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HURDLE TECHNOLOGIES: MICROBIAL INACTIVATION by PULSED ELECTRIC FIELDS DURING MILK PROCESSING a Thesis Presented to the Faculty O

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HURDLE TECHNOLOGIES: MICROBIAL INACTIVATION by PULSED ELECTRIC FIELDS DURING MILK PROCESSING a Thesis Presented to the Faculty O HURDLE TECHNOLOGIES: MICROBIAL INACTIVATION BY PULSED ELECTRIC FIELDS DURING MILK PROCESSING A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by OSCAR RODRIGUEZ GONZALEZ In partial fulfillment of requirements for the degree of Doctor of Philosophy December, 2010 ©Oscar Rodriguez Gonzalez, 2010 ABSTRACT HURDLE TECHNOLOGIES: MICROBIAL INACTIVATION BY PULSED ELECTRIC FIELDS DURING MILK PROCESSING Oscar Rodriguez Gonzalez Advisor: University of Guelph, 2010 Dr. Mansel W. Griffiths The application of non-thermal processes pulsed electric fields (PEF) and cross-flow micro-filtration (CFMF) continuous to be studied with the purpose of controlling microorganisms in milk. Trends suggesting increased adoption include the study of Food Safety Objectives as a safety criterion, the promotion of sustainable processing, and the implementation of hurdle strategies. While the advance of gentle processing is counteracted by the risk of enhanced resistance due to microbial stress response, several techniques can be applied to quantitatively assess its impact. The objective of this project was to evaluate the effectiveness of microbial inactivation by PEF and CFMF at various steps of milk processing including shelf-life, its comparison with high temperature short time (HTST) pasteurization, and the quantitative assessment of the cross protection resistance to PEF of Escherichia coli O157:H7. Some differences in mesophilics inactivation were observed in milks (fat contents between 1.1% and 3.1%). Increasing the PEF inlet temperature decreased the treatment time by three or two-fold. The combination of CFMF/PEF yielded similar microbial reductions as CFMF/HTST. Higher inactivation of the coliforms was achieved in homogenized cream (12% fat) compared to non-homogenized. The linear relation between electrical conductivity and nutrient content (fat and solids content) was established. In a parallel study the PEF/CFMF sequence resulted in higher inactivation of mesophilics compared to CFMF/PEF and HTST. The shelf life was acceptable for CFMF/PEF and HTST after 7 days, while enterics and psychrotrophs grew more after PEF/CFMF, thermodurics did after HTST. The growth and stress of Escherichia coli O157:H7 in lactose containing broths was monitored by absorbance and fluorescence expression of stress reporters. Growth was explained using a secondary model, and stress response using mechanistic and probabilistic models. PEF inactivation was evaluated following the Weibull distribution after the cells reached stationary phase or maximum fluorescence expression. Similar resistances were observed within the cells grown in lactose broth at 10, 25 or 40°C, as within stressed cells (starved or cold shocked). Cells grown at 45 °C were more resistant compared to the cells grown in acid, high salt concentration while the ones grown at cold temperatures were the weakest. ACKNOWLEDGMENTS To all my colleagues, advisors, family, and friends for their support. To the Dairy Farmers of Ontario and the Natural Sciences and Engineering Research Council for their financial assistance. i! ! ABBREVIATIONS Abbreviation Significance AA Acetic acid ABS Absorbance AIC Akaike information criterion ANOVA Analysis of variance ATCC American Type Culture Collection ATP Adenosine triphosphate BIC Bayesian information criterion CFMF Cross-flow microfiltration CFU Colony forming units CIPEC Canadian Industry Program for Energy Conservation ESL Extended shelf life FIL-IDF International Dairy Federation FSO Food safety objective G/NG Growth/no growth GFP Green fluorescence protein GWP Global warming potential HEST High electric field short time HHP Hydrostatic high pressure HPH High pressure homogenization HPHT High pressure high temperature ii! ! HSP Heat shock proteins HTST High temperature short time LA Lactic acid LACB Lactose broth LB Luria Berthani Broth, Miller MF Microfiltration MPN Most probable number MSE Mean square errors MTS Manothermosonication NACMCF National Advisory Committee on Microbiological Criteria for Foods NF Nanofiltration OD Optical density PATS Pressure assisted thermal sterilization PC Performance criterion PEF Pulsed electric fields PFN Pulse forming network PI Process integration PI Propidium iodide PL Pulsed light PO Performance objective RFEF Radio frequency electric fields RNA Ribonucleic acid RO Reverse osmosis iii! ! SEM Standard error of the mean TAL Thin agar layer TAMC Total aerobic mesophilic counts TEBC Total enteric bacteria counts TLABC Total lactic acid bacteria counts TMP Trans-membrane potential TMYC Total moulds and yeasts counts TP Traditional pasteurization TPC Total psychrotroph counts TSB Tryptic soy broth TSBL Tryptic soy broth with added lactose (0.5%) TSC Total staphylococci counts TTMC Total thermoduric mesophilic counts TTPC Total thermoduric psychotroph counts TTTC Total thermoduric thermophilic counts UF Ultrafiltration UHT Ultra high temperature UTMP Uniform trans-membrane pressure WeLL Weibullian-log logistic model iv! ! NOMENCLATURE Symbol Significance units a decay rate kV/mm ABS absorbance - Af Accuracy factor - - aw water activity b number of bacteria at the end of time interval log CFU/ml B number of bacteria at the beginning of time interval log CFU/ml b inactivation rate !s-1 bE regression coefficient for electric field - bt regression coefficient for treatment time - C storage capacity or charging capacitance F Cp specific heat capacity kJ/kg°C cr cell radius m ctrl non-transformed cells - d electrode gap m DX°C decimal reduction time (at given temperature X°C) s E electric field strength kV/m EC0 critical value of E for 100% survival kV/mm E critical E value constant C00 kV/mm f repetition rate or frequency pulses per second, Hz f(t) density function - v! ! Fat fat fraction % FSO food safety objective CFU/l G generation time min, h g rate constant - gfpUV plasmid containing cells - H0 the initial level of the hazard CFU/l I current signals A i intercept of relation between % fat and electrical %fat Fat conductivity intercept of relation between % solids and electrical i %sol Sol conductivity k constant factor - k (E,Tin) inactivation constant, - k1,2,3…8 dimensionless empirical parameters - log (N/N0) reduction in log survivor count - m shape parameter - !" mass flow rate kg/s n number of pulses - N the number of observations used in the calculation - Obs observed - P pressure Pa p shape parameter - pH pH h-1 PO performance objective - vi! ! Pre predicted - q concentration of limiting substrate - R response ratio - r rate of response - RC PEF chamber resistance ! RFI relative fluorescence intensity - S surviving fraction - s slope of relation between % fat and electrical S / m %fat Fat conductivity SFI specific fluorescence intensity - Sol solids fraction % s slope of relation between % solids and electrical S / m %sol Sol conductivity t time s T temperature °C tc the mean of the microbial population resistance s Tc temperature level of inactivation onset °C tC0 critical value of tt for 100% survival s Tin inlet temperature °C TMP trans-membrane potential V tt treatment time "s U charging voltage kV V Volume l !" volumetric flow rate m3/h vii! ! v rate of increase of the limiting substrate - 3 VC volume of PEF treatment chamber m w total applied energy kJ wpl specific energy per pulse kJ/pulse wsp specific energy input kJ/kg, kJ/l x microbial population log CFU/ml ! scale parameter - " shape parameter - ! gamma function - # pH parameter - $P pressure rise Pa $T temperature rise °C pump and motor efficiency, 0.85 for positive pumps, and % - 0.65 for efficient centrifugal pumps & upper asymptote log CFU/ml ' electrical conductivity of the product S/m ( growth rate h-1 -1 (max maximum growth rate h ) density kg/l * maximum slope of the inactivation curve s-1 +I CFU/l total (cumulative) increase of the hazard on a log scale the total (cumulative) reduction of the hazard on a log +R CFU/l scale log time at which the maximum slope is reached " s (position of the curve) viii! ! !w pulse width s -1 -1 ! Ratkowsky parameter °C h angle between the electric field and the point of interest ! - in the membrane (cos ! = 1 at the poles) " a form factor (3/2) for spherical cells - # lower asymptote log CFU/ml " lower asymptote log CFU/ml $ temperature parameter - " water activity parameter - ix! ! ! TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………………………...i ABBREVIATIONS…………………………………………………………………………....….ii NOMENCLATURE………………………………………………………………………….......v 1. Introduction ............................................................................................................... 1 1.1. Non-thermal dairy processing ............................................................................... 1 1.1.1. Advances in critical milk processes ................................................................ 2 1.1.2. Microfiltration .................................................................................................. 5 1.1.3. Emerging technologies ................................................................................... 7 1.1.4. Regulatory status of pasteurization technologies .......................................... 11 1.1.5. Sustainable dairy processing ........................................................................ 14 1.2. Pulsed electric fields ........................................................................................... 17 1.3. Combined processes .......................................................................................... 25 1.3.1. Advanced
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