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Heat resistance of Saccharomyces cerevisiae strains isolated from spoiled peach puree
ELSEVIER
International Journal of Food Microbiology 23 (1994) 209-213
International Journal of Food Microbiology
Short C o m m u n i c a t i o n
Heat resistance of Saccharomyces cerevisiae strains isolated from spoiled peach puree Salvador Garza a,*, j. Antonio Teixid6 a, Vicente Sanchis a, Inmaculada Vifias a, Santiago Conddn b a Departamento de Tecnolog[a de Alimentos, ETSE Agraria, Universidad de Lleida, Alcalde Rovira Route 177, 25198 Lleida, Spain b Departamento de Tecnolog[a y Bioqu[mica de los Alimentos, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain
Received 24 November 1993; revision received 11 February 1994; accepted 21 March 1994
Abstract
The heat resistance of three Saccharomyces cerevisiae strains isolated from spoiled peach puree, in McIlvaine buffer (pH 4 and 7) and peach puree (pH 3.9) was studied. The D60-values in buffer at pH 7 were 0.75, 1.32 and 0.14 min for the strains 173, 180 and 325, respectively. The pH of the buffer did not influence the heat resistance of the three strains studied. The thermal sensitivity for all strains assayed was higher when peach puree was used with D60-values of 0.53, 0.20 and 0.10 min for the strains 173, 180 and 325, respectively. The menstrua used had limited influence on the z-values, varying between 3 (strain 173, pH 4) and 4 (strain 325, peach puree). Keywords." Saccharomyces cerevisiae; Peach puree; Heat Resistance; Yeast
1. Introduction S a c c h a r o m y c e s cerevisiae is one o f the most important spoilage agents in fruits p r o d u c t s ( D e a k and Beuchat, 1993). M a n y environmental factors can have a significant influence on the microbial heat resistance (Beuchat, 1982). T h e effect of p H is one of the most important ( C o n d 6 n and Sala, 1992). However, there is limited information on the influence of p H on the thermal resistance of yeasts and the results o b t a i n e d are sometimes contradictory.
* Corresponding author. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00031-Z
210
S. Garza et aL / International Journal of Food Microbiology 23 (1994) 209-213
The aim of this study was to evaluate the heat resistance at real pasteurization temperatures of Saccharomyces cerevisiae strains isolated from spoiled peach puree using peach puree (pH 3.9) and McIlvaine buffer (pH 7 and 4) as heating menstrua. 2. Materials and methods
Microorganisms. The microorganisms used throughout this investigation (strains
173, 180 and 325) were isolated from spoiled peach puree and were identified by Centraalbureau voor Schimmelcultures (Delft, Netherlands) as Saccharomyces cerevisiae. Stock cultures were maintained on potato dextrose agar (Oxoid) slants at 4°C. The yeasts were grown at 28 °C for 48 h on malt extract agar (Oxoid) and harvested by flooding the agar surface with sterile, McIlvaine buffer, pH 7 (Dawson et al., 1969). The suspensions were centrifuged and washed five times in the same buffer. After the last centrifugation, the pellet was suspended again in McIlvaine buffer (pH 7) to obtain a concentration of c a . 10 9 yeast cells per ml, and stored at 0-5°C until used. The yeast cell concentration was estimated by microscopy. Heating menstruum. Mcllvaine buffer of pH 4 and 7 was prepared as described by
Dawson et al., (1969). Peach puree (pH 3.9) was obtained from Indulerida S.A. (Lleida, Spain) and was prepared from peeled, pitted, homogenized peaches. Heat treatments. Heat resistance determinations were carried out in a thermoresis-
tometer TR-SC (Zaragoza University, Zaragoza, Spain) as described by Cond6n et al., (1989). When the desired temperature was obtained (_+ 0.05°C), the menstruum (350 ml of McIlvaine buffer or peach puree) was inoculated with 0.2 ml of the yeast suspension under constant agitation (1500-2000 rev/min). Samples of 0.1 ml were then removed at intervals, transferred to tubes containing 15 ml of melted Sabouraud dextrose agar (Difco) at 45°C and pour plated. Enumeration of survivors. Colony forming units (CFU) were counted (Cond6n et
al., 1987) after incubation for 48 h at 28°C. D t and z-values. Decimal reduction times (Dt-values) were calculated by regression
analysis of the straight line portion of the survival curve (log~0 of survivors vs. time). Only survival curves with a coefficient of correlation (r o > 0.94) and with more than three values in the straight portion of the curve were used. Survival curves with a straight portion including less than 1 log cycle were rejected. The z-values were determined by regression analysis of log D t vs. the corresponding heating temperatures. Correlation coefficient and the comparison of slopes of decimal reduction time curves were carried out as described by Steel and Torrie (1960).
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
211
3. Results and discussion Tables
1, 2 a n d 3 s h o w D t a n d
resistance
of the
compared
to
obtained
three
yeast
published
results
at the lowest pH
z-values
strains
for the yeast strains studied.
using
(Torok
Mcllvaine
and
are in accordance
King,
buffer
1991).
In
at pH general,
with the investigations
The
heat
7 is h i g h
of Put and De
Table 1 Heat resistance data for Saccharomyces cerevisiae 173 isolated from spoiled peach puree Heatin g mediu m
Heating temperature (oc)
D-value (min)
Correlation coefficient a
McIlvaine buffer pH 7
58 60 62 64
3.00 0.75 0.23 0.08
-
McIlvaine buffer pH 4
58 60 62 64 58
5.68 1.18 0.22 0.06 2.95
- 0.999 -0.998 - 0.992 - 0.994 - 0.987
Peach puree
60 62 64
0.53 0.13 0.05
- 0.996 - 0.993 - 0.982
0.998 0.992 0.999 0.987
Equation of the T D T curve
Correlation coefficient b
z (°C)
Log D = 15.93 - 0.27T
- 0.994
3.75
Log D = 1 9 . 7 9 - 0 . 3 3 T
-0.998
3.04
Log D = 1 7 . 8 6 - 0.30T
- 0.994
3.32
For Survival Curve regression analysis. b For T h ermal D e a t h Time (TDT) regression analysis. a
Table 2 Heat resistance data for Saccharomyces cerevisiae 180 isolated from spoiled peach puree Heating m ediu m
Heating temperature (oc)
D-value (min)
Correlation coefficient a
Mcllvaine buffer pH 7
58 60 62 64
1.49 1.32 0.24 0.06
-
0.996 0.942 0.989 0.990
Mcllvaine buffer pH 4
58 60 62 64 58
1.53 0.68 0.14 0.04 0.67
-
0.993 0.996 0.998 0.996 0.988
Peach puree
60 62
0.20 0.05
- 0.996 - 0.991
Equ a t i on of the TDT curve
Correlation coefficient b
z (°C)
Log D = 15.45 - 0.25T
- 0.961
3.85
Log D = 15.45 - 0.26T
- 0.994
3.82
Log D = 16.76 - 0.29T
- 0.999
3.44
a For Survival Curve regression analysis. b For Thermal D e a t h Time (TDT) regression analysis.
as
D60-values
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
212
Table 3 Heat resistance data for Saccharomyces cerevisiae 325 isolated f r o m spoiled peach puree Heating
Heating
D-value
Correlation
Equation of the
Correlation
medium
temperature
(min)
coefficient a
T D T curve
coefficient b
56 58 62 64
2.36 0.56 0.03 0.01
0.998 -0.992 - 0.991 - 0.975
Log D = 1 7 . 1 4 - 0 . 3 0 T
-0.999
3.33
56 58 60 62
3.44 0.60 0.13 0.05
- 0.994 - 0.990 -0.991 - 0.999
L o g D = 17.49 - 0.30T
- 0.995
3.28
56
0.90
- 0.998
58
0.18
- 1.000
0.989
4.00
60 62
0.10 0.03
- 0.993 - 0.986
z (°C)
(°C) Mcllvaine
buffer pH 7 McIlvaine
buffer pH 4
Peach puree
L o g D = 13.85 - 0 . 2 5 T
a F o r Survival C u r v e regression analysis. b F o r Thermal Death T i m e ( T D T ) regression analysis.
Jong (1980) who studied the heat resistance of 120 yeast strains and showed Saccharomyces cereuisiae to be one of the most thermo-resistant. At pH 4 and in peach puree, a significant difference in heat resistance among the yeast strains studied was observed. The D60-value at pH 4 of the strain 325 was nine times lower than the D60-value for strain 173, and five times lower than for strain 180. For the same strain the heating menstruum pH did not have any great influence on the heat resistance. Results which are in accordance with Splittstoesser et al., (1975) who concluded that pH had limited effect on the thermoresistance of S. cerevisiae.
No significant differences (P < 0.01) in the z-values using buffer at pH 7, buffer at pH 4 and peach puree at pH 3.9 were observed (Tables 1-3). Similar results were obtained by other researchers (Barillere, 1981; Splittstoesser et al., 1975). The z-values obtained in the present study with the three Saccharomyces cerevisiae strains are similar to those reported for grape juice (Aref and Cruess, 1934), wine (Splittstoesser et al., 1975) and buffer at pH 3.2 (Barillere, 1981)
References Aref, H. and Cruess, W . V . (1934) A n investigation of the thermal death point of Saccharomyces ellipsoideus. J. Bact. 27, 4 4 3 - 4 5 2 . B e u c h a t , L . R . (1982) E f f e c t s of environmental stress in recovery media on colony f o r m a t i o n by sublethally heat-injured Saccharomyces cerevisiae. Trans. Br. Mycol. Soc. 78, 5 3 6 - 5 4 0 . Barillere, J.M. (1981) Etude de la thermor~sistance des soches de levures et de bact6ries lactiques isol6es de vins; influence du degr6 alcoolique et de la teneur en SO 2. Th~se D o c t e u r - I n g e n i e u r , E N S A de Montpellier, 196 pp.
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
213
Cond6n, S., Lopez, P., Oria, R. and Sala, F.J. (1989) Thermal death determination: design and evaluation of a thermoresistometer. J. Food Sci. 54, 451-457. Cond6n, S., Oria, R. and Sala, F.J. (1987) Heat resistance of microorganisms: an improved method for survival counting. J. Microbiol. Meth. 7, 37-44. Cond6n, S. and Sala, F.J. (1992) Heat resistance of Bacillus subtilis in buffer and foods of different pH.J. Food Prot. 55, 605-608. Dawson, R.M.C, Elliot, C., Elliot, W. and Hand Jones, K.M. (1969) Data for Biochemical Research. Clarendon, Oxford, 654 pp. Deak, T. and Beuchat, L.R. (1993) Yeasts associated with fruit juice concentrates. J. Food Prot. 56, 777-782. Put, H.M.C. and De Jong, J. (1980) The heat resistance of selected yeasts causing spoilage of canned soft drinks and fruit products. In: F.A. Skinner, S.M. Passmore, R.R. Davenport (editors), Biology and Activities of Yeasts. Academic Press, New York, pp. 181-213. Splittstoesser, D.F., Lenk, L.L., Wilkinson, M. and Stamer, J.R. (1975) Influence of wine composition on the heat resistance of potential spoilage organisms. Appl. Microbiol. 30, 369-373. Steel, R.G. and Torrie, J.H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York, pp. 161-180. Torok, T. and King, A.D. (1991) Thermal inactivation kinetics of food-borne yeast. J. Food Sci. 56, 6-9.
International Journal of Food Microbiology 23 (1994) 209-213
International Journal of Food Microbiology
Short C o m m u n i c a t i o n
Heat resistance of Saccharomyces cerevisiae strains isolated from spoiled peach puree Salvador Garza a,*, j. Antonio Teixid6 a, Vicente Sanchis a, Inmaculada Vifias a, Santiago Conddn b a Departamento de Tecnolog[a de Alimentos, ETSE Agraria, Universidad de Lleida, Alcalde Rovira Route 177, 25198 Lleida, Spain b Departamento de Tecnolog[a y Bioqu[mica de los Alimentos, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain
Received 24 November 1993; revision received 11 February 1994; accepted 21 March 1994
Abstract
The heat resistance of three Saccharomyces cerevisiae strains isolated from spoiled peach puree, in McIlvaine buffer (pH 4 and 7) and peach puree (pH 3.9) was studied. The D60-values in buffer at pH 7 were 0.75, 1.32 and 0.14 min for the strains 173, 180 and 325, respectively. The pH of the buffer did not influence the heat resistance of the three strains studied. The thermal sensitivity for all strains assayed was higher when peach puree was used with D60-values of 0.53, 0.20 and 0.10 min for the strains 173, 180 and 325, respectively. The menstrua used had limited influence on the z-values, varying between 3 (strain 173, pH 4) and 4 (strain 325, peach puree). Keywords." Saccharomyces cerevisiae; Peach puree; Heat Resistance; Yeast
1. Introduction S a c c h a r o m y c e s cerevisiae is one o f the most important spoilage agents in fruits p r o d u c t s ( D e a k and Beuchat, 1993). M a n y environmental factors can have a significant influence on the microbial heat resistance (Beuchat, 1982). T h e effect of p H is one of the most important ( C o n d 6 n and Sala, 1992). However, there is limited information on the influence of p H on the thermal resistance of yeasts and the results o b t a i n e d are sometimes contradictory.
* Corresponding author. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00031-Z
210
S. Garza et aL / International Journal of Food Microbiology 23 (1994) 209-213
The aim of this study was to evaluate the heat resistance at real pasteurization temperatures of Saccharomyces cerevisiae strains isolated from spoiled peach puree using peach puree (pH 3.9) and McIlvaine buffer (pH 7 and 4) as heating menstrua. 2. Materials and methods
Microorganisms. The microorganisms used throughout this investigation (strains
173, 180 and 325) were isolated from spoiled peach puree and were identified by Centraalbureau voor Schimmelcultures (Delft, Netherlands) as Saccharomyces cerevisiae. Stock cultures were maintained on potato dextrose agar (Oxoid) slants at 4°C. The yeasts were grown at 28 °C for 48 h on malt extract agar (Oxoid) and harvested by flooding the agar surface with sterile, McIlvaine buffer, pH 7 (Dawson et al., 1969). The suspensions were centrifuged and washed five times in the same buffer. After the last centrifugation, the pellet was suspended again in McIlvaine buffer (pH 7) to obtain a concentration of c a . 10 9 yeast cells per ml, and stored at 0-5°C until used. The yeast cell concentration was estimated by microscopy. Heating menstruum. Mcllvaine buffer of pH 4 and 7 was prepared as described by
Dawson et al., (1969). Peach puree (pH 3.9) was obtained from Indulerida S.A. (Lleida, Spain) and was prepared from peeled, pitted, homogenized peaches. Heat treatments. Heat resistance determinations were carried out in a thermoresis-
tometer TR-SC (Zaragoza University, Zaragoza, Spain) as described by Cond6n et al., (1989). When the desired temperature was obtained (_+ 0.05°C), the menstruum (350 ml of McIlvaine buffer or peach puree) was inoculated with 0.2 ml of the yeast suspension under constant agitation (1500-2000 rev/min). Samples of 0.1 ml were then removed at intervals, transferred to tubes containing 15 ml of melted Sabouraud dextrose agar (Difco) at 45°C and pour plated. Enumeration of survivors. Colony forming units (CFU) were counted (Cond6n et
al., 1987) after incubation for 48 h at 28°C. D t and z-values. Decimal reduction times (Dt-values) were calculated by regression
analysis of the straight line portion of the survival curve (log~0 of survivors vs. time). Only survival curves with a coefficient of correlation (r o > 0.94) and with more than three values in the straight portion of the curve were used. Survival curves with a straight portion including less than 1 log cycle were rejected. The z-values were determined by regression analysis of log D t vs. the corresponding heating temperatures. Correlation coefficient and the comparison of slopes of decimal reduction time curves were carried out as described by Steel and Torrie (1960).
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
211
3. Results and discussion Tables
1, 2 a n d 3 s h o w D t a n d
resistance
of the
compared
to
obtained
three
yeast
published
results
at the lowest pH
z-values
strains
for the yeast strains studied.
using
(Torok
Mcllvaine
and
are in accordance
King,
buffer
1991).
In
at pH general,
with the investigations
The
heat
7 is h i g h
of Put and De
Table 1 Heat resistance data for Saccharomyces cerevisiae 173 isolated from spoiled peach puree Heatin g mediu m
Heating temperature (oc)
D-value (min)
Correlation coefficient a
McIlvaine buffer pH 7
58 60 62 64
3.00 0.75 0.23 0.08
-
McIlvaine buffer pH 4
58 60 62 64 58
5.68 1.18 0.22 0.06 2.95
- 0.999 -0.998 - 0.992 - 0.994 - 0.987
Peach puree
60 62 64
0.53 0.13 0.05
- 0.996 - 0.993 - 0.982
0.998 0.992 0.999 0.987
Equation of the T D T curve
Correlation coefficient b
z (°C)
Log D = 15.93 - 0.27T
- 0.994
3.75
Log D = 1 9 . 7 9 - 0 . 3 3 T
-0.998
3.04
Log D = 1 7 . 8 6 - 0.30T
- 0.994
3.32
For Survival Curve regression analysis. b For T h ermal D e a t h Time (TDT) regression analysis. a
Table 2 Heat resistance data for Saccharomyces cerevisiae 180 isolated from spoiled peach puree Heating m ediu m
Heating temperature (oc)
D-value (min)
Correlation coefficient a
Mcllvaine buffer pH 7
58 60 62 64
1.49 1.32 0.24 0.06
-
0.996 0.942 0.989 0.990
Mcllvaine buffer pH 4
58 60 62 64 58
1.53 0.68 0.14 0.04 0.67
-
0.993 0.996 0.998 0.996 0.988
Peach puree
60 62
0.20 0.05
- 0.996 - 0.991
Equ a t i on of the TDT curve
Correlation coefficient b
z (°C)
Log D = 15.45 - 0.25T
- 0.961
3.85
Log D = 15.45 - 0.26T
- 0.994
3.82
Log D = 16.76 - 0.29T
- 0.999
3.44
a For Survival Curve regression analysis. b For Thermal D e a t h Time (TDT) regression analysis.
as
D60-values
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
212
Table 3 Heat resistance data for Saccharomyces cerevisiae 325 isolated f r o m spoiled peach puree Heating
Heating
D-value
Correlation
Equation of the
Correlation
medium
temperature
(min)
coefficient a
T D T curve
coefficient b
56 58 62 64
2.36 0.56 0.03 0.01
0.998 -0.992 - 0.991 - 0.975
Log D = 1 7 . 1 4 - 0 . 3 0 T
-0.999
3.33
56 58 60 62
3.44 0.60 0.13 0.05
- 0.994 - 0.990 -0.991 - 0.999
L o g D = 17.49 - 0.30T
- 0.995
3.28
56
0.90
- 0.998
58
0.18
- 1.000
0.989
4.00
60 62
0.10 0.03
- 0.993 - 0.986
z (°C)
(°C) Mcllvaine
buffer pH 7 McIlvaine
buffer pH 4
Peach puree
L o g D = 13.85 - 0 . 2 5 T
a F o r Survival C u r v e regression analysis. b F o r Thermal Death T i m e ( T D T ) regression analysis.
Jong (1980) who studied the heat resistance of 120 yeast strains and showed Saccharomyces cereuisiae to be one of the most thermo-resistant. At pH 4 and in peach puree, a significant difference in heat resistance among the yeast strains studied was observed. The D60-value at pH 4 of the strain 325 was nine times lower than the D60-value for strain 173, and five times lower than for strain 180. For the same strain the heating menstruum pH did not have any great influence on the heat resistance. Results which are in accordance with Splittstoesser et al., (1975) who concluded that pH had limited effect on the thermoresistance of S. cerevisiae.
No significant differences (P < 0.01) in the z-values using buffer at pH 7, buffer at pH 4 and peach puree at pH 3.9 were observed (Tables 1-3). Similar results were obtained by other researchers (Barillere, 1981; Splittstoesser et al., 1975). The z-values obtained in the present study with the three Saccharomyces cerevisiae strains are similar to those reported for grape juice (Aref and Cruess, 1934), wine (Splittstoesser et al., 1975) and buffer at pH 3.2 (Barillere, 1981)
References Aref, H. and Cruess, W . V . (1934) A n investigation of the thermal death point of Saccharomyces ellipsoideus. J. Bact. 27, 4 4 3 - 4 5 2 . B e u c h a t , L . R . (1982) E f f e c t s of environmental stress in recovery media on colony f o r m a t i o n by sublethally heat-injured Saccharomyces cerevisiae. Trans. Br. Mycol. Soc. 78, 5 3 6 - 5 4 0 . Barillere, J.M. (1981) Etude de la thermor~sistance des soches de levures et de bact6ries lactiques isol6es de vins; influence du degr6 alcoolique et de la teneur en SO 2. Th~se D o c t e u r - I n g e n i e u r , E N S A de Montpellier, 196 pp.
S. Garza et al. / International Journal of Food Microbiology 23 (1994) 209-213
213
Cond6n, S., Lopez, P., Oria, R. and Sala, F.J. (1989) Thermal death determination: design and evaluation of a thermoresistometer. J. Food Sci. 54, 451-457. Cond6n, S., Oria, R. and Sala, F.J. (1987) Heat resistance of microorganisms: an improved method for survival counting. J. Microbiol. Meth. 7, 37-44. Cond6n, S. and Sala, F.J. (1992) Heat resistance of Bacillus subtilis in buffer and foods of different pH.J. Food Prot. 55, 605-608. Dawson, R.M.C, Elliot, C., Elliot, W. and Hand Jones, K.M. (1969) Data for Biochemical Research. Clarendon, Oxford, 654 pp. Deak, T. and Beuchat, L.R. (1993) Yeasts associated with fruit juice concentrates. J. Food Prot. 56, 777-782. Put, H.M.C. and De Jong, J. (1980) The heat resistance of selected yeasts causing spoilage of canned soft drinks and fruit products. In: F.A. Skinner, S.M. Passmore, R.R. Davenport (editors), Biology and Activities of Yeasts. Academic Press, New York, pp. 181-213. Splittstoesser, D.F., Lenk, L.L., Wilkinson, M. and Stamer, J.R. (1975) Influence of wine composition on the heat resistance of potential spoilage organisms. Appl. Microbiol. 30, 369-373. Steel, R.G. and Torrie, J.H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York, pp. 161-180. Torok, T. and King, A.D. (1991) Thermal inactivation kinetics of food-borne yeast. J. Food Sci. 56, 6-9.