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Byssochlamys nivea inactivation in pineapple juice and nectar using high pressure cycles
Journal of Food Engineering 95 (2009) 664–669
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Byssochlamys nivea inactivation in pineapple juice and nectar using high pressure cycles Elisa Helena da Rocha Ferreira a, Amauri Rosenthal b,*, Verônica Calado a, Jorge Saraiva c, Sónia Mendo d a
Federal University of Rio de Janeiro, Chemistry and Biochemistry Processes Post Graduation, Rio de Janeiro, RJ, Brazil Embrapa Labex Europe, University of Burgundy, GPMA, 1, Esplanade Erasme, F21000 Dijon, France c Aveiro University, Department of Chemistry, 3810-193 Aveiro, Portugal d Aveiro University, Department of Biology, 3810-193 Aveiro, Portugal b
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 29 May 2009 Accepted 20 June 2009 Available online 17 July 2009 Keywords: High pressure Byssochlamys nivea Pineapple juice
a b s t r a c t The aim of this work was to assess the inactivation of Byssochlamys nivea ascospores in pineapple juice and nectar by combining pressure sequences involving high pressure cycles with relatively mild thermal processing. The effect of 550 and 600 MPa sustained pressures (holding time of 15 min), combinations of sustained pressures and pressure pulses (holding time of 10 s), and pressure cycles (two, three and five cycles of 550 and 600 MPa for 7.5, 5 and 3 min, respectively), at 20, 40, 60, 70, 80 and 90 °C were compared. B. nivea ascospores were inactivated by applying sustained a pressure of 600 MPa at 90 °C for 5 min (juice) and 15 min (nectar), and three and five cycles of pressure at 600 MPa and 80 °C for nectar with holding time of 5 and 3 min, respectively, and in all pressure cycles for juice. In general, pressure cycles were more effective for inactivating B. nivea ascospores than the application of sustained high pressures. Ó 2009 Published by Elsevier Ltd.
1. Introduction Ascospores of Byssochlamys fulva, B. nivea, Neosartorya fischeri and Talaromyces flavus are extremely heat resistant and frequently associated with the deterioration of thermally treated fruit products (Hocking and Pitt, 1984; Beuchat, 1986). The sexual reproduction of B. nivea is provided by ascospores which are produced in saclike specialized terminal cells called asci. Normally, in one ascus eight ascospores are produced (Beuchat and Rice, 1979). Elevate heat resistance is caused by ascospores which possess high resistance to pH variation, and environments with high sugar, fat and acids concentrations (Tournas, 1994). The soil is the principal contamination source of B. nivea. The ascospores can remain dormant, survive the commercial pasteurization conditions normally applied to fruit products, and spoil such products by germinating and growing even under reduced oxygen conditions (Kotzekidou, 1997; Obeta and Ugwuanyi, 1995). The ascospores’ dormancy can be interrupted by sub-lethal stresses, such as mild to highly intense heat treatments depending on the microorganism, leading to the so called spores’ activation, which allows further germination and growth in favorable conditions (Beuchat and Rice, 1979; Beuchat, 1986; Conner and Beuchat,
* Corresponding author. Tel.: +33 3 80396656; fax: +33 3 80396898. E-mail address: [email protected] (A. Rosenthal). 0260-8774/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2009.06.053
1987; Beuchat, 1988; Splittstoesser et al., 1993). Dijksterhuis and Teunisse (2004) associated the ascospores activation to cell wall damage without inactivation and verified that pressure treatments between 400 and 800 MPa could activate Taloramyces macrosporus ascospores. The growth of B. nivea can produce CO2 which inflates fruit juice packages, and causes visual deterioration of the juice due to pectinolytic enzymes activity (Ugwuanyi and Obeta, 1999). Besides, some strains of Byssochlamys are mycotoxigenic due to their ability to produce byssochlamic acid, patulin and byssotoxin A (Beuchat and Rice, 1979). Pineapple nectar can be susceptible to filamentous mold contamination (Masson, 2004). Some fungi, specially ascospores producing molds, are very often able to survive even to more severe thermal treatments. The thermal conditions required to inactivate B. nivea ascospores in fruit products generally affect the sensory quality (Beuchat and Rice, 1979; Tournas, 1994; Butz et al., 1996). Preservation by high hydrostatic pressure in combination with mild temperature can possibly be an alternative for fruit juice processing (Palou et al., 1998). Previous studies showed that high pressure preserves the overall sensory quality of pineapple (Laboissiére et al., 2007) and passion fruit (Marcellini et al., 2006) juices in comparison to pasteurization. The principal aim of this work is to evaluate whether a combination of the application of novel high pressure sequences with the use of moderate temperatures can be used to inactivate ascospores in pineapple drinks.
E. H. d. R. Ferreira et al. / Journal of Food Engineering 95 (2009) 664–669
Combined treatments of high pressure and thermal processing both generally in very high intensity have been reported to achieve spore forming microorganism inactivation in food products. A combination of high pressure and temperature can increase the wall permeability and burst the ascospores. Maggi et al. (1994), Butz et al. (1996) and Palou et al. (1998) demonstrated that pressures above 600 MPa and temperatures greater than 60 °C could inactivate B. nivea ascospores. Palou et al. (1998) studied the effect of continuous pressure application (689 MPa for 5, 15 and 25 min) and the application of oscillatory pressures (one, three or five cycles at 689 MPa with holding time of 1 s) on ascospores of B. nivea in apple and cranberry juices having two different water activity values (0.94 and 0.98) at 21 and 60 °C. No inactivation was observed at 21 °C but a 4 log reduction was observed following the application of 3 or 5 cycles at 60 °C to both juices at a higher water activity. Butz et al. (1996) verified that ascospores of B. nivea were only inactivated above 600 MPa in combination with temperatures above 60 °C and Palou et al. (1998) suggested that inactivation was more effective when pressure treatments are repeated in close succession (pressure cycles). The use of simultaneous application of pressure and temperature to destroy spores of microorganisms is expected to gain much interest in the near future, since US Food and Drug Administration has recently approved the so called pressure assisted thermal processing (PATP) as a possible sterilization process (Anonymous, 2009; Barbosa-Cánovas and Juliano, 2008). This paper aims to assess the effect of constant pressure (550 and 600 MPa) followed or not by pressure pulses (holding time of 10 s) and the effect of pressure cycles (two, three and five cycles at 550 and 600 MPa for 7.5, 5 and 3 min, respectively) at different temperatures (20, 40, 60, 70, 80 and 90 °C) on the inactivation of B. nivea ascospores in pineapple nectar and juice.
2. Materials and methods 2.1. The Byssochlamys nivea The mold B. nivea used in this investigation was isolated from Brazilian fresh strawberry by Aragão (1989) and identified according to standard procedures (Pitt and Hocking, 1985). Ascospores were obtained after 30 days of the mold growth at 30 °C on malt extract agar (Merck) and produced according to Pitt and Hocking (1985). Ascospores were harvested using 25 mL of sterile water. Hyphal fragments were removed by filtering the suspension through sterile glass cotton. The ascospores suspension was collected in a sterile bottle containing glass pearls and then homogenized in vortex for 15 min to liberate the ascospores from the asci. Microscopic observation revealed a high proportion (>90%) of free ascospores.
2.2. Pineapple juice and nectar Commercial pasteurized pineapple juice (Aw 0.93, pH 3.7, 13°Brix, without adding sucrose) and nectar (Aw 0.94, pH 3.7, 12°Brix, with sucrose addition) were used. B. nivea ascospores suspension was inoculated to give an initial concentration (N0) of 105– 106 ascospores/mL. The samples (5 mL) were filled into sterile flexible plastic bags (low-density polyethylene) and pressurized. The total carbohydrate content in pineapple juice and nectar was 10 g/100 mL and 12.5 g/100 mL, respectively. Water activity (Aw) was determined using a rotronic hygrometer (Hygroskop DV-2). Analyses were performed in triplicate to yield mean values and standard error.
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2.3. High pressure treatments Different high pressure sequences were applied to the materials: sustained pressures and combinations of sustained pressures and pressure cycles. These treatments were carried out at temperatures between 20 and 90 °C as showed in Tables 1 and 2. The equipment used (Unipress Equipment, Model U33, Poland) consisted of a 100 mL pressure vessel (35 mm diameter and 100 mm height), which was surrounded by an external jacket connected to a thermostatic bath in order to control the temperature. A mixture of propylene glycol and water (1:1) constituted the pressurization fluid. The temperature of pressure transfer liquid and samples (pineapple juice and nectar) were set at the processing temperature (Tables 1 and 2) before applying the pressure sequence. Immediately after pressurization, the samples were removed and cooled down in ice bath. Unpressurized samples were used as control and measurements were duplicated. The temperature of the pressurization fluid was recorded along the treatment by a thermocouple inserted in the basis of the chamber. Laboissiére et al. (2005) verified that the compression heating of pineapple juice was in average 1.4 °C/100 MPa. However, in this study it was assumed that the sample temperature was equivalent to the pressurization fluid temperature due to process controlling and monitoring limitations. Iron bars were inserted inside the chamber to decrease the fluid volume required to the process and as a consequence avoiding great temperature reduction (loss).
2.4. Sustained pressure treatments Treatments at 600 MPa for 15 min were evaluated at 20, 40, 60, 70, 80 and 90 °C. The inoculum was found to be totally inactivated (Fig. 2) in both products at 90 °C. Then, the inactivation levels at 600 MPa and 90 °C for 5, 10 and 15 min were verified. The time taken to reach 550 and 600 MPa (i.e. come up time) was around 1 min and the decompression time was 10 s.
2.5. Pressure cycles Fig. 1 and Table 1 shows the combination of pressure cycles applied in pineapple juice and nectar. Also, three combinations of sustained pressures and pressure cycles were applied as shows Table 2. The pause between high pressure cycles treatments was set at 1 min (Fig. 1).
2.6. Determination of ascospores counts The initial count (N0) of B. nivea ascospores and the numbers surviving the pressure treatments were determined by spread plating on malt extract agar (Merck). Two plates were used for each dilution and incubated for 3–7 days at 30 °C. Complete inactivation was assumed to have happened when no observable cell growth occurred in the considered condition.
2.7. Statistical analyses Statistical analyses were carried out using Statistica 8.0. The significance differences between the results were calculated by analyses of variance (ANOVA). Differences at p < 0.05 were considered to be significant.
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Table 1 Experimental conditions evaluated in sustained pressures and pressure cycles runs. Pressure (MPa)
Temperature (°C)
Number of cycles
Holding time (min)
Representation
550
20, 40 and 60
1 2 3 5
15 7.5 5 3
1 150 2 7.50 3 50 5 30
600
20, 40, 60, 70 and 80
1 2 3 5
15 7.5 5 3
1 150 2 7.50 3 50 5 30
Table 2 Sequences involving combinations of sustained pressures and pressure cycles. Pressure (MPa)
Temperature (°C)
Sequences of treatments
550 and 600
20
Sustained pressure for 15 min. Two pressure pulses of 10 s. Two pressure pulses of 10 s.
Representation Two pressure pulses of 10 s. Sustained pressure for 15 min. Five pressure pulses of 3 min.
1 150 + 2 1000 2 1000 + 1 150 2 1000 + 5 30
3. Results 3.1. Sustained pressure treatments The combination of pressure and temperature applied determines the extent of B. nivea ascospores inactivation. Fig. 2 clearly shows that the extent of inactivation increases with temperature at 600 MPa for both pineapple juice and nectar. By using a LSD (Least Square Difference) test, it is possible to affirm that there were not significant differences in ascospores inactivation with temperature, except at 80 °C, considering 5% of significance. This may happen because of the higher sucrose concentration in pineapple nectar that could protect the ascospores during the processes. Maggi et al. (1994) observed that treatments at 800 MPa and 50 °C for 4 min or 900 MPa and 50 °C for 2 min were enough to reduce the counts of thermal resistant ascospores by 4 log cycles while Butz et al. (1996) demonstrated that continuous treatments at 700 and 800 MPa at 60–70 °C rapidly inactivated B. nivea ascospores. Butz et al. also noted that no inactivation occurred at 0 and 40 °C. Herein, we found a reduction of 5.6 log cycles in pineapple nectar at 90 °C and 5.7 log cycles in pineapple juice at 80 and
Fig. 2. Inactivation of Byssochlamys nivea ascospores at 600 MPa for 15 min at different temperatures in pineapple juice (h) and pineapple nectar (j). (N0) is the initial count of Byssochlamys nivea ascospores.
90 °C. Temperatures of 40 and 60 °C decrease the count only by 0.5 log cycles in pineapple nectar, while at 70 °C the decrease is 2.0 log cycles for juice and 1.5 log cycles for nectar. Spore count increases in juice following treatments at 20 °C which can be attributed to the ascospores liberation from the ascus
Fig. 1. Pressure cycles applied in pineapple juice and nectar. (a) Sustained pressure of 550 or 600 MPa for 15 min. (b) Combination of two cycles of 7.5 min at 550 or 600 MPa. (c) Combination of three cycles of 5 min at 550 or 600 MPa. (d) Combination of five cycles of 3 min at 550 or 600 MPa: (1) come up time (1 min); (2) holding time; (3) depressurization (10 s); (4) pause time (1 min).
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or the ascospores activation. Similar results were found by Palou et al. (1998) when ascospores of B. nivea were treated at 689 MPa for 15 and 25 min at 21 °C and by Maggi et al. (1994) at 900 MPa for 20 min at 20 °C. The results show that the application of heat or high pressure could break up the dormant state in spores of fungi and ensure the germination. Similarly to mild heating considering the elevate spores resistance, intermediate intense high pressure activate the fungi spores, i.e. interrupts the dormant state by supplying appropriate conditions for initiating spores germination. The effects of applying sustained pressure of 600 MPa at 90 °C for 5, 10 and 15 min are shown in Fig. 3. In pineapple juice, a reduction of 6 log cycles was observed after 5 min of processing, while the same extent of reduction was only observed after 10 min in the case of pineapple nectar. The effects of time and type of drink (juice or nectar) were statistically significant at 5% of significance level. Probably the higher sucrose concentration increases the ascospores resistance in nectar. The difference between ascospores inactivation in juice and nectar can be attributed to a higher baroprotective effect of sucrose that is present at higher concentration in the nectar. The same effect happened when it was used the treatment of 600 MPa and 80 °C for 15 min (Fig. 2) and also observed by Koseki and Yamamoto (2007).
3.2. Pressure cycles Palou et al. (1998) observed no inactivation of B. nivea ascospores when pressure cycles (one, three or five cycles at 689 MPa with holding time of 1 s) were applied at 21 °C. Figs. 4–6 shows that inactivation of B. nivea ascospores is more noticeable in treatments involving cyclic variations of pressure whilst maintaining the same overall treatment time (i.e. five cycles lasting 3 min each or three cycles lasting 5 min each, as opposed to a single cycle of pressure for 15 min). At 20 °C, Fig. 4 shows that at 550 MPa an effective pressure sequence for inactivation of B. nivea ascospores is the one involving a single cycle of pressure for 15 min, followed by the application of two 10-s pressure pulses after decompression to atmospheric pressure. This is indeed more effective than following the same sequence at 600 MPa and treatments at 550 and 600 MPa for 15 min at all temperatures (Figs. 5 and 6), and may be attributed to increase the ascospores fragility after the first single cycle at 550 MPa for 15 min, becoming easier the inactivation with the two pulses after on. However, same treatments at 40 and 60 °C (data not shown) demonstrated activation on B. nivea ascospores. The same ascospores activation might explain the results observed for a single 15 min pressure cycle at 600 MPa. It is clear that the best treatment applied in pineapple juice and nectar at 20 °C
Fig. 3. Inactivation of Byssochlamys nivea ascospores with processing time at 600 MPa and 90 °C in pineapple juice (h) and pineapple nectar (j).
Fig. 4. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles at 20 °C. Maximum pressure: 550 (h) and 600 MPa (j). (N0) is the initial count of Byssochlamys nivea ascospores.
Fig. 5. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles, maximum pressure = 550 MPa at different temperatures (20, 40 and 60 °C). (N0) is the initial count of Byssochlamys nivea ascospores.
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each at 80 °C, it was possible to completely inactivate the ascospores in both products, meaning that no viable cell was detected after the treatment (Fig. 6). This is consistent with Butz et al. (1996) who noted that B. nivea ascospores could survive any treatment, including cycling pressure exposure up to 500 MPa and 70 °C for several hours. Palou et al. (1998) noted that pressure sequences involving cyclic compression and decompression at 689 MPa at 60 °C could inactivate heat resistant molds in diluted fruit juices (Aw 0.98), but could not inactivated the same molds in juices with lower Aw. The present study has succeeded in inactivating B. nivea ascospores by 5.7 log cycles in pineapple juice (Aw 0.93) and nectar (Aw 0.94), by using sequences involving high pressure cycles up to 600 MPa at 80 °C. 4. Conclusion
Fig. 6. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles, maximum pressure = 600 MPa at different temperatures (20, 40, 60, 70 and 80 °C). (N0) is the initial count of Byssochlamys nivea ascospores.
(Fig. 4) was five cycles of 3 min in both high pressure treatments (550 and 600 MPa). There were statistical differences among the treatments (pressure level and cycles number), and the ascospores inactivation in juice and nectar was statistically equivalent, considering 5% of significance. The most effective sequence involving 550 MPa was the use of five cycles of 3 min each for all three temperatures used. Such combination resulted in count reductions as high as 3.9 logs in juice and 4.5 logs in nectar at 40 °C (Fig. 5). The statistical difference appears only at temperature level and cycles number, being the ascospores inactivation in juice and nectar again statistically equal. Contrary to the report by Palou et al. (1998), treatments at 60 °C resulted in lower inactivation than at 20 °C, possibly because of ascospore activation at 550 MPa. At 600 MPa (Fig. 6) the same happened in treatments of two cycles of 7.5 min and 3 cycles of 5 min at 60 °C, however treatment of five cycles of 3 min at 60 °C inactivated more than at 20 °C. It was also observed that at 20 °C only treatments at 600 MPa for 15 min (Fig. 6) activated B. nivea ascospores, and so that treatments at 550 MPa (Fig. 5) showed to be more effective on ascospores inactivation. At 600 MPa (Fig. 6), with only one pressure cycle of 15 min, inactivation was only noticeable at 80 °C in pineapple juice. When the temperature increased to 80 °C, all treatment sequences resulted in inactivation in the juice, whereas only the sequences involving pressure cycles (three cycles of 5 min and 5 cycles of 3 min) proved to be effective in the nectar. As mentioned earlier, this observation can be attributed to the protective effect of the prevailing higher sucrose concentration in nectar on the spores. At 20, 40, 60 and 70 °C it was not observed any statistical difference in ascospores inactivation in both samples (juice and nectar), except at 80 °C. None of the sequences at 20, 40, 60 and 70 °C resulted in inactivation of the ascospores for both products. Only by combining three cycles of 5 min each or five cycles for 3 min
For the inactivation of ascospores in pineapple juice and nectar, sequences involving the use of pressure cycles were found to be more effective than the use of sustained high pressures. The results showed that the application of sustained pressure at 600 MPa and 90 °C for 15 min, and pressure cycles at 600 MPa and 80 °C for three cycles of 5 min or five cycles of 3 min could inactivated 105–106 CFU/mL of B. nivea ascospores in pineapple juice (Aw 0.93) and nectar (Aw 0.94). Considering that the normal levels of contamination in fruits destined for processing can be as high as 105 CFU/mL of yeast or molds in some places and manufacturing conditions, the best treatments investigated in this research appear to be sufficient for pasteurization. This study has succeeded in inactivation B. nivea ascospores by 5.7 log cycles in pineapple juice (Aw 0.93) and nectar (Aw 0.94), by using sequences involving high pressure cycles up to 600 MPa at 80 °C. At the present, most industries in Brazil apply thermal treatments at 98–104 °C for 30 s for tropical juices and nectars, and commonly high pressure treatments in other places vary from 300 to 500 MPa for 90 to 180 s to similar products. It is necessary other studies to compare the nutritional, sensorial and economic issues, since the pressure process required to inactivate ascospores of B. nivea in pineapple juice and nectar needs combination with high temperatures (above 80 °C) and long treatments (15 min in sustained pressure or cyclic variations of pressure, whilst maintaining the same overall treatment time of 15 min). Acknowledgements The experimental part of this research was realized in cooperation with Chemistry and Biology Departments of Aveiro University under the financial support of the EMBRAPA, FAPERJ and CAPES. Laboratory assistance of Marise Oliveira at University of Aveiro and Luana Tashima at Embrapa Food Technology is acknowledged. References Anonymous, 2009. The PATS process paves the way for advanced processing of next-generation shelf-stable foods, says national research consortium.
E. H. d. R. Ferreira et al. / Journal of Food Engineering 95 (2009) 664–669 Beuchat, I.R., Rice, S.I., 1979. Byssochlamys spp. and their importance in fruits processes. Advances in Food Research 25, 237–288. Butz, P., Funtenberguer, T., Haberditzl, T., Tauscher, B., 1996. High pressure inactivation of Byssochlamys nivea ascospores and other heat resistant molds. Lebensmittel Wissenschaftund Technologie 29, 404–410. Conner, D.E., Beuchat, L.R., 1987. Heat resistance of ascospores of Neosartorya fischeri as affected by sporulation and heating medium. International Journal of Food Microbiology 4, 303–312. Dijksterhuis, J., Teunisse, P.G.M., 2004. Dormant ascospores of Taloramyces macrosporus are activated to germinate after treatment with ultra high pressure. Journal of Applied Microbiology 96, 162–169. Hocking, A.D., Pitt, J.I., 1984. Food spoilage fungi. II. Heat resistant fungi. CSIRO Food Research Quarterly 44 (4), 73–82. Koseki, S., Yamamoto, K., 2007. Water activity of bacterial suspension media unable to account for the baroprotective effect of solute concentration on the inactivation of Listeria monocytogenes by high hydrostatic pressure. International Journal of Food Microbiology 115, 43–47. Kotzekidou, P., 1997. Heat resistance of Byssochlamys nivea, Byssochlamys fulva and Neosartorya fischeri isolated from canned tomato paste. Journal of Food Science 62 (2), 410–437. Laboissiére, L.H.E.S., Barros, A.M., Menezes, E.M.S., Rosenthal, A., Camargo, L.M.A.Q., Deliza, R., 2005. Temperature profile of passion fruit, pineapple and açaí (Euterpe oleracea Mart.) purées processed using high pressure (HP). In: The 7th Fruit, Nut and Vegetable Production Engineering Symposium 2005, Montpellier. CD-ROM. Laboissiére, L.H.E.S., Deliza, R., Barrosmarcellini, A.M., Rosenthal, A., Camargo, L.M.A.Q., Junqueira, R.G., 2007. Effects of high hydrostatic pressure (HHP) on sensory characteristics of yellow passion fruit juice. Innovative Food Science & Emerging Technologies 8, 469–477.
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Maggi, A., Gola, S., Spotti, E., Rovere, P., Mutti, P., 1994. Trattamenti ad alta pressione di ascospore di muffe termoresistenti e di patulina in nettare di albicocca e in accqua. Industria Conserve 69, 26–29. Marcellini, A.M.B., Deliza, R., Laboissiére, L.H.E.S., Camargo, L.M.A.Q., Rosenthal, A., 2006. Does high pressure really preserve pineapple juice sensory attributes? In: The Fourth International Conference on High Pressure Bioscience and Biotechnology, Tsukuba. Program and Abstracts. pp. 14. Masson, L.M.P., 2004. Evaluation os pasteurization process of pineapple juice (Ananas comosus L. Merr.) regarding the inactivation of thermal resistant moulds. Dissertation (Master Degree of Food Science and Technology). Federal University of Rio de Janeiro, Rio de Janeiro, Brasil, pp. 81. Obeta, J.A., Ugwuanyi, J.O., 1995. Heat-resistant fungi in Nigerian heat-processed fruit juices. International Journal of Food Science and Technology 30, 587– 590. Palou, E., López-malo, A., Barbosa-Cánovas, G.V., Welti-Chanes, J., Davidson, P.M., Swanson, B.G., 1998. Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates. Letters in Applied Microbiology 27, 375–378. Pitt, J.I., Hocking, A.D., 1985. Fungi and Food Spoilage. Academic Press, Sydney. pp. 413. Splittstoesser, D.F., Nielsen, P.V., Churey, J.J., 1993. Detection of viable ascospores of Neosartorya fischeri. Journal of Food Protection 56, 599–603. Tournas, V., 1994. Heat resistance fungi of importance to the food and beverage industry. Critical Reviews in Microbiology 20, 243–263. Ugwuanyi, J.O., Obeta, J.A.N., 1999. Pectinolytic and cellulolytic activities of heat resistant fungi and their macerating effects on mango and African mango. Journal of the Science of Food and Agriculture 79, 1054–1059.
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Byssochlamys nivea inactivation in pineapple juice and nectar using high pressure cycles Elisa Helena da Rocha Ferreira a, Amauri Rosenthal b,*, Verônica Calado a, Jorge Saraiva c, Sónia Mendo d a
Federal University of Rio de Janeiro, Chemistry and Biochemistry Processes Post Graduation, Rio de Janeiro, RJ, Brazil Embrapa Labex Europe, University of Burgundy, GPMA, 1, Esplanade Erasme, F21000 Dijon, France c Aveiro University, Department of Chemistry, 3810-193 Aveiro, Portugal d Aveiro University, Department of Biology, 3810-193 Aveiro, Portugal b
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 29 May 2009 Accepted 20 June 2009 Available online 17 July 2009 Keywords: High pressure Byssochlamys nivea Pineapple juice
a b s t r a c t The aim of this work was to assess the inactivation of Byssochlamys nivea ascospores in pineapple juice and nectar by combining pressure sequences involving high pressure cycles with relatively mild thermal processing. The effect of 550 and 600 MPa sustained pressures (holding time of 15 min), combinations of sustained pressures and pressure pulses (holding time of 10 s), and pressure cycles (two, three and five cycles of 550 and 600 MPa for 7.5, 5 and 3 min, respectively), at 20, 40, 60, 70, 80 and 90 °C were compared. B. nivea ascospores were inactivated by applying sustained a pressure of 600 MPa at 90 °C for 5 min (juice) and 15 min (nectar), and three and five cycles of pressure at 600 MPa and 80 °C for nectar with holding time of 5 and 3 min, respectively, and in all pressure cycles for juice. In general, pressure cycles were more effective for inactivating B. nivea ascospores than the application of sustained high pressures. Ó 2009 Published by Elsevier Ltd.
1. Introduction Ascospores of Byssochlamys fulva, B. nivea, Neosartorya fischeri and Talaromyces flavus are extremely heat resistant and frequently associated with the deterioration of thermally treated fruit products (Hocking and Pitt, 1984; Beuchat, 1986). The sexual reproduction of B. nivea is provided by ascospores which are produced in saclike specialized terminal cells called asci. Normally, in one ascus eight ascospores are produced (Beuchat and Rice, 1979). Elevate heat resistance is caused by ascospores which possess high resistance to pH variation, and environments with high sugar, fat and acids concentrations (Tournas, 1994). The soil is the principal contamination source of B. nivea. The ascospores can remain dormant, survive the commercial pasteurization conditions normally applied to fruit products, and spoil such products by germinating and growing even under reduced oxygen conditions (Kotzekidou, 1997; Obeta and Ugwuanyi, 1995). The ascospores’ dormancy can be interrupted by sub-lethal stresses, such as mild to highly intense heat treatments depending on the microorganism, leading to the so called spores’ activation, which allows further germination and growth in favorable conditions (Beuchat and Rice, 1979; Beuchat, 1986; Conner and Beuchat,
* Corresponding author. Tel.: +33 3 80396656; fax: +33 3 80396898. E-mail address: [email protected] (A. Rosenthal). 0260-8774/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2009.06.053
1987; Beuchat, 1988; Splittstoesser et al., 1993). Dijksterhuis and Teunisse (2004) associated the ascospores activation to cell wall damage without inactivation and verified that pressure treatments between 400 and 800 MPa could activate Taloramyces macrosporus ascospores. The growth of B. nivea can produce CO2 which inflates fruit juice packages, and causes visual deterioration of the juice due to pectinolytic enzymes activity (Ugwuanyi and Obeta, 1999). Besides, some strains of Byssochlamys are mycotoxigenic due to their ability to produce byssochlamic acid, patulin and byssotoxin A (Beuchat and Rice, 1979). Pineapple nectar can be susceptible to filamentous mold contamination (Masson, 2004). Some fungi, specially ascospores producing molds, are very often able to survive even to more severe thermal treatments. The thermal conditions required to inactivate B. nivea ascospores in fruit products generally affect the sensory quality (Beuchat and Rice, 1979; Tournas, 1994; Butz et al., 1996). Preservation by high hydrostatic pressure in combination with mild temperature can possibly be an alternative for fruit juice processing (Palou et al., 1998). Previous studies showed that high pressure preserves the overall sensory quality of pineapple (Laboissiére et al., 2007) and passion fruit (Marcellini et al., 2006) juices in comparison to pasteurization. The principal aim of this work is to evaluate whether a combination of the application of novel high pressure sequences with the use of moderate temperatures can be used to inactivate ascospores in pineapple drinks.
E. H. d. R. Ferreira et al. / Journal of Food Engineering 95 (2009) 664–669
Combined treatments of high pressure and thermal processing both generally in very high intensity have been reported to achieve spore forming microorganism inactivation in food products. A combination of high pressure and temperature can increase the wall permeability and burst the ascospores. Maggi et al. (1994), Butz et al. (1996) and Palou et al. (1998) demonstrated that pressures above 600 MPa and temperatures greater than 60 °C could inactivate B. nivea ascospores. Palou et al. (1998) studied the effect of continuous pressure application (689 MPa for 5, 15 and 25 min) and the application of oscillatory pressures (one, three or five cycles at 689 MPa with holding time of 1 s) on ascospores of B. nivea in apple and cranberry juices having two different water activity values (0.94 and 0.98) at 21 and 60 °C. No inactivation was observed at 21 °C but a 4 log reduction was observed following the application of 3 or 5 cycles at 60 °C to both juices at a higher water activity. Butz et al. (1996) verified that ascospores of B. nivea were only inactivated above 600 MPa in combination with temperatures above 60 °C and Palou et al. (1998) suggested that inactivation was more effective when pressure treatments are repeated in close succession (pressure cycles). The use of simultaneous application of pressure and temperature to destroy spores of microorganisms is expected to gain much interest in the near future, since US Food and Drug Administration has recently approved the so called pressure assisted thermal processing (PATP) as a possible sterilization process (Anonymous, 2009; Barbosa-Cánovas and Juliano, 2008). This paper aims to assess the effect of constant pressure (550 and 600 MPa) followed or not by pressure pulses (holding time of 10 s) and the effect of pressure cycles (two, three and five cycles at 550 and 600 MPa for 7.5, 5 and 3 min, respectively) at different temperatures (20, 40, 60, 70, 80 and 90 °C) on the inactivation of B. nivea ascospores in pineapple nectar and juice.
2. Materials and methods 2.1. The Byssochlamys nivea The mold B. nivea used in this investigation was isolated from Brazilian fresh strawberry by Aragão (1989) and identified according to standard procedures (Pitt and Hocking, 1985). Ascospores were obtained after 30 days of the mold growth at 30 °C on malt extract agar (Merck) and produced according to Pitt and Hocking (1985). Ascospores were harvested using 25 mL of sterile water. Hyphal fragments were removed by filtering the suspension through sterile glass cotton. The ascospores suspension was collected in a sterile bottle containing glass pearls and then homogenized in vortex for 15 min to liberate the ascospores from the asci. Microscopic observation revealed a high proportion (>90%) of free ascospores.
2.2. Pineapple juice and nectar Commercial pasteurized pineapple juice (Aw 0.93, pH 3.7, 13°Brix, without adding sucrose) and nectar (Aw 0.94, pH 3.7, 12°Brix, with sucrose addition) were used. B. nivea ascospores suspension was inoculated to give an initial concentration (N0) of 105– 106 ascospores/mL. The samples (5 mL) were filled into sterile flexible plastic bags (low-density polyethylene) and pressurized. The total carbohydrate content in pineapple juice and nectar was 10 g/100 mL and 12.5 g/100 mL, respectively. Water activity (Aw) was determined using a rotronic hygrometer (Hygroskop DV-2). Analyses were performed in triplicate to yield mean values and standard error.
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2.3. High pressure treatments Different high pressure sequences were applied to the materials: sustained pressures and combinations of sustained pressures and pressure cycles. These treatments were carried out at temperatures between 20 and 90 °C as showed in Tables 1 and 2. The equipment used (Unipress Equipment, Model U33, Poland) consisted of a 100 mL pressure vessel (35 mm diameter and 100 mm height), which was surrounded by an external jacket connected to a thermostatic bath in order to control the temperature. A mixture of propylene glycol and water (1:1) constituted the pressurization fluid. The temperature of pressure transfer liquid and samples (pineapple juice and nectar) were set at the processing temperature (Tables 1 and 2) before applying the pressure sequence. Immediately after pressurization, the samples were removed and cooled down in ice bath. Unpressurized samples were used as control and measurements were duplicated. The temperature of the pressurization fluid was recorded along the treatment by a thermocouple inserted in the basis of the chamber. Laboissiére et al. (2005) verified that the compression heating of pineapple juice was in average 1.4 °C/100 MPa. However, in this study it was assumed that the sample temperature was equivalent to the pressurization fluid temperature due to process controlling and monitoring limitations. Iron bars were inserted inside the chamber to decrease the fluid volume required to the process and as a consequence avoiding great temperature reduction (loss).
2.4. Sustained pressure treatments Treatments at 600 MPa for 15 min were evaluated at 20, 40, 60, 70, 80 and 90 °C. The inoculum was found to be totally inactivated (Fig. 2) in both products at 90 °C. Then, the inactivation levels at 600 MPa and 90 °C for 5, 10 and 15 min were verified. The time taken to reach 550 and 600 MPa (i.e. come up time) was around 1 min and the decompression time was 10 s.
2.5. Pressure cycles Fig. 1 and Table 1 shows the combination of pressure cycles applied in pineapple juice and nectar. Also, three combinations of sustained pressures and pressure cycles were applied as shows Table 2. The pause between high pressure cycles treatments was set at 1 min (Fig. 1).
2.6. Determination of ascospores counts The initial count (N0) of B. nivea ascospores and the numbers surviving the pressure treatments were determined by spread plating on malt extract agar (Merck). Two plates were used for each dilution and incubated for 3–7 days at 30 °C. Complete inactivation was assumed to have happened when no observable cell growth occurred in the considered condition.
2.7. Statistical analyses Statistical analyses were carried out using Statistica 8.0. The significance differences between the results were calculated by analyses of variance (ANOVA). Differences at p < 0.05 were considered to be significant.
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Table 1 Experimental conditions evaluated in sustained pressures and pressure cycles runs. Pressure (MPa)
Temperature (°C)
Number of cycles
Holding time (min)
Representation
550
20, 40 and 60
1 2 3 5
15 7.5 5 3
1 150 2 7.50 3 50 5 30
600
20, 40, 60, 70 and 80
1 2 3 5
15 7.5 5 3
1 150 2 7.50 3 50 5 30
Table 2 Sequences involving combinations of sustained pressures and pressure cycles. Pressure (MPa)
Temperature (°C)
Sequences of treatments
550 and 600
20
Sustained pressure for 15 min. Two pressure pulses of 10 s. Two pressure pulses of 10 s.
Representation Two pressure pulses of 10 s. Sustained pressure for 15 min. Five pressure pulses of 3 min.
1 150 + 2 1000 2 1000 + 1 150 2 1000 + 5 30
3. Results 3.1. Sustained pressure treatments The combination of pressure and temperature applied determines the extent of B. nivea ascospores inactivation. Fig. 2 clearly shows that the extent of inactivation increases with temperature at 600 MPa for both pineapple juice and nectar. By using a LSD (Least Square Difference) test, it is possible to affirm that there were not significant differences in ascospores inactivation with temperature, except at 80 °C, considering 5% of significance. This may happen because of the higher sucrose concentration in pineapple nectar that could protect the ascospores during the processes. Maggi et al. (1994) observed that treatments at 800 MPa and 50 °C for 4 min or 900 MPa and 50 °C for 2 min were enough to reduce the counts of thermal resistant ascospores by 4 log cycles while Butz et al. (1996) demonstrated that continuous treatments at 700 and 800 MPa at 60–70 °C rapidly inactivated B. nivea ascospores. Butz et al. also noted that no inactivation occurred at 0 and 40 °C. Herein, we found a reduction of 5.6 log cycles in pineapple nectar at 90 °C and 5.7 log cycles in pineapple juice at 80 and
Fig. 2. Inactivation of Byssochlamys nivea ascospores at 600 MPa for 15 min at different temperatures in pineapple juice (h) and pineapple nectar (j). (N0) is the initial count of Byssochlamys nivea ascospores.
90 °C. Temperatures of 40 and 60 °C decrease the count only by 0.5 log cycles in pineapple nectar, while at 70 °C the decrease is 2.0 log cycles for juice and 1.5 log cycles for nectar. Spore count increases in juice following treatments at 20 °C which can be attributed to the ascospores liberation from the ascus
Fig. 1. Pressure cycles applied in pineapple juice and nectar. (a) Sustained pressure of 550 or 600 MPa for 15 min. (b) Combination of two cycles of 7.5 min at 550 or 600 MPa. (c) Combination of three cycles of 5 min at 550 or 600 MPa. (d) Combination of five cycles of 3 min at 550 or 600 MPa: (1) come up time (1 min); (2) holding time; (3) depressurization (10 s); (4) pause time (1 min).
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or the ascospores activation. Similar results were found by Palou et al. (1998) when ascospores of B. nivea were treated at 689 MPa for 15 and 25 min at 21 °C and by Maggi et al. (1994) at 900 MPa for 20 min at 20 °C. The results show that the application of heat or high pressure could break up the dormant state in spores of fungi and ensure the germination. Similarly to mild heating considering the elevate spores resistance, intermediate intense high pressure activate the fungi spores, i.e. interrupts the dormant state by supplying appropriate conditions for initiating spores germination. The effects of applying sustained pressure of 600 MPa at 90 °C for 5, 10 and 15 min are shown in Fig. 3. In pineapple juice, a reduction of 6 log cycles was observed after 5 min of processing, while the same extent of reduction was only observed after 10 min in the case of pineapple nectar. The effects of time and type of drink (juice or nectar) were statistically significant at 5% of significance level. Probably the higher sucrose concentration increases the ascospores resistance in nectar. The difference between ascospores inactivation in juice and nectar can be attributed to a higher baroprotective effect of sucrose that is present at higher concentration in the nectar. The same effect happened when it was used the treatment of 600 MPa and 80 °C for 15 min (Fig. 2) and also observed by Koseki and Yamamoto (2007).
3.2. Pressure cycles Palou et al. (1998) observed no inactivation of B. nivea ascospores when pressure cycles (one, three or five cycles at 689 MPa with holding time of 1 s) were applied at 21 °C. Figs. 4–6 shows that inactivation of B. nivea ascospores is more noticeable in treatments involving cyclic variations of pressure whilst maintaining the same overall treatment time (i.e. five cycles lasting 3 min each or three cycles lasting 5 min each, as opposed to a single cycle of pressure for 15 min). At 20 °C, Fig. 4 shows that at 550 MPa an effective pressure sequence for inactivation of B. nivea ascospores is the one involving a single cycle of pressure for 15 min, followed by the application of two 10-s pressure pulses after decompression to atmospheric pressure. This is indeed more effective than following the same sequence at 600 MPa and treatments at 550 and 600 MPa for 15 min at all temperatures (Figs. 5 and 6), and may be attributed to increase the ascospores fragility after the first single cycle at 550 MPa for 15 min, becoming easier the inactivation with the two pulses after on. However, same treatments at 40 and 60 °C (data not shown) demonstrated activation on B. nivea ascospores. The same ascospores activation might explain the results observed for a single 15 min pressure cycle at 600 MPa. It is clear that the best treatment applied in pineapple juice and nectar at 20 °C
Fig. 3. Inactivation of Byssochlamys nivea ascospores with processing time at 600 MPa and 90 °C in pineapple juice (h) and pineapple nectar (j).
Fig. 4. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles at 20 °C. Maximum pressure: 550 (h) and 600 MPa (j). (N0) is the initial count of Byssochlamys nivea ascospores.
Fig. 5. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles, maximum pressure = 550 MPa at different temperatures (20, 40 and 60 °C). (N0) is the initial count of Byssochlamys nivea ascospores.
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each at 80 °C, it was possible to completely inactivate the ascospores in both products, meaning that no viable cell was detected after the treatment (Fig. 6). This is consistent with Butz et al. (1996) who noted that B. nivea ascospores could survive any treatment, including cycling pressure exposure up to 500 MPa and 70 °C for several hours. Palou et al. (1998) noted that pressure sequences involving cyclic compression and decompression at 689 MPa at 60 °C could inactivate heat resistant molds in diluted fruit juices (Aw 0.98), but could not inactivated the same molds in juices with lower Aw. The present study has succeeded in inactivating B. nivea ascospores by 5.7 log cycles in pineapple juice (Aw 0.93) and nectar (Aw 0.94), by using sequences involving high pressure cycles up to 600 MPa at 80 °C. 4. Conclusion
Fig. 6. Inactivation of Byssochlamys nivea ascospores in pineapple juice (a) and pineapple nectar (b) by high pressure cycles, maximum pressure = 600 MPa at different temperatures (20, 40, 60, 70 and 80 °C). (N0) is the initial count of Byssochlamys nivea ascospores.
(Fig. 4) was five cycles of 3 min in both high pressure treatments (550 and 600 MPa). There were statistical differences among the treatments (pressure level and cycles number), and the ascospores inactivation in juice and nectar was statistically equivalent, considering 5% of significance. The most effective sequence involving 550 MPa was the use of five cycles of 3 min each for all three temperatures used. Such combination resulted in count reductions as high as 3.9 logs in juice and 4.5 logs in nectar at 40 °C (Fig. 5). The statistical difference appears only at temperature level and cycles number, being the ascospores inactivation in juice and nectar again statistically equal. Contrary to the report by Palou et al. (1998), treatments at 60 °C resulted in lower inactivation than at 20 °C, possibly because of ascospore activation at 550 MPa. At 600 MPa (Fig. 6) the same happened in treatments of two cycles of 7.5 min and 3 cycles of 5 min at 60 °C, however treatment of five cycles of 3 min at 60 °C inactivated more than at 20 °C. It was also observed that at 20 °C only treatments at 600 MPa for 15 min (Fig. 6) activated B. nivea ascospores, and so that treatments at 550 MPa (Fig. 5) showed to be more effective on ascospores inactivation. At 600 MPa (Fig. 6), with only one pressure cycle of 15 min, inactivation was only noticeable at 80 °C in pineapple juice. When the temperature increased to 80 °C, all treatment sequences resulted in inactivation in the juice, whereas only the sequences involving pressure cycles (three cycles of 5 min and 5 cycles of 3 min) proved to be effective in the nectar. As mentioned earlier, this observation can be attributed to the protective effect of the prevailing higher sucrose concentration in nectar on the spores. At 20, 40, 60 and 70 °C it was not observed any statistical difference in ascospores inactivation in both samples (juice and nectar), except at 80 °C. None of the sequences at 20, 40, 60 and 70 °C resulted in inactivation of the ascospores for both products. Only by combining three cycles of 5 min each or five cycles for 3 min
For the inactivation of ascospores in pineapple juice and nectar, sequences involving the use of pressure cycles were found to be more effective than the use of sustained high pressures. The results showed that the application of sustained pressure at 600 MPa and 90 °C for 15 min, and pressure cycles at 600 MPa and 80 °C for three cycles of 5 min or five cycles of 3 min could inactivated 105–106 CFU/mL of B. nivea ascospores in pineapple juice (Aw 0.93) and nectar (Aw 0.94). Considering that the normal levels of contamination in fruits destined for processing can be as high as 105 CFU/mL of yeast or molds in some places and manufacturing conditions, the best treatments investigated in this research appear to be sufficient for pasteurization. This study has succeeded in inactivation B. nivea ascospores by 5.7 log cycles in pineapple juice (Aw 0.93) and nectar (Aw 0.94), by using sequences involving high pressure cycles up to 600 MPa at 80 °C. At the present, most industries in Brazil apply thermal treatments at 98–104 °C for 30 s for tropical juices and nectars, and commonly high pressure treatments in other places vary from 300 to 500 MPa for 90 to 180 s to similar products. It is necessary other studies to compare the nutritional, sensorial and economic issues, since the pressure process required to inactivate ascospores of B. nivea in pineapple juice and nectar needs combination with high temperatures (above 80 °C) and long treatments (15 min in sustained pressure or cyclic variations of pressure, whilst maintaining the same overall treatment time of 15 min). Acknowledgements The experimental part of this research was realized in cooperation with Chemistry and Biology Departments of Aveiro University under the financial support of the EMBRAPA, FAPERJ and CAPES. Laboratory assistance of Marise Oliveira at University of Aveiro and Luana Tashima at Embrapa Food Technology is acknowledged. References Anonymous, 2009. The PATS process paves the way for advanced processing of next-generation shelf-stable foods, says national research consortium.
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