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Resistance of vegetative cells and ascospores of heat resistant mould Talaromyces avellaneus to the high pressure treatment in apple juice
Journal of Food Engineering 61 (2004) 541–543 www.elsevier.com/locate/jfoodeng
Resistance of vegetative cells and ascospores of heat resistant mould Talaromyces avellaneus to the high pressure treatment in apple juice erovsk Michal Voldrich *, Jaroslav Dobi as, Lydie Tich a, Miroslav C y, Jana Kr atka Department of Food Preservation and Meat Technology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technick a 3, 166 28 Prague 6, Czech Republic Received 1 August 2002
Abstract Thermoresistant moulds including Talaromyces avellaneus are frequent microbial contamination of fruit juices and purees, which are supposed as food products suitable for pressure pasteurisation. The effect of high pressure treatment on survival of vegetative cells and ascospores of T. avellaneus was studied. The vegetative cells T. avellaneus were very sensitive to high pressure, the sufficient pressure treatment for the reduction by six orders was 200 MPa at 17 C for 60 min or 300 MPa at 17 C for 5 min. In apple juice the ascospores of T. avellaneus were relatively resistant to the high pressure treatment, the treatment of 600 MPa at 17 or 25 C for 60 min reduced their concentration by two or three orders, respectively. Sufficient inactivation rate was obtained when the combination of pressurisation and gentle heating was used, the total reduction (by six orders) was reached at 600 MPa and 60 C after 60 min. 2003 Elsevier Ltd. All rights reserved. Keywords: High pressure treatment; Tallaromyces avellaneus; Fruit products; Ascospores; Resistance
1. Introduction Heat resistant fungi often cause spoilage of pasteurised and canned fruit products. Mild thermal processing can usually inactivate most acidophile microorganisms that are only slightly heat resistant. Some fungi, especially ascospores producing moulds, are able to survive more severe heat treatment. Talaromyces avellaneus is ranked among them. It can be found as a natural contamination in fruit products. T. avellaneus belongs to the perfect fungus forming three different kinds of cells–– vegetative hyphae, asexually derived conidiospores and sexually derived ascospores. The ascospores are known to be very stable against heating (King & Halbrook, 1987; King & Whitehand, 1990; Tournas, 1994). Baroresistance of moulds in fruit juices was studied by several authors (Cheftel, 1992; Ifuku, Takahashi, & Yamasaki, 1993; Maggi, 1994) and high pressure treatment seems to be a very promising possibility of their inactivation. Generally, the pressure treatment at 450– 600 MPa and 25–50 C for 5–30 min have been found to
*
Corresponding author. Tel.: +420-224353012; fax: 233337337. E-mail address: [email protected] (M. Voldrich).
+420-
0260-8774/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00224-3
cause significant inactivation of moulds, yeasts as well as bacteria in fruit products. As T. avellaneus have been found in processed fruit juices in Czech Republic the aim of this study was to determine the resistance of strains isolated from these drinks.
2. Material and methods 2.1. Moulds T. avellaneus (Thom and Turesson) C.R. Benjamin was isolated from processed apple juice. Culture was grown at 30 C on Malt Extract Agar (Imuna Sariske Michalany, Slovakia). Vegetative cells (mycelium and conidiospores) were harvested after 1 week cultivation. Ascospores were taken after 8 weeks cultivation. To break asci and get free ascospores the raw material was shaken with glass beads in ultrasonic bath and filtered through a sterile glass wool. For the separation of ascospores from vegetative cells the centrifugation in 34% CsCl solution for 20 min at 1100g was used. The white sediment in the bottom of the tube contained more than 90% of single ascospores, which could be distinguished
M. Voldrich et al. / Journal of Food Engineering 61 (2004) 541–543
2.2. High-pressure devices Laboratory press (Department of Physics of Czech Academy of Science in Prague, Czech Republic): one chamber (i.d. 22 mm, capacity 53 ml), the pressure transmitting medium mineral oil OL3. Press type CYX 6/0103 (Zdas a.s., Czech Republic): one chamber (i.d. 70 mm, capacity 1.2 l), the pressure transmitting medium was water.
8.00 7.00 6.00
(log CFU/ml)
from conidiospores by phase contrast microscopy. The suspension of ascospores was twice washed by sterile distilled water and concentration determined by counting in B€ urker cell. The ascospores suspension was then stored in refrigerator at 4 C and before test it was diluted with sterile apple juice.
viabillity of vegetative cells
542
5.00 4.00 3.00 2.00 1.00 0.00 0.00
10.00
20.00
30.00
40.00
50.00
60.00
t (min) device A
device B
detection
Fig. 1. Baroinactivation of vegetative cells of T. avellaneus at 200 MPa and 17 C in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press, device B ¼ press CYX 6/0103, detection ¼ detection limit.
The ascospore suspension (2 ml) was filled into sterile flexible plastic bags (low-density polyethylene film thickness 80 lm) and pressurised. The number of surviving microorganisms was determined by the surfacespatula method. For cultivation Rose Bengal Agar Base (HiMedia, India) was used. Plates were counted after 3 days incubation at 30 C. The results were presented in colony forming units (CFU) per ml. Two series of tests were done, in each of them five parallel samples were analysed for the determination of vegetative cells as well as ascospores baroresistance. The mean value (x) and the standard deviation (SD) were calculated for each of the followed parameters.
Numbre of surviving ascospores (log CFU/ml)
2.3. Treatment and cultivation 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 0
10
20 600 MPa
30 t (min) 500 MPa
40 400 MPa
50
60
300 MPa
Fig. 2. Baroinactivation of ascospores T. avellaneus at different pressures and 17 C in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press.
3. Results Vegetative cells. The results of baroresistance of vegetative cells of tested strains of T. avellaneus in apple juice are given in Fig. 1. It is obvious, that their sensitivity to high pressure is comparable with that of other vegetative cells of moulds or yeast as it is referred in available literature (Maggi, 1994; Tournas, 1994). In our case the pressure 200 MPa at 17 C caused the decrease of concentration of viable vegetative cells from initial level 6 · 106 CFU/ml to less than 1 · 101 CFU/ml, i.e. nearly by six orders, after 60 min. Ten and twenty minutes treatment at given conditions reduced the number of viable vegetative cells by three and five orders, respectively. The influence of the treatment at 100 MPa at the same conditions was less severe and caused the vegetative cell inactivation by four orders after 60 min. It can be supposed, that high pressure and high temperature would accelerate mould inactivation. The single experiment at 600 MPa and 17 C under the same conditions as mentioned above caused nearly complete
inactivation of vegetative cells of T. avellaneus, i.e. reduction by six orders in 5 min. From the Fig. 1 it can be also seen, that inactivation efficiency of both presses was similar. There was no significant difference using StudentÕs test. Ascospores. The results on high pressure treatment of ascospores of T. avellaneus are listed in Fig. 2. The ascospores are baroresistant in contrast with vegetative cells, e.g. 60 min treatment of 600 MPa at 17 C caused decrease of two orders only. The higher pressure was used the faster ascospores destruction was observed. The same experiments were done at 25 and 60 C. While stability of ascospores at 25 C was rather close to that at 17 C, at 60 C the inactivation efficiency of high pressure was much higher. Obtained data of ascospores baroresistance were used for D–p diagram construction (see Fig. 3). D value (decimal reduction time) represents time necessary for reduction of viable ascospores concentration by one order. While D value at 300 MPa and 17 C was 85 min, at 600 MPa and 17 C it decreased to
M. Voldrich et al. / Journal of Food Engineering 61 (2004) 541–543
great destruction of cells after action of 600 MPa at 17 C for 60 min is apparent.
100
D (min)
543
10
4. Conclusions
1 250
300
350
400
450
500
550
600
650
p (MPa) 17 °C
25 °C
60 °C
Fig. 3. D–p diagram of inactivation of T. avellaneus ascospores in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press.
32 min. At 60 C D value dropped from 65 min at 300 MPa to nearly 10 min at 600 MPa. The effect of high pressure treatment on T. avellaneus ascospores is also obvious from Figs. 4 and 5. While in Fig. 4 the fresh ascospores can be seen, from Fig. 5 the
There is significant difference in baroresistance of vegetative cells and ascospores of T. avellaneus. While first are quite sensitive to high pressure action, ascospores are much more resistant. Considering the common levels of contamination of freshly prepared fruit juices or purees at about 103 –105 CFU/ml of yeast or moulds the treatment using 300–500 MPa for 5–20 min seems to be sufficient for sterilization. When the presence of ascosporogenic thermoresistant moulds is expected, the use of higher pressure is necessary, i.e. treatment at 600– 700 MPa for longer time or its combination with other factors, usually with gentle heating.
Acknowledgements The experimental part of this work was realised in cooperation with Food Research Institute Prague (CZ) under the financial support of the Czech Ministry of Agriculture projects EP 9026 and EP 0960006258. The authors thank to Dr. Zdenka Jasenska (IPCM Bratislava, Slovakia) for identification of Talaromyces avellaneus strains and to Dr. Olga Kofro nov a (IM CAS Prague, Czech republic) for preparing of electron microscope photos.
Fig. 4. Vegetative cells and ascospores T. avellaneus not pressurised (photoelectron microscope, 4500·, Olga Kofro nova, IM CAS Prague).
Fig. 5. Vegetative cells and ascospores T. avellaneus pressurised using 600 MPa at 17 C for 60 min, device B (photo electron raster microscope, 4500·, Olga Kofro nova, IM CAS Prague).
References Cheftel, J. C. (1992). Effects of high hydrostatic pressure on food constituents: an overview. In C. Balny, R. Hayashi, K. Heremans, & P. Masson (Eds.), High pressure and biotechnology (pp. 195–209). London: Colloque INSERM/John Libbey Eurotext Ltd. Ifuku, Y., Takahashi, Y., & Yamasaki, S. (1993). New developments on the Japanese fruit juice/drink market––(part 1). Fruit Processing, 1, 19–22. King, A. D., Jr., & Halbrook, W. U. (1987). Ascospore heat resistance and control measures for Talaromyces flavus isolated from fruit juice concentrate. Journal of Food Science, 52(5), 1252–1254, 1266. King, A. D., Jr., & Whitehand, L. C. (1990). Alteration of Talaromyces flavus heat resistance by growth conditions and heating medium composition. Journal of Food Science, 55(3), 830–832. Maggi, A. (1994). High pressure treatments of ascospores of heat resistant moulds and patulin in apricot nectar and water. Industria Conserve, 69(1), 26–29. Tournas, V. (1994). Heat resistant fungi of importance to the food and beverages industry. Critical Reviews in Microbiology, 20(2), 243– 263.
Resistance of vegetative cells and ascospores of heat resistant mould Talaromyces avellaneus to the high pressure treatment in apple juice erovsk Michal Voldrich *, Jaroslav Dobi as, Lydie Tich a, Miroslav C y, Jana Kr atka Department of Food Preservation and Meat Technology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technick a 3, 166 28 Prague 6, Czech Republic Received 1 August 2002
Abstract Thermoresistant moulds including Talaromyces avellaneus are frequent microbial contamination of fruit juices and purees, which are supposed as food products suitable for pressure pasteurisation. The effect of high pressure treatment on survival of vegetative cells and ascospores of T. avellaneus was studied. The vegetative cells T. avellaneus were very sensitive to high pressure, the sufficient pressure treatment for the reduction by six orders was 200 MPa at 17 C for 60 min or 300 MPa at 17 C for 5 min. In apple juice the ascospores of T. avellaneus were relatively resistant to the high pressure treatment, the treatment of 600 MPa at 17 or 25 C for 60 min reduced their concentration by two or three orders, respectively. Sufficient inactivation rate was obtained when the combination of pressurisation and gentle heating was used, the total reduction (by six orders) was reached at 600 MPa and 60 C after 60 min. 2003 Elsevier Ltd. All rights reserved. Keywords: High pressure treatment; Tallaromyces avellaneus; Fruit products; Ascospores; Resistance
1. Introduction Heat resistant fungi often cause spoilage of pasteurised and canned fruit products. Mild thermal processing can usually inactivate most acidophile microorganisms that are only slightly heat resistant. Some fungi, especially ascospores producing moulds, are able to survive more severe heat treatment. Talaromyces avellaneus is ranked among them. It can be found as a natural contamination in fruit products. T. avellaneus belongs to the perfect fungus forming three different kinds of cells–– vegetative hyphae, asexually derived conidiospores and sexually derived ascospores. The ascospores are known to be very stable against heating (King & Halbrook, 1987; King & Whitehand, 1990; Tournas, 1994). Baroresistance of moulds in fruit juices was studied by several authors (Cheftel, 1992; Ifuku, Takahashi, & Yamasaki, 1993; Maggi, 1994) and high pressure treatment seems to be a very promising possibility of their inactivation. Generally, the pressure treatment at 450– 600 MPa and 25–50 C for 5–30 min have been found to
*
Corresponding author. Tel.: +420-224353012; fax: 233337337. E-mail address: [email protected] (M. Voldrich).
+420-
0260-8774/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00224-3
cause significant inactivation of moulds, yeasts as well as bacteria in fruit products. As T. avellaneus have been found in processed fruit juices in Czech Republic the aim of this study was to determine the resistance of strains isolated from these drinks.
2. Material and methods 2.1. Moulds T. avellaneus (Thom and Turesson) C.R. Benjamin was isolated from processed apple juice. Culture was grown at 30 C on Malt Extract Agar (Imuna Sariske Michalany, Slovakia). Vegetative cells (mycelium and conidiospores) were harvested after 1 week cultivation. Ascospores were taken after 8 weeks cultivation. To break asci and get free ascospores the raw material was shaken with glass beads in ultrasonic bath and filtered through a sterile glass wool. For the separation of ascospores from vegetative cells the centrifugation in 34% CsCl solution for 20 min at 1100g was used. The white sediment in the bottom of the tube contained more than 90% of single ascospores, which could be distinguished
M. Voldrich et al. / Journal of Food Engineering 61 (2004) 541–543
2.2. High-pressure devices Laboratory press (Department of Physics of Czech Academy of Science in Prague, Czech Republic): one chamber (i.d. 22 mm, capacity 53 ml), the pressure transmitting medium mineral oil OL3. Press type CYX 6/0103 (Zdas a.s., Czech Republic): one chamber (i.d. 70 mm, capacity 1.2 l), the pressure transmitting medium was water.
8.00 7.00 6.00
(log CFU/ml)
from conidiospores by phase contrast microscopy. The suspension of ascospores was twice washed by sterile distilled water and concentration determined by counting in B€ urker cell. The ascospores suspension was then stored in refrigerator at 4 C and before test it was diluted with sterile apple juice.
viabillity of vegetative cells
542
5.00 4.00 3.00 2.00 1.00 0.00 0.00
10.00
20.00
30.00
40.00
50.00
60.00
t (min) device A
device B
detection
Fig. 1. Baroinactivation of vegetative cells of T. avellaneus at 200 MPa and 17 C in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press, device B ¼ press CYX 6/0103, detection ¼ detection limit.
The ascospore suspension (2 ml) was filled into sterile flexible plastic bags (low-density polyethylene film thickness 80 lm) and pressurised. The number of surviving microorganisms was determined by the surfacespatula method. For cultivation Rose Bengal Agar Base (HiMedia, India) was used. Plates were counted after 3 days incubation at 30 C. The results were presented in colony forming units (CFU) per ml. Two series of tests were done, in each of them five parallel samples were analysed for the determination of vegetative cells as well as ascospores baroresistance. The mean value (x) and the standard deviation (SD) were calculated for each of the followed parameters.
Numbre of surviving ascospores (log CFU/ml)
2.3. Treatment and cultivation 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 0
10
20 600 MPa
30 t (min) 500 MPa
40 400 MPa
50
60
300 MPa
Fig. 2. Baroinactivation of ascospores T. avellaneus at different pressures and 17 C in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press.
3. Results Vegetative cells. The results of baroresistance of vegetative cells of tested strains of T. avellaneus in apple juice are given in Fig. 1. It is obvious, that their sensitivity to high pressure is comparable with that of other vegetative cells of moulds or yeast as it is referred in available literature (Maggi, 1994; Tournas, 1994). In our case the pressure 200 MPa at 17 C caused the decrease of concentration of viable vegetative cells from initial level 6 · 106 CFU/ml to less than 1 · 101 CFU/ml, i.e. nearly by six orders, after 60 min. Ten and twenty minutes treatment at given conditions reduced the number of viable vegetative cells by three and five orders, respectively. The influence of the treatment at 100 MPa at the same conditions was less severe and caused the vegetative cell inactivation by four orders after 60 min. It can be supposed, that high pressure and high temperature would accelerate mould inactivation. The single experiment at 600 MPa and 17 C under the same conditions as mentioned above caused nearly complete
inactivation of vegetative cells of T. avellaneus, i.e. reduction by six orders in 5 min. From the Fig. 1 it can be also seen, that inactivation efficiency of both presses was similar. There was no significant difference using StudentÕs test. Ascospores. The results on high pressure treatment of ascospores of T. avellaneus are listed in Fig. 2. The ascospores are baroresistant in contrast with vegetative cells, e.g. 60 min treatment of 600 MPa at 17 C caused decrease of two orders only. The higher pressure was used the faster ascospores destruction was observed. The same experiments were done at 25 and 60 C. While stability of ascospores at 25 C was rather close to that at 17 C, at 60 C the inactivation efficiency of high pressure was much higher. Obtained data of ascospores baroresistance were used for D–p diagram construction (see Fig. 3). D value (decimal reduction time) represents time necessary for reduction of viable ascospores concentration by one order. While D value at 300 MPa and 17 C was 85 min, at 600 MPa and 17 C it decreased to
M. Voldrich et al. / Journal of Food Engineering 61 (2004) 541–543
great destruction of cells after action of 600 MPa at 17 C for 60 min is apparent.
100
D (min)
543
10
4. Conclusions
1 250
300
350
400
450
500
550
600
650
p (MPa) 17 °C
25 °C
60 °C
Fig. 3. D–p diagram of inactivation of T. avellaneus ascospores in apple juice (10.8% (w/w), pH 3.45). Device A ¼ laboratory press.
32 min. At 60 C D value dropped from 65 min at 300 MPa to nearly 10 min at 600 MPa. The effect of high pressure treatment on T. avellaneus ascospores is also obvious from Figs. 4 and 5. While in Fig. 4 the fresh ascospores can be seen, from Fig. 5 the
There is significant difference in baroresistance of vegetative cells and ascospores of T. avellaneus. While first are quite sensitive to high pressure action, ascospores are much more resistant. Considering the common levels of contamination of freshly prepared fruit juices or purees at about 103 –105 CFU/ml of yeast or moulds the treatment using 300–500 MPa for 5–20 min seems to be sufficient for sterilization. When the presence of ascosporogenic thermoresistant moulds is expected, the use of higher pressure is necessary, i.e. treatment at 600– 700 MPa for longer time or its combination with other factors, usually with gentle heating.
Acknowledgements The experimental part of this work was realised in cooperation with Food Research Institute Prague (CZ) under the financial support of the Czech Ministry of Agriculture projects EP 9026 and EP 0960006258. The authors thank to Dr. Zdenka Jasenska (IPCM Bratislava, Slovakia) for identification of Talaromyces avellaneus strains and to Dr. Olga Kofro nov a (IM CAS Prague, Czech republic) for preparing of electron microscope photos.
Fig. 4. Vegetative cells and ascospores T. avellaneus not pressurised (photoelectron microscope, 4500·, Olga Kofro nova, IM CAS Prague).
Fig. 5. Vegetative cells and ascospores T. avellaneus pressurised using 600 MPa at 17 C for 60 min, device B (photo electron raster microscope, 4500·, Olga Kofro nova, IM CAS Prague).
References Cheftel, J. C. (1992). Effects of high hydrostatic pressure on food constituents: an overview. In C. Balny, R. Hayashi, K. Heremans, & P. Masson (Eds.), High pressure and biotechnology (pp. 195–209). London: Colloque INSERM/John Libbey Eurotext Ltd. Ifuku, Y., Takahashi, Y., & Yamasaki, S. (1993). New developments on the Japanese fruit juice/drink market––(part 1). Fruit Processing, 1, 19–22. King, A. D., Jr., & Halbrook, W. U. (1987). Ascospore heat resistance and control measures for Talaromyces flavus isolated from fruit juice concentrate. Journal of Food Science, 52(5), 1252–1254, 1266. King, A. D., Jr., & Whitehand, L. C. (1990). Alteration of Talaromyces flavus heat resistance by growth conditions and heating medium composition. Journal of Food Science, 55(3), 830–832. Maggi, A. (1994). High pressure treatments of ascospores of heat resistant moulds and patulin in apricot nectar and water. Industria Conserve, 69(1), 26–29. Tournas, V. (1994). Heat resistant fungi of importance to the food and beverages industry. Critical Reviews in Microbiology, 20(2), 243– 263.