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Non-thermal microbial inactivation in waste brine using high-voltage low-energy electrical pulses
Innovative Food Science & Emerging Technologies 2 Ž2001. 251᎐259
Non-thermal microbial inactivation in waste brine using high-voltage low-energy electrical pulses S.Y. Ho, G.S. MittalU School of Engineering, Uni¨ ersity of Guelph, Guelph, ON, Canada N1G 2W1 Accepted 19 June 2001
Abstract A novel low-energy pulsed electrical system was used to pasteurize waste brine from smokehouses. The microbial reduction was modeled as a sigmoid function of electrical field strength Ž E f . and number of pulses Ž n.. The E f value was a more important factor in microbial reduction than n. For a 50-ml batch, optimum process conditions were: E f s 60 kVrcm and 1 F n G 5 for no microbial contamination. For continuous flow mode, the maximum flow rate was 18 lrh at a 0.5-Hz pulse frequency. Microbial, physical and chemical properties and shelf life were consistent with batch processing. The log reductions for Staphylococcus and Micrococcus were ) 2.01" 0.03 in batch and ) 2.28" 0.01 in continuous processing at the above-mentioned conditions. 䊚 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pulsed electrical field; Non thermal processing; Recycling brine
1. Introduction High voltage pulsed electrical field for pumpable food pasteurization has been investigated extensively by researchers in Canada, USA, Germany, Japan and a few other countries ŽHo & Mittal, 2000.. Numerous experimental studies have shown that the pulsed electric field can induce moderate to significant microbial inactivation in various aqueous solutions ŽHo, Mittal, Cross & Griffiths, 1995.. Engineers and physicists have used pulsed power in various defense applications, in the steel industry for continuously casting molten steel into blooms and billets ŽCenanovic & Maureira, 1989., in electrohydraulic rock breaking, spark drilling, and seismic sounding ŽGray, Moeny, Beckes & Davis, 1987; Touryan, Moeny, Aimone & Benze, 1989., in lightning and static electricity simulation ŽHebert, Herbert,
U
Corresponding author. Tel.: q1-519-824-4120, ext. 2431; fax: q1-519-836-0227. E-mail address: [email protected] ŽG.S. Mittal..
Walko, Schneider & Serrano, 1987. and in various studies of the breakdown strengths of water and water mixtures ŽZahn, Ohki, Fenneman, Gripshover & Gehman, 1986; Mcleod and Gehman Jr., 1987.. Brine Žsodium chloride solution. is used by meat processors in bacon and wiener processing. After cooking in the smokehouse, the meat product is chilled using brine to wash off excess fat and smoke from the product, to provide excess salt at the product’s surface, and to cool the product enough to go through slicing and packaging along the processing line. Brine at y6 to y8 ⬚C is pumped from a loading tank and sprayed onto the product. The wash-off brine is then collected, recycled back to the tank, and reused. Currently, in many plants, the changeover rate of the brine is every 3᎐4 days. During this period, contamination of the brine Žand thus, the product. is common. Operations such as preparing and loading of the brine to the tank, or pumping and recycling the brine through pipes and spray nozzles contaminate the brine. The total plate count of waste brine can be ) 1000 cfurml ŽMittal &
1466-8564r01r$ - see front matter 䊚 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 4 3 - 1
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S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259
Choudhry, 1997.. The waste brine was reported to contain both gram-positive cocci and gram-negative rod-shaped bacteria, mainly consisting of Micrococcus and Staphylococcus species, with a small number of Pseudomonas and lactic acid bacteria ŽMittal & Choudhry, 1997.. The use of an in-line high-voltage electric pulse system to pasteurize waste brine from the chilling operation was tested in this research. The objectives were to investigate microbial survivability in waste brine as a function of electric field strength and number of pulses applied in the batch and continuous treatment systems, and to identify the optimum conditionŽs. for maximum microbial reduction.
2. Materials and methods 2.1. Waste brine Waste brine from a bacon smokehouse cooking was provided for this study by a meat plant in Scarborough, Ontario, Canada. Waste brine was collected into sterile containers. The brine solution was stored at y7⬚C ŽIsotemp cooler, Fisher Scientific Co., Toronto, Canada.. The salt content of the filtered waste brine was determined by calculating the difference in mass before and after drying in a Speed Vac concentrator ŽSavant Speed Vac-SVC 100H, Instruments Inc., Farmingdale, N.Y.., which was attached to a freeze drier ŽLabconco Trivac freeze drier-D8A, LeyboldHerraus Vacuum Products Inc., Mellon, PA.. that controlled the temperature and vacuum. Electrical conductivity of the brine was also determined by using a conductivity meter ŽCole-Parmer Oakton, Niles, IL, model WD-35607-10.. 2.2. Treatment system and conditions A bench-top pulsed field treatment system was used to process brine in batch and continuous modes ŽHo et al., 1995.. The system consists of a 30-kV d.c. high voltage pulse generator, a circular treatment chamber and devices for pumping and recording. Fig. 1 shows the block diagram of the unit. The 110-V a.c. is raised in voltage through a high voltage transformer, and then rectified. The d.c. high voltage supply then charges up the 0.12-F capacitor through a series of 6 M⍀ resistors Žthe time constant s 0.72 s.. The pulse generator emits a train of 5 V pulses, and the trigger circuit serves to convert that to 500 V pulses using a silicon control rectifier ŽSCR.. The pulse frequency can be adjusted with the choice of a single pulse Žmanual., or an autotriggering system of 0.09᎐0.5 Hz. The uniqueness of this pulsar is that pulses of low-energy and of instant-charge-reversal shape are generated. Conven-
Fig. 1. Block diagram of the pulsed field treatment system.
tional pulsar generates pulses of much higher energy and of square or exponential decay or bipolar shape. The circular treatment chamber Ž25.0 cm diameter. has two circular and parallel stainless steel electrodes Ž16.5 cm diameter.. The insulation, Delrin, was constructed to have close physical contact with the electrodes. The distance between the electrodes can be adjusted by inserting Delrin circular plates Ž14.5 cm diameter. with five different thicknesses: 0.08, 0.16, 0.3, 0.6 or 0.9 cm. Thus, the process volume can be varied between 13.2 and 148.6 ml per batch. To eliminate air bubbles in the treatment chamber, the sample entered into the chamber from the bottom through a channel in the chamber under a 85-kPa vacuum. A digital oscilloscope Žmodel TDS 340, Tektronix Inc., Beaverton, OR. and a high voltage probe Žmodel P6015A, Tektronix Inc., Beaverton, OR. are connected to the system so that each pulse across the chamber can be monitored. The batch system can be used in continuous mode by pumping the sample through the horizontal chamber from one end and collected at the other. The flow rate can be controlled by adjusting the position of needle valves on the treatment chamber. In this research, the maximum process capacity used was 18 lrh. The brine was pumped from feed tank into treatment chamber from one end with a pressure pump at 186 kPa Žabsolute.. The sample was collected after 5 min running into sterile plastic containers. Sterile containers were placed on ice with NaCl to minimize microbial growth and to maintain temperature at y7 " 1⬚C before and after treatment. For all experiments, constant process parameters were: electrode gaps 3 mm, pulse frequency s 0.5 Hz and process Žtreatment . temperature s y7 " 1⬚C. Voltage supplied by the d.c. source s 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 18 and 21 kV, and pulses applieds 1, 5, 10, 20 and 30. Flow rates in continuous mode were 9 Ž10 pulses applied. and 18 lrh Žfive pulses applied.. All experiments were repeated at least three times, in the reverse experimental order as the previous set. This was to minimize the variation in count due to microbial growth with time.
S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259
2.3. Microbial procedure Before and after electrical treatment, microbial plate counts of colony forming units Žcfu. on selected media indicated the reduction of the microbial cells under different process conditions. Previous results ŽMittal & Choudhry, 1997. on microbial analysis of waste brine from the same meat plant indicated that approximately 76% of the microbial population contained Staphylococcus and Micrococcus, and it was necessary to use agar with NaCl for getting higher microbial count. Hence, in this study, only the total plate count, and Staphylococcus and Micrococcus were measured. The microbial level of the untreated and treated samples was measured by platting and incubating ŽHotpack incubator; Hot Pack, Waterloo, Ontario. at 30⬚C for 48 h for a total plate count on agar with 10% NaCl, or agar with Mannitol salt at 37⬚C for 36 h for Staphylococcus and Micrococcus. Microbial plating of samples was done twice: once before the experiment Žuntreated., and once after the experiment Žtreated.. This was to ensure that the time length of the experiment had no effect on microbial reduction. For each test set, a control with all the steps followed but no electrical treatment was carried out. This was to ensure that the sanitization had no effect on the killing rate. For selflife studies Žmicrobial count., samples were stored at y7⬚C and plated every 3᎐4 days for 3 weeks.
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bomb. The procedure was repeated six times: 3-min soak and 3-min purge. Water was circulated through the CPD bomb at 40᎐45⬚C and 100᎐1350 psi for 10 min. Pressure was released, and the filtrate was removed and gold plated. The sample was studied by SEM Žmodel S-570, Hitachi, Tokyo, Japan; 10᎐15 kV accelerating voltage.. The magnification Ž13 000 = ., resolution and tilt angle were adjusted. Then photographs were taken. 2.6. Data analysis Statistical analysis was performed on the data ŽSYSTAT, 1992.. The microbial reduction was modeled as a function of electrical field strength and number of pulses applied ŽEqs. Ž1. ᎐ Ž3... The microbial plate count Žbefore and after pulse treatment with three replications. were transformed into statistical files and fitted using the technique of non-linear regression modeling ŽSYSTAT, 1992.: a Quasi-Newton estimation method was used, with 0.10 as the parameter starting value; iterations continued until the maximum iteration limit Ž999. was reached. 2.7. Microbial inacti¨ ation models Peleg Ž1995. reported that the microbial inactivation curve had a characteristic sigmoid shape, and established a model based on Fermi’s equation:
2.4. Other tests and calculations The electrical conductivity of the sample using a hand-held conductivity meter ŽCole-Parmer Oakton, Niles, IL, model WD-35607-10., pH using a pH meter ŽFisher Scientific, Whitby, Ontario, Canada, model 925. and sample temperature immediately before and after treatments ŽFisher Scientific Digi-thermo, Whitby, Ontario, Canada, model 15-077. were measured. Energy consumption ŽJrml. was calculated as 0.5 C E 2 nrV⬘, where C is the charging capacitor, F; E is voltage applied, V; n is number of pulses applied, and V⬘ is the sample volume treated, ml. 2.5. Scanning electron microscopy (SEM) The sample was filtered through a polycarbonate membrane filter Ž0.2᎐0.4 m., and filtrate was transferred to Sorensen’s phosphate buffer solution Ž1 part Na 2 HPO4 to 1 part KH 2 PO4 .. Then 2% glutaraldehyde was added to the PO4 buffer, and kept for 30 min at room temperature. The filtrate was dehydrated in a series of ethanol solution: 50, 70, 90, 95 and 3 = 100% at 15-min time intervals. Water was circulated through the CPD bomb at 15⬚C. The filtrate was put into specimen basket, and the bomb was filled with buffer. The filtrate was dried by flushing CO 2 through the
S Ž E f ,n . s
1 E f y Ec Ž n . 1 q exp ac Ž n .
Ž1.
where S s surviving fraction of microorganisms, NrNo; n s number of pulses applied; E f s electrical field strength applied, kVrcm; Ec s critical electrical field strength where the survival fraction is 0.50 or the inflection point of S, kVrcm; a c s a parameter indicating the steepness of the survival curve around Ec , kVrcm; Nos initial microbial level; and N s microbial level after treatment. Ec Ž n. and a c Ž n. were described by exponential decay functions, as follows: Ec Ž n . s Ec o ⭈ exp Ž yk 1 ⭈ n .
Ž2.
a c Ž n . s a o ⭈ exp Ž yk 2 ⭈ n .
Ž3.
where Ec o and a o s constants, kVrcm; and k 1 and k 2 s constants, dimensionless. Peleg Ž1995. used data from Castro, BarbosaCanovas & Swanson Ž1993. and showed that the data had a good fit Ž R 2 s 0.97᎐0.999. with Eq. Ž1.. Experimental results from Ho et al. Ž1995. seemed to support this model as well. Zhang, Chang, Barbosa-Canovas &
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S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259
Swanson Ž1994. used the Hulsheger, Potel and Niemann Ž1981. model to test the survival fraction of E. coli, S. aureus and S. cere¨ isiae in a semisolid model food containing potato dextrose agar. Our work did not support the Hulsheger et al. Ž1981. model.
3. Results and discussion 3.1. Properties of waste brine The salt content of the brine was 26.00" 0.02% wrw, the water content was 73.5" 0.03%, and rest was traces of protein and fat. The electrical conductivity was 300 " 20 mSrm. The specific gravity was 1.25" 0.01 and pH was 7.0" 0.1. All properties were unaffected under all test conditions. Temperature change was also negligible due to small energy input during treatment. A typical pulse waveform is shown in Fig. 2 using waste brine. For batch processing, the mean initial total plate count was 887 " 186 cfurml and the mean initial Staphylococcus and Micrococcus count was 1798 " 103 cfurml. For continuous processing, the mean initial total plate count was 998 " 85 cfurml, and mean initial Staphylococcus and Micrococcus count was 1910 " 44 cfurml. 3.2. Batch system Model parameters of Eqs. Ž1. ᎐ Ž3. are listed in Tables 1 and 2. Table 3 lists the statistical analyses on the model parameters. Results from Table 3 indicate that there is a good correlation between the individual microbial reduction estimation and the overall model parameters Ž R 2 s 0.994᎐1, P- 0.01.. The individual typical survival curves Žraw data and fitted model. are depicted in Figs. 3 and 4 in terms of survival fraction. Similar plots were also obtained for other conditions Žnot shown.. A three-dimensional plot of Eq. Ž1. is shown in Fig. 5 for total plate count. In general, microbial results are in good agreement with the model. Statistically corrected R 2 is 0.981 Žstandard plate count. and 0.989 Žfor Staphylococcus and Micrococcus. for the
Fig. 2. Pulse waveform measured across the chamber containing waste brine in the batch treatment system at 15 kV voltage supply and 3 mm electrode gap. Scale: vertical 2 kVrdivision, horizontal 500 nsrdivision.
overall model Ž R 2 is in the range of 0.981᎐0.995 for the individual curves. ŽANOVA for this is not given.. Results from both total plate count and StaphylococcusrMicrococcus illustrate a sigmoidal relationship between microbial survivability and the applied electric field strength at all number of pulses applied ŽFigs. 3 and 4.. This is clearly in support of Peleg’s findings Ž1995.. Peleg obtained Ec in the range of 6.7᎐21.2 kVrcm and a c in the range of 1.6᎐3.1 kVrcm for various pure culture organisms in buffer solutions. For waste brine, Ec is in the range of 14᎐31 kVrcm and a c is in the range of 6.7᎐9.0 kVrcm. By definition, Ec Table 1 Overall parameters of Peleg model for PEF batch treating of waste brine using Eqs. Ž2. and Ž3. Parameters
Staphylococcus and Micrococcus
Total plate count
Ec o ŽkVrcm. k1 ao ŽkVrcm. k2
31.232 0.012 6.889 y0.005
25.727 0.020 6.821 0.006
Table 2 Peleg model parameters for PEF batch treating of waste brine expressed in terms of number of pulses applied using Eq. Ž1. Pulses applied, n
1 5 10 20 30
Staphylococcus and Micrococcus
Total plate count
Ec ŽkVrcm.
ac ŽkVrcm.
Ec ŽkVrcm.
ac ŽkVrcm.
30.859 29.413 27.700 24.568 21.790
7.924 8.063 8.242 8.614 9.004
25.218 23.279 21.063 17.245 14.119
7.780 7.619 7.424 7.050 6.697
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Table 3 Statistical analyses on the Peleg model parameters for PEF batch treating of waste brine using Eq. Ž1.
2
R Standard error of estimate Mean sum of squares Žregression. DF Žerror. P) T
Staphylococcus and Micrococcus
Total plate count
Ec
ac
Ec
ac
1 0.665 3650.292 4 - 0.01
0.994 0.747 346.716 4 - 0.01
0.997 1.297 2132.898 4 - 0.01
0.998 0.323 266.954 4 - 0.01
Fig. 3. Microbial survival fraction Ž NrNo. for total plate count in batch mode at various electrical fields Ž E . and number of pulses applied Ž n..
represents the critical electric field strength where the survival fraction is 0.50 Žreflection point on curve., and a c represents a parameter indicating the steepness of the survival curve around Ec . It seems that the value of Fig. 5. Three-dimensional microbial survival Ž NrNo. plot of waste water for total plate count using Eq. Ž1.. Es electrical field and n s number of pulses.
Fig. 4. Microbial survival fraction Ž NrNo. for Staphylococcus and Micrococcus in batch mode at various electrical fields Ž E . and number of pulses applied Ž n..
Ec and a c would increase with the increase in the number of microbial species Žor the relative resistance . in the fluid medium. Previous work ŽHo et al., 1995. on a pure culture of Pseudomonas fluorescens ŽM3r6. suspended in various fluid conditions indicated an either ‘kill-or-no-kill’ scenario, with 15᎐35 kVrcm as the applied critical electric field strength. This can be interpreted as a sigmoidal survival curve with a moderate Ec value Ž14᎐30 kVrcm. and a small a c value Ž7᎐9 kVrcm.. On the other hand, various species of StaphylococcusrMicrococcus have been identified in the waste brine, along with other microbes such as Pseudomonas, and lactic acid bacteria ŽMittal & Choudhry, 1997.. As a result, Ec and a c increase, with values for StaphylococcusrMicrococcus significantly Ž PF 0.05. higher than values for total plate
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count ŽTable 2. Ž Ec s 22᎐31 kVrcm vs. 14᎐25 kVrcm, and a c s 7.9᎐9.0 vs. 6.7᎐7.8 kVrcm.. By comparing Ec and a c values of different pure microbial cultures to the values of a product which is usually comprised of many mixed species, it is possible to identify the relative quantity and physiological state of each species or, more importantly, the presence of any interactions between or among species in the product under pulse treatment. This helps to identify the optimum operating conditionŽs. when the lethality of a specific species is intended. More work is needed in this area. From Table 4 ŽANOVA., the electric field strength applied is a far more important factor in microbial reduction than the number of pulses applied. This indirectly supports the idea of transmembrane potential theory ŽHo & Mittal, 1996. where a critical potential is required to develop on the membrane surface for cell lysis, and that the number of pulses applied only affects the area being electroporated. Based on the results, no colony forming units Ž- 10 cfurml. were detected on either agar media when the product underwent the following treatments, where E f is electrical field applied and n is the number of pulses: E f s 50 kVrcm, 10 F n F 30 « minimal energy consumption s 2.7 Jrml at n s 10 for a 50-ml batch; and E f s 60 kVrcm, 1 F n F 5 « minimal energy consumption s 0.39 Jrml at n s 1 for a 50-ml batch.
1.
2.
The log reductions for total plate count were ) 1.95 " 0.08, and for Staphylococcus and Micrococcus count were ) 2.26" 0.02. Option 2 clearly has economical advantages over option 1. However, option 1 may probably provide a better safety net for quality assurance due to large number of pulses applied, especially in a continuous operation as missing of a few pulses will not have significant effect on microbial decay. No detectable physical ŽpH, electrical conductivity, specific gravity. nor thermal changes Žnegligible change in temperature. occurred under those conditions. Using the same treatment system but a sightly different waste brine Ž22.5% NaCl, wrw., Mittal and Choudhry Ž1997. identified E f s 50 kVrcm and n s 15 as the optimum
treatment condition. The results of this research seem to be comparable with previous work. Selected samples, both before and after pulse treatment using the optimum condition Ž60 kVrcm, one and five pulses., were analyzed by scanning electron microscopy ŽSEM. for visual damage to the microorganisms. Results are shown in Figs. 6, 7 and 8. It is evident that microbes under pulse treatment are disrupted and ruptured while microbes without pulse treatment remain fully intact. The figures also provide support for the transmembrane potential theory. Selected samples were treated with the optimal treatment condition Ž E f s 60 kVrcm and n s 5. and subjected to shelf-life studies. The storage temperature was y7⬚C Žnormal storage temperature used., and samples were plated every 3᎐4 days for 3 weeks. Again, no colony-forming units Ž- 10 cfurml. were detected on either agar media throughout the test period. Together with the microbial and SEM data, it seems that the pulsed field treatment employed in this research caused irreversible damage to the microbes being tested, with negligible physical ŽpH, electrical conductivity, and specific gravity. and thermal changes Žas temperature increase was negligible. to the waste brine. 3.3. Continuous treatment system Waste brine was treated at various field strength and two flow rates. The log reductions for total plate count at the flow rates of 9 and 18 lrh were ) 2.01" 0.03 with an initial count of 998 " 85 cfurml, and for the Staphylococcus and Micrococcus count were ) 2.28" 0.01 with an initial count of 1910 " 44 cfurml in both cases. No significant change in microbial decay was noted when constant flow rate and process conditions were used. Fig. 9 shows the microbial survival fraction as a function of electrical field strength and flow rates, with Eq. Ž1. Žbatch condition. plotted for comparison. Although some pulses were occasionally missed Ž- 5%., the pulse waveforms Žpeak voltage and pulse width. under continuous treatment remained stable and were similar to those obtained under batch processing. This ensured that the number of pulses applied to the brine
Table 4 Analysis of variance for the PEF process parameters to treat waste brine in batch system Source
Pulses applied Field strength Error U
PF 0.05. P- 0.01.
UU
d.f.
4 12 48
Staphylococcus and Micrococcus
Total plate count Mean sum of squares
F Ratio
Mean sum of squares
F Ratio
0.052 0.670 0.005
11.507U UU 148.564
0.036 0.693 0.003
11.995U UU 233.393
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- 10 cfurml, i.e. ) 2.01" 0.05 log reductions., the energy consumption was: E f s 60 kVrcm, n s 5 at 18 lrh, n s 10 at 9 lrh « energy required s 1.94 Jrml.
4. Conclusions and summary of results There was no effect on pH and electrical conductivity of the waste brine due to high voltage pulsed field treatment. No microbial contamination was detected after waste brine was subjected to pulsed field treatment in batch or continuous flow systems. Shelf life studies indicated no microbial growth for at least 3 weeks. The pulsed field treatment caused irreversible damage to microbes. Optimum process conditions in batch system were: E f s 60 kVrcm and 1 F n G 5 with an energy consumption of 0.39 Jrml at n s 1. The microbial reduction was modeled as a function of E f and n in sigmoid function. The E f value was a far more important factor in microbial reduction than n.
Fig. 6. Magnified view Ž=30 000. of microbes in waste brine without pulsed field treatment using scanning electron microscopy.
passing through the treatment chamber was sufficient. Microbial results were consistent with batch processing. A - 10-cfurml plate count was obtained when the samples underwent pulse treatment at optimum conditions. Again, the flow rate Žnumber of pulses applied. and process temperature appeared not to be significant at the test range. The relatively small standard deviation ŽS.D.; not given. indicates that the results from separate trials are in good agreement with each other. Fig. 10 shows the log reductions of Staphylococcus and Micrococcus at various applied fields and two flow rates. Thus, the results obtained from the continuous mode are in agreement with those obtained from the batch mode, indicating that the optimum conditions specified are effective, and that the treatment system can be used in a batch or continuous mode of operation. Although the degree of mixing in the chamber was significant, as indicated by the deviation of data points from Eq. Ž1. at various number of pulses applied, the impact on microbial reduction was not. From Figs. 9 and 10, the optimum electric field strength for waste brine would still be 60 kVrcm at 9 or 18 lrh flow rate, as established from the batch condition. Using the optimum treatment conditions Žwhere plate count gives
Fig. 7. Magnified view Ž=30 000. of microbes in waste brine after pulsed field treatment using scanning electron microscopy. Batch treatment conditions employed were 60 kVrcm electrical field strength, 3 mm electrode gap, 0.5 Hz pulse frequency, and one pulse applied.
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Fig. 10. Microbial decay Žlog N . for Staphylococcus and Micrococcus in waste brine at various flow rates and electrical fields Ž E .. At 9 lrh, n s 10, and at 10 lrh, n s 5.
the area being electroporated. In continuous mode at 9 lrh Ž n s 10. and 18 lrh Ž n s 5. and 0.5 Hz pulse frequency, microbial, pH and electrical conductivity and shelf life results were consistent with batch processing, and the energy consumption was 1.94 Jrml.
Fig. 8. Magnified view Ž=20 000᎐30 000. of microbes in waste brine after pulsed field treatment using scanning electron microscopy. Batch treatment conditions employed were 60 kVrcm electrical field strength, 3 mm electrode gap, 0.5 Hz pulse frequency, and five pulses applied.
This supported the transmembrane potential theory where a critical potential is required to develop on membrane surface for cell lysis, and that n only affects
Fig. 9. Microbial survival fraction Ž NrNo. for total plate count in waste brine at various flow rates and electrical fields Ž E .. Predicted data are based on parameters from Tables 1 and 2, and Eq. Ž1.. At 9 lrh, n s 10, and at 10 lrh, n s 5.
Acknowledgements Funding of this research by the Ontario Ministry of Agriculture, Food and Rural Affairs is greatly appreciated. References Castro, A. J., Barbosa-Canovas, G. V., & Swanson, B. G. Ž1993.. Microbial inactivation of foods by pulsed electric fields. Journal of Food Processing and Preser¨ ation, 17, 47᎐73. Cenanovic, M. B., & Maureira, H. A. Ž1989.. Application of pulsed power in the steel industry. Proceedings of 7th IEEE Pulsed Power Conference, 65᎐68. Gray, E. W., Moeny, W. M., Beckes, B. R., & Davis, B. B. Ž1987.. Pulsed power fracturing of rock. Proceedings of 6th IEEE Pulsed Power Conference, 330᎐335. Hebert, J. L., Herbert, M. P., Walko, L. C., Schneider, J. G., & Serrano, A. Ž1987.. Pulsed power applications in aircraft lightning qualification testing. Proceedings of 6th IEEE Pulsed Power Conference, 326᎐329. Ho, S. Y., Mittal, G. S., Cross, J. D., & Griffiths, M. W. Ž1995.. Inactivation of P. fluorescens by high voltage electric pulses. Journal of Food Science, 60 Ž6., 1337᎐1340. Ho, S. Y., & Mittal, G. S. Ž1996.. Electroporation of cell membrane: a review. Critical Re¨ iews in Biotechnology, 16 Ž4., 349᎐362. Ho, S., & Mittal, G. S. Ž2000.. High voltage pulsed electrical field liquid food pasteurization. Food Re¨ iew International Žin press.. Hulsheger, H., Potel, J., & Niemann, E. G. Ž1981.. Killing of bacteria with electric pulses of high field strength. Radiation and En¨ ironmental Biophysics, 20, 53᎐65. Mcleod, A. R., & Gehman Jr., V. H. Ž1987.. Water breakdown measurements of stainless steel and aluminium alloys for long-
S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259 charging times. Proceedings of 6th IEEE Pulsed Power Conference, 57᎐59. Mittal, G. S., & Choudhry, M. Ž1997.. Pulsed electric field sterilization of waste brine solution. In R. Jowitt, Engineering and Food at ICEF 7 Žpp. O13᎐16.. Sheffield, UK: Academic Press. Peleg, M. Ž1995.. A model of microbial survival after exposure to pulsed electric fields. Journal of Science in Food and Agriculture, 67, 93᎐99. SYSTAT Ž1992.. SYSTAT for Windows, version 5. Evanston, IL: SYSTAT, Inc. Touryan, K. J., Moeny, W. M., Aimone, C. T., & Benze, J. W. Ž1989..
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Electrohydraulic rock fracturing by pulsed power generated focused shocks. Proceedings of 7th IEEE Pulsed Power Conference, 69᎐72. Zahn, M., Ohki, Y., Fenneman, D. B., Gripshover, R. J., Gehman, V. H. Ž1986.. Dielectric properties of water and waterrethylene glycol mixtures for use in pulsed power system design. Proceedings of IEEE, 74Ž9., 1182᎐1221. Zhang, Q., Chang, F. J., Barbosa-Canovas, G. V., & Swanson, B. G. Ž1994.. Inactivation of microorganisms in a semisolid model food using high voltage pulsed electric fields. Food Science and Technology, 27 Ž6., 538᎐543.
Non-thermal microbial inactivation in waste brine using high-voltage low-energy electrical pulses S.Y. Ho, G.S. MittalU School of Engineering, Uni¨ ersity of Guelph, Guelph, ON, Canada N1G 2W1 Accepted 19 June 2001
Abstract A novel low-energy pulsed electrical system was used to pasteurize waste brine from smokehouses. The microbial reduction was modeled as a sigmoid function of electrical field strength Ž E f . and number of pulses Ž n.. The E f value was a more important factor in microbial reduction than n. For a 50-ml batch, optimum process conditions were: E f s 60 kVrcm and 1 F n G 5 for no microbial contamination. For continuous flow mode, the maximum flow rate was 18 lrh at a 0.5-Hz pulse frequency. Microbial, physical and chemical properties and shelf life were consistent with batch processing. The log reductions for Staphylococcus and Micrococcus were ) 2.01" 0.03 in batch and ) 2.28" 0.01 in continuous processing at the above-mentioned conditions. 䊚 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pulsed electrical field; Non thermal processing; Recycling brine
1. Introduction High voltage pulsed electrical field for pumpable food pasteurization has been investigated extensively by researchers in Canada, USA, Germany, Japan and a few other countries ŽHo & Mittal, 2000.. Numerous experimental studies have shown that the pulsed electric field can induce moderate to significant microbial inactivation in various aqueous solutions ŽHo, Mittal, Cross & Griffiths, 1995.. Engineers and physicists have used pulsed power in various defense applications, in the steel industry for continuously casting molten steel into blooms and billets ŽCenanovic & Maureira, 1989., in electrohydraulic rock breaking, spark drilling, and seismic sounding ŽGray, Moeny, Beckes & Davis, 1987; Touryan, Moeny, Aimone & Benze, 1989., in lightning and static electricity simulation ŽHebert, Herbert,
U
Corresponding author. Tel.: q1-519-824-4120, ext. 2431; fax: q1-519-836-0227. E-mail address: [email protected] ŽG.S. Mittal..
Walko, Schneider & Serrano, 1987. and in various studies of the breakdown strengths of water and water mixtures ŽZahn, Ohki, Fenneman, Gripshover & Gehman, 1986; Mcleod and Gehman Jr., 1987.. Brine Žsodium chloride solution. is used by meat processors in bacon and wiener processing. After cooking in the smokehouse, the meat product is chilled using brine to wash off excess fat and smoke from the product, to provide excess salt at the product’s surface, and to cool the product enough to go through slicing and packaging along the processing line. Brine at y6 to y8 ⬚C is pumped from a loading tank and sprayed onto the product. The wash-off brine is then collected, recycled back to the tank, and reused. Currently, in many plants, the changeover rate of the brine is every 3᎐4 days. During this period, contamination of the brine Žand thus, the product. is common. Operations such as preparing and loading of the brine to the tank, or pumping and recycling the brine through pipes and spray nozzles contaminate the brine. The total plate count of waste brine can be ) 1000 cfurml ŽMittal &
1466-8564r01r$ - see front matter 䊚 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 4 3 - 1
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Choudhry, 1997.. The waste brine was reported to contain both gram-positive cocci and gram-negative rod-shaped bacteria, mainly consisting of Micrococcus and Staphylococcus species, with a small number of Pseudomonas and lactic acid bacteria ŽMittal & Choudhry, 1997.. The use of an in-line high-voltage electric pulse system to pasteurize waste brine from the chilling operation was tested in this research. The objectives were to investigate microbial survivability in waste brine as a function of electric field strength and number of pulses applied in the batch and continuous treatment systems, and to identify the optimum conditionŽs. for maximum microbial reduction.
2. Materials and methods 2.1. Waste brine Waste brine from a bacon smokehouse cooking was provided for this study by a meat plant in Scarborough, Ontario, Canada. Waste brine was collected into sterile containers. The brine solution was stored at y7⬚C ŽIsotemp cooler, Fisher Scientific Co., Toronto, Canada.. The salt content of the filtered waste brine was determined by calculating the difference in mass before and after drying in a Speed Vac concentrator ŽSavant Speed Vac-SVC 100H, Instruments Inc., Farmingdale, N.Y.., which was attached to a freeze drier ŽLabconco Trivac freeze drier-D8A, LeyboldHerraus Vacuum Products Inc., Mellon, PA.. that controlled the temperature and vacuum. Electrical conductivity of the brine was also determined by using a conductivity meter ŽCole-Parmer Oakton, Niles, IL, model WD-35607-10.. 2.2. Treatment system and conditions A bench-top pulsed field treatment system was used to process brine in batch and continuous modes ŽHo et al., 1995.. The system consists of a 30-kV d.c. high voltage pulse generator, a circular treatment chamber and devices for pumping and recording. Fig. 1 shows the block diagram of the unit. The 110-V a.c. is raised in voltage through a high voltage transformer, and then rectified. The d.c. high voltage supply then charges up the 0.12-F capacitor through a series of 6 M⍀ resistors Žthe time constant s 0.72 s.. The pulse generator emits a train of 5 V pulses, and the trigger circuit serves to convert that to 500 V pulses using a silicon control rectifier ŽSCR.. The pulse frequency can be adjusted with the choice of a single pulse Žmanual., or an autotriggering system of 0.09᎐0.5 Hz. The uniqueness of this pulsar is that pulses of low-energy and of instant-charge-reversal shape are generated. Conven-
Fig. 1. Block diagram of the pulsed field treatment system.
tional pulsar generates pulses of much higher energy and of square or exponential decay or bipolar shape. The circular treatment chamber Ž25.0 cm diameter. has two circular and parallel stainless steel electrodes Ž16.5 cm diameter.. The insulation, Delrin, was constructed to have close physical contact with the electrodes. The distance between the electrodes can be adjusted by inserting Delrin circular plates Ž14.5 cm diameter. with five different thicknesses: 0.08, 0.16, 0.3, 0.6 or 0.9 cm. Thus, the process volume can be varied between 13.2 and 148.6 ml per batch. To eliminate air bubbles in the treatment chamber, the sample entered into the chamber from the bottom through a channel in the chamber under a 85-kPa vacuum. A digital oscilloscope Žmodel TDS 340, Tektronix Inc., Beaverton, OR. and a high voltage probe Žmodel P6015A, Tektronix Inc., Beaverton, OR. are connected to the system so that each pulse across the chamber can be monitored. The batch system can be used in continuous mode by pumping the sample through the horizontal chamber from one end and collected at the other. The flow rate can be controlled by adjusting the position of needle valves on the treatment chamber. In this research, the maximum process capacity used was 18 lrh. The brine was pumped from feed tank into treatment chamber from one end with a pressure pump at 186 kPa Žabsolute.. The sample was collected after 5 min running into sterile plastic containers. Sterile containers were placed on ice with NaCl to minimize microbial growth and to maintain temperature at y7 " 1⬚C before and after treatment. For all experiments, constant process parameters were: electrode gaps 3 mm, pulse frequency s 0.5 Hz and process Žtreatment . temperature s y7 " 1⬚C. Voltage supplied by the d.c. source s 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 18 and 21 kV, and pulses applieds 1, 5, 10, 20 and 30. Flow rates in continuous mode were 9 Ž10 pulses applied. and 18 lrh Žfive pulses applied.. All experiments were repeated at least three times, in the reverse experimental order as the previous set. This was to minimize the variation in count due to microbial growth with time.
S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259
2.3. Microbial procedure Before and after electrical treatment, microbial plate counts of colony forming units Žcfu. on selected media indicated the reduction of the microbial cells under different process conditions. Previous results ŽMittal & Choudhry, 1997. on microbial analysis of waste brine from the same meat plant indicated that approximately 76% of the microbial population contained Staphylococcus and Micrococcus, and it was necessary to use agar with NaCl for getting higher microbial count. Hence, in this study, only the total plate count, and Staphylococcus and Micrococcus were measured. The microbial level of the untreated and treated samples was measured by platting and incubating ŽHotpack incubator; Hot Pack, Waterloo, Ontario. at 30⬚C for 48 h for a total plate count on agar with 10% NaCl, or agar with Mannitol salt at 37⬚C for 36 h for Staphylococcus and Micrococcus. Microbial plating of samples was done twice: once before the experiment Žuntreated., and once after the experiment Žtreated.. This was to ensure that the time length of the experiment had no effect on microbial reduction. For each test set, a control with all the steps followed but no electrical treatment was carried out. This was to ensure that the sanitization had no effect on the killing rate. For selflife studies Žmicrobial count., samples were stored at y7⬚C and plated every 3᎐4 days for 3 weeks.
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bomb. The procedure was repeated six times: 3-min soak and 3-min purge. Water was circulated through the CPD bomb at 40᎐45⬚C and 100᎐1350 psi for 10 min. Pressure was released, and the filtrate was removed and gold plated. The sample was studied by SEM Žmodel S-570, Hitachi, Tokyo, Japan; 10᎐15 kV accelerating voltage.. The magnification Ž13 000 = ., resolution and tilt angle were adjusted. Then photographs were taken. 2.6. Data analysis Statistical analysis was performed on the data ŽSYSTAT, 1992.. The microbial reduction was modeled as a function of electrical field strength and number of pulses applied ŽEqs. Ž1. ᎐ Ž3... The microbial plate count Žbefore and after pulse treatment with three replications. were transformed into statistical files and fitted using the technique of non-linear regression modeling ŽSYSTAT, 1992.: a Quasi-Newton estimation method was used, with 0.10 as the parameter starting value; iterations continued until the maximum iteration limit Ž999. was reached. 2.7. Microbial inacti¨ ation models Peleg Ž1995. reported that the microbial inactivation curve had a characteristic sigmoid shape, and established a model based on Fermi’s equation:
2.4. Other tests and calculations The electrical conductivity of the sample using a hand-held conductivity meter ŽCole-Parmer Oakton, Niles, IL, model WD-35607-10., pH using a pH meter ŽFisher Scientific, Whitby, Ontario, Canada, model 925. and sample temperature immediately before and after treatments ŽFisher Scientific Digi-thermo, Whitby, Ontario, Canada, model 15-077. were measured. Energy consumption ŽJrml. was calculated as 0.5 C E 2 nrV⬘, where C is the charging capacitor, F; E is voltage applied, V; n is number of pulses applied, and V⬘ is the sample volume treated, ml. 2.5. Scanning electron microscopy (SEM) The sample was filtered through a polycarbonate membrane filter Ž0.2᎐0.4 m., and filtrate was transferred to Sorensen’s phosphate buffer solution Ž1 part Na 2 HPO4 to 1 part KH 2 PO4 .. Then 2% glutaraldehyde was added to the PO4 buffer, and kept for 30 min at room temperature. The filtrate was dehydrated in a series of ethanol solution: 50, 70, 90, 95 and 3 = 100% at 15-min time intervals. Water was circulated through the CPD bomb at 15⬚C. The filtrate was put into specimen basket, and the bomb was filled with buffer. The filtrate was dried by flushing CO 2 through the
S Ž E f ,n . s
1 E f y Ec Ž n . 1 q exp ac Ž n .
Ž1.
where S s surviving fraction of microorganisms, NrNo; n s number of pulses applied; E f s electrical field strength applied, kVrcm; Ec s critical electrical field strength where the survival fraction is 0.50 or the inflection point of S, kVrcm; a c s a parameter indicating the steepness of the survival curve around Ec , kVrcm; Nos initial microbial level; and N s microbial level after treatment. Ec Ž n. and a c Ž n. were described by exponential decay functions, as follows: Ec Ž n . s Ec o ⭈ exp Ž yk 1 ⭈ n .
Ž2.
a c Ž n . s a o ⭈ exp Ž yk 2 ⭈ n .
Ž3.
where Ec o and a o s constants, kVrcm; and k 1 and k 2 s constants, dimensionless. Peleg Ž1995. used data from Castro, BarbosaCanovas & Swanson Ž1993. and showed that the data had a good fit Ž R 2 s 0.97᎐0.999. with Eq. Ž1.. Experimental results from Ho et al. Ž1995. seemed to support this model as well. Zhang, Chang, Barbosa-Canovas &
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S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259
Swanson Ž1994. used the Hulsheger, Potel and Niemann Ž1981. model to test the survival fraction of E. coli, S. aureus and S. cere¨ isiae in a semisolid model food containing potato dextrose agar. Our work did not support the Hulsheger et al. Ž1981. model.
3. Results and discussion 3.1. Properties of waste brine The salt content of the brine was 26.00" 0.02% wrw, the water content was 73.5" 0.03%, and rest was traces of protein and fat. The electrical conductivity was 300 " 20 mSrm. The specific gravity was 1.25" 0.01 and pH was 7.0" 0.1. All properties were unaffected under all test conditions. Temperature change was also negligible due to small energy input during treatment. A typical pulse waveform is shown in Fig. 2 using waste brine. For batch processing, the mean initial total plate count was 887 " 186 cfurml and the mean initial Staphylococcus and Micrococcus count was 1798 " 103 cfurml. For continuous processing, the mean initial total plate count was 998 " 85 cfurml, and mean initial Staphylococcus and Micrococcus count was 1910 " 44 cfurml. 3.2. Batch system Model parameters of Eqs. Ž1. ᎐ Ž3. are listed in Tables 1 and 2. Table 3 lists the statistical analyses on the model parameters. Results from Table 3 indicate that there is a good correlation between the individual microbial reduction estimation and the overall model parameters Ž R 2 s 0.994᎐1, P- 0.01.. The individual typical survival curves Žraw data and fitted model. are depicted in Figs. 3 and 4 in terms of survival fraction. Similar plots were also obtained for other conditions Žnot shown.. A three-dimensional plot of Eq. Ž1. is shown in Fig. 5 for total plate count. In general, microbial results are in good agreement with the model. Statistically corrected R 2 is 0.981 Žstandard plate count. and 0.989 Žfor Staphylococcus and Micrococcus. for the
Fig. 2. Pulse waveform measured across the chamber containing waste brine in the batch treatment system at 15 kV voltage supply and 3 mm electrode gap. Scale: vertical 2 kVrdivision, horizontal 500 nsrdivision.
overall model Ž R 2 is in the range of 0.981᎐0.995 for the individual curves. ŽANOVA for this is not given.. Results from both total plate count and StaphylococcusrMicrococcus illustrate a sigmoidal relationship between microbial survivability and the applied electric field strength at all number of pulses applied ŽFigs. 3 and 4.. This is clearly in support of Peleg’s findings Ž1995.. Peleg obtained Ec in the range of 6.7᎐21.2 kVrcm and a c in the range of 1.6᎐3.1 kVrcm for various pure culture organisms in buffer solutions. For waste brine, Ec is in the range of 14᎐31 kVrcm and a c is in the range of 6.7᎐9.0 kVrcm. By definition, Ec Table 1 Overall parameters of Peleg model for PEF batch treating of waste brine using Eqs. Ž2. and Ž3. Parameters
Staphylococcus and Micrococcus
Total plate count
Ec o ŽkVrcm. k1 ao ŽkVrcm. k2
31.232 0.012 6.889 y0.005
25.727 0.020 6.821 0.006
Table 2 Peleg model parameters for PEF batch treating of waste brine expressed in terms of number of pulses applied using Eq. Ž1. Pulses applied, n
1 5 10 20 30
Staphylococcus and Micrococcus
Total plate count
Ec ŽkVrcm.
ac ŽkVrcm.
Ec ŽkVrcm.
ac ŽkVrcm.
30.859 29.413 27.700 24.568 21.790
7.924 8.063 8.242 8.614 9.004
25.218 23.279 21.063 17.245 14.119
7.780 7.619 7.424 7.050 6.697
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Table 3 Statistical analyses on the Peleg model parameters for PEF batch treating of waste brine using Eq. Ž1.
2
R Standard error of estimate Mean sum of squares Žregression. DF Žerror. P) T
Staphylococcus and Micrococcus
Total plate count
Ec
ac
Ec
ac
1 0.665 3650.292 4 - 0.01
0.994 0.747 346.716 4 - 0.01
0.997 1.297 2132.898 4 - 0.01
0.998 0.323 266.954 4 - 0.01
Fig. 3. Microbial survival fraction Ž NrNo. for total plate count in batch mode at various electrical fields Ž E . and number of pulses applied Ž n..
represents the critical electric field strength where the survival fraction is 0.50 Žreflection point on curve., and a c represents a parameter indicating the steepness of the survival curve around Ec . It seems that the value of Fig. 5. Three-dimensional microbial survival Ž NrNo. plot of waste water for total plate count using Eq. Ž1.. Es electrical field and n s number of pulses.
Fig. 4. Microbial survival fraction Ž NrNo. for Staphylococcus and Micrococcus in batch mode at various electrical fields Ž E . and number of pulses applied Ž n..
Ec and a c would increase with the increase in the number of microbial species Žor the relative resistance . in the fluid medium. Previous work ŽHo et al., 1995. on a pure culture of Pseudomonas fluorescens ŽM3r6. suspended in various fluid conditions indicated an either ‘kill-or-no-kill’ scenario, with 15᎐35 kVrcm as the applied critical electric field strength. This can be interpreted as a sigmoidal survival curve with a moderate Ec value Ž14᎐30 kVrcm. and a small a c value Ž7᎐9 kVrcm.. On the other hand, various species of StaphylococcusrMicrococcus have been identified in the waste brine, along with other microbes such as Pseudomonas, and lactic acid bacteria ŽMittal & Choudhry, 1997.. As a result, Ec and a c increase, with values for StaphylococcusrMicrococcus significantly Ž PF 0.05. higher than values for total plate
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count ŽTable 2. Ž Ec s 22᎐31 kVrcm vs. 14᎐25 kVrcm, and a c s 7.9᎐9.0 vs. 6.7᎐7.8 kVrcm.. By comparing Ec and a c values of different pure microbial cultures to the values of a product which is usually comprised of many mixed species, it is possible to identify the relative quantity and physiological state of each species or, more importantly, the presence of any interactions between or among species in the product under pulse treatment. This helps to identify the optimum operating conditionŽs. when the lethality of a specific species is intended. More work is needed in this area. From Table 4 ŽANOVA., the electric field strength applied is a far more important factor in microbial reduction than the number of pulses applied. This indirectly supports the idea of transmembrane potential theory ŽHo & Mittal, 1996. where a critical potential is required to develop on the membrane surface for cell lysis, and that the number of pulses applied only affects the area being electroporated. Based on the results, no colony forming units Ž- 10 cfurml. were detected on either agar media when the product underwent the following treatments, where E f is electrical field applied and n is the number of pulses: E f s 50 kVrcm, 10 F n F 30 « minimal energy consumption s 2.7 Jrml at n s 10 for a 50-ml batch; and E f s 60 kVrcm, 1 F n F 5 « minimal energy consumption s 0.39 Jrml at n s 1 for a 50-ml batch.
1.
2.
The log reductions for total plate count were ) 1.95 " 0.08, and for Staphylococcus and Micrococcus count were ) 2.26" 0.02. Option 2 clearly has economical advantages over option 1. However, option 1 may probably provide a better safety net for quality assurance due to large number of pulses applied, especially in a continuous operation as missing of a few pulses will not have significant effect on microbial decay. No detectable physical ŽpH, electrical conductivity, specific gravity. nor thermal changes Žnegligible change in temperature. occurred under those conditions. Using the same treatment system but a sightly different waste brine Ž22.5% NaCl, wrw., Mittal and Choudhry Ž1997. identified E f s 50 kVrcm and n s 15 as the optimum
treatment condition. The results of this research seem to be comparable with previous work. Selected samples, both before and after pulse treatment using the optimum condition Ž60 kVrcm, one and five pulses., were analyzed by scanning electron microscopy ŽSEM. for visual damage to the microorganisms. Results are shown in Figs. 6, 7 and 8. It is evident that microbes under pulse treatment are disrupted and ruptured while microbes without pulse treatment remain fully intact. The figures also provide support for the transmembrane potential theory. Selected samples were treated with the optimal treatment condition Ž E f s 60 kVrcm and n s 5. and subjected to shelf-life studies. The storage temperature was y7⬚C Žnormal storage temperature used., and samples were plated every 3᎐4 days for 3 weeks. Again, no colony-forming units Ž- 10 cfurml. were detected on either agar media throughout the test period. Together with the microbial and SEM data, it seems that the pulsed field treatment employed in this research caused irreversible damage to the microbes being tested, with negligible physical ŽpH, electrical conductivity, and specific gravity. and thermal changes Žas temperature increase was negligible. to the waste brine. 3.3. Continuous treatment system Waste brine was treated at various field strength and two flow rates. The log reductions for total plate count at the flow rates of 9 and 18 lrh were ) 2.01" 0.03 with an initial count of 998 " 85 cfurml, and for the Staphylococcus and Micrococcus count were ) 2.28" 0.01 with an initial count of 1910 " 44 cfurml in both cases. No significant change in microbial decay was noted when constant flow rate and process conditions were used. Fig. 9 shows the microbial survival fraction as a function of electrical field strength and flow rates, with Eq. Ž1. Žbatch condition. plotted for comparison. Although some pulses were occasionally missed Ž- 5%., the pulse waveforms Žpeak voltage and pulse width. under continuous treatment remained stable and were similar to those obtained under batch processing. This ensured that the number of pulses applied to the brine
Table 4 Analysis of variance for the PEF process parameters to treat waste brine in batch system Source
Pulses applied Field strength Error U
PF 0.05. P- 0.01.
UU
d.f.
4 12 48
Staphylococcus and Micrococcus
Total plate count Mean sum of squares
F Ratio
Mean sum of squares
F Ratio
0.052 0.670 0.005
11.507U UU 148.564
0.036 0.693 0.003
11.995U UU 233.393
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- 10 cfurml, i.e. ) 2.01" 0.05 log reductions., the energy consumption was: E f s 60 kVrcm, n s 5 at 18 lrh, n s 10 at 9 lrh « energy required s 1.94 Jrml.
4. Conclusions and summary of results There was no effect on pH and electrical conductivity of the waste brine due to high voltage pulsed field treatment. No microbial contamination was detected after waste brine was subjected to pulsed field treatment in batch or continuous flow systems. Shelf life studies indicated no microbial growth for at least 3 weeks. The pulsed field treatment caused irreversible damage to microbes. Optimum process conditions in batch system were: E f s 60 kVrcm and 1 F n G 5 with an energy consumption of 0.39 Jrml at n s 1. The microbial reduction was modeled as a function of E f and n in sigmoid function. The E f value was a far more important factor in microbial reduction than n.
Fig. 6. Magnified view Ž=30 000. of microbes in waste brine without pulsed field treatment using scanning electron microscopy.
passing through the treatment chamber was sufficient. Microbial results were consistent with batch processing. A - 10-cfurml plate count was obtained when the samples underwent pulse treatment at optimum conditions. Again, the flow rate Žnumber of pulses applied. and process temperature appeared not to be significant at the test range. The relatively small standard deviation ŽS.D.; not given. indicates that the results from separate trials are in good agreement with each other. Fig. 10 shows the log reductions of Staphylococcus and Micrococcus at various applied fields and two flow rates. Thus, the results obtained from the continuous mode are in agreement with those obtained from the batch mode, indicating that the optimum conditions specified are effective, and that the treatment system can be used in a batch or continuous mode of operation. Although the degree of mixing in the chamber was significant, as indicated by the deviation of data points from Eq. Ž1. at various number of pulses applied, the impact on microbial reduction was not. From Figs. 9 and 10, the optimum electric field strength for waste brine would still be 60 kVrcm at 9 or 18 lrh flow rate, as established from the batch condition. Using the optimum treatment conditions Žwhere plate count gives
Fig. 7. Magnified view Ž=30 000. of microbes in waste brine after pulsed field treatment using scanning electron microscopy. Batch treatment conditions employed were 60 kVrcm electrical field strength, 3 mm electrode gap, 0.5 Hz pulse frequency, and one pulse applied.
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Fig. 10. Microbial decay Žlog N . for Staphylococcus and Micrococcus in waste brine at various flow rates and electrical fields Ž E .. At 9 lrh, n s 10, and at 10 lrh, n s 5.
the area being electroporated. In continuous mode at 9 lrh Ž n s 10. and 18 lrh Ž n s 5. and 0.5 Hz pulse frequency, microbial, pH and electrical conductivity and shelf life results were consistent with batch processing, and the energy consumption was 1.94 Jrml.
Fig. 8. Magnified view Ž=20 000᎐30 000. of microbes in waste brine after pulsed field treatment using scanning electron microscopy. Batch treatment conditions employed were 60 kVrcm electrical field strength, 3 mm electrode gap, 0.5 Hz pulse frequency, and five pulses applied.
This supported the transmembrane potential theory where a critical potential is required to develop on membrane surface for cell lysis, and that n only affects
Fig. 9. Microbial survival fraction Ž NrNo. for total plate count in waste brine at various flow rates and electrical fields Ž E .. Predicted data are based on parameters from Tables 1 and 2, and Eq. Ž1.. At 9 lrh, n s 10, and at 10 lrh, n s 5.
Acknowledgements Funding of this research by the Ontario Ministry of Agriculture, Food and Rural Affairs is greatly appreciated. References Castro, A. J., Barbosa-Canovas, G. V., & Swanson, B. G. Ž1993.. Microbial inactivation of foods by pulsed electric fields. Journal of Food Processing and Preser¨ ation, 17, 47᎐73. Cenanovic, M. B., & Maureira, H. A. Ž1989.. Application of pulsed power in the steel industry. Proceedings of 7th IEEE Pulsed Power Conference, 65᎐68. Gray, E. W., Moeny, W. M., Beckes, B. R., & Davis, B. B. Ž1987.. Pulsed power fracturing of rock. Proceedings of 6th IEEE Pulsed Power Conference, 330᎐335. Hebert, J. L., Herbert, M. P., Walko, L. C., Schneider, J. G., & Serrano, A. Ž1987.. Pulsed power applications in aircraft lightning qualification testing. Proceedings of 6th IEEE Pulsed Power Conference, 326᎐329. Ho, S. Y., Mittal, G. S., Cross, J. D., & Griffiths, M. W. Ž1995.. Inactivation of P. fluorescens by high voltage electric pulses. Journal of Food Science, 60 Ž6., 1337᎐1340. Ho, S. Y., & Mittal, G. S. Ž1996.. Electroporation of cell membrane: a review. Critical Re¨ iews in Biotechnology, 16 Ž4., 349᎐362. Ho, S., & Mittal, G. S. Ž2000.. High voltage pulsed electrical field liquid food pasteurization. Food Re¨ iew International Žin press.. Hulsheger, H., Potel, J., & Niemann, E. G. Ž1981.. Killing of bacteria with electric pulses of high field strength. Radiation and En¨ ironmental Biophysics, 20, 53᎐65. Mcleod, A. R., & Gehman Jr., V. H. Ž1987.. Water breakdown measurements of stainless steel and aluminium alloys for long-
S.Y. Ho, G.S. Mittal r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 251᎐259 charging times. Proceedings of 6th IEEE Pulsed Power Conference, 57᎐59. Mittal, G. S., & Choudhry, M. Ž1997.. Pulsed electric field sterilization of waste brine solution. In R. Jowitt, Engineering and Food at ICEF 7 Žpp. O13᎐16.. Sheffield, UK: Academic Press. Peleg, M. Ž1995.. A model of microbial survival after exposure to pulsed electric fields. Journal of Science in Food and Agriculture, 67, 93᎐99. SYSTAT Ž1992.. SYSTAT for Windows, version 5. Evanston, IL: SYSTAT, Inc. Touryan, K. J., Moeny, W. M., Aimone, C. T., & Benze, J. W. Ž1989..
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Electrohydraulic rock fracturing by pulsed power generated focused shocks. Proceedings of 7th IEEE Pulsed Power Conference, 69᎐72. Zahn, M., Ohki, Y., Fenneman, D. B., Gripshover, R. J., Gehman, V. H. Ž1986.. Dielectric properties of water and waterrethylene glycol mixtures for use in pulsed power system design. Proceedings of IEEE, 74Ž9., 1182᎐1221. Zhang, Q., Chang, F. J., Barbosa-Canovas, G. V., & Swanson, B. G. Ž1994.. Inactivation of microorganisms in a semisolid model food using high voltage pulsed electric fields. Food Science and Technology, 27 Ž6., 538᎐543.