RESISTANCE OF YEASTS 2999 growth of Z. billii FRR1292 in 2 mm benzoic cid cused reduction in size of <5% (9). The biochemicl bsis for the difference in permebilities mong species is presently unknown. Degrdtion nd prtil removl of the cell wll in Z. billii did not increse the permebility to propnoic cid (9), suggesting tht the cell membrne rther thn the cell wll ws the prime permebility brrier. Anlysis of the dt of Kneko et l. (3) for lipid composition, s well s the present dt for MIC, 1P, nd 11R, for six yest species for which there were corresponding dt showed n increse in phosphtidylcholine nd phosphtidylserine levels nd decrese in the phosphtidylethnolmine level with incresing MIC, 1P, nd 11R. However, the reltionships were not sttisticlly significnt (P >.15). Among smller number of species, no reltionships between the ftty cid composition nd MIC were evident. The observtion tht resistnce to wek-cid preservtives (but not to methylprben) is relted to permebility to undissocited propnoic cid supports conclusions, drwn from growth yield nd dpttion dt (8), tht the opertion of the preservtive elimintion system is n importnt component in enhncing preservtive tolernce. The mjor physiologicl vrible determining differences in preservtive resistnce mong species ppers to be the permebility of the membrne to preservtive, but differences in the V/A rtio of cells, s well s differences in their energy metbolism nd intrinsic sensitivity to preservtive, my lso contribute. LITERATURE CITED 1. Bltsours, G. D., nd N. G. Polymencos Chemicl preservtives s inhibitors of yest growth. J. Food Sci. 28: Cole, M. B., nd M. H. J. Keenn Effects of wek cids nd externl ph on the intrcellulr ph of Zygoscchromyces bilii, nd its implictions in wek-cid resistnce. Yest 3: Kneko, H., M. Hosohr, M. Tnk, nd T. Itoh Lipid composition of 3 species of yest. Lipids 11: Kreger, D. R Observtions on cell wlls of yests nd some other fungi by X-ry diffrction nd solubility tests. Biochim. Biophys. Act 13: McMurrough, I., nd A. H. Rose Effect of growth rte nd substrte limittion on the composition nd structure of the cell wll of Scchromyces cerevisie. Biochem. J. 15: Wrth, A. D Mechnism of resistnce of Scchromyces bilii to benzoic, sorbic nd other wek cids used s food preservtives. J. Appl. Bcteriol. 43: Wrth, A. D Resistnce of yest species to benzoic nd sorbic cids nd to sulfur dioxide. J. Food Prot. 48: Wrth, A. D Effect of benzoic cid on the growth yield of yests differing in their resistnce to preservtives. Appl. Environ. Microbiol. 54: Wrth, A. D Trnsport of benzoic nd propnoic cids by Zygoscchromyces bilii. J. Gen. Microbiol. 135: Wrth, A. D Reltionships between the resistnce of yests to cetic, propnoic, nd benzoic cids nd to methyl prben nd ph. Int. J. Food Microbiol. 8: Downloded from on October 26, 218 by guest