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2021 40 Santiago Condón Abanto Adding Value to Ready-toCrustacean Products: Process optimization for "entire" crustaceans using novel technologies Departamento Director/es Producción Animal y Ciencia de los Alimentos Raso Pueyo, Javier Lyng, James G. Álvarez LAnzarote, Ignacio © Universidad de Zaragoza Servicio de Publicaciones ISSN 2254-7606 Santiago Condón Abanto ADDING VALUE TO READY-TO-CRUSTACEAN PRODUCTS: PROCESS OPTIMIZATION FOR "ENTIRE" CRUSTACEANS USING NOVEL TECHNOLOGIES Director/es Producción Animal y Ciencia de los Alimentos Raso Pueyo, Javier Lyng, James G. Álvarez LAnzarote, Ignacio Tesis Doctoral Autor 2019 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Adding Value to Ready-to-eat Crustacean Products: Process optimization for “entire” and “particulate” crustaceans using novel technologies by Santiago Condón-Abanto, Dipl. Ing (Agr.), B.Sc., M.Sc. UNIVERSITY COLLEGE DUBLIN UNIVERSIDAD DE ZARAGOZA AN COLÁISTE OLLSCOILE, BAILE ÁTHA CLIATH A thesis submitted to University College Dublin and Universidad de Zaragoza for the degree of Doctor of Philosophy (PhD) in Food Science at UCD Dublin, School of Agriculture & Food science, Belfield, Dublin 4, Ireland and Universidad de Zaragoza, Facultad de Veterinaria, Zaragoza, España Principal Supervisor: Co-Supervisor: Prof. James Lyng Dr. Cristina Arroyo Prof. Ignacio Álvarez Prof. Javier Raso Declaration I hereby declare that the work herein submitted for the degree of Doctor of Philosophy of the National University of Ireland, Dublin is the result of my own investigation, except where the reference is made to the publish literature. I also declare that the material submitted in this thesis has not been accepted for any other degree and currently is only submitted to the University of Zaragoza as agreeded in the joint-PhD contract signed between both instittutions (Annex I). I authorise the National University of Ireland, Dublin and University of Zaragoza to lend this thesis to institutions or individuals for the purpose of scholarship research. I further the National University of Ireland, Dublin to reproduce this thesis by photocopying or byany other means in total or in part at the request of other institutions for the purpose of scholarly research, subjected to the normal conditions of acknowledgement. --------------------------------- Santiago Condón-Abanto D. Ignacio Álvarez Lanzarote y D. Javier Raso Pueyo, Porfesor titular y Catedrático respectivamente, del Área de Tecnología de los Alimentos de Departamento de Producción Animal y Ciencia de los Alimentos de la Facultad de Veterinaria de la Universidad de Zaragoza, Certifican: Que la Tesis Doctoral titulada “Adding Value to Ready-to-eat Crustacean Products: Process optimization for “entire” and “particulate” crustaceans using novel technologies” de la que es autor Santiago Condón Abanto, ha sido realizada bajo su dirección y en cotutela con el University College Dublin, donde el profesor James G. Lyng y la Dr. Cristina Arroyo han actuado como supervisores, y que su contenido corresponde con el Proyecto de Tesis aprobado en su momento y cumple con las condiciones requeridas para optar al grado de Doctor por la Universidad de Zaragoza. Fdo.: Dr. Ignacio Álvarez Lanzarote Fdo.: Dr. Javier Raso Pueyo Table of contents TABLE OF CONTENTS ABBREVIATIONS ...................................................................................................... I LIST OF FIGURES ...................................................................................................III LIST OF TABLES ...................................................................................................... X LIST OF PUBLICATIONS ...................................................................................... XIII CONFERENCE AND WORKSHOP PRESENTATIONS .............................................. XIV ACKNOWLEDGEMENT ........................................................................................... XV ABSTRACT ............................................................................................................ XVI RESUMEN ........................................................................................................... XVIII Chapter 1 General introduction and literature review ..................................................................... 1 1.1 FOOD PRESERVATION .................................................................................. 4 1.1.1 Food spoilage agents ............................................................................ 4 1.1.2 Food preservation strategies ................................................................ 8 1.2. HEAT TREATMENTS FOR FOOD PROCESSING ............................................ 10 1.2.1 Pasteurization ...................................................................................... 11 1.2.2 Sterilization .......................................................................................... 12 1.3 NOVEL TECHNOLOGIES FOR FOOD PROCESSING ...................................... 13 1.3.1 Ionizing radiation (IR) ......................................................................... 17 1.3.2 Ultrasound (US) ................................................................................... 21 1.4 PROCESS OPTIMIZATION ........................................................................... 25 1.4.1 Modelling in the food industry ............................................................ 27 1.4.2 Heat process optimization ................................................................... 36 1.5 EDIBLE CRAB-BROWN CRAB ( CANCER PAGURUS ) .................................... 41 1.6 OBJECTIVES ................................................................................................ 54 Chapter 2 Microbial characterization of edible crab ( Cancer pagurus ) ............................................ 56 2.1 ABSTRACT/RESUMEN ................................................................................. 57 2.2 INTRODUCTION .......................................................................................... 61 2.3 MATERIAL AND METHODS .......................................................................... 63 2.3.1 Raw material ........................................................................................ 63 2.3.2 Crab sample preparation ..................................................................... 64 2.3.2.1 Microbiota in raw crab ....................................................................... 64 2.3.2.2 Microbial shelf-life experiments .......................................................... 64 Table of contents 2.3.2.3 Microbiota in lab-cooked and commercial crab products ....................... 64 2.3.3 Microbial analyses and growth media ................................................. 65 2.3.4 Isolation and identification ................................................................. 67 2.3.5 Heat resistance experiments ............................................................... 68 2.3.5.1 Culture preparation and treatment media ........................................... 68 2.3.5.2 Heat treatments ............................................................................... 69 2.3.6 Data and statistical analysis ................................................................ 70 2.3.6.1 Shelf-life determination ..................................................................... 70 2.3.6.2 Thermal resistance determination ...................................................... 71 2.4 RESULTS AND DISCUSSION ....................................................................... 72 2.4.1 Microbiological profile of raw crab meats ........................................... 72 2.4.2 Microbiological shelf-life of cooked crabs ........................................... 74 2.4.3 Microbial growth modelling (Primary model) ..................................... 75 2.4.4 Effect of storage temperature (Secondary model) ............................. 78 2.4.5 Shelf-life determination (Tertiary model) ........................................... 81 2.4.6 Effect of storage temperature on microbial flora present in cooked crab ……. ............................................................................................................. 84 2.4.7 Bacterial groups present in lab-cooked and commercial samples of ready-to-eat brown crab .................................................................................... 86 2.4.8 Bacterial species isolated in brown crab ............................................. 89 2.4.9 Thermal resistance of the main vegetative cells isolated from readyto-eat brown crab ............................................................................................... 91 2.4.10 Heat treatments for ready-to-eat brown crab .................................... 96 2.5 CONCLUSIONS ............................................................................................ 99 Chapter 3 The use of ultrasound technology in the first cooking step of ready-to-eat whole brown crab (Cancer pagurus) ............................................................................................. 101 3.1 ABSTRACT/RESUMEN ............................................................................... 102 3.2 INTRODUCTION ........................................................................................ 104 3.3 MATERIALS AND METHODS ...................................................................... 105 3.3.1 Raw material and cooking conditions ............................................... 105 3.3.2 Heat transfer study ............................................................................ 106 3.3.2.1 Heat penetration curves .................................................................. 106 3.3.2.2 Lethality and F value ....................................................................... 108 3.3.3 Microbiological examination of fresh crab meat .............................. 108 3.3.4 Mass transfer study ........................................................................... 109 List of Figures IV Figure 1.14: Example of crabs during cooking (left) and immediately after cooking with no washing (right). ......................................................... 49 Figure 2.1: Log10 counts obtained in different selective and non-selective agars (see Table 2.2) for white (white bars) and brown (grey bars) raw crab meat. TVCm, Total viable mesophilic counts; TVCp, Total viable psychrophilic counts; L.H, Long and Hammer agar. ND = not detectable. ......................................................................................... 73 Figure 2.2: Evolution of TVCm over storage time (hours) at 2°C (), 5°C (), 7°C (), and 10°C () for white (A) and brown (B) meat obtained from ready-to-eat whole brown crabs cooked (75°C for 45 minutes) and packed under vacuum. ................................................................. 76 Figure 2.3: Measured Vs predicted counts of TVCm at all storage temperatures in white (A) and brown (B) meat (estimated from Eq. 2.6 and 2.7) ............................................................................................. 83 Figure 2.4: Log10 counts obtained for Bacillus spp., H2S producing bacteria, Enterobacteriaceae, Lactic Acid Bacteria, Pseudomonas spp., Staphylococcus spp., Total Viable Counts, Total Psychrophilic Aerobic Counts and total psychrophilic bacteria (L.H) for white (A) and brown (B) meat of cooked crab store at 4°C (grey bars) and 10°C (white bars) at the end of the shelf-life (calculated from Eq. 2.6). .................... 85 Figure 2.5: Relative percentage of in lab-cooked white and brown crab meat and various commercial samples at the end of their shelf-life. ............... 88 Figure 2.6: Log10 reductions in the populations of Staphylococcus spp. (A), Pseudomonas spp. (B), Carnobacterium spp. (C), Shewanella spp. (D) and Corynebacterium spp. (E) following a heat treatment at 60°C for 90 seconds. ........................................................................................ 92 Figure 2.7: Time (min) necessary to reduce the population of different species by 6 Log10 cycles (6 D values) in pH 7 McIlvaine citratephosphate buffer at 60°C. ................................................................... 95 Figure 2.8: Theoretical thermal death time curves (TDT) to achieve 6 Log10 reductions of Kocuria atrinae (black line) and Listeria monocytogenes (red line). The latter based on the recommended treatments by FDA (FDA, 2011). ...................................................................................... 97 Figure 2.9: Theoretical TDT lines to achieve 6 Log10 reductions of the spore forming bacteria isolated from crab based on data presented in chapter 6 and heat treatments required to achieve similar reductions of C. botulinum non-proteolytic spores based on the recommended List of Figures V treatments by FDA ( F 90ºC7/10ºC = 10 min; F 90ºC9ºC = 31 min; F 90ºC8.6ºC = 57 min) (FDA, 2011). .......................................................................... 97 Figure 3.1: Scheme of the location of the three thermocouples during the heat penetration experiments: abdomen (1), mandibular (2) and claw (3). Image adapted from reference (BIM, 2017a). .............................. 107 Figure 3.2: Time-temperature profiles over a cooking process at 75°C in the claw (dashed line), mandibula (dotted line) and abdomen (block line) of a 500g crab in a conventional cooking process without ultrasound. .. 108 Figure 3.3: Relationship between crab’s weight (from 300 to 870g) and the total surface of the crab’s carapace (cm2). ......................................... 115 Figure 3.4: F 70ºC7.5ºC value (min) applied during the cooking process in the crab's cold spot (i.e. abdomen), with (dashed line) and without (block line) ultrasound for the (A) small, (B) medium and (C) large crabs. The horizontal dotted line represents the target F 70ºC7.5ºC of two minutes. .... 117 Figure 3.5: Microbial load over conventional (grey bars) and ultrasound assisted cooking processes (black bars) in (A) white meat and (B) brown meat. Dotted line shows the detection limit for the counts. ....... 118 Figure 3.6: (A) Turbidity (OD515) and (B) conductivity (µS/cm) values for the cook water during the cooking of brown crabs in water at 75°C with (black bars) and without (grey bars) ultrasound. ................................ 120 Figure 3.7: Picture of the crab’s exudate after a conventional cooking (A) and ultrasound-assisted cooking (B). ................................................. 121 Figure 4.1: Release rate of Cd (% of Cd released per min) in crabs treated at 50°C (A), 65°C (B) and 80°C (C) with () and without () ultrasound. 137 Figure 4.2: Percentage of Cd removed in crabs treated at different temperatures with or without ultrasound. Bars with different letters indicate statistically significant differences (P≤0.05). .......................... 139 Figure 5.1: Time/temperature profiles during thermal pasteurization in a shower retort at 105°C in the crab’s claw (block line), mandibula (dotted lines) and abdomen (dotted line) of a 790g crab. Grey line shows the thermal profile of the autoclave. ........................................ 147 Figure 5.2: Diagram of the tubes used to study of the colour degradation kinetics of crab white meat. .............................................................. 151 List of Figures VI Figure 5.3: Effect of crab weight on the heat penetration parameters fh (A) and j (B), estimated from the fitting of Ball & Olson Equation to the thermal profiles obtained in the crab cold-spot. Figure 5.3A also shows the regression line (block line) as well as the 95% confidence intervals (dotted lines). .................................................................................. 154 Figure 5.4: Effect of the processing time on the evolution of ΔE* of crab white meat at 95°C (), 105°C () and 115°C (). Figure also shows the data fitting lines to Equation 5.10C. Error bars represent the standard deviation of three replicates. ............................................... 159 Figure 5.5: Effect of the treatment temperature on the colour change rate ( k ) (A) and maximum colour change developed ( Ymax ) (B) on crab white meat. Error bars show the standard deviation of three replicates.161 Figure 5.6: Correlation between Browning index (BI*) and total colour change (ΔE*) after different thermal treatments................................. 162 Figure 5.7: Quality score associated with crab white meat colour change based on the Irish crab producers perception. Number 4 indicates the maximum quality and 1 the lowest. ................................................... 163 Figure 5.8: Theoretical optimisation graph for the pasteurization process of ready-to-eat whole edible crab based on Equations 5.12 and 5.13. Grey lines represent the quality boundaries between “good” and “acceptable” (dashed line) and between “acceptable” and “nonacceptable” (block line). Black lines represents the minimum processing conditions (time/temperature) required for crabs of different weights (from bottom to top 400, 500, 600, 700 and 800 grams) to achieve an adequate F value based on the inactivation of C. botulinum type E ( F 90ºC10ºC=10 min). .................................................. 164 Figure 5.9: Observed versus predicted values for the thermal profiles (A) and white meat colour degradations (B) of crabs of different weights that were pasteurised under the conditions defined in Figure 5.5. .............. 165 List of Figures VII Figure 6.1: Diagram of the MTS resistometer. A, MTS resistometer main unit; B, ultrasound generator; C, heating/cooling water bath; D, agitation motor; E, Main vessel; F, treatment chamber; G, temperature data logger; 1, two way valve; 2, mano-reducer; 3, pressure/vacuum valve; 4, treatment chamber thermocouple; 5, cooling inlet (only MS); 6, main vessel filling empty tube; 7, ultrasound probe housing; 8, ultrasound probe; 9, treatment chamber filling valve; 10, solenoid sampling valve; 11, automatic injection syringe; 12, agitation shaft; 13, heating element; 14, cooling outlet (only MS); 15, main vessel pressure inlet; 16, main vessel thermocouple; 17 and 18 bottom and top caps. .......................................................................................... 175 Figure 6.2: Survival curves of Bacillus mycoides to heat (A) at 80°C (), 85°C (), 90°C () and 95°C () and mano-sonication (B) () and mano-thermo-sonication (B) at 50°C (), 65°C (), 80°C () and 90°C () in pH 6.8 citrate-phosphate buffer. ..................................... 178 Figure 6.3: Survival curves of Psychrobacillus psychrodurans to heat (A) at 90°C (), 93°C (), 96°C () and 100°C () and mano-sonication (B) () and mano-thermo-sonication (B) at 80°C (), 85°C (), 90°C () and 95°C () in pH 6.8 citrate-phosphate buffer. ........................ 178 Figure 6.4: Survival curves of Bacillus weihenstephanensis to heat (A) at 102.5°C (), 105°C (), 107.5°C () and 110°C () and manosonication (B) () and mano-thermo-sonication (B) at 80°C (), 85°C (), 90°C () and 95°C () in pH 6.8 citrate-phosphate buffer. ........ 178 Figure 6.5: Influence of the temperature on the shoulder length calculated with the Geeraerd log-linear regression plus shoulder model for B. weihenstephanensis (A) and P. psychrodurans (B) by heat (open symbols) and MTS (solid symbols) in pH 6.8 citrate phosphate buffer. Dotted line represents the theoretical DRT curves to MTS calculated with Equation 6.6. ............................................................................ 179 Figure 6.6: Influence of temperature on the 4 D values calculated with the Geeraerd log-linear regression plus shoulder model or log-linear model for B. mycoides (), P. psychrodurans () and B. weihenstephanensis () inactivation by heat in pH 6.8 citrate-phosphate buffer. ............... 180 Figure 6.7: Influence of temperature on the 4 D values calculated with the Geeraerd log-linear regression plus shoulder model or log-linear model for B. mycoides (), P. psychrodurans () and B. weihenstephanensis () inactivation by MS/MTS in pH 6.8 citrate-phosphate buffer. .......... 182 List of Figures VIII Figure 6.8: Percentage of synergism calculated with Equation 6.6 for the inactivation by MTS of 4 log10 cycles (4 D values calculated with Geeraerd log-linear regression plus shoulder model) of B. mycoides (block line), P. psychrodurans (dotted line) and B. weihenstephanensis (dashed line). ................................................................................... 184 Figure 7.1: Survival curves to electron beam ionizing radiation at room temperature of B. mycoides (A), B. weihenstephanensis (B) and P. psychrodurans (C) in citrate-phosphate buffer of pH 7 and water activity (aw) of >0.99 (), 0.90 () and 0.80 (). Error bars represent standard deviation of three replicates. ............................................... 200 Figure 7.2: Survival curves to electron beam ionizing radiation at room temperature of B. mycoides (A), B. weihenstephanensis (B) and P. psychrodurans (C) in citrate-phosphate buffer of pH 5.5 and water activity (aw) of >0.99 (), 0.90 () and 0.80 (). Error bars represent standard deviation of three replicates. ............................................... 201 Figure 7.3: Survival curves to electron beam ionizing radiation at room temperature of B. mycoides (A), B. weihenstephanensis (B) and P. psychrodurans (C) in citrate-phosphate buffer of pH 4 and water activity (aw) of >0.99 (), 0.90 () and 0.80 (). Error bars represent standard deviation of three replicates. ............................................... 202 Figure 7.4: Effect of the water activity (aw) on the dose necessary to reduce 6 Log10 cycles of B. mycoides (A), B. weihenstephanensis (B) and P. psychrodurans (C) at pH 7.0 (), 5.5 () and 4.0 (). Error bars represent standard deviation of three replicates. ................................ 205 Figure 7.5: Survival curves to electron beam ionizing radiation at room temperature of B. mycoides (), B. weihenstephanensis () and P. psychrodurans () in crab’s white meat (A) and brown meat (B). Error bars represent standard deviation of three replicates. ......................... 207 Figure 7.6: Specific resistance of B. mycoides (black bars), B. weihenstephanensis (grey bars) and P. psychrodurans (white bars) to different inactivation technologies in citrate-phosphate buffer of pH 7.0 and aw >0.99 (data for MS, MTS and Heat are adapted from Chapter 6). ................................................................................................... 212 Figure 8.1: Maximum pasteurization temperatures allow to achieve the target F 90°C10°C= 10 min minmaintaining “good quality” (green line) and “acceptable quality” (blue line) depending on the crab weight, based on the equations developed in Chapter 5. .......................................... 226 List of Figures IX Figure 8.2: Theoretical optimisation graph for the pasteurization of ready-toeat whole edible crab based on Equations 5.12 and 5.13 for crabs of 600g for the inactivation of 6 Log10 reductions of C. botulinum nonproteolytic type E based on U.S. FDA recommendations and B. weihenstephanensis based on the thermal resistance obtained in this PhD Thesis (Chapter 6). .................................................................... 227 Figure 8.3: Log of the time required to achieve 6 Log10 reductions of the population of B. weihenstephanensis by heat (black bars) and MTS (white bars) at different temperatures in pH 6.8 McIlvaine citratephosphate buffer. ............................................................................. 230 Figure 8.4: Required radiation dose to reduce 6 Log10 cycles the population of the main three bacterial spores isolated from edible crab in the two types of crab meat............................................................................ 231 Figure 8.5: Flow diagram of the different alternative processes proposed, using different alternative technologies. ............................................. 234 List of Tables X LIST OF TABLES Table 1.1: Brief description of the novel thermal and non-thermal technologies for food processing. ........................................................ 15 Table 1.2: List of authorized food products and maximum irradiation dose in different EU countries. ........................................................................ 21 Table 1.3: Volume and value of landings of edible crab in the main producers countries in 2013. ............................................................................... 44 Table 2.1: Summary of commercial crab products evaluated in this study. ...... 63 Table 2.2: Culture media and incubation conditions used during this investigation. ...................................................................................... 66 Table 2.3: Lag phase ( λ ) in hours and maximum growth rate ( μmax ) in hours-1 estimated with the modified Gompertz Equation (Zwietering et al ., 1990) for total viable counts in white and brown crab meat from readyto-eat whole brown crab pack under vacuum and stored at 2°C, 5°C, 7°C and 10°C. .................................................................................... 78 Table 2.4: Parameters of the square root model (Ratkowsky et al ., 1982) for the maximum growth rate and Lag phase for total viable counts growth on ready-to-eat brown crab pack under vacuum. ...................... 80 Table 2.5: Equations based on the final Equation which define the shelf-life of ready-to-eat whole brown crab pack under vacuum depending on the storage temperature. .................................................................... 82 Table 2.6: Vegetative bacterial species isolated from cooked ready-to-eat brown crab ( Cancer pagurus ) processed under laboratory conditions and various commercial products identified by 16S rRNA sequencing. .... 90 Table 2.7: Thermal resistance ( D T and z values) of a selection of non-spore forming bacterial species isolated from cooked crab meat. .................... 94 Table 3.1: Heat penetration parameters ( fh and j , dimensionless) arising from the application of the Ball & Olson model to the heat penetration curves in the cold spot of brown crabs ( Cancer pagurus ) of different weights and sizes cooked with or without ultrasound in water at 75°C. 112 Table 3.2: First order Equations correlating fh values (dimensionless) with the weight of brown crabs (g) during conventional and ultrasoundassisted cooking. .............................................................................. 114 List of Tables XI Table 3.3: Salt content (%) and moisture content (%) in white and brown crab meat cooked with or without ultrasound in water and water with 5% NaCl. Values represent mean value ± standard error. For both salt and moisture content, ANOVA followed by Tukey’s tests were performed for white and brown meat independently. .......................... 122 Table 4.1: DRC-e ICP-MS operating conditions ............................................ 131 Table 4.2: Total samples weights (dry weight) and moisture content (%) of white and brown meats for each crab used in this investigation. The theoretical initial amount of Cd was calculated based on Equation 4.2. 133 Table 4.3: Measured cadmium (Cd) concentrations in white and brown meats, both raw and after treatments at different temperatures with and without ultrasound (35 kHz, 100%, 200W, 60 minutes) and the maximum cadmium concentration detected in water after each treatment. Different letters indicate statistically significant differences within columns (P≤0.05). .................................................................. 134 Table 4.4: First order Equations which correlate the percentage of cadmium extracted with the treatment time during the different processes applied. ............................................................................................ 138 Table 5.1: Heat penetration parameters ( fh and j , dimensionless) arising from the fitting of the Ball & Olson Equation to the heat penetration curves in the cold-spot of various edible crabs ( Cancer pagurus ), of different weights and sizes, during thermal pasteurization in a shower retort at different temperatures. ........................................................ 148 Table 5.2: Average values of moisture, water holding capacity and colour of white and brown meat after retort pasteurization ( F 90ºC10ºC=10 min) at different temperatures. ..................................................................... 156 Table 5.3: Colour parameters of crab’s white meat at different times during heat processes at different temperatures. .......................................... 160 Table 6.1: Heat resistance parameters from the fitting of the Geeraerd loglinear plus shoulder model or log-linear model to the survival curves of Bacillus mycoides , Bacillus weihenstephanensis and Psychrobacillus psychrodurans spores treated in pH 6.8 citrate-phosphate buffer. ....... 177 Table 6.2: MS/MTS resistance parameters from the fitting of the Geeraerd log-linear plus shoulder model to the survival curves of Bacillus mycoides , Bacillus weihenstephanensis and Psychrobacillus psychrodurans spores treated in pH 6.8 citrate-phosphate buffer. ....... 181 List of Tables XII Table 7.1: Electron beam ionization resistance parameters obtained from the fitting of the Geeraerd log-linear plus shoulder model (Equation 7.1) to the survival curves of B. mycoides , B. weihenstephanensis and P. psychrodurans in citrate-phosphate buffers of different pH and aw. ..... 203 Table 7.2: Electron beam ionization resistance parameters obtained from the fitting of the Geeraerd log-linear plus shoulder model (Equation 7.1) to the survival curves of B. mycoides , B. weihenstephanensis and P. psychrodurans in white and brown crab meats. .................................. 206 List of Publications XIII LIST OF PUBLICATIONS Peer-reviewed articles  Condón-Abanto, S., Arroyo, C., Álvarez, I., Brunton, N., Whyte, P., & Lyng, J. G. (2018). An assessment of the application of ultrasound in the processing of ready-to-eat whole brown crab ( Cancer pagurus ). Ultrasonics Sonochemistry, 40, Part A, 497-504.  Condón-Abanto, S., Arroyo, C., Álvarez, I., Condón, S., & Lyng, J. G. (2016). Application of ultrasound in combination with heat and pressure for the inactivation of spore forming bacteria isolated from edible crab ( Cancer pagurus ). International Journal of Food Microbiology, 223, 9-16.  Condón-Abanto, S., Pedrós-Garrido, S., Cebrián, G., Raso, J., Condón, S., Lyng, J. G., & Álvarez, I. (2018). Crab-meat-isolated psychrophilic spore forming bacteria inactivation by electron beam ionizing radiation. Food Microbiology, 76, 374-381.  Condón-Abanto, S., Raso, J., Arroyo, C., Lyng, J.G., Condón, S. and Álvarez, I. (2018). Evaluation of the potential of ultrasound technology combined with mild temperatures to reduce cadmium content of edible crab ( Cancer pagurus ). Ultrasonics Sonochemistry, 48, 550-554. Articles submitted  Condón-Abanto, S., Raso, J., Arroyo, C., Lyng, J.G. and Álvarez, I. Qualitybased thermokinetic optimization of ready-to-eat whole edible crab ( Cancer pagurus ) pasteurization treatments. Submitted to Food and bioprocess technology. (First review 4/7/2018). Articles in preparation  Condón-Abanto, S., Lian F., Arroyo, C., Álvarez, I. and Lyng, J.G. Effect of storage temperature on microbial shelf-life and microbiological profiles of ready-to-eat Irish whole brown crab ( Cancer pagurus ).  Condon-Abanto, S., Pedrós-Garrido, S., Alvarez, I., Whyte, P., Lyng, J.G. and Arroyo, C. Isolation, identification and thermal resistance of the microbiota present in ready-to-eat brown crab ( Cancer pagurus ) meat. Chapter 1 1 Chapter 1 General introduction and literature review Chapter 1 2 Due to their composition, crustaceans such as brown crab are considered to be a healthy choice for consumers due to their high-quality protein, amino acid composition (Gökoðlu and Yerlikaya, 2003; Maulvault, et al ., 2012) and their low saturated fat content (Barrento et al ., 2008a, 2009b). However, its consumption is also associated with certain health risks such as high cholesterol levels in some edible parts, allergic reactions and contamination with toxic elements (Maulvault et al ., 2012). Either way in case of edible crab ( Cancer pagurus ) a continuous increase in European landings has occurred over the last 60 years, increasing from 11,000 tonnes to over 48,000 tonnes, giving a total value of in excess of 57 million euros which endorses the increased interest in this crustacean. To date the three main European producers of edible crab are the United Kingdom, Ireland and France (Eurostat, 2018). Edible crab production in Ireland started in 1980s and over the last three decades has suffered a significant increase, with Ireland being now one of the top three producers of this crustacean in the world with productions ranging between 5,000 and 8,000 tonnes per year, which has a net value of over 8 million Euros. In terms of production, edible crab is the second most exploited species in Ireland. This data shows the importance of this crustacean as a valuable commodity for the Irish fishing industry (BIM, 2014, 2017a). Out of the total production of edible crab in Ireland, 42% is sold as fresh live crab and the remaining 58% is sold in various ready-to-eat formats. From processed crab only 1% is presented as transformed product with the majority simply presented as cooked whole crab, cooked claws or cooked meat (both white meat from crab appendages or brown meat from the carapace) which is either frozen or chilled (Eurostat, 2018). One of the main challenges for the crab industry is to reduce losses associated with transportation. In the case of exports of edible crab as fresh live product, losses of up to a 50% can occur from crab deaths or damage during transportation, while the export in a ready-to-eat format avoids such losses during transportation while also adding Chapter 1 3 value to the final product (Barrento, et al ., 2008a; 2010b; Uglow, et al ., 1986). The main steps required in the production of ready-to-eat edible crab products are: (1) Initial cooking (e.g. boiling water for 20-30 minutes): During this step a series of compounds (e.g. proteins and crab dirt) are released from the crab and attach to the carapace which makes a subsequent washing of the crabs prior to packaging essential. (2) Cooling/washing: This step is necessary to complete the crab meat coagulation and is applied first using jets of water for dirt removal which is followed by cooling in a cold room. After cooling crabs are selected depending on their final destination to be sold as either whole cooked, extracted meats, claws, etc. (3) Finally a second pasteurization step is commonly applied to products following their packagaing to ensure the microbiological safety of the final inpack products. Although these steps are generally common across the crab industry, the conditions used in each step are varying among companies leading to a wide range of variability in the quality of the final products. Additionally due to the microbiological risks associated with this kind of product as mentioned, crustacean consumption is also associated with other health risks due to high cholesterol levels in some edible parts, allergic reactions and contamination with toxic elements (Maulvault et al ., 2012). Particularly, in edible crab high levels of Cadmium (Cd) have been reported, especially in the brown meat (Barrento et al ., 2009b, 2009c; Bolam et al ., 2016; Maulvault et al ., 2012, 2013). In the EU, the Cd content for crabs is only regulated for the white meat, which is located in the crab appendages (claws and legs), with 0.5 mg/kg being the maximum permitted limit (EU, Chapter 1 4 2006, 2011). However, several studies have warned of the risk of exceeding the total weekly intake of Cd set by EFSA (2009) (2.5 μg/kg body weight) in consumers from countries where brown meat is commonly consumed (Bolam et al ., 2016; Maulvault et al ., 2013; Noël et al ., 2011; Wiech et al ., 2017). As opposed to the EU, where there is no regulation concerning Cd levels in brown crab meat, some other countries such as China have recently started to impose limits considering all crab edible parts instead of white meat only. So, in a near future this problem could represent a great issue for the edible crab processing industry. 1.1 FOOD PRESERVATION Most foodstuffs are derived from plants or animals. The inherent biological nature of such products means that once harvested or slaughtered these products undergo a series of changes, which modify the original characteristics of the product and ultimately these lead to spoilage. These changes can be physical or chemical in nature but can also be induced by the metabolic activity of ubiquitous microbial contaminants. The ability to preserve foods and extend their shelf life has always been a key area that has developed with Human evolution. Since ancient times, and for many centuries, with no understanding of the underlying causes of food spoilage, a series of preservation methods were developed to ensure a constant availability of food. Many of these methods are still used today. The evolution of these preservation techniques came from experience which was in many instances influenced by the geographic locations of sub-populations. From these ancient times many food preservation methods including drying, smoking, salting, pickling, freezing (in cold climates) or fermentation (in warm climates where spoilage occurred at a faster rate) have evolved. 1.1.1 Food spoilage agents Food spoilage agents are influenced by many external factors such as temperature, humidity, oxygen concentration, light and storage temperature Chapter 1 5 (Casp and Abril, 2003; Potter and Hotchkiss, 1995). Independent of the effect of these parameters on microbial growth/survival, significant changes can be also induced on microbial growth due to intrinsic characteristics of the food matrix. Overall, the main causes of food degradation can be classed as chemical or biological. Chemical degradation. The two main reactions producing food alterations in this group are non-enzymatic browning and lipid oxidation/rancidity. Non-enzymatic browning, also known as the Maillard reaction, involves a series of complex reactions between reducing sugars and proteins present in foods, which modify odour and flavour of foods, producing dark brown pigments with associated losses in protein (due to denaturation) and nutritional value (Pérez-Locas and Yaylayan, 2010). Additionally when these reactions are very intense toxic substances can potentially be produced (Cheriot et al., 2009). The changes produced due to these reactions might be desirable, such as in the case of a roast appearance or flavour in meat but they can also be undesirable, as in case of a darkened colour or an off flavour in sterilized milk. The rate of development of Maillard reactions is influenced by a number of external factors with heat processing conditions (i.e. time and temperature) being the most important. Thus the Maillard reaction is a key factor in food processes such as cooking, pasteurization, sterilization and dehydration where products are exposed to high temperatures for long times (Martins et al., 2000). Another important chemical cause of food degradation is fat rancidity (Velasco et al., 2010). These reactions are responsible for nutritional value losses and the formation of undesirable volatile compounds. The two main reactions occurring within this group are hydrolytic rancidity and oxidative rancidity. The first one is due to the action of lipases which releases fatty acids from triglycerides and phospolipids; while the second one refers to the action of oxygen and lipoxygenases on unsaturated fatty acids. These reactions are Chapter 1 6 also influenced by a number of external factors such as temperature, presence of catalysts, available oxygen concentration and the type of fatty acid among other things (Waraho et al ., 2011). Biological degradation. Biological deterioration is quantitatively more important than chemical due to the higher frequency of its occurrence and the severity of the consequences produced (i.e. food poisoning and/or spoilage). Within this group enzymes and microorganisms are the main causative agents though parasites and viruses are also of significant importance (Potter and Hotchkiss, 1995). The enzymatic activity in all living cells is precisely controlled. In the case of foods, enzymatic activity continues after harvesting or slaughtering producing different changes in foodstuffs. Some of this enzymatic activity is desirable; leading in some cases to positive changes such as fruit maturation or meat tenderization. However, beyond certain limits continued enzymatic action leads to the decomposition of tissues which in turn facilitates further deterioration by microorganisms. Food spoilage due to the action of microorganisms is a very variable phenomenon, since it is dependent on type and number of microbial species present which in turn depends on the type of food matrix, preservation conditions, including storage temperature and the presence or absence of oxygen (Blackburn, 2006). The action of microorganisms, due to their different metabolic processes, leads to the degradation of foods making them unfit for consumption. A wide range of microorganisms are related in different ways to foods, some of which are natural contaminants of foods while others are consciously or sub-consciously introduced in the food chain (Garg et al ., 2016). The three main groups of microorganisms found in foodstuffs are those which (a) produce, preserve or develop characteristic flavours (i.e. processing aids), (b) cause food spoilage due to organoleptic changes or (c) are pathogenic and lead to food safety issues Chapter 1 7 Although, the total amount of food lost due to the action of both biological and chemical spoilage is uncertain, is estimated that one third of total global food produced for human consumption is lost or wasted (FAO, 2011). From these total losses, it is is estimated that about 25% of these losses is directly related to the activity of microorganisms (Petruzzi et al ., 2017). Despite the losses produced by microorganisms these biological agents are of even greater concern for the food industry due to their potential as a serious public health hazard (Ravishankar and Bai, 2015). In addition to the economic problems created by microbial spoilage, the presence of some of these microorganisms in addition to the presence of viruses and parasites is a constant threat to public health and a significant impediment to socio-economic development worldwide. It has been estimated that in 2010, the consumption of contaminated foods produced over 600 million illnesses and over 400,000 deaths worldwide (Havelaar et al ., 2017). Figure 1.1A and 1.1B shows the percentages attributed to the different food contaminants (viruses, bacteria, parasites and chemicals). As Figures 1.1A and 1.1B show, about 60% of the total illness and deaths related to food are produced by microorganisms, which emphasises the importance of this biological contaminant for the food industry. A wide range of bacteria can be hosted in food. The main vegetative threats are Listeria spp., Salmonella enterica, Campylobacter spp., Escherichia coli , Shigella spp., Staphylococcus aureus , Vivrio cholera and Brucella spp. while the spore forming bacteria of public health significance are Clostridium botulinum , Clostridium perfringens and Bacillus cereus (Havelaar et al ., 2017). From these microorganisms, spore forming bacteria are of special interest in food technology due to their ability to generate forms which are resistant to food preservation processes. Chapter 1 8 Figure 1.1: Percentage of (A) total foodborne illnesses and (B) total foodborne deaths produced by parasites (green), viruses (blue), chemicals (purple) and microorganisms (red). Data extracted from Havelaar et al ., (2015). 1.1.2 Food preservation strategies In light of these biological and chemical challenges the main objectives of food preservation could be summarized as: (i) ensuring the product safety maintaining appropriate organoleptic conditions during its shelf-life; (ii) minimizing the impact of the preservation process on product quality (Blackburn, 2006). Thanks to current knowledge about the afore-mentioned food spoilage agents, their mechanism of action and the influence of other factors on their behaviour, the main strategies used for food preservation are (Rahaman, 2007a): Inhibition or Inactivation/elimination of the contamination. These preservation strategies constitute the basis of modern food technology and can be applied using different methods and technologies. a. Inhibition (i.e. reducing microbial metabolic activity and the rate of enzymatic and chemical reactions). The inhibition methods used to reduce the microbial activity and enzymatic/chemical reactions include: 1) Reduction of water activity (aw) by elimination of water or addition of solutes. The reduction of aw in a food matrix prolongs the microbial growth lag phase, reduces the exponential growth rate and also the maximum microbial load attained at the stationary phase. The main advantages of this process is its simplicity and low cost. However, its principal disadvantage is its significant 23% 60% 17% A 14% 60% 22% 4% B Chapter 1 9 impact on the organoleptic characteristics of the final product depending of the system used to reduce aw (Rhaman and Labuza, 2007). 2) Reduction of storage temperature. This preservation method can be applied at two levels, chilling or freezing. Food chilling involves temperatures ranging from -1 to 15°C, and allows a shelf-life extension with reduced effects on food properties. The main inconvenience of chilling is the capability of certain pathogenic microorganisms, such as Listeria spp. and Yersinia spp., to grow at chilling temperatures (Walker and Betts, 2008). On the other hand, freezing leads to a reduction in product temperature, typically in the range of -18°C to -30°C. At these temperatures no microorganism is capable of reproducing and therefore chemical reactions become the dominant deterioration mechanism (Rhaman and Velez-Ruiz, 2007). 3) Reduction of redox potential. This method is based on the reduction of oxygen concentration, which in turn leads to a reduction or inhibition of aerobic microbial activity which in turn extends the shelf-life of food. The main disadvantage of this method is that the absence of oxygen promotes the development of anaerobic species, such C. botulinum (both proteolytic and non-proteolytic species) which is considered to be one of the most hazardous microorganisms in food, because of the severity of the associated poisoning. This method is applied during the packaging of food by: physical removal of air by vacuum packaging; chemical absorption of oxygen using oxygen scavengers; or by changing the gas atmosphere surrounding the food using inert or modified atmospheres (Gorris and Peppelenbos, 2007). 4) Reduction of pH (Acidification). The reduction of pH acts by destroying some enzymes located in cell envelopes and also by modifying the cytoplasmic pH, which in turn significantly reduces microbial development. From a technical perspective, food acidification can be achieved by adding acids to the food or by promoting the action of certain fermentative processes. The main advantage of this process is its simplicity and relatively low cost. However, similar to reductions in aw , this process also produces significant changes on the properties and appearance of food matrixes (Rahaman, 2007b). Chapter 1 10 5) Addition of bacteriostatic agents. Traditionally this technique consisted of the addition of a wide range of chemical additives with antimicrobial properties, many of which were synthetic as opposed to natural compounds. Despite the efficacy of this method, current consumer perception of these additives has changed and nowadays its use as food preservation method is declining in popularity. Nowadays, the food industry is looking for more natural alternatives with antimicrobial activity, such as bacteriocins, organic acids, essential oils, etc., to replace the use of synthetic chemical additives (Smid and Gorris, 2007). These preservation strategies are efficient at controlling microbial/chemical/enzymatic activity but do not eliminate them. They have advantages in terms of their simplicity or in terms of their low impact on the quality of the food material. However, the main limitation of these strategies is their inability to ensure food safety by eliminating the causative agents. Therefore, there effectiveness is highly dependent upon the initial quality of the raw material (Rahaman, 2007a). b. Inactivation/elimination of contaminating sources (i.e. microorganisms or spoilage agents present in/on raw materials). Although the inhibition strategies can extend the shelf-life, are not able to eliminate the hazard. For this reason another group of preservation strategies which have the potential to eliminate the pathogens and spoilage agents, thereby ensuring foods safety, exists. Within these strategies, methods based on physical principles are the most important. Within that group thermal processing can be considered the most widely used (Rahaman, 2007a). 1.2. HEAT TREATMENTS FOR FOOD PROCESSING The use of heat in food preparation dates back to the discovery of fire by man. In more recent times, it is fitting the person who is often credited as being the first food technologist is Nicolas Appert who came up with the concept of heating foods in sealed jars to extend their shelf-life in 1809 Chapter 1 17 1.3.1 Ionizing radiation (IR) The use of ionizing radiation for food preservation can be considered as a re-emergent technology (Molins, 2001). Food irradiation was proposed in the 19th century and since then a wide range of research has been performed to evaluate the potential of this technology for microbial inactivation (De Lara et al ., 2002; Jeong and Kang, 2017; Sarrıas et al ., 2003) and assess its influence on food properties (Byun et al ., 2000, 2008; Lee et al ., 2001). Despite the fact that it shows great potential for food decontamination, the use of this technology at an industrial scale remains stalled in the field of the food processing in several countries, due to poor consumer perception of irradiated foods. This confused perception is generally a result of a lack of information, misinformation and misleading imagery and also the confusion among consumers of the difference between irradiated food and radioactive contamination. Food ionization consists of the exposure of packaged or unpackaged foodstuffs to ionizing radiation for macro-organisms (e.g. insects, parasites) or microbial destruction in the food products (Odueke et al ., 2016). The three main sources of ionizing radiation for food processing, are gamma rays produced from cobalt (60Co) (1.17 and 1.33 MeV) or Cesium (137Cs) (0.662 MeV), generators of electron beam (max. energy 10 MeV) and X rays (max. energy 5MeV) (Codex Alimentarius Commission, 1984). Apart from their differing origins these irradiation sources have different energy and penetration depths. In food processing the usefulness of electron beam irradiation is limited by its low penetration depth while X rays are limited by their low energetic efficiency. By contrast, lambda rays (ϒ) have an elevated energy and penetration depth, which makes them more suited for processing larger food masses. However, the use of Y rays is limited because its main sources are radioisotopes. Chapter 1 18 Nowadays the main applications of this technology in the food industry are focused on the inhibition of vegetable germination; disinfection and disinsection in cereals, fruits, meats and fish; retardation of fruit and vegetable ripeness; shelf-life extension of raw meat and fish; pathogenic microbial inactivation; and sterilisation of spices and meals for immunocompromissed people and also for space travel (Crawford and Ruff, 1996; Odueke et al ., 2016). In food ionization the main parameter to take into account is the total dose absorved by the product. The irradiation dose is the total amount of energy absorbed by the irradiated product. The dose applied is measured in Gray (Gy) or kiloGray (kGy). In terms of energy 1 Gray equals to 1 Joule absorbed per kilogram of irradiated product. From a practical point of view three different ranges of doses exist for food processing: low, medium and high dose (Farkas, 2006; Molins, 2001; Odueke et al ., 2016):  Radurization or low-dose treatments involve doses below 1kGy. These treatments are used for germination, inhibition, delay of ripeness, disinfestation and parasites destruction.  Radicidation or medium dose treatments involve doses between 1 and 10kGy. These treatments are useful for the inactivation of spoilage and pathogenic vegetative cells but not for bacterial spores.  Rappertization or high dose treatments involve doses over 10kGy. These treatments are focused on the destruction of microorganisms up to levels of sterilization. The effects of ionizing radiations on biological material are the sum of its direct and indirect effects (Figure 1.2). The direct effect is a consequence of the physical changes produced on the molecules as a result of their absorption of energy. The energy absorption is proportional to the molecular weight which makes DNA one of the main target molecules due its larger size and complexity. For this reason the irradiation lethal dose Chapter 1 19 varies among different organisms depending on the complexity of their DNA (viruses (10-200kGy) Bacterial spores (>10-50kGy), Vegetative cells (>0.510kGy), Insects and parasites (>0.01-0.5kGy) and Mammals (>0.005kGy)). The indirect effect is related to the formation of reactive oxygen species (ROS) due to the direct action of radiation on cell components or the media surrounding, water for instance (Farkas, 2006; Lomax et al ., 2002; Sutherland et al ., 2000; Yokova et al ., 2008). Figure 1.2: Scheme of the direct and indirect effects of irradiation on DNA Despite the great potential of irradiation for food decontamination this process may also produce some undesirable changes on foodstuffs by both direct and indirect mechanisms; being water, proteins, other nitrogen compounds and aromatic compounds some of the most affected by irradiation treatments. The effects induced depend among other things on the irradiation dose, treatment temperature, oxygen availability and moisture. Some strategies to reduce the impact of these adverse effects on foodstuffs are the reduction of temperature (freezing temperatures), reduction of oxygen availability, addition of free radicals scavengers and dose reduction (Jay et al ., 2005). Chapter 1 20 Currently, a number of organisations worldwide have accepted this technology as a safe alternative technology for food decontamination (WHO, FDA). The World Health Organization has established 10kGy as the maximum dose for food processing without any adverse effect on food matrixes (WHO, 1981). Though, a later study concluded that no limiting dose is required (WHO, 1999). Either way, nowadays more than 60 countries worldwide have regulations regarding the use of ionizing radiation for food products (IAEA, 2017). In fact, the joint FAO/IAEA (International Atomic Energy Agency) Division of Nuclear Techniques in Food and Agriculture estimates that approximately 700,000 tonnes of food were irradiated in 2013 (IAEA, 2015). In the European Union (EU), only a total of 5,690.1 tonnes of food was irradiated in 2015, with Belgium being the most active country with a total of 3,916.9 tonnes of food irradiated in the same year (EU, 2016). Only dried aromatic herbs, spices and vegetable seasoning are authorised in the EU to be treated with ionizing radiation up to a maximum dose of 10kGy (EU, 1999). However, some countries within the EU have an extended list of food authorized to be treated with this technology. Table 1.2 shows the different foods and the maximum doses authorised in these EU countries. Chapter 1 21 Table 1.2: List of authorized food products and maximum irradiation dose in different EU countries (EU, 2009). BE, Belgium; CZ, Czech Republic; FR, France; IT, Italy; NL, Netherlands; PL, Poland; UK, United Kingdom. 1.3.2 Ultrasound (US) Ultrasound technology consists of sonic waves with frequencies above the threshold of human hearing (16-18 kHz). These ultrasonic waves are defined and characterized by their frequency and their wavelength. Waves with frequencies between 20 and 40 kHz are defined as high-energy or Chapter 1 22 high-power ultrasound, whereas those whose frequency ranges between 40 kHz and 1 MHz are known as low-power ultrasound (Mason et al ., 1996). These two types of ultrasonic waves have very different applications. Highpower ultrasound is used in the food industry for different purposes and is generally applied in processes which involve heat transfer, mass transfer or both. On the other side low-power ultrasound is used for quality analysis and process control purposes (e.g. sensors). Ultrasonic power is defined as the energy transmitted by the wave per second (W), ultrasonic intensity as the power per surface unit (W/cm2) and ultrasonic power density as energy per volume of treated product (W/L) (Meullemiestre et al ., 2017). When an ultrasonic wave is propagated through a liquid media, it creates alternating compression and expansion cycles. When the expansion cycle has the capacity to exceed intermolecular forces and to reduce the absolute pressure below vapour pressure, small bubbles are formed. Over the course of subsequent expansion/compression cycles, the bubbles expand and contract. A rectified diffusion process leads to the growth of bubbles until they reach resonance size. Then, over the course of the next acoustic cycle (which corresponds to one oscilation in Figure 1.3), the bubbles grow to a maximum size and then collapse. As a consequence of that implosion, molecules violently collide with each other, giving rise to shock waves and creating spots of very high temperature and pressure (Mason et al ., 1996). These extreme conditions usually induce water sonolysis, resulting in the emergence of highly reactive radicals. This phenomenon is known as transient cavitation, when generated by high power ultrasound. Cavitation is considered the main mechanism by which this form of ultrasound enhances heat and mass transfer phenomena (Kim et al ., 2004; Zhou et al ., 2002), though other effects such us acoustic streaming, which is a physical force that produces an additional mass flow (Solovchuk et al ., 2011) are also involved (Legay et al ., 2011). A scheme of the cavitation process is shown in Figure 1.3. Chapter 1 23 Figure 1.3: Scheme of the evolution of cavitation bubbles during the application of power ultrasound. Source (Adapted from Abbas et al ., 2013). The effects of ultrasound on heat transfer has been extensively studied since the 1990s in model systems such as water, metal tubes, metal balls, etc. (Huamao et al ., 1997; Hyun et al ., 2005; Oh et al ., 2002; Zhou et al ., 2002). Furthermore its ability to enhance heat transfer in foods, mainly in processed meat products, during cooking processes (Alarcon-Rojo et al ., 2015; Reynolds et al ., 1978; Vimini et al ., 1983) has been investigated. Also the theoretical advantages of ultrasonic cavitation have been assessed in different processes related with the food industry, with cooking, marinating and extraction evaluated extensively (Alarcon-Rojo et al ., 2015; Cárcel et al ., 2007; Khan et al ., 2010; Luengo et al ., 2014; Ma et al ., 2009; Mason et al ., 1996; McDonnell et al ., 2014; Turhan et al ., 2013; Vimini et al ., 1983) and the usefulness of the application of ultrasound to enhance freezing, drying, degassing, filtration, demoulding, defoaming, emulsification, oxidation and cutting have also been explored (Ashokkumar, 2015; Chandrapala et al ., 2012; Chemat et al ., 2011). Besides the applications related to heat and mass transfer processes, US technology is one of the new microbial inactivation technologies that has been suggested as an alternative to conventional heat treatments (US FDA, Chapter 1 24 2000). As mentioned before, today most authors (Condón et al ., 2011; Lee et al ., 2009; Wu et al ., 2015) agree that high power ultrasound inactivates cells via envelope breakdown as a consequence of shock waves produced due to cavitation phenomena. As a general rule the US resistance of bacterial cells decreases with size and is greater in coccoid-shaped bacteria (Alliger, 1975; Condón et al ., 2005). Furthermore, Gram-positive bacteria are usually more resistant than Gram-negative bacteria; with yeast and moulds presenting intermediate resistance (Condón et al ., 2011; Jambrak et al ., 2017; López Malo et al ., 2005), while bacterial spores are almost not affected by US treatments (Jambrak et al ., 2017; Sanz et al ., 1985). Most published data indicate that the bactericidal efficacy of ultrasound is low (Jambrak et al ., 2017; Lee et al ., 2013; Meullemiestre et al ., 2017). Therefore, most researchers have tried to improve the efficacy of the process by designing combined processes to enhance the overall lethal efficacy of ultrasound (Lee et al ., 2013; López-Malo et al ., 2005; Raso et al ., 1998a). Some of the combined US processes proposed to date to increase the lethal effect of ultrasound have been classified (Chemat et al ., 2011; Piyasena et al ., 2003; Sala et al ., 1995) as thermosonication (TS, combination of ultrasound and heat), manosonication (MS, combination of ultrasound and pressure), and manothermosonication (MTS, combination of ultrasound, pressure and heat). The increment of the lethal effect of ultrasound by raising the temperature of the treatment media (TS) has been observed on bacterial vegetative cells (Lee et al ., 2009), yeast (Abid et al ., 2014; Bermudes-Aguirre and Barbosa-Canovas, 2012) and on bacterial spores (Garcia et al ., 1989; Milly et al ., 2007). It has been suggested that higher temperatures sensitize cell envelopes to shock waves originated by cavitation (Lee et al ., 2009; Wu et al ., 2015). In some cases the lethal effect of TS increases until reaching a maximum at a certain temperature following which the ultrasound effect decreases. This Chapter 1 25 decreasing behaviour has been associated with an increase of the vapour pressure of the medium (Knorr et al ., 2004; Sala et al ., 1995), which should facilitate cavitation, but while also reducing the intensity of cavitation implosion, as the vapour within the bubbles acts as a cushion (Alliger, 1975). When boiling temperatures are reached, cavitation ceases. On the other side, the inactivation rate of vegetative cells due to MS increases when static pressure is raised (Lee et al ., 2009, 2013; Mañas et al ., 2000). An increase in hydrostatic pressure makes the formation and growth of bubbles more difficult and requires higher energy consumption for their production, but the resultant cavities release more energy when they implode, thereby increasing the lethal effect. When pressure is too high in comparison with ultrasonic power, cavitation formation is inhibited and transient cavitation ceases. This explains that the effect of pressure increases the bactericidal effectiveness of US until reaching a threshold, above which the effect of pressure progressively decreases until it vanishes (Guzel et al ., 2014; Pagán et al ., 1999b; Raso et al ., 1998a). MTS process was designed in 1992 (Spanish Patent No. 9200686) with the aim of overcoming the limitations of TS and MS, while making use of their advantages. The authors hypothesized that pressure would increase cavitation intensity and compensate for changes in the vapour pressure of the medium as a result of heating, thereby permitting an advantageous use of thermosonication even at high temperatures (Sala et al ., 1995). Data published over the last 20 years confirms the validity of this hypothesis to inactivate vegetative microbial cells, spores and also enzymes. 1.4 PROCESS OPTIMIZATION The previous section outlined in more detail the main technologies that were explored in the present PhD Thesis. The current section provides an overview about the area of process optimisation and their usage. Optimization is defined as the action of making the best or most effective Chapter 1 26 use of a situation or resource (Oxford dictionary, 2018). From the perspective of food processing, process optimization can be defined as the selection of the process parameters which will achieve a safe/stable product, while maintaining the best possible nutritional and organoleptic characteristics and maximizing the profit. The origins of food process optimization can be attributed to the discoveries of Bigelow, Easty and Mayer in the 1920s, in the laboratories of the National Canners association in U.S. These researchers were investigating microbial destruction kinetics by heat. The modern concept of optimization, which combines microbial inactivation and food quality, was not developed until 1940s when Esselen proved the logarithmic destruction of vitamins subjected to heat, discovering the differences on the thermal sensitivities between microorganisms and vitamins. Nowadays the role of process optimization is to minimize product deterioration, maximize profits while, at the same time, ensure the hygienic and safety requirements imposed by national or international regulations. Nowadays, as a general rule in the food industry process optimization is dictated by food safety requirements (Valdramidis et al ., 2012) with quality having a secondary role. Food process optimization requires an appropriate knowledge of the process and the variables involved on it. Figure 1.4 shows the main information and steps required to perform food process optimization. However, obtaining all required information is a slow and costly procedure which most of the times makes it a big challenge. A reasonable alternative is to predict, based on mathematical models the impact of the process variables on the characteristics of the product and optimize the process based on these predictions (Ling et al ., 2015; Stoforos, 1995). Chapter 1 33 Figure 1.5: Example of a validation graph. Dashed line represents the bisectrix which correspond to a perfect fit. The dashed line in Figure 1.5 represents the perfect fit. The points furthest from it represent the least accurate predictions. From Figure 1.5 it is also possible to define if the model overestimates or underestimates its predictions. If the points are located above the line, the model would overestimate on its predictions while if the points are located in the area under the line the model would underestimate on its predictions. This procedure is commonly done in conjunction with the analysis of residual errors which indicates the differences between predicted and real values. Although the graphical evaluation is simple and useful, a mathematical analysis is also necessary in order to evaluate the robustness and goodness of the model fit. The most common used parameters are: a) Coefficient of determination (R2). This parameter provides a measure of how well real values are represented by the model, based on the proportion of total variation of outcomes explained by the model. It is calculated as shown in Equation 1.1. 0 2 4 6 8 10 0 2 4 6 8 10 Real values Predicted values Chapter 1 34 𝑅2=( ∑ (𝑋𝑖−𝑋)(𝑌𝑖−𝑌) 𝑛 𝑖=1 √∑ (𝑋𝑖−𝑋)2 𝑛 𝑖=1 ∑(𝑌𝑖−𝑌)2 𝑛 𝑖=1 )2 (Eq. 1.1) Where Xi represents real values values, 𝑋 the average of the real values, Yi represents predicted values, 𝑌 the average of predicted values and n is the total number of observations. R2 values close to 1 indicate a better correlation between predicted and observed values. b) Root mean square error (RMSE). This parameter quantifies the quadratic error of the differences between predicted and real values. It is calculated as shown Equation 1.2. 𝑅𝑀𝑆𝐸=√∑(𝑋𝑖−𝑋0)2 𝑛 (Eq. 1.2) Where Xi represents predicted values, X0 observed values and n is the total number of observations. Small values of this parameter involve higher certainty in the models prediction. However, models with high RMSE values still can predict accurately as this parameter depends on the order of magnitude of the values predicted and observed. c) Standard error of prediction (SEP). This parameter is similar to RMSE, but quantifies the error in relative terms, so its value is not dependent on the magnitude of the data. It is calculated as shown Equation 1.3. 𝑆𝐸𝑃=100 𝑋0√∑(𝑋𝑖−𝑋0)2 𝑛 (Eq. 1.3) Chapter 1 35 Where 𝑋0 represents the average of the observed values and the others are defined in RMSE . d) Bias factor (Bf) and Accuracy factor (Af). These parameters were defined to assess the goodness of fit of the predictive models in a rapid way and provide indicators of the reliability of the models (Ross, 1996). These parameters are calculated following Equations 1.4 and 1.5 respectively. 𝐵𝑓=10(∑log(𝑋 𝑝𝑟𝑒𝑑. 𝑋 𝑜𝑏𝑠. ⁄) 𝑛) (Eq. 1.4) 𝐴𝑓=10√(∑(log(𝑋 𝑝𝑟𝑒𝑑. 𝑋 𝑜𝑏𝑠. ⁄))2 𝑛) (Eq. 1.5) Where Xpred. represents the predicted values, Xobs. represents the observed values and n is the number of observations. The Bf indicates if the model overestimates or underestimates in its predictions. Values of Bf <1 indicates that observed values are smaller than the predicted and values of Bf >1 indicates that the observed values are bigger than the predicted. In the case of the perfect fit of the data, Bf is equal to 1. Big values of Af indicates less accuracy in the predictions while, when the correlation between observed and predicted values is perfect Af becomes equal to 1. Ideally the value of Af should be 1 but generally increases between 0.1 and 0.15 for each variable, included in the model, are accepted (i.e. Af values between 1.2 and 1.3 are acceptable for a model which considers two or more variables) (Ross et al ., 2000). To date one of the main examples of the application of modelling to the processing of the food industry is the design and optimization of heat processes. Chapter 1 36 1.4.2 Heat process optimization The American canning industry began sometime after 1810 and is considered to be the origin of modern food technology. A greater understanding of the canning process was developed due to the findings of Louis Pasteur in 1865 and further advances in knowledge of microbial inactivation and vitamin degradation by heat during 1920s and 1940s. As mentioned previously, the primary objective of food processing is the production of safe products which will not pose a potential hazard to public health. Therefore, the first step in the optimisation of a process is to evaluate and characterise the effect of that process on the main target microorganisms present in the product to be treated (Stofors et al ., 1995). Form the experiences of Bigelow and Esty in 1920 it was concluded that microbial inactivation by heat follows a first-order kinetic at a constant temperature. Although, the techniques have evolved during the years and other inactivation models have been reported to describe microbial inactivation by heat (Cebrián et al ., 2017), still nowadays the findings of Bigelow and Esty are being used in the food industry for the design of thermal processes. Due to the exponential reduction of microbial population by heat, when the logarithm of microbial count is represented versus treatment time a straight line is observed as shown in Figure 1.6. This new knowledge gave rise to the concept of the D T value, which can be defined as the time necessary, at a constant temperature, to reduce the microbial count by one Log10 cycle (i.e. to reduce the total microbial population by 90% or by a factor of 10). This parameter can be calculated as the negative inverse of the slope of the line which describes the microbial inactivation expressed as Log of microbial numbers vs treatment time. This concept could be considered as the primary model used to define thermal processes. Chapter 1 37 Figure 1.6: Effect of treatment time on the microbial count subjected to a heat treatment at a constant temperature. Source (Cebrián et al ., 2017). In addition Bigelow (1921) demonstrated the exponential effect of the treatment temperature on the D T value, which gave rise to the concept of the z value. This concept is defined as the required temperature rise (°C) to bring about a 1 log change in the D T value (i.e. reduce or increase the D T by 90% or by a factor of 10). Similar to the calculation of the D T, z values can be calculated graphically as the inverse of the slope of the line correlating the Log10 of D T values and the treatment temperature (Figure 1.7). Figure 1.7: Effect of treatment temperature on the microbial inactivation rate subjected to a heat treatment. Source (Cebrián et al ., 2017). The concept of the z value can be considered as a secondary model used to define a thermal process. But, contrary to the observations with D T values, the concept of the z value has been proven to be a more robust and useful model to correlate the treatment temperature with the parameters of other primary models used to describe the heat inactivation curves, examples including the Weibull distribution (Fernández et al ., 2007; Hassani et al ., 2005) or the Geeraerd Equation (Arroyo et al ., 2011). Time (min) Time (min) Microbial count Log microbial count Dt Chapter 1 38 D T and z are correlated by Equation 1.6 (This Equation could be considered as a tertiary model used to calculate the impact of a thermal process in terms of microbial inactivation), which allows the definition of the D T values at any treatment temperature (T) by knowing a reference D T of the target microorganism and also its z value. These concepts form the basis of classical thermobacteriology and forms basis of heat process optimization. 𝐷𝑇=𝐷𝑇𝑟𝑒𝑓10(𝑇𝑟𝑒𝑓−𝑇)𝑧 ⁄ (Eq. 1.6) Similar to what has been described for microbial inactivation, the kinetics of heat induced chemical reactions in foods can be studied and modelled. However, the key reaction which defines the quality of the product varies from product to product and from consumer to consumer. For example with cooked beef, tenderness is generally the key factor affecting consumer acceptance but consumers’ attitude vary in terms of the choice of well-done or rare steak. For milk, colour and nutritional value may be key factors while in juices the flavour and enzyme inactivation may be key reactions. Additionally, a general model to describe these degradation kinetics (i.e. primary model) does not exist and different order of reactions has been observed (i.e. zero-order, first-order, pseudo-firstorder, second-order kinetics) (Ling et al ., 2015). Despite the differences in primary models between microbial inactivation and quality degradation, process optimization is possible because the effect of treatment temperature remains constant (i.e. primary model parameters vary exponentially due to temperature changes). So an equivalent to z value, known as Q10 value, can be defined. Conceptually, this parameter relates to quality changes and has the same meaning as z value for microbial inactivation; though Q10 values for chemical reactions are generally higher than those reported for z , (e.g. 10ºC for microbial inactivation vs 30ºC for chemical reactions) which is the basis of thermal Chapter 1 39 process optimization. This difference in z values between chemical and microbial inactivation is exploited in UHT processing where a 30°C increase in processing temperature may produce a 1000 fold increase in the rate of microbial inactivation but only a 10 fold change in the rate of chemical reactions. The development of kinetic models for both, microbial inactivation and quality degradation are two of the main components in the optimization of heat processes. However, the applicability of these tertiary models is limited to treatments performed at constant temperatures, while in most heat processes within the food industry the product temperature is not constant but varies throughout the process (Holdsworth and Simpson, 2007). Therefore, the last required information, in the optimization of a heat process is the characterization of the time temperature profile of the product in order to integrate the combined effect of all temperatures attained during the treatment time. The time temperature profile of a foodstuff during heat processing consists of, in its simplest form, a heating, holding and cooling cycle and can be determined experimentally, by placing thermocouples within a product, or can be estimated mathematically by predictive models (Stoforos, 1995). The advantage of the experimental procedure is that the results obtained will be more robust, for subsequent calculations, but in contrast, the applicability of the data is solely focused on the specific conditions used to obtain the data. Additionally, data collection is a costly and time consuming procedure. On the other side the use of mathematical models to predict the time temperature profile during processing has the advantage that it allows the definition and evaluation of different process variables (the number and nature of those variables is dependent on the type of model used) but also the relatively reduced amount of experimental data required to generate the model. By contrast, the conclusions extracted have higher uncertainty. Chapter 1 40 Once all the required information has been obtained the next step in heat process optimization is to calculate the process conditions in terms of time and temperature to achieve a certain limit of microbial inactivation and quality characteristics (Figure 1.4). To do this, two equivalent parameters are generally described: F T z for microbial inactivation and C T z for quality changes (Holdsworth and Simpson, 2007). Conceptually, F and C values are similar than D T but, these two values represent the equivalent treatment time at a reference temperature to reach a certain level of microbial inactivation or quality level respectively. Results calculated for a process, in terms of C value, are generally compared to a target F value which represents the time required to achieve a target Log10 reduction of the most pertinent pathogen in the product. Although, the C T z value is also critical in terms of product quality and nutritional value it is less frequently considered, as the main focus is product safety. These two values represent the whole thermal effect accrued during the heat process. Figure 1.8 shows as example the theoretical graph for the optimization of a thermal process related to microbial inactivation (i.e. F T z ) and two different quality parameters (i.e. C T z values). In the graph, the black line represents the minimum equivalent time-temperature combinations necessary in order to achieve an adequate level of microbial inactivation. The blue line represents the minimum equivalent time-temperature combinations required in order to achieve a minimum quality target and the red line represents the maximum tolerable equivalent time-temperature combinations in order to keep a specific quality attribute under a defined limit. Based on Figure 1.8, the optimum conditions in this example are those which accomplish all product requirements, represented by the green area. In the example, only one microbiological and two quality variables have been considered, but in this type of optimization graph it is possible to include even more lines representing other variables such as enzymatic inactivation or even the economic cost of the process. Chapter 1 41 Figure 1.8: Theoretical lines for the minimum microbial inactivation required (black line), the maximum quality degradation accepted (red line) and the minimum quality required (blue line) depending on the treatment temperature. The green area represents the timetemperature combinations which accomplish all product requirements (safety and quality in this example). 1.5 EDIBLE CRAB-BROWN CRAB ( CANCER PAGURUS ) The development of the present PhD thesis is part of a larger project focused on Irish brown crab ( Cancer pagurus ). Therefore, the following section describes the particular characteristics of this foodstuff and its production process. Cancer pagurus , commonly known as edible crab or brown crab, is a species of crab found in the North Sea, North Atlantic Ocean and in the Mediterranean Sea. It is a robust crab of a reddish-brown colour, having an oval carapace with a characteristic "pie crust" edge and black tips to the claws (Figure 1.9). C. pagurus is a nocturnal predator, targeting a range of molluscs and crustaceans. The maximum age of brown crab is around 20 years and the maximum sizes registered for the carapace width are 265mm for males and 225mm for females and the maximum registered weight is 3kg. For fishing the minimum size accepted, of this crab, is 130140mm depending on the region it is captured and measured perpendicular to the anteroposterior midline of the carapace (Council regulation (EC) No. Treatment time Treatment temperature Microbial inactivation requirements Limit of quality retention (i.e. loss of nutritional value) Minimum quality required (i.e. cook value) Chapter 1 42 850/98, as ammended). This corresponds to a weight of about 350 grams, and it takes about 5 or 6 years for crab larvae to reach this size. Figure 1.9: Female exemplar of edible crab ( Cancer pagurus ) Like all decapods an edible crab has ten appendages attached to its thorax, the first being a pair of claws. In case of edible crab both appendages and carapace contain edible tissues with commercial interest. Muscle meat or white meat, is mainly located in claws and legs but also some of this meat type is located in the thorax. Brown meat is obtained exclusively from the carapace of the female exemplars and mainly consists of hepatopancreas and gonads. The composition of each meat type varies between crabs but also depends on the maturity stage, season and fishing area. In case of white meat the total fat content ranges from 0.2 to 0.6% while the protein content varies between 16.4 and 20.5%. On the other side, in brown meat the total fat varied between 3.1-3.6% and protein content from 19.2-25.5%; while for hepatopancreas the fat content ranges between 10.0 and 16.6% and protein content ranges from 9.4 to 15.8% (Barrento et al ., 2009b, 2010a). Chapter 1 49 are removed. During cooking a series of compounds are released from the crab, mainly proteins. These released compounds attach to the carapace during cooking, as shown in Figure 1.14, which makes it essential to wash the crabs prior packaging. For this reason the first part of cooling is carried out using water jets, for cleaning and finalised in a cold room. The entire process has a duration of approximately 3-4 hours. On the other side, cooling is very important because allows the completion of meat coagulation, which facilitates its removal from the carapace and avoids losses (Edwards, 2001). Figure 1.14: Example of crabs during cooking (left) and immediately after cooking with no washing (right). Immediately after cooling crabs, which will be sold as ready-to-eat cooked whole are packaged for sale (premium products) or frozen. Most commonly, a second heat treatment is applied, to ensure the microbiological safety of the product. Crabs which are destined for manufacture of crab based products or packaged crab meat are transferred to a meat picking area. At this stage, white meat, from the body claws and legs and brown meat, from the body of female crabs, is removed. The main inconvenience of this process is that the main procedure to remove the meat is hand picking, although compressed air jets are sometimes used to assist removal of meat from the legs (Edwards, 2010). Either way this step takes up a significant portion of processing time. Finally, when the various Chapter 1 50 types of meat have been removed, they are packed together or separately, depending on the intended final destination and pasteurised. In more elaborated products, which require a greater level of processing (e.g. pate, chowder, bouillion, etc.) a second pasteurization is mandatory due to the high risk of contamination/recontamination (Adams, 2000; Dima et al ., 2015; Ghazala and Trenholm, 1996). Due to the nature of crab edible tissues (high water activity, moderate salinity and moderate pH) it provides an excellent environment for the development of various pathogens, which can be associated with foodborne diseases due to improperly processed/preserved/stored low-acid chilled foods. According to the U.S. Department of Health and Human Services of the Food and Drug Administration (FDA, 2011) the most important foodborne non-spore-forming pathogens, associated with these kinds of products are Listeria monocytogenes, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Escherichia coli, Salmonella spp. , Shigella spp. , Staphylococcus aureus, Campylobacter jejuni and Yersinia enterocolitica (FDA, 2011; Gram and Huss, 1996). For all of these vegetative bacteria, Listeria monocytogenes is considered as the target microorganisms in pasteurisation treatments, since it is generally considered to be the most heat resistant vegetative cell and is able to multiply at chill temperatures (FDA, 2011). L. monocytogenes may be transmitted to humans via contaminated foods (Brackett and Beuchat, 1990). Its development is dependent on intrinsic factors such as the product pH, water activity and salt content and extrinsic factors such as cooking/storage temperatures and gas atmosphere and packaging type (FSAI, 2005). L. monocytogenes is able to grow in a range of pH from 4.2 to 9.5 and at temperatures from -1.5C to 45C and at aw of up to 0.9. It is documented that they are able to survive in environments down to -18C and pH values as low as 4.3 (FSAI, 2005). Chapter 1 51 Pasteurized crabmeat has been shown to be an adequate support for the growth of L. monocytogenes at 1C and temperature rises up to 5C dramatically increase its growth rate (González et al ., 2013). Multiple potential reasons for the presence of L. monocytogenes have been identified, insufficient thermal processing, cross contamination from raw crab, factory environment or food-handlers. According to Regulation (EC) No 2073/2005 ready-to-eat foods able to support the growth of L. monocytogenes , such as ready-to-eat edible crab and derived products, must comply with the following food safety criterion: absence of the pathogen in 25 g of product before the food has left the immediate control of the food business operator (FBO) who has produced it. However, if the FBO is able to demonstrate, to the satisfaction of the competent authority, that the product will not exceed the limit of 100 cfu/g throughout the shelflife, then this latter criterion would apply (100 cfu/g throughout the shelflife). For pasteurisation treatments, achieving a six logarithmic reduction of the target microorganism is accepted as an adequate level of inactivation to avoid foodborne disease (FDA, 2011). In the case of L. monocytogenes , its prevalence in raw seafood is estimated as 1 to 103 colony forming units per gram (CFU/g) in up to 8% of these products. Only 1% of these products are contaminated with levels greater than 103 CFU/g while levels greater than 106 CFU/g are not observed. However, for the majority of seafood (91%), the contamination level of L. monocytogenes is generally lower than 1 CFU/g (FDA, 2011). If this pathogen is considered as the target microorganisms for pasteurization the recommended treatment is a F 70°C7.5°C of two minutes or an equivalent treatment, in order to achieve a proper inactivation level of L. monocytogenes i.e. in excess of 6 Logs (CCFRA, 1996; CFA, 2006; ECFF, 1996; FDA, 2011). Additionally to the vegetative cells enumerated before, ready-to-eat crab based products and seafood in general, represent a serious hazard due Chapter 1 52 to the presence of pathogenic spore forming bacteria, namely: Clostridium perfringens , Clostridium botulinum and Bacillus spp. which are the main three spore forming bacteria associated with seafood products (FDA, 2011). Among these, group II (non-proteolytic) Clostridium botulinum types B, F and specially type E, are of particular concern to the seafood industry. C. botulinum non-proteolytic type E is known to be a naturally occurring marine organism which can also grow at refrigeration temperatures as low at 2C (Corner et al ., 1989) and it has been also associated with human botulism cases (Lindström et al ., 2006). In addition, it has been reported that the prevalence of group II (non-proteolytic) C. botulinum type E in fish and other seafood may be as high as 40-70% (Baker, 1990; Huss et al ., 1974; Johannsen, 1963). However, despite its high prevalence in fish and seafood, its counts in raw foods are typically not very high, varying from less than 1 spore/kg (Baker, 1990; Lindström et al ., 2006; Tanasugarn, 2006) to 102 spores/kg (Hielm et al ., 1998; Lindström et al ., 2006). For all these reasons C. botulinum non-proteolytic type E is considered as a major concern when designing a pasteurization process for ready-to-eat seafood based product (FDA, 2011; Silva and Gibbs, 2010). Generally as it is considered for L. monocytogenes, a pasteurization process which ensures a 6 Log10 reduction of the population of spores of C. botulinum nonproteolytic type E is considered adequate for any of these product types (FDA, 2011). Considering the thermal resistance of C. botulinum non-proteolytic type E the most recommended heat treatment to be applied is an F 90°C7or10°C of ten minutes, or equivalent considering z of 10°C for treatment temperatures ≥90°C or 7°C for treatment temperatures <90°C (CFA, 2006; ECFF, 2006; FDA, 2011). These treatments are recommended for all readyto-eat refrigerated products packed in the absence of oxygen, including fish and fishery products. However, in case of crab meat-based products, more severe heat treatments have been proposed. It is known that the presence Chapter 1 53 of the enzyme lysozyme improves the recovery of heat-treated Clostridium spp. spores and therefore, a higher heat resistance is observed for this species (Peck and Fernandez, 1995; Scott and Bernard, 1982). Moreover, it has also been reported that this enzyme is present in fish and seafood at level of up to 200 μg/g (Lie et al ., 1989; Lund and Notermans, 1992). For this reason, two more severe alternative treatments were proposed by the FDA for crab meat F 90ºC9ºC = 31 min (Blue crab, Callinectes sapidus ) and F 90ºC8.6ºC = 57 min (Dungeness crab, Metacarcinus magister ) to achieve a satisfactory reduction of Clostridium botulinum spores in crab meat (FDA, 2011; Peterson et al ., 1997, 2002). Additionally to the microbiological risks mentioned, crustacean consumption is also associated with other health risks due to high cholesterol levels in some edible parts, allergic reactions and contamination of toxic elements (Maulvault et al ., 2012). Particularly, in edible crab high levels of Cadmium (Cd) have been reported, especially in brown meat (Barrento et al ., 2009b, 2009c; Bolam et al ., 2016; Maulvault et al ., 2012, 2013). Noël et al . (2011) reported Cd levels from 0.78 to 27.6 mg/kg of wet weight (ww) and from 6.38 to 61.8 mg/kg (ww) in the brown meat of common crabs ( Cancer pagurus ), captured in open or coastal areas of the Atlantic North East respectively, landed in Ireland and United Kingdom. Also, Bolam et al. (2016) analysed Cd levels in the brown meat of Cancer pagurus , sampled throughout the UK, and observed Cd concentrations between 0.11 and 26 mg/kg (ww) and Maulvault et al. (2012) reported Cd concentrations between 5.0 to 8.4 mg/kg (ww) in the brown meat of crabs caught in the Scotland coasts. Besides, Wiech et al. (2017) reported Cd concentrations from 5.7 to 12 mg/kg (ww) after different processes and significantly higher Cd levels of up to 44 mg/kg (ww) in crabs from Northern Norway. In the EU, the Cd content for crabs is only regulated for the white meat from crab appendages (claws and legs); with 0.5 mg/kg being the Chapter 1 54 maximum permitted limit (EU, 2006, 2011). However, several studies have warned of the risk of exceeding the total weekly intake of Cd set by EFSA (2009) (2.5 μg/kg body weight) in consumers from countries where brown meat is commonly consumed, such as in Spain and Portugal (Bolam et al ., 2016; Maulvault et al ., 2013; Noël et al ., 2011; Wiech et al ., 2017). The main challenge in this regard is that the processing techniques currently used in the crab industry hardly affect the original concentration of Cd present in the crab (Maulvault et al ., 2012; Wiech et al ., 2017), which is directly dependant on the fishing area where they originated from. There is not specific EU regulation regarding the Cd content in crab’s brown meat, tough other countries such as China have started to regulate those. Therefore, in a near future Cd could represent a great issue for the edible crab processing industry. Considering the background presented in this literature review regarding food preservation, process optimization and edible crab the main objectives of the present PhD Thesis could be summarize in: 1.6 OBJECTIVES The main objectives of the present PhD Thesis were:  To evaluate the effect of the storage temperature on the shelf-life in raw crab, commercial crab products and readyto-eat whole crab cooked under mild temperatures and packed under vacuum.  To identify the main microorganisms present at the end of the microbiological shelf-life of raw crab, commercial crab products and ready-to-eat whole crab cooked under mild temperatures and packed under vacuum.  To determine the thermal resistance of the main microorganisms present in raw crab, commercial crab Chapter 1 55 products and ready-to-eat whole crab cooked under mild temperatures and packed under vacuum.  To evaluate the effectiveness of the heat treatments recommended by U.S. Food and Drug Administration for this type of products.  To evaluate the potential advantages of the incorporation of ultrasound technology in the initial cooking step of edible crab to improve heat and mass transfer processes (e.g. reduce cooking time, cleaning and cadmium reduction).  To optimise the second pasteurization step applied in the production of ready-to-eat edible crab using a quality index degradation kinetic approach.  To evaluate the potential use of alternative technologies (i.e. mano-sonication, mano-thermo-sonication and ionizing radiation) to inactivate the main spore forming bacteria isolated from ready-to-eat edible crab. Chapter 2 56 Chapter 2 Microbial characterization of edible crab ( Cancer pagurus ) From this chapter two articles are in preparation for their submission. Condón-Abanto, S., Lian F., Arroyo, C., Álvarez, I. and Lyng, J.G. Effect of storage temperature on microbial shelf-life and microbiological profiles of readyto-eat Irish whole brown crab ( Cancer pagurus ). In preparation Condón-Abanto, S., Pedrós-Garrido, S., Alvarez, I., Whyte, P., Lyng, J.G. and Arroyo, C. Isolation, identification and thermal resistance of the microbiota present in ready-to-eat brown crab ( Cancer pagurus ) meat. In preparation Chapter 2 57 2.1 ABSTRACT/RESUMEN ABSTRACT In the past, the brown crab ( Cancer pagurus ) market mainly consisted of fresh live product. However, the opening of new markets (e.g. China and USA) has now created a demand for ready-to eat products. This in turn creates an opportunity to add value but also affords manufacturers the chance to improve existing commercial processes (e.g. use of milder heat treatments to produce a higher quality product). However, an important consideration in all of this is product shelf-life following these new processes and the microbiota present in these types of product. For this reason, one of the aims of the present study was to assess the effect of storage temperature on the shelf-life and microbiological profiles of ready-to-eat whole crab cooked under mild temperatures and packed under vacuum. A tertiary model based on the modified Gompertz Equation and the root square model, for mesophilic bacterial growth at different storage temperatures was developed. The end of shelf-life was considered as the point where the mesophilic bacterial load reached 6 Log10-cycles. Once the shelf-life was determined and the effect of the storage temperature was quantified, a deeper study of the natural microbiota present in raw and cooked brown crab meat and in some commercial brown crab products was performed. Finally, the thermal resistance of the main microorganisms identified in crab meat samples were determined, in terms of D T and z values and the effectiveness of the recommended heat treatments by U.S. Food and Drug Administration for this type of products was assessed. Shelf-lifes of 362, 257, 204 and 145 hours for white meat and 492, 340, 265 and 183 hours in case of brown meat, were calculated under storage temperatures of 2, 5, 7 and 10°C respectively. A linear relationship was found between the storage temperature (°C) and the Log10 of shelf-life regardless of the meat type, though white meat was found as the limiting component which defined the shelf-life of ready-to-eat brown crab. In all cases a bigger heterogeneity of microbiota was detected in the case of brown meat. In raw Chapter 2 58 meat the main microbial groups detected were Pseudomonas spp., then Bacillus spp. which was present in a similar concentration to Staphylococcus spp . In cooked crab the microbial groups present in the greatest proportion were Bacillus spp. and Staphylococcus spp ., although their proportions varied depending on the storage temperature. The 18 bacterial genus and 31 species isolated were identified by 16S rRNA sequencing. Carnobacterium divergens and Shewanella baltica were the most frequently isolated non-spore forming species while Bacillus mycoides , Bacillus weihenstephanensis and Psychrobacillus psychrodurans were the most frequently detected spore forming bacteria. In the thermal resistance characterization study, Kocuria atrinae showed the highest level of resistance among all vegetative cells, though the recommended treatment for Listeria monocytogenes (F70ºC7.5ºC = 2 min) proved to be effective to inactivate this microorganism. For spore forming species, the most severe treatment recommended to inactivate Clostridium botulinum (F90ºC8.6ºC = 57 min in Dungeness crab ( Metacarcinus magister )), proved to be sufficient to achieve a 6 Log10 reduction of B. mycoides and P. psychrodurans but not B. weihenstephanensis. Overall, this research provides valuable information necessary to design processes for the production of safe cooked brown crab products and highlights the importance of Bacillus spp. in this crustacean. Chapter 2 65 regular refrigerator (Whirpool mod. WMT552) (7±2°C) up to the end of their shelf-life (date indicated in the label by the producer). After storage, meats from lab-cooked crabs were extracted aseptically and aliquots of ten grams of each type of meat were prepared and used for microbiological analysis. Each sample was sampled at least in duplicate. In case of commercial samples, on the last day of their shelf-life, samples were opened and two aliquots of 10 grams each were prepared and used for microbial analysis. 2.3.3 Microbial analyses and growth media For the microbiological analyses, samples were taken from the refrigerator on the last day of their shelf-life. Ten grams of crab meat of each sample were diluted in 90mL of Maximum Recovery Diluent (MRD) (Oxoid, Hampshire, UK) and mixed thoroughly for 2 min with a stomacher (model 400 circulator, Seward Stomacher, UK) at 300rpm. Subsequently, ten-fold dilution series were prepared in MRD and aliquots were plated on a range of different agars to evaluate the main microbial groups present in the samples. The agars used in this investigation are summarized in Table 2.2. For the enumeration of colonies grown in/on the plates at least two replicates of each sample were performed, considering only the plates with counts between 10 and 300 CFU. Experiments were performed in triplicate on different working days. Chapter 2 66 Agar Brand Bacterial Group Incubation temp. (°C) Incubatio n time (h) Atmosphere Observations TSA Oxoid TVCm 25 48 Aerobic +0.5% NaCl/ pour plate TSA Oxoid TVCp 10 168 Aerobic +0.5% NaCl/ pour plate L.H. recipe TVCp 10 168-240 Aerobic Pour plate CFC Oxoid Pseudomonas spp. 24 48 Aerobic Spread plate VRBGA Oxoid Enterobacteriaceae 37 48 Aerobic Double layer/pour plate MRS Oxoid Lactic acid bacteria 25 72 Anaerobic Pour plate BP LAB Staphylococcus spp. 35 24 Aerobic Spread plate PEMBA Oxoid Bacillus spp. 37 24 Aerobic Spread plate Iron agar Lyngby Oxoid H2S producing bacteria 30 24 Aerobic Pour plate/ Black colonies SPS Sigma-Aldrich Clostridium spp. 37 48 Anaerobic Black colonies Oxford Oxoid Listeria spp. 30 48 Aerobic Whitish colonies/ Pagadala et al ., 2012 Table 2. 2: Culture media and incubation conditions used during this investigation. TSA, Tryptic Soy Agar; L.H, Long and Hammer’s Agar; CFC, Cetrimide Fusidin Cephaloridine agar; VRBGA, Violet Red Bile Glucose Agar; MRS, Man Rogosa Sharpe agar; BP, Baird-Parker agar; PEMBA, Bacillus cereus agar base supplemented with polymyxin B; SPS Sulfite Polymyxin Sulfadizine agar. TVCm, Total Viable Mesophilic Counts; TVCp, Total Viable Psychrophilic Counts. Chapter 2 67 2.3.4 Isolation and identification After enumeration, a number of well defined colonies were randomly selected from the different agar plates. Each colony was used to inoculate a test tube containing 5mL of Tryptic Soy Broth (TSB) (Oxoid) supplemented with 0.6% (w/v) of Yeast Extract (YE) (Oxoid) and 0.5% (w/v) of NaCl (TSBsupplemented) and incubated at 25°C under either aerobic or anaerobic conditions until turbidity was detected. Following incubation, a loopful from each tube was streaked on a plate of Tryptic Soy Agar (TSA) (Oxoid) supplemented with 0.6% (w/v) of Yeast Extract (YE) (Oxoid) and 0.5% (w/v) of NaCl (TSA-supplemented), and incubated for 24–72h at 25°C under aerobic or anaerobic conditions. A single colony was then used to inoculate test tubes containing 5mL of TSB-supplemented and re-incubated in similar conditions. Once turbidity was detected, 0.1 mL from the tubes were transferred into a 200mL flask containing 50mL of TSB-supplemented and incubated as before. Bacterial populations were examined periodically by microscopy using a Neubauer chamber. When bacterial cultures reached the stationary phase of growth (i.e. stable counts for 3 consecutive days), two stock cryovials were prepared, coded and stored at -80°C. A total of 92 visually-distinct types of colonies, 28 from raw crab meat (both white and brown), 40 from cooked meats and 24 from commercial products were cultured for identification. For the identification work using 16S rRNA sequencing, bacterial populations were recovered by inoculating 5mL TSBsupplemented tubes with a loop-full from the cryovials and incubated at 25°C under either aerobic or anaerobic conditions until turbidity was detected. Growth was sub-cultured from each tube, by plating onto a TSA-supplemented media and incubated at 25°C under either aerobic or anaerobic conditions for 24-72h. A single colony was then transferred into a 5mL TSB-supplemented tube and incubated at 25°C under the conditions described previously. When turbidity was detected, 0.1mL was then transferred in to 50mL TSBsupplemented. Once the stationary phase was reached (using a Neubauer Chapter 2 68 chamber) two aliquots of 1mL were prepared in sterile Eppendorf tubes and a stock cryovial from each culture was prepared. One of the aliquots was stored at 4°C and the other one was used for 16S rRNA sequencing. Samples for 16S rRNA sequencing were prepared as follows. A 1 mL aliquot of each bacterial culture was centrifuged at 10,000rpm for 5 min at 4°C in an Eppendorf centrifuge (model 5417 R, Eppendorf AG 22331, Hamburg, Germany). The supernatant was discarded and the pellet was re-suspended in sterile distilled water inside a laminar flow cabinet. This washing process was repeated twice. After washing, pellets were re-suspended in 500μL of lysis buffer (Fisher Scientific, New Hampshire, United States) and 200μL were transferred to a 96 deep-well plate and sealed. The lysed cells were sent under refrigeration to a commercial laboratory (Eurofins genomics Ltd.) for the sequencing. Sequences (forward and reverse) for each bacterial culture were received in AB1 and SEQ file formats and compared against the US National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 2.3.5 Heat resistance experiments After the identification of the microbial species isolated, their heat resistance was evaluated. 2.3.5.1 Culture preparation and treatment media Microorganisms were first resuscitated from the stock cryovials as described in section 2.3.4. Then, a 0.1mL aliquot of an overnight pre-culture was inoculated into 200mL Duran bottles containing 50mL of sterile TSBsupplemented containing 6mm diameter sterile glass beads (approximately 30g) to prevent the formation of cell aggregates. These were incubated at 25°C in a shaking thermostatic bath at 130rpm (mod. LSB18, Grant instruments Ltd., Cambridge, UK) until cultures reached the stationary phase. All microorganisms were incubated for 48h except for Staphylococcus spp. which required 72h to reach the stationary phase. Chapter 2 69 2.3.5.2 Heat treatments In this study, thermal resistance experiments were performed in pH 7.0 McIlvaine citrate-phosphate buffer (Dawson et al ., 1974) which is similar to the pH of crab meat. Firstly, the most heat resistant species within the same genus was determined by means of the end-point method. The procedure used was as follows: 1mL of the stationary phase cultures was re-suspended in tubes containing 9mL of citrate-phosphate buffer in order to achieve an initial count of approximately 108 CFU/mL. Tubes were then submerged in a thermostatic bath (mod. LSB18, Grant instruments Ltd., Cambridge, UK) at 60°C for 90 seconds and immediately cooled in iced water. Finally, aliquots of each serial dilution were pour plated in TSA-supplemented agar and incubated at 25°C for 72 h. Further incubation times did not provide higher counts (data not shown). Once the most heat resistant microbial species within the same genus was determined (those showing lower Log10 reductions after the 60ºC/90s treatment), their decimal reduction time values ( D T) and z values were determined using a thermoresistometer TR-SC (Condón et al ., 1989, 1993). This instrument consists of a 350mL stainless steel vessel containing the treatment medium and equipped with an electrical heater and a refrigeration coil system for maintaining a constant temperature, an agitation device to ensure uniform distribution and temperature homogeneity of the medium and inoculum and ports for injecting the microbial suspension and for sample extraction. Once the target temperature attained stability (±0.1°C), 0.2mL of the corresponding microbial suspension was injected with a sterile syringe into the treatment medium. After inoculation, 0.1mL samples were collected at different times and immediately pour plated in TSA-supplemented agar. Survival curves (log10 number of survivors versus treatment time) were obtained at different temperatures ranging from 54 to 69°C and the corresponding D T values (min) were calculated. In order to calculate z values (°C), four temperatures were evaluated for each bacterial culture. All thermal experiments were carried out at least in triplicate on different working days. Chapter 2 70 2.3.6 Data and statistical analysis 2.3.6.1 Shelf-life determination Gompertz Equation modified by Zwietering et al ., (1990) (Equation 2.1) was fitted to the TVCm growth at different temperatures (2, 5, 7 and 10°C): 𝑦=𝐴∗𝑒𝑥𝑝{−𝑒𝑥𝑝[1+𝜇𝑚𝑎𝑥∗𝑒 𝐴∗(𝜆−𝑡)]} (Eq. 2.1) where, y indicates the Log10 of the count reached at the different times, A indicates the increased Log10 cycles from initial count (y0) to stationary phase ( ymax ) and t is the storage time expressed in hours; μmax represents the maximum growth rate (h-1) observed in the linear part of the growth curve, λ represents the duration of the lag phase (h) and e equals to 2.718. The estimated values for λ and μmax were further expressed as a function of the storage temperature by using the Square Root Model (Ratkowsky et al ., 1982) (Equations 2.2 and 2.3): √𝜇𝑚𝑎𝑥=𝑏𝜇∗(𝑇−𝑇minμ) (Eq. 2.2) √1 𝜆=𝑏𝜆∗(𝑇−𝑇minλ) (Eq. 2.3) where b is a constant, T (°C) is the storage temperature and Tmin is the respective theoretical minimum temperature of growth (°C), estimated by extrapolation of the regression line when √𝜇𝑚𝑎𝑥 or √1 λ = 0. All models were fitted to the data by using a non-linear regression and least square approach with GraphPad PRISM® 5.0 software (GraphPad software, Inc., San Diego, CA, USA). The same software was used to perform the statistical analyses: t -test and one-way ANOVA followed by the post hoc Tukey test. Differences were considered significant when P≤0.05. Chapter 2 71 2.3.6.2 Thermal resistance determination Survival curves at a constant temperature (T) were obtained by plotting the Log10 fraction of survivors (survivors after a certain treatment time t divided by the microbial concentration at t =0) versus the treatment time ( t , min). In cases where the profile of the survival curve appeared as a downward concave shape, the Geeraerd log-linear regression plus shoulder model was used (Geeraerd et al ., 2000). To fit the model to the survival curves, the Geeraerd and Van Impe inactivation model fitting tool (GInaFiT) was used (Geeraerd et al ., 2005). The model (Equation 2.4) describes the survival curves through two parameters: the shoulder length ( Sl ), defined as the time preceding the exponential decay, and the inactivation rate ( kmax ) which corresponds to the slope of the exponential portion of the survival curve. N0=N0e-kmax t(ekmax Sl 1+ (ekmax Sl-1)e-kmax t) (Eq. 2.4) where N0 represents the initial count. For survival curves with log-linear profiles, Bigelow and Esty model (Bigelow & Esty, 1920), which is also included in the GInaFiT software, was used to calculate the exponential inactivation rate ( kmax ) in min-1. The traditional decimal reduction time value ( D T value) in minutes was then calculated with Equation 2.5. D T = 2.303/ kmax (Eq. 2.5) The z value (°C), which represents the temperature increase required to reduce D T values by 10 fold was then calculated from the negative inverse of the regression line of the Thermal Death Time curves (TDT) obtained when Log10 D T values are represented versus treatment temperature. Chapter 2 72 Based on the thermal resistance parameters ( D T and z values), an equivalent treatment to reduce 6 Log10 cycles of the microbial population were also calculated and compared with those proposed for L. monocytogenes in case of vegetative cells and C. botulinum in case of spores, based on the recommended treatments by FDA for fishery products (FDA, 2011). t-test and one-way ANOVA were performed with the GraphPad PRISM 5.0 software (GraphPad software, Inc., San Diego, USA) and differences were considered significant if P≤0.05. The standard deviations ( SD ) are given in the Figures as error bars. 2.4 RESULTS AND DISCUSSION 2.4.1 Microbiological profile of raw crab meats Experimental counts for crab raw fresh meats obtained on the different specific agars are shown in Figure 2.1. Overall, the counts obtained in this research for crab raw meats are in accordance with those previously reported for different raw unprocessed shell fish, which are between 2.5 and 3 Log10 cycles (Gornik et al ., 2011; Robson et al ., 2007). Differences in counts between the two types of meat (i.e. white vs brown) were observed for all bacterial groups. Interestingly, higher counts were detected in brown meat. Faghri et al ., (1984) reported similar differences in counts depending on the crab’s tissue (muscle, gills and hemolymph), obtaining lower counts in crab muscle, which corresponds to white meat than in case of gills and hemolymph. These higher counts in brown meat can be explained by the fact that crabs do not have a closed structured circulatory system so the haemolymph, located inside the crab body, could be a reservoir of bacteria (ICMSF, 2005). In both types of crab meat the psychrophilic counts were higher than for mesophilic bacteria. In case of mesophilic bacteria the counts were 1.2 and 2.5 Log10 cycles for white and brown meat, respectively, while for the psychrophilic bacteria the counts in PCA (Plate Count Agar, Oxoid) were 1.8 and 3.0 Log10 cycles. However, when a specific agar was used for marine psychrophilic bacteria (L.H agar) the counts were 2.1 and 3.7 Log10 cycles in white and brown meat, respectively, i.e. 0.3 Chapter 2 73 and 0.7 Log10 cycles higher compared with the non-selective PCA. This mismatch was also observed by Broekaert et al ., (2011) who reported increases in the counts between 0.3 and 1.1 Log10 cycles when L.H agar was used instead of PCA. Figure 2.1: Log10 counts obtained in different selective and non-selective agars (see Table 2.2) for white (white bars) and brown (grey bars) raw crab meat. TVCm, Total viable mesophilic counts; TVCp, Total viable psychrophilic counts; L.H, Long and Hammer agar. ND = not detectable. In case of white meat, only Pseudomonas spp. and Staphylococcus spp. were detected and the counts were 1.8 and 1.4 Log10 cycles, respectively. The heterogeneity of the flora detected in brown meat was larger than in white meat. Also in brown meat Pseudomonas spp. was the genus detected in a higher proportion with a total count of 3.5 Log10 cycles while Staphylococcus spp. and Bacillus spp. counts were 2.9 and 3 Log10 cycles respectively. The main difference observed between meats were the significant counts observed for Bacillus spp. (2.9 Log10 cycles) and the presence of H2S producing bacteria (0.9 Log10 cycles) and LAB (1.2 Log10 cycles) in brown meat. Overall, results obtained in this section are in agreement with most published data on different crab species and shellfish. But interestingly, our study also revealed a significant presence of Bacillus spp. in brown meat though Bacillus spp. S prod. bacteria 2 HEnterobacteriaceae Lactic Acid Bacteria Pseudomonas spp. Staphylococcus spp. m TVC p TVC (L.H) p TVC Clostridium spp. L. monocytogenes 0 1 2 3 4 ND ND ND ND ND ND Log10 CFU/g Chapter 2 74 not in white meat. Some authors have also reported the presence of this spore forming bacteria in the intestinal content of Dungeness crab (Lee and Pfeifer, 1975). Linton et al ., (2003) also described the presence of Bacillus spp. in different shellfish after a high hydrostatic pressure process, though some other authors did not find this microorganism in raw shellfish (Boziaris et al ., 2011; Faghri et al ., 1984; Gornik et al ., 2011). The presence of Bacillus spp. could be expected since different authors have reported its presence but mainly in processed products (Gram and Huss, 1996; Linton et al ., 2003). This could be attributable to the low counts found in raw products, which will make them unable to compete with other bacteria present in higher proportions. Nonetheless, the fact that they are spore-forming bacteria with a “far-fromnegligible” resistance to conservation processes gives them an important role in the spoilage of processed seafood products. The absence of H2S producing bacteria and LAB in white meat is also in accordance with the study of Boziaris et al ., (2011) with lobsters. Although these two bacterial groups along with the Enterobacteriaceae family are considered of significant relevance in the degradation of fresh fish (Gram and Huss, 1996; Gram and Dalgaard, 2002; Jay, 2000), their importance in brown crab seems to be minor due to the lower counts observed here compared to reports on other species. It is also interesting to note that in this research, neither Listeria spp . nor Clostridium spp . were detected in any of the samples analysed. This could be attributable to insufficient sample size or due to the small counts and relatively low prevalence of these microorganisms in crab as indigenous bacteria (Fledhusen, 2000; Rocourt et al ., 2000; Pagadala et al ., 2012). 2.4.2 Microbiological shelf-life of cooked crabs In this section the effect of the storage temperature on the shelf-life of crabs cooked at a mild temperature (75°C) for the required time to apply at least an equivalent F 70ºC7.5ºC= 2 min (45 min) as determined in Chapter 3, was evaluated. To determine the effect of storage temperature on the shelf-life of this seafood product, the Gompertz Equation modified by Zwietering et al ., Chapter 2 81 differently depending on the type of meat. b values, but not Tmin , reported here are in the range of those reported by Koutsuomanis (2001) and Koutsoumanis et al ., (2001) for Pseudomonas spp. in gilt-head seabream. The differences between the calculated b values and those reported in the literature would be attributable, as in case of μmax , to differences in the microbiota present in the product. Nevertheless, the differences found between bλ values for the crab meat types would need further explanation. In this research the shelf-life model was built from the results obtained from a mild cooked crab. As shown in Chapter 3 the crab’s cold spot is located in the abdomen. Due to the different heat penetration rates observed, between crab parts, significant differences on the total F value applied exists between the two types meats, so a difference on the microbiota selected after the treatment could exist. Although, the differences between meat compositions could also contribute, as has been discussed previously. 2.4.5 Shelf-life determination (Tertiary model) Finally, based on the parameters extracted from the primary (modified Gompertz Equation) and secondary (Root Square Model) models, two different final Equations were developed to calculate the microbial growth in white (Eq. 2.6) and brown meat (Eq. 2.7). 𝑦=𝐴×𝑒𝑥𝑝{−𝑒𝑥𝑝[1+[0.0058×(𝑇−(−24.9))]2×𝑒 𝐴∗[(0.014×((𝑇−(−3.7)))2−𝑡]]} (Eq. 2.6) 𝑦=𝐴×𝑒𝑥𝑝{−𝑒𝑥𝑝[1+[0.0061×(𝑇−(−21.1))]2×𝑒 𝐴∗[(0.0096×(𝑇−(−4.4)))2−𝑡]]} (Eq. 2.7) Where y indicates the Log10 cycles of the counts, A indicates the difference between the counts (Log10 cycles) in the stationary phase ( ymax ) and the initial counts (y0), t is the storage time expressed in hours and T represent the Chapter 2 82 storage temperature (°C). Figure 2.3 shows the comparison between observed and predicted values calculated from Equations 2.6 and 2.7 as well as R2 and RMSE as indicators of the accuracy of the tertiary Equations for white (A) and brown (B) meat. Based on Equations 2.6 and 2.7 the shelf-life of white and brown meat were calculated, considering 6 Log10 cycles as the end of the shelflife. A log-linear relationship was found between the shelf-life and the storage temperature for both meat types as Dabadé et al ., (2015) previously reported for tropical shrimps. Table 2.5 shows the Equations which describe the effect of the storage temperature on the shelf-life of each meat type. In this case, no differences were found between the slopes obtained for each meat type meaning that the relative effect of the storage temperature on the shelf-life was independent of the type of meat. From the Equations shown in Table 2.5, a shelf-life of 12 and 16 days were calculated at 4°C, while 6 and 7 days were defined as the shelf-life limit at 10°C for white and brown meat, respectively. According to these results, the readyto-eat edible crab ( Cancer pagurus ) shelf-life seems to be limited by the white meat, when crabs are processed in a single step cooking at mild temperatures. Table 2.5: Equations which define the shelf-life of ready-to-eat whole brown crab packed under vacuum as a factor of the storage temperature. Meat type Equation White Log10 Shelf-life (h) = -0.0497 * T + 2.6588 Brown Log10 Shelf-life (h) = -0.0537 * T + 2.8001 Chapter 2 83 Figure 2.3: Measured Vs predicted counts of TVCm at all storage temperatures in white (A) and brown (B) meat (estimated from Eq. 2.6 and 2.7) A 0 2 4 6 8 10 0 2 4 6 8 10 R2=0.94 RMSE = 0.82 Measured values Predicted values B 0 2 4 6 8 10 0 2 4 6 8 10 R2=0.91 RMSE = 0.97 Measured values Predicted values Chapter 2 84 2.4.6 Effect of storage temperature on microbial flora present in cooked crab Based on the developed model (Section 2.4.5), a study of the main microbiota present at the end of the shelf-life i.e. after 12 days at 4°C and after 6 days at 10°C was performed. Similar to raw meats (Figure 2.1), Figure 2.4 shows the microbiological profiles for cooked crab at the end of shelf life, using different selective and non-selective agars (Table 2.2) for white (A) and brown (B) crab meat. In general, no differences between counts were observed at 4 and 10°C in the case where the same bacterial group was detected at both temperatures; except for Bacillus spp. in white meat and Staphylococcus spp. in brown meat. This result supports the effectiveness of the model developed in the previous section. The maximum counts registered for TVCm (Figure 2.4) were 5.3 and 4.8 Log10 CFU/g in white meat and 5.9 and 6.0 Log10 CFU/g in brown meat at 4 and 10°C, respectively. As it was observed with the raw meats (Figure 2.1), in the case of cooked crab meats, the psychrophilic bacteria counts were higher and the highest counts were detected in L.H agar (6.1 and 4.9 Log10 CFU/g in the case of white meat and 6.4 and 6.1 Log10 cycles in the case of brown meat). For white meat (Figure 2.4A), H2S producing bacteria were only found in the meat stored at 10°C in a level of 2.7 Log10 CFU/g. In the case of Pseudomonas spp . no differences were detected between the absolute counts at 4 and 10°C (2.5 Log10 CFU/g). Similarly, no differences between counts were observed between Staphylococcus spp . counts at 4 and 10°C (3.4 Log10 CFU/g). Only in the case of Bacillus spp . significant differences were observed in counts 3.9 Log10 at 10°C and 5.3 Log10 at 4°C. For brown meat (Figure 2.4B), H2S producing bacteria, Enterobacteriaceae and LAB (Lactic Acid Bacteria) were only detected when the meat was stored at 10°C. The presence of Staphylococcus spp. was dependent on the storage temperature: at 4°C this bacteria count was 3.2 Log10 cycles and 5.7 Log10 cycles for samples stored at 10°C. On the other Chapter 2 85 side, in case of Bacillus spp . , no significant differences were detected between the counts at 4 and 10°C (between 5.5 and 6 Log10 CFU/g). Figure 2.4: Log10 counts obtained for Bacillus spp., H2S producing bacteria, Enterobacteriaceae , Lactic Acid Bacteria, Pseudomonas spp., Staphylococcus spp., Total Viable Counts, Total Psychrophilic Aerobic Counts and total psychrophilic bacteria (L.H) for white (A) and brown (B) meat of cooked crab store at 4°C (grey bars) and 10°C (white bars) at the end of the shelf-life (calculated from Eq. 2.6). A Bacillus spp. S prod. bacteria 2 HEnterobacteriaceae Lactic Acid Bacteria Pseudomonas spp. Staphylococcus spp. m TVC p TVC (L.H) p TVC Clostridium spp. L. monocytogenes 0 2 4 6 84C 10C ND ND NDNDND Log10 CFU/g B Bacillus spp. S prod. bacteria 2 HEnterobacteriaceae Lactic Acid Bacteria Pseudomonas spp. Staphylococcus spp. TVCm TVCp TVCp (L.H) Clostridium spp. L. monocytogenes 0 2 4 6 84C 10C ND ND ND ND ND Log10 CFU/g Chapter 2 86 Based on the results of this section, Bacillus spp . proved to be the species of major significance in ready-to-eat whole brown crab followed by Staphylococcus spp. especially at higher storage temperatures. The presence of other SSO bacteria, which is considered of importance, was practically nonexistent. Ward et al ., (1977) also reported the presence of Bacillus spp. in pasteurized crab meat stored at 3.3 °C. However the same authors also reported an important relevance of Pseudomonas spp., which has not been detected in this research. Lorentzen et al ., (2014) also reported the importance of H2S producing bacteria and Pseudomonas spp. in cooked King crab clusters stored at 4°C. The presence of Pseudomonas spp. as important SSO in cooked brown crab was also reported by Anacleto et al ., (2011). But other authors have also reported the importance of Bacillus spp . in processed fish and fishery products (Gram and Huss, 1996; Linton et al ., 2003). However, this is the first work which shows the importance of Staphylococcus spp. as species of significance in a processed seafood stored at 10ºC. The discrepancies observed with previous studies, where the presence of Staphylococcus spp. has not been highlighted, could be attributable to different raw materials, the temperatures used in the experimental plan or the different treatments applied. In the present study a thermal treatment specifically designed to inactivate L. monocytogenes in seafood, based on the US FDA recommendations, was applied. This would explain the low counts for vegetative bacteria cells and the strong presence of Bacillus spp. as those are able to produce heat resistant spore forms. It is also noteworthy to mention that in the majority of the published studies the thermal treatment conditions applied are not reported hence the differences in the microbiota could be also associated to the different thermal treatments applied. 2.4.7 Bacterial groups present in lab-cooked and commercial samples of ready-to-eat brown crab Once a suitable model to describe the shelf-life of ready-to-eat brown crab meat samples depending on the storage temperature has been defined, the Chapter 2 87 microbiota of a number of crabs cooked, as described in section 3.2, packed under vacuum and stored at 7±1°C were analysed on the last day of their shelflife (defined based on previous results). Additionally, the microbial groups of a number of commercial samples (see Table 2.1) were also analysed at the end of their shelf-life, as indicated in the label. The percentages calculated in this section are calculated based on the presumptive counts in specific agars using Equation 2.8. Representativity %= Count in specific agar Count in non−specific agar (𝑇𝑉𝐶𝑚)×100 (Eq. 2.8) For laboratory processed crab meat stored for 7 days (i.e. the end of their shelf-life as defined by the equations developed in section 2.4.5), the maximum TVCm counts observed were 5.1 and 7.1 log10 CFU/g for white and brown meat, respectively. In contrast to raw meat, the highest counts were detected for TVCm instead of TVCp. The proportions of the different bacterial groups detected are presented in Figure 2.5. For white meat, the main genera detected were: Bacillus spp . (14%), Staphylococcus spp. (13%), Pseudomonas spp . (0.7%) and H2S producing bacteria (0.1%), while undefined bacterial groups represented 71% of the total microbiota, showing the limitations of presumptive counts in selective agars. For brown meat, unidentified bacterial groups made up 9%, with Bacillus spp. most frequently detected (59.4%) followed by Staphylococcus spp. (29.7%). Furthermore, Enterobacteriaceae , LAB and Pseudomonas spp. were detected in low proportions corresponding to 0.3%, 0.5% and 0.2%, respectively. As for raw crab meat, H2S producing bacteria, Listeria spp. and Clostridium spp. were not detected in either white or brown cooked meats samples. The TVCm counts enumerated in commercial samples at the end of their shelf-life were 8.3, 2.1, 6.8, 5.4, 6.7 and 3.6 Log10 CFU/g for samples A, B, C, D, E and F, respectively (see Table 2.1). These results highlight substantial variability in TVCm counts among commercial crab products. The highest counts were detected in particulate white meat packed under modified atmosphere Chapter 2 88 conditions, where the main bacteria detected were H2S producing bacteria (77.6%), followed by Bacillus spp. (4.9%) and Pseudomonas spp . (3.8%), while LAB and Staphylococcus spp. represented only 1% of the total microbial concentration, as shown in Figure 2.5. Ready-to-eat whole crab claws (sample B) were found to have the lowest counts at the end of shelf-life. In samples C and F no specific bacterial groups were detected with the selective agars used. Staphylococcus spp. represented 20% of the flora in product E, while Pseudomonas spp. and H2S producing bacteria were detected in proportions of 16.5% and 5% respectively in sample D (Figure 2.5). Figure 2.5: Relative percentage of in lab-cooked white and brown crab meat and various commercial samples at the end of their shelf-life. The heterogeneity observed among the commercial samples may be related to the different processes used by individual companies within the crab processing sector. Differences in the microbiota present in meat cooked in a single step under controlled conditions versus commercial could be related to the fact that commercial crab products are pasteurised after packaging and they are generally designed to target non-proteolytic Clostridium botulinum type E (Edwards and Early, 2001). Therefore, the low presence of sporeforming bacteria??? is not surprising and this is likely the reason why neither Listeria nor Clostridium were detected. white meat brown meat Commercial A Commercial B Commercial C Commercial D Commercial E Commercial F 0 10 20 30 40 50 60 70 80 90 100 Bacillus spp. H2S prod. Bacteria Enterobacteriaceae Lactic acid bacteria Pseudomonas spp. Staphylococcus spp. others species % Chapter 2 89 2.4.8 Bacterial species isolated in brown crab Table 2.6 shows the main bacterial species identified using 16S rRNA sequencing in crab and crab products. Interestingly, in raw crab meat 14% of the isolates identified were confirmed as Shewanella baltica in both brown and white meat even though no H2S producing bacteria were detected in iron Lyngby agar, which is expected to isolate this species as it is a sulphite producing bacteria. However, the detection of this bacterium is not surprising given that they are considered a common specific spoilage organism (SSO) in seafood. Carnobaterium divergens was identified in 28.5% of the bacterial isolates and was the most frequently isolated bacterium irrespective of crab meat type even though the LAB counts (the group to which it belongs) were very low in MRS agar. The remaining bacterial species isolated from crab meat represented from 3.5 to 7% of the total bacteria isolated in raw crab meats and included several species of Staphylococcus , Pseudomonas , Psychrobacter , Kocuria and different genera such us Enterococcus , Dietzia , Vibrio , Planococcus , Brochotrix , Oerskovia , Chryseobacterium , Corynebacterium , Providencia and Pseudoalteromonas . For commercial samples, a number of vegetative cells were identified, though Staphylococcus spp., Carnobacterium spp. and Corynebacterium spp. were the dominant species identified. In case of spore-forming bacteria, only Psychrobacillus psychrodurans and Bacillus weihnestephanensis were identified, with P. psychrodurans being the most prevalent in commercial samples. For crab meats cooked in a single step under laboratory conditions, a number of vegetative genera were also detected including Pseudomonas and Staphylococci . For spore-forming bacteria, Bacillus mycoides , B. weihenstephanensis and P. psychrodurans were the most prevalent species recovered with proportions of 35%, 30% and 7.5% identified, respectively. Chapter 2 90 Table 2.6: Vegetative bacterial species isolated from cooked ready-to-eat brown crab ( Cancer pagurus ) processed under laboratory conditions and various commercial products and identified by 16S rRNA sequencing. Vegetative Cells Sample Type Bacterial genus species White meat Brown meat Commercial meat Raw Processed Raw Processed Processed Staphylococccus equorum 1 1 1 - - capitis 1 1 - - - xylosus 1 - - - 3 fluorettii - - - - 1 Shewanella baltica 2 1 1 - 1 Carnobacterium divergens 4 1 4 1 2 funditum - - - - 1 Enterococcus aquimarinus - - - - 1 Dietzia maris - - 1 - - Pseudomonas deceptionensis - 1 - - - lundensis - 1 - 1 - fluorescens - - - 1 - synxantha - - 1 - - psychrophila - 1 - - - Psychrobacter fozii 1 - 1 - - maritimus - - 1 - - articus - - 1 - - faecalis - - 1 - - Vibrio anguillarum - - 1 - - Planococcus halocryophilus - - 1 - - Brochotrix thermosphacta - - - 1 - Oerskovia turbata - - 1 - - Kocuria salsiccia - - 1 - - atrinae - - - - 1 Chryseobacterium humi - - 1 - - Corynebacterium testudinoris - - - - 3 Providencia heimbachae - - - - 1 Pseudoalteromonas elyakovii - - 1 - - Spore forming Bacillus mycoides - 9 - 5 - weihenstephanensis - 8 1 4 1 Psychrobacillus psychrodurans - - - 3 9 Chapter 2 97 Figure 2.8: Theoretical thermal death time curves (TDT) to achieve 6 Log10 reductions of Kocuria atrinae (black line) and Listeria monocytogenes (red line). The latter based on the recommended treatments by FDA (FDA, 2011). Figure 2.9: Theoretical TDT lines to achieve 6 Log10 reductions of the spore forming bacteria isolated from crab based on data presented in chapter 6 and heat treatments required to achieve similar reductions of C. botulinum non-proteolytic spores based on the recommended treatments by FDA ( F 90ºC7or10ºC = 10 min; F 90ºC9ºC = 31 min; F 90ºC8.6ºC = 57 min) (FDA, 2011). A 50 60 70 80 90 -3 -2 -1 0 1 2 3Kokuria L. monocytogenes Treatment temperature (C) Log10 6DT(min) 70 80 90 100 110 120 -3 -2 -1 0 1 2 3 4 F907or10= 10min F908.6= 57min F909= 31min B. mycoides B. weihenstephanensisP. psychrodurans Treatment temperature (C) Log10 6DT(min) 70 80 90 100 110 120 -3 -2 -1 0 1 2 3 4 F90C7or10C= 10min F90C8.6C= 57min F90C9C= 31min B. mycoides B. weihenstephanensisP. psychrodurans Treatment temperature (C) Log10 6DT(min) Chapter 2 98 On the other side, Figure 2.9 shows the thermal treatments proposed for crab and crab based products considering C. botulinum non-proteolytic type E as the target microorganism. F 90°C= 10 min or equivalent, considering z values of 7°C for temperatures below 90°C, or 10°C for temperatures over 90°C. As it can be seen, this treatment would not reliably provide a reduction of 6 log10 cycles for B. mycoides, the most heat sensitive spore isolated from crab samples as it is shown in section 6.4.1. However, some researchers consider crab meat as an exception with regard to the inactivation of Clostridium spores due to its high concentration of lysozyme (Peterson et al ., 1997). It is known that the presence of lysozyme improves the recovery of heat-treated Clostridium spores and therefore its heat resistance (Peck and Fernadez, 1995; Scott and Bernard, 1982). It has also been reported that this enzyme is present in fish and seafood up to levels of 200 μg/g (Lie et al ., 1989; Lund and Notermans, 1992). For this reason, two more severe alternative treatments were proposed by the FDA for crab meat: F 90ºC9ºC= 31 min (Blue crab, Callinectes sapidus ) and F 90ºC8.6ºC= 57 min (Dungeness crab, Metacarcinus magister ) in order to achieve a satisfactory reduction of Clostridium spores (FDA, 2011; Peterson et al ., 1997, 2002). These two alternative treatments are also represented in Figure 2.9. As the Figure shows, if the most severe recommended treatment, i.e. for Dungeness crab meat ( F 90ºC8.6ºC= 57 min), is to be applied to brown crab, an adequate reduction of two of the three spore formers isolated in this thesis ( B. mycoides and P. psychrodurans ) would be achieved. However, this treatment would not be sufficient to achieve a similar reduction in the population of B. weihenstephanensis spores, which would need treatment times 10-fold longer. Van Asselt and Zwietering (2006) reported similar differences when comparing the thermal resistance of various Clostridium botulinum and Bacillus cereus strains . In 1998 Bacillus weihenstephanensis was proposed as a new species to accommodate some of the psychrotrophic strains of Bacillus cereus (Lechner et al ., 1998). This spore-forming species is able to grow at temperatures of 5°C or less and the ability to produce emetic and diarrheal toxins, normally associated Chapter 2 99 with B. cereus , has also been reported (Stenfors and Granum, 2001; Stenfors et al ., 2002; Thorsen et al ., 2006, 2009). As a result, the isolation of this species in brown crab combined with its high heat resistance (higher than nonproteolytic species of C. botulinum ) highlights their potential significance in terms of food safety. However, no disease associated with Bacillus spp. in brown crab products have been reported, probably due to products being packaged under vacuum, which can hinder the growth of B. wiehentephanensis (Samapundo et al ., 2011). Overall, the results of this investigation showed that spores naturally present in brown crab meat could be more thermo-tolerant than the non-proteolytic strains of C. botulinum . Therefore, heat treatments designed for these spore-forming bacteria may not be sufficient to reduce populations to safe levels. 2.5 CONCLUSIONS From the obtained results in this chapter it can be concluded that Bacillus spp . , Pseudomonas spp. and Staphylococcus spp. are the main microbial groups present in raw white and brown crab meat. From the shelf-life study it can be concluded that the storage temperature has a greater effect on the lag phase than on the growth rate of the TVCm during storage. A log-linear relationship was found between the storage temperature and the logarithm of shelf-life (hours) for both meat types (i.e. white and brown). Bacillus spp. and Staphylococcus spp . proved to be the bacterial groups present in a major proportion at the end of the shelf-life of the product. However, their proportions changed considerably depending on the storage temperature. Raw brown crab meat shows a greater heterogeneity in microbiota than white meat. In case of raw meat, Carnobacterium divergens and Sewanella baltica were the most frequently isolated microorganisms regardless of the meat type, while for cooked meat stored at 7-8°C, B. mycoides , B. weihenstephanensis and P. psychrodurans were the most frequently detected in all commercial meats and crabs cooked under controlled conditions at mild temperatures in the Chapter 2 100 laboratory. For vegetative cells, Kocuria atrinae showed the highest level of heat resistance among non-spore forming bacterial species isolated from brown crab meat, though importantly, the heat treatment recommended by the FDA to inactivate L. monocytogenes ( F 70ºC7.5ºC= 2 min) proved to be sufficient to inactivate it. In contrast, the most severe heat treatment recommended to inactivate non-proteolytic species of C. botulinum was enough to reduce 6 Log10 cycles of B. mycoides and P. psychrodurans but not B. weihenstephanensis, which would require a treatment time 10-fold longer. Overall, this section provides significant knowledge about the microbiota and the shelf-life of readyto-eat brown crab ( Cancer pagurus ), a product of ample market but underestimated from a scientific point of view and highlights the importance of Bacillus spp. although additional studies would be required to determine the potential food safety risks associated with B. weihenstephanensis in brown crab based products. Chapter 3 101 Chapter 3 The use of ultrasound technology in the first cooking step of ready-to-eat whole brown crab (Cancer pagurus) This chapter is as publish in Ultrasonics Sonochemistry, with some minor modifications to avoid duplication between chapters. Condón-Abanto, S., Arroyo, C., Álvarez, I., Brunton, N., Whyte, P., & Lyng, J. G. (2018). An assessment of the application of ultrasound in the processing of ready-to-eat whole brown crab ( Cancer pagurus ). Ultrasonics Sonochemistry, 40, Part A, 497-504. Chapter 3 102 3.1 ABSTRACT/RESUMEN ABSTRACT This study assesses the potential of incorporating ultrasound as a processing aid in the production of whole cooked brown crab ( Cancer pagurus ). The FDA recommended heat treatment to reduce Listeria monocytogenes by 6 Log10 cycles in this product is a F 70ºC7.5ºC of 2 min. An equivalent F value was applied at 75°C in presence and absence of ultrasound in water alone or in water with 5% w/v NaCl added. Heat penetration, turbidity and conductivity of the cook water and also salt and moisture content of the crab meat (both white and brown meat) were determined. Ultrasound assisted cooking allowed a reduction of the cooking time by up to 15% while still maintaining an F 70ºC7.5ºC of 2 min. Ultrasound also enhanced the rate and total amount of compounds released from the crab, which suggests that crabs cooked in the presence of ultrasound would be expected to be cleaner. Ultrasound also proved to be effective in reducing the salt content but hardly affected the final moisture content of the crab meat. Chapter 3 103 RESUMEN Este estudio se realizó para evaluar el potencial de la incorporación de la tecnología de los ultrasonidos como coadyuvante en la producción de buey de mar ( Cancer pagurus ) cocinado entero listo para el consumo. El tratamiento recomendado por la FDA para inactivar 6 ciclos logarítmicos de Listeria monocytogenes en este tipo de productos es un F 70ºC7.5ºC de 2 minutos. Se aplicó un F equivalente a 75°C en agua y agua con un 5% p/v de NaCl utilizando o no ultrasonidos durante el tratamiento. Se evaluó la penetración de calor y se midió la turbidez y conductividad eléctrica del agua de cocinado y el contenido de sal y humedad de ambos tipos de carne (blanca y marrón) de cangrejo tras los distintos tratamientos. El tratamiento con ultrasonidos permitió reducir hasta un 15% el tiempo total de tratamiento manteniendo un F 70ºC7.5ºC equivalente de 2 minutos. La aplicación de ultrasonidos durante el cocinado también mejoró la velocidad de salida de compuestos del cangrejo así como la cantidad total de estos compuestos, lo cual sugiere que los cangrejos cocinados en presencia de ultrasonidos deberían de estar más limpios. Los ultrasonidos fueron también efectivos para reducir el contenido de sal de la carne de cangrejo, pero prácticamente no afectaron a su contenido de humedad. En conclusión, los resultados obtenidos en este capítulo permitirían reducir el tiempo de cocción así como eliminar la etapa de lavado del proceso tradicional del cangrejo con las consiguientes consecuencias económicas y de calidad del producto. Chapter 3 104 3.2 INTRODUCTION As mentioned before the production of ready-to-eat edible crab involve a series of general steps which have not evolved in line with current technological developments. Many producers still use traditional techniques and define their own cooking conditions in terms of time and temperature which leads to heterogeneity in the quality of marketable products (e.g. over or undercooking). The size of these companies is usually small and their investment in technology and process optimisation is generally low. However, novel processing technologies such as ultrasound have many benefits to offer them. For example, ultrasound technology is widely used in the food industry to enhance heat and mass transfer processes (Chandrapala, et al ., 2012; Chemat, et al ., 2011) which could have great relevance and be easily adopted to improve traditional immersion cooking processes used in the production of ready-to-eat crab. High-intensity ultrasound involves intensities greater than 1 W/cm2 and is performed at frequencies ranging from 18 to 100 kHz. Cavitation is considered the main mechanism by which this form of ultrasound enhances heat and mass transfer phenomena (Kim, et al ., 2004) though other effects such as acoustic streaming are also involved (Legay, et al ., 2011). The effects of ultrasound on heat transfer have been extensively studied since the 1990s in model systems such as water, metal tubes, metal balls, etc. (Huamao, et al ., 1997; Hyun, et al ., 2005; Oh, et al ., 2002) and its ability to enhance heat transfer in foods, mainly in processed meat products, during cooking processes has also been proven (Alarcon-Rojo, et al ., 2015; Vimini, et al ., 1983). The potential for ultrasound to assist different processes such as extraction (Khan, et al ., 2012; Luengo, et al ., 2014; Ma, et al ., 2009), cooking (Mason, et al ., 1996) and marinating (Cárcel, et al ., 2007; McDonnell, et al ., 2014; Turshan, et al ., 2013; Vinimi, et al ., 1983) have been assessed in vegetables tissues, meats and fish. However, its effects on the industrial heat processing of ready-to-eat crustaceans products have never been explored. Therefore, the aim of this Chapter 3 105 study was to assess the potential of ultrasound for the cooking of brown crab by accelerating heat and mass transfer processes. The increase of heat penetration should lead to a reduction in cooking times which in turn should enhance product quality whilst ensuring adequate levels of safety. In addition, the production of ready-to-eat brown crab involves a cleaning/cooling step which is needed to remove crab dirt and cook exudate deposits before packing. This step takes 3-4 h and constitutes a microbiological risk due to a possible recontamination of the product, hence requiring a subsequent pasteurization step with the sole purpose of eliminating microbial contamination (Ghazala and Trenholm, 1996; Pagadala, et al ., 2012). The ability of ultrasound to enhance mass transfer could also be used to remove dirt and exudate from crab shells during cooking thus eliminating or reducing the severity of the subsequent pasteurization ultimately resulting in greater yields, less energy input and a milder heattreated higher quality product. Therefore the objective of this research was to evaluate the potential improvements induced by the application of ultrasound in the cooking process of ready-to-eat whole brown crab, with particular reference to the benefits of associated heat and mass transfer phenomena. 3.3 MATERIALS AND METHODS 3.3.1 Raw material and cooking conditions All performed experiments were carried out with female crabs with weights ranging from 375 to 732g, landed in Ireland in the winter of 2014. Those were obtained from a local fishmonger and maintained alive at 4ºC in dry conditions for a maximum of 48 h. After storage, crabs were adequately euthanized (Roth and Øines, 2010) while maintaining the integrity of the carapace. Before cooking, each crab was characterised by measuring weight (grams) and dimensions (cm2) (assuming that the crab shape was oval, the area of which was multiplied by two in order to account for both sides of the crab). After cooking the two types of crab meat, white and brown, were evaluated Chapter 3 106 separately. The white meat is the meat located in claws and legs and the brown meat inside the carapace. The distinction between the two types of meat was considered due to their different composition and market value. Cooking experiments with and without ultrasound were performed immediately after euthanasia in an ultrasonic bath (Guyson mod. KS MK3 525, North Yorkshire, UK) with a tank capacity of 55L, a maximum ultrasound power of 900 W and a heating power of 2000 W. All trials were carried out by using the maximum volume of water (55L). Once the temperature of the water reached 75ºC, eight crabs were submerged and cooked for 45 min. Preliminary experiments (data not shown) showed that 45 min was a sufficient cooking time to apply an equivalent F 70ºC7.5ºC of 2 min, which corresponds to the FDA recommended heat treatment for ready-to-eat seafood products. This heat treatment ensures the inactivation of at least 6 Log10 cycles of Listeria monocytogenes , the target microorganism in pasteurised seafood products (FDA, 2011). For those experiments applying ultrasound, the maximum ultrasonic power of the tank was used, 900W (ultrasonic energy consumption). In order to standardize the cooking conditions as much as possible, the total weight of all batches ranged from 4.0 to 4.3kg. At least three independent replicates of each cooking conditions were performed on different working days. 3.3.2 Heat transfer study To assess the effect of ultrasound on the heat transfer phenomena in crabs two different comparisons, based on mathematical models, were carried out. 3.3.2.1 Heat penetration curves Heat penetration curves were obtained by placing a K type thermocouple (Alhborn, Holzikirchen, Germany) in the abdomen of the crab which corresponds to its cold spot (Figure 3.1) which had been previously identified in preliminary experiments (Figure 3.2). Chapter 3 113 Table 3.1 Continued Weight (g) Cooking type Length (cm) Width (cm) fh j R2 RMSE 300.0 Conventional 13.0 8.0 18.45 1 0.95 4.19 348.0 Conventional 14.0 9.0 21.44 1.05 0.97 1.54 375.5 Conventional 14.0 9.0 31.48 1.21 0.99 1.15 388.0 Conventional 14.0 8.5 20.68 1 0.95 3.47 398.0 Conventional 14.5 9.0 29.09 1.11 0.99 0.31 404.0 Conventional 14.5 8.5 26.45 1.02 0.99 0.55 411.4 Conventional 15.0 9.5 22.79 1.07 0.99 0.59 428.0 Conventional 14.0 9.5 17.12 1 0.98 1.09 432.0 Conventional 14.5 9.0 24.15 1.47 0.99 1.01 455.4 Conventional 14.5 9.0 33.99 1.4 0.99 0.91 462.8 Conventional 15.0 10.5 27.68 1.48 0.99 0.69 472.0 Conventional 16.0 9.0 27.55 1.27 0.99 0.03 478.0 Conventional 14.5 9.0 30.12 1.36 0.99 0.21 484.0 Conventional 15.0 9.0 34.14 1.23 0.99 0.39 500.0 Conventional 16.5 10.5 35.45 1.58 0.99 0.47 538.0 Conventional 17.0 10.5 37.88 1.13 0.99 0.19 553.6 Conventional 16.0 10.0 40.62 1.11 0.99 0.31 586.9 Conventional 16.5 10.0 35 1.06 0.99 1.62 614.6 Conventional 16.0 10.0 39.91 1.47 0.99 0.48 630.5 Conventional 16.5 11.0 43.4 1.09 0.99 0.24 640.7 Conventional 18.8 11.0 43.67 1.5 0.99 0.47 714.0 Conventional 18.0 11.5 41.35 1.4 0.99 0.69 732.7 Conventional 17.5 11.0 42.99 1.05 0.99 0.30 799.4 Conventional 17.5 11.0 40.91 1.06 0.99 1.91 816.8 Conventional 19.0 12.5 42.65 1.28 0.98 1.60 829.7 Conventional 18.0 11.0 40.97 1.29 0.99 0.64 869.7 Conventional 19.0 13.5 64.32 1 0.90 0.06 Chapter 3 114 In addition, a linear relationship between fh values and crab weight was noted in each cooking process indicating that heat penetration in the crab cold spot is weight-dependent. Table 3.2 includes the first order Equations which correlate the increase in fh values with the increase of the weight of the crab in both cooking processes. Significantly different slopes were observed between Equations (P≤0.05), indicating that the crab weight affected heat penetration to a differing extent depending on the cooking process. When ultrasound was used to assist the cooking the slope was 2.6-fold smaller, meaning that the weight of the crab had a much smaller effect on increases in fh value. Hence, the larger the crab the greater the impact of ultrasound in enhancing the heating rate as indicated in Table 3.2. Table 3.2: First order Equations correlating fh values (dimensionless) with the weight of brown crabs (g) during conventional and ultrasound-assisted cooking. Cooking type Equation RMSE Bf Af Conventional 𝑓ℎ=0.053× 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) +4.9 5.57 1.01 1.14 Ultrasound-assisted 𝑓ℎ=0.020×𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) + 20.4 4.23 1.01 1.12 Much of the work published to date attributes the ultrasonic enhancement of heat transfer to the formation of cavitation bubbles (Wong and Chon, 1969), although other authors have also suggested that improvements in convection heat transfer may also be due to acoustic streaming (Gould, 1966; Hyun, et al ., 2005). Either way, it is generally accepted that ultrasonically induced heating is a result of energy dissipation from the accumulation of cavitation bubbles at the interface of the submerged body (Baffigi and Bartoli, 2012; Kiani, et al ., 2012). Additionally it is accepted that the number and density of cavitation bubbles can play an important role in the heat transfer caused by ultrasound and also that the cavitation of bubbles increases the micro-convection effect at the product surface (Kim, et al ., 2004). In relation to the surface area, some researchers have reported a relationship between the weight and dimensions (width and length) of the Chapter 3 115 carapace of a crab (Klaoudatos, et al ., 2013). In the present study a linear relationship (represented by Equation 3.5) was found between the surface area (cm2) of the carapace and the crab weight (g) (Figure 3.3). Figure 3.3: Relationship between crab’s weight (from 300 to 870 g) and the total surface of the crab’s carapace (cm2). 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 (𝑐𝑚2)=0.362× 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) + 54.83 ( R 2 = 0.85) (Eq. 3.5) This relationship indicates that heavier crabs had higher carapace surface areas (Figure 3.3). Therefore the greater impact of the ultrasonic field on the reduction of fh values in heavier crabs could be attributed to their larger surface areas and as a consequence a greater amount of cavitation bubbles around their surface. The effectiveness of a heat treatment in terms of microbial inactivation is given by the applied F value. As indicated before, the cooking processes applied in the current study were designed following FDA recommendations (FDA, 2011) to ensure a 6 Log10 reduction of L. monocytogenes in seafood products ( F 70ºC7.5ºC = 2 min). A cooking process of 45 min in water at 75°C was previously demonstrated to achieve this minimum recommended F value in all 0200 400 600 800 1000 0 100 200 300 400 500 weight (g) surface (cm2) Chapter 3 116 crabs irrespective of their weight (data not shown). For each cooking process, either with or without ultrasound, the actual equivalent F 70ºC7.5ºC value applied was calculated based on the corresponding heat penetration curves. Figures 3.4A, 3.4B and 3.4C show the F 70ºC7.5ºC values attained during the cooking of crab at 75°C without ultrasound (block line) and with ultrasound (dashed line) in small, medium and large crabs, respectively. Figure 3.4 also shows the threshold for the target F 70ºC7.5ºC of 2 min (horizontal dotted line). The F value applied in the ultrasound-assisted cooking was 2.5-, 3.2and 2.2-fold higher than the conventional heat-only cooking for the small, medium and large crabs, respectively. In other words, the cooking time was reduced by 15.5% (from 45 to 38 min), 16.9% (from 45 to 37.4 min) and 12.7% (from 45 to 39.3 min), respectively, while applying the same F value when ultrasound was used during cooking. Chapter 3 117 Figure 3.4: F 70ºC7.5ºC value (min) applied during the cooking process in the crab's cold spot (i.e. abdomen), with (dashed line) and without (block line) ultrasound for the (A) small, (B) medium and (C) large crabs. The horizontal dotted line represents the target F 70ºC7.5ºC of two minutes. A 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 C ooking tim e (m in) F707 .5 value (m in) Ta rget F707.5 B 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 C ooking tim e (m in) F707 .5 value (m in) Ta rget F707.5 C 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 C ooking tim e (m in) F707 .5 value (m in) Ta rget F707.5 Chapter 3 118 The efficacy of the two cooking processes was also evaluated by enumerating total bacterial levels in white (Figure 3.5A) and brown crab meat (Figure 3.5B). After 5 min of ultrasound-assisted cooking the microbial load in white meat was 0.6 Log10 cycles lower than in samples which had undergone regular cooking (2.4 vs 1.8 Log10 cycles), while brown meat needed a 10 min longer treatment to achieve similar reductions (2.3 vs 1.8 log10 cycles). These results indicate that the microbial reductions in crab are significantly higher in products exposed to an ultrasound assisted cooking. In addition, the effect of ultrasound alone was evaluated by measuring microbial loads in white and brown meat treated at temperatures below 30ºC for 45 min. In this case no differences were detected in the microbial loads regardless of the meat type ( p >0.05). Figure 3.5: Microbial load over conventional (grey bars) and ultrasound assisted cooking processes (black bars) in (A) white meat and (B) brown meat. Dotted line shows the detection limit for the counts. A 0 5 10 15 30 45 0 1 2 3 4 5Regular cooking Ultrasonic cooking Cooking time (min) Log cfu/g B 0 5 10 15 30 45 0 1 2 3 4 5Regular cooking Ultrasonic cooking Cooking time (min) Log cfu/g Chapter 3 119 Some authors have suggested that the higher microbial inactivation levels often observed when ultrasound and heat are combined (i.e. thermosonication) is due to additive or synergistic effects between the two technologies (Arroyo, et al ., 2012; Raso, et al ., 1998). However, in the case of crab cooking, when ultrasound was applied at low temperatures the microbial loads did not decrease. Therefore, the greater microbial reduction observed could be attributed to the fact that ultrasound improved heat penetration in the crab rather than as a result of the effect of ultrasound itself. For example, as a consequence of more rapid heat penetration the cumulative F value increased at a higher rate which would result in greater reduction in microbial loads during ultrasonic cooking. Furthermore, ultrasound may have sub-lethally damaged bacterial cell envelopes thus reducing heat tolerance and therefore resulting in greater microbial reduction values compared to those samples that received the heat only treatment (Sala, et al ., 1995). 3.4.2 Mass transfer Ultrasound technology is widely used in the food industry to improve processes involving mass transfer phenomena such as cleaning, extraction, brining, pickling, marinating and curing (Chemat, et al ., 2011; McDonnell, et al ., 2014). In this section the effect of ultrasound on the mass transfer phenomena occurring during the cooking of crabs is quantified using two different methods. 3.4.2.1 Method 1: Turbidity and conductivity of cook water Figure 3.6A illustrates the mean cook water turbidity value after cooking with and without ultrasound. It is very clear that the turbidity of the cook water increased more rapidly after 15 min when ultrasound was used to assist the process. After 40 min of cooking with ultrasound the turbidity of the cook water reached a maximum of 1.04 absorbance units which constitute a 113.7% increase compared to turbidity in conventional cook water. The conductivity of the cook water (Figure 6B) also increased in the presence of ultrasound after 10 min indicating a faster rate of ionic compound release from the crab. The Chapter 3 120 maximum increase in cook water conductivity was reached after 35 min of cooking, and was 55.7% higher than values observed in water used for the cooking without ultrasound. Regular commercial practice in the Irish crab industry involves a cleaning/cooling step with fresh water immediately after cooking. This step is critical in terms of microbial safety as recontamination can potentially occur. As a result, immediately after the cleaning/cooling step crabs are packed and pasteurized (Edwards and Early, 2001; Lalitha and Thampuran, 2012). If the turbidity of the cook water is considered as an indicator of the removal of dirt from the crab’s surface, as Figure 3.7 suggests, the results obtained with ultrasound indicate that the use of this technology may have the potential for eliminating the cleaning step as this ultrasonically induced cleaning would be done concurrenly with the cooking. Figure 3.6: (A) Turbidity (OD515) and (B) conductivity (µS/cm) values for the cook water during the cooking of brown crabs in water at 75°C with (black bars) and without (grey bars) ultrasound. A 0510 15 20 25 30 35 40 45 0.0 0.5 1.0 1.5 Cooking time (min) Abs (515 nm) B 0510 15 20 25 30 35 40 45 0 200 400 600 Cooking time (min) conductivity (S/cm) Chapter 3 121 Figure 3.7: Picture of the crab’s exudate after a conventional cooking (A) and ultrasoundassisted cooking (B). 3.4.2.2 Method 2: Salt content of the crab meat The salt content in both white and brown crab meat was measured to assess the potential of ultrasound to transfer substances from the cook water into the crab meat. The initial salt content for raw crab was 1.43% in the white meat, which is located mainly in claws and legs, and 0.89% in the brown meat, which is located inside the carapace. Salt content before and after cooking with and without ultrasound and in the presence or absence of 5% NaCl in the cook water is shown in Table 3.3. When the crabs were cooked in water without NaCl added the final salt content in white meat was reduced by 33.6% using regular cooking and by 46.1% for the ultrasound-assisted cooking. When 5% NaCl was added to the cook water, the salt content in white meat remained stable after regular cooking and was reduced by 21.1% following ultrasound assisted cooking. For brown meat, the salt content remained the same when crabs were cooked either with or without ultrasound in water without 5% NaCl added as opposed to white meat. However, when crabs were cooked in water with 5% NaCl added, the salt content in brown meat slightly increased from 0.89% (raw) up to 1.34% in the absence of ultrasound and remained unchanged with the application of ultrasound. Our results suggest that ultrasound did not facilitate the uptake of Chapter 3 122 salt during cooking in water with 5% NaCl added. This effect may be due to the physical barrier of the crab carapace which could act as a resonant box on which cavitation bubbles are produced on the internal surfaces. These bubbles could create micro-currents which could aid in the release of the salt from the meat to the cook water even against an osmotic gradient. The effect on white meat when crabs were cooked in water without NaCl added suggests that ultrasound enhances the release of salt from the meat. Some authors observed a similar effect with enhanced extraction when vegetable tissues such as tomato peel were treated with ultrasound (Luengo, et al ., 2014; Riera, et al ., 2004). Other studies assessing the effectiveness of ultrasound for accelerating the marinating of meats reported contrasting findings to those in the current study (Cárcel, et al ., 2007; Mason, et al ., 1996; McDonnell, et al ., 2014) showed that the content of salt in slices of pork tenderloin increased when they were soaked in a saturated salt solution for 45 min with increasing ultrasound intensity. Also Siró et al ., (2009) showed a significant improvement in salt difussion in pork loins when ultrasound was applied. A similar effect was observed by Turhan et al ., (2013) who reported enhanced rates of marinating in anchovies when ultrasound was used. Table 3.3: Salt content (%) and moisture content (%) in white and brown crab meat cooked with or without ultrasound in water and water with 5% NaCl. Values represent mean value ± standard error. For both salt and moisture content, ANOVA followed by Tukey’s tests were performed for white and brown meat independently. Raw: Raw meat, CC: Conventional cooking, USC: Ultrasound-assisted cooking. Different superscript letters indicate significant differences (P≤0.05). White meat Brown meat Water Water with 5% NaCl Water Water with 5% NaCl Salt content (%) Raw 1.43 (0.03)c 1.43 (0.03)c 0.89 (0.03)a 0.89 (0.03)a CC 1.06 (0.03)b 1.33 (0.03)c 0.96 (0.12)a 1.34 (0.02)b USC 0.84 (0.02)a 1.17 (0.05)b 0.98 (0.02)a 0.81 (0.04)a Moisture content (%) Raw 75.83 (3.85)b 75.83 (3.85)b 49.08 (0.54)a 49.08 (0.54)a CC 75.02 (1.65)b 60.75 (0.92)a 56.61 (1.44)b 45.20 (0.20)a USC 70.08 (0.89)ab 69.98 (2.85)ab 56.32 (1.70)b 63.60 (1.51)c Chapter 8 225 Moreover and in parallel to the colour change kinetics study, the quality perception of the white meat was correlated with a ΔE* parameter during a series of focus groups with the main edible crab producers of Ireland. Based on the obtained results white meat was classified, as “good quality” (ΔE* ≤ 7), “acceptable quality” (7< ΔE* <9) and “non-acceptable quality” (ΔE* ≥ 9) based on their colour (Figure 5.7 in Chapter 5). One of the main advantages of this study is that, the Equations developed for the colour change, could be easily transferred to other crab products such as picked white meat or crab claws. Using the limits established above in terms of the ΔE* parameter, the thermal profile of crab claws and crab’s “cold-spot”, an optimization graph was constructed (Figure 5.8 in Chapter 5). As observed, depending on its weight, different heat treatments were required to achieve the goal of F 90°C7or10°C= 10min, in the crab’s “cold-spot”. To evaluate the effect of the crab weight on the pasteurization parameters, Figure 8.1 shows the maximum temperatures at which it would be possible to pasteurize whole cooked crabs to maintain a “good quality” or “acceptable quality” in their white meat, based on the required time at each temperature to achieve an appropriate level of C. botulinum non-proteolytic type E inactivation. In this Figure all temperatures represented by the green line or below would produce crabs with a “good quality” in terms of white meat colour, while the temperatures in the area between green and blue line would produce an “acceptable quality”. Also in the Figure it can be observed that increases in the weight of the crab suppose a reduction of the maximum pasteurization temperatures, to maintain a certain quality, in a proportion of 1°C for an increase of 100g. All of these calculi are referred to the quality associated with the colour of white meat. Chapter 8 226 Figure 8.1: Maximum pasteurization temperatures allow to achieve the target F 90°C10°C = 10 minmaintaining “good quality” (green line) and “acceptable quality” (blue line) depending on the crab weight, based on the equations developed in Chapter 5. The effect of crab weight on pasteurization parameters is a consequence of the different thermal profiles of crab claws (where the white meat is located) and crab abdomen (where the “cold-spot” was defined) as is represented Figure 5.1 (Chapter 5). While the crab weight did not affect the thermal profile of the claws, because small differences between claws exist regardless the size of the crabs. The impact of crab weight has a big impact on the thermal profile of the “cold-spot” (i.e. crab’s abdomen). Therefore for the pasteurization of bigger crabs a reduction of the pasteurization temperature is required, based on the colour change kinetics described, due to the longer pasteurization time necessary to achieve the pasteurization objective in the “cold-spot”. These results endorse, as in case of cooking, the importance of the classification step prior to processing in order to avoid the over-heating or under-heating within crabs in the same batch. In based of the results obtained in this PhD Thesis considering microbial and quality aspects already discussed with respect of the FDA recommended pasteurization treatment, a theoretical optimisation graph for the pasteurization of ready-to-eat whole edible crab has been generated. This graph has been generated thanks to the equations developed in Chapter 5 (i.e. Eq. 5.9 and Eq. 300 400 500 600 700 800 900 85 90 95 100 105 110 115 T max. Good T max. Acceptable Crab weight (g) Max. past. Tmp. (C) Chapter 8 227 5.12) and Chapter 3 (Eq. 3.2 and Eq. 3.3). It has to be pointed out that the mathematical Equations developed in Chapter 5 allow the definition of treatment conditions for whichever the target microorganism may be and not only centred on C. botulinum non-proteolytic type E. Figure 8.2 compares the treatments required for C. botulinum ( F 90°C10°C= 10 min) vs. those proposed for B. weihenstephanensis ( F 105°C7.6°C= 6 min) with the impact on the quality of white meat. Figure 8.2: Theoretical optimisation graph for the pasteurization of ready-to-eat whole edible crab based on Equations 5.12 and 5.13 for crabs of 600g for the inactivation of 6 Log10 reductions of C. botulinum non-proteolytic type E based on U.S. FDA recommendations and B. weihenstephanensis based on the thermal resistance obtained in this PhD Thesis (Chapter 6). As Figure 8.2 shows, with B. weihenstephanensis considered as the target microorganism the pasteurization parameters (i.e. time/temperature combinations) required to achieve a similar inactivation level to C. botulinum non-proteolytic type E (i.e. 6 Log10 cycles) would produce considerable changes in the colour of crab white meat. In fact, based on Figure 8.2 in the range of temperatures from 90 to 120°C, the required times to reduce the population of B. weihenstephanensis by 6 Log10 cycles will produce products with only “acceptable” quality. If a “good” quality is to be maintained, the pasteurization 80 90 100 110 120 130 1.0 1.5 2.0 2.5 B. weihenstephanensis Acceptable Good excellent C botulinu, Treatment temperature (C) Log10 pasteruization time (min) No pasteurize “Good” Pasteurize C. botulinum “Good” Pasteurize C. botulinum “Acceptable” Pasteurize B. weihenstephanensis “Acceptable” Pasteurize B. weihenstephanensis “No Acceptable” Chapter 8 228 temperatures must be ≤90°C. However, due to the high thermal resistance of B. weihenstephanensis , these treatments would require an excessively long treatment time (i.e. over 3 hours). These results suggest the need of looking for strategies which enable to improve the lethal effectiveness of the current heat treatments or to find technologies which guaranty the safety and stability of the defined heat pasteurization treatments while minimally impacting crab quality. As mentioned in Chapter 1 several alternative technologies could be employed to reduce or avoid the negative heat induced impact when imposing heat treatments for the inactivation of spores. During the development of this PhD Thesis two of these alternative technologies, ultrasound in combination with pressure and heat and electron beam ionizing radiation, were explored in terms of their potential to inactivate the main bacterial spores isolated form edible crab. As previously discussed, ultrasound has the potential to improve the cooking and cleaning of edible crab. Also in Chapter 3 the potential lethality of ultrasound during cooking was discussed (Figure 3.5 A and B). The results obtained suggest that ultrasound can increase microbial inactivation during cooking. However, under atmospheric pressures and the low ultrasonic power density employed in the present study, this effect is more likely due to ultrasonically induced improvements in heat transfer. Using the same ultrasonic conditions, but at room temperature (i.e. 20°C) no microbial inactivation was observed. Under certain conditions ultrasound is considered as one of the new alternative microbial inactivation technologies to conventional heat treatments (U.S. FDA, 2000) when applied at temperatures similar to those used in the present study. However, for effective microbial inactivation the ultrasonic power density required is considerably higher than that employed in the present study and also ultrasound is generally applied under pressure. Due to the low bactericidal efficacy of ultrasound at room temperature (Lee et al ., 2013; Meullemiestre et al ., 2017; Jambrak, et al ., 2017), most researchers have tried to design combined processes to enhance the overall lethal efficacy of Chapter 8 229 ultrasound (López-Malo et al ., 2005; Lee et al ., 2013; Raso et al ., 1998a). Some of the combined ultrasound processes proposed to date which increase the lethal effect of ultrasound are thermosonication (TS), manosonication (MS) and manothermosonication (MTS) (Chemat et al ., 2011; Piyasena et al ., 2003; Sala et al ., 1995). Results obtained in Chapter 3 showed that the applied TS treatment (i.e. cooking at 75°C in presence of ultrasound in an ultrasonic bath) did not produce a substantial increase of microbial inactivation, though MS and MTS treatments proved to be effective for the inactivation of spores isolated from crab, in liquid media (Chapter 6). It has been reported that the bactericidal efficacy of ultrasound is directly correlated with the ultrasonic power transferred to the media (Mañas et al ., 2000). In Chapter 6 all trials were carried out in a specific device specially designed to evaluate the bactericidal potential of TS, MS and MTS treatments, which allows the application of ultrasonic power densities ranging between 1600 and 8000W/L at 20kHz. In Chapter 6 an ultrasonic power of 4800W/L and 20kHz was used to evaluate the potential of MS and MTS treatments while for the TS treatment, which was applied in an ultrasonic bath for crab cooking at atmospheric pressure, a maximum ultrasonic power density of 10W/L and 35kHz was used. Although, ultrasonic frequency could have an influence on the microbial inactivation, the difference on the applied ultrasonic power would more than likely be the main reason why in case of TS no bactericidal effect was observed while MS and MTS treatments proved to be effective for bacterial spore inactivation. Although the results obtained in Chapter 6 show great potential for MTS treatment (using temperatures over 80°C and an ultrasonic input power of 4800 W/L) to inactivate the main bacterial spores isolated from crab, to this process is generally only considered for pumpable liquids and is not readily adaptable for whole crab, though its advantages could be exploited only for liquid products such as crab soups. However, the high ultrasonic energy required for bacterial spore inactivation using MTS treatments, (4800W/L) Chapter 8 230 compared to other ultrasound applications such as assisted freezing (from 7.3 to 25.89W/L) (Li and Sun, 2002) or marinating (from 800 to 1200W/L) (Turhan et al ., 2013) makes its industrial application difficult due to the high energies involved. Either way, considering the results obtained for bacterial spores inactivation using ultrasound in combination with heat plus pressure (MTS) (Chapter 6), if enough ultrasonic power is applied during the pasteurization process , with B. weihenstephanensis considered as a target microorganism, a substantial reduction, of over the 80%, of the pasteurization time could be reached as shows Figure 8.3. Figure 8.3: Log of the time required to achieve 6 Log10 reductions of the population of B. weihenstephanensis by heat (black bars) and MTS (white bars) at different temperatures in pH 6.8 McIlvaine citrate-phosphate buffer. Besides MTS technology, in this PhD Thesis Electron Beam Ionizing radiation (EBI) was also evaluated. Initially, this technology would have more viability, if legally permitted, since it could be applied to cooked whole crab and crab products in pack. As it is discussed in Chapter 7 one of the major advantages of ionizing radiation is its capability to inactivate the main bacterial spores present in crab at low temperatures (even frozen) which in turn would avoid the adverse effects produced during heat pasteurization. From the results shown in Chapter 7, Figure 8.4 shows the required dose to reduce the population of the main three bacterial spores isolated from edible crab by 6 Log10 cycles. Based on Figure 8.4, ionization treatments below 8kGy appear to 80 85 90 95 0 1 2 3 4 5Heat treatment MTS treatment Treatment temperature (C) Log 6D (min) Chapter 8 231 be sufficient to achieve a proper inactivation level of the three main spores isolated from edible crab, identified in this PhD Thesis. The results obtained also showed that despite the different composition and chemical characteristics of the two types of crab meat (Anacleto et al ., 2011; Barrento et al ., 2010a), the resistance of the bacterial spores was barely affected by the meat type. Therefore, if the penetration depth, which depends on the radiation source, is sufficient these results indicate that a homogeneous inactivation will be produced during the ionization treatment within the crab. Additionally, the specific resistances of the bacterial spore species in crab meat were similar to those detected in lab media at similar pH (7) and aw (>0.99). Therefore, considering the results obtained in the present PhD thesis, the radiation dose calculated in lab media for a specific microorganism could be used as reference to calculate the necessary treatments to achieve a certain level of safety or stability for a specific foodstuff. Figure 8.4: Required radiation dose to reduce 6 Log10 cycles the population of the main three bacterial spores isolated from edible crab in the two types of crab meat. Considering that the maximum permitted radiation dose for foods is10 kGy (WHO 1981), the results obtained endorsed the usefulness of this technology for the pasteurization of edible crab, since an adequate level of inactivation of Data 1 B. mycoides B. weihenstephanensis P. psychrodurans 0 2 4 6 8 10 White meat Brown meat 6D irradiation dose (kGy) Chapter 8 232 the bacterial spores isolated from crab could be reached by applying radiation doses below 10kGy. However, the downside is that although more than 60 countries worldwide now have regulations regarding the use of ionizing radiation for food products (IAEA, 2017) its use in Europe is still very limited for food pasteurization especially for seafood products. Only Belgium, Czech Republic and United Kingdom have authorized the irradiation of fish and seafood and only up to a maximum dose of 3kGy. It has been also reported that a radiation dose ≤ 2kGy produces a significant extension of the shelf-life of different crab products though; treatments over this dose might produce significant impact on the sensory perception of these products (Chen et al ., 1996; ICGFI, 1998). Therefore in order to finalise this study, future work would involve a sensory evaluation to determine the impact of irradiation on the crab meat quality and the maximum applicable doses to avoid possible undesirable changes on its sensory characteristics, though this was beyond the scope of the present study. Considering the results obtained during the development of this PhD Thesis, a number of different alternative processes have emerged which can improve the production of ready-to-eat edible crab and crab based products. Figure 8.5 shows these processes for crab products based on different alternative technologies at different stages in crab processing. As Figure 8.5 shows, different alternative processes can be applied for the production of ready-to-eat crab products using novel technologies. Blue shadow boxes represent the traditional steps currently applied in the crab industry while, white boxes represent the possible alternative applications, at different stages of the production line. On the other side, black lines connect the common steps applied to all crab products or early processing stage, independent of its format. While green lines connect the specific processes depending on the different formats i.e. whole cooked crab (Green block lines), picked white and brown meats or claws (Green dashed lines) or transformed crab products (Green dotted line). Chapter 8 233 On one side, the results obtained in this PhD Thesis proved the possibility of improving the traditional processes considering the obtained data regarding the microbiota present in edible crab (Chapter 2), cooking process (Chapter 3) and the effect of second pasteurization on quality characteristics of edible crab. Regarding the use of the different alternative technologies the results obtained in this PhD Thesis showed that ultrasound technology could be used at different stages with different goals. This technology could be applied in the early processing stages as a pre-treatment prior to cooking to reduce the Cd content of the crab (Chapter 4) or could be also implemented in the cooking process (Chapter 3) where, on one side would enhance heating rates in the crab coldspot and on the other side would allow to avoid the washing step, after cooking, which in turn would limit the recontamination of the cooked crab (Figure 8.5). Additionally ultrasound technology could be also used in combination with heat and pressure (MTS) (Chapter 6), in certain type of crab based products such as crab soups. However, the down side of this technology for pasteurization purposes is the elevated energy required, which makes very difficult its industrial application. , Another technical option for edible crab pasteurization is electron beam ionizing radiation, which has been shown in this PhD Thesis (Chapter 7) to be an effective technology for inactivating the main bacterial spores isolated from edible crab. Unfortunately, while it is very effective, its use for seafood pasteurization is currently not permitted in most EU jurisdictions. In countries where its use is approved a maximum treatment dose of 3kGy is all that is permitted though the results obtained in Chapter 7 of the present study suggest that treatments of between 6 and 8kGy are necessary in order to attain sufficient inactivation of bacterial spores isolated from crab. Thus, new strategies based on either the optimization of traditional processes or on the retrofitting of alternative technologies into traditional processes, could be used to for process intensification and for enhancing competitiveness in the crab processing sector. Chapter 8 234 Shells discarded Crab transport to processing plant Crab classification Crab slaughter Crab Ultrasonic Cooking/Cleaning Ultrasonic pre-treatment (Cd removal) Crab Cooking Crab Cooling/Cleaning Crab Cooling Shelling Processing/Transformation Retort pasteurization Radicidation MTS pasteurization (Liquid products) Aseptic packaging End products storage and market Crab/Meats/Transformed products Packaging Entire crab processing Claws and crab meats processing Transformed crab products processing Figure 8. 5: Flow diagram of the different alternative processes proposed, using different alternative technologies. Chapter 9 241 mycoides and P. psychrodurans respectively. Also from the results obtained it can be deducted that an agreement between spore resistance in crab meats and lab media, with similar characteristics in pH and aw, exist which opens the possibility to extrapolate the results obtained in-vitro to different ready-to-eat crab based products Overall, from the inactivation results obtained it can be concluded that the resistance variability among the main three spore formers species against heat treatments is much higher than the variability observed for MTS or EBI treatments. EBI treatment showed the lowest variability among bacterial spores resistance (20%) followed by MS (70%), MTS (440%) and finally heat (4400%). Therefore, the use of these alternative technologies would reduce the potential risk associate with the pasteurization of products due to the wrong choice of the target microorganism. The results presented in this PhD thesis point out the possibility of improving the production of ready-to-eat brown crab and brown crab based products by introducing different technologies at different stages of the production process or by optimizing current practices, to solve the challenges associated with the processing of this type of products. Chapter 10 242 Chapter 10 Resumen y Conclusiones Chapter 10 243 La industria alimentaria está en continua competencia y evolución, lo que requiere una gran actividad innovadora apoyada de una gran labor de investigación y desarrollo. En el caso del sector pesquero y más concretamente en la producción de buey de mar y sus productos listos para el consumo, la I+D+i se ha convertido en una parte fundamental. El desarrollo de la presente Tesis Doctoral da respuesta a muchas necesidades del sector permitiendo obtener avances considerables para el sector al introducir nuevas tecnologías de procesado en su cadena productiva. En la producción de buey de mar y sus productos listos para el consumo se llevan a cabo diversas etapas que a continuación se detallan y en las que se pueden abordar nuevos desarrollos. Así, el procesado requiere de los siguientes pasos: una vez sacrificado el cangrejo, estos son cocinados, generalmente en agua hirviendo durante 20-30 minutos (primer tratamiento térmico). Tras el cocinado, se realiza un proceso de enfriamiento y lavado necesario para retirar la suciedad y los exudados del caparazón del cangrejo, producidos durante el cocinado. Una vez enfriados los cangrejos que van a ser destinados a la venta como enteros, estos son envasados, generalmente a vacío, y se les aplica un segundo tratamiento térmico, en este caso de pasteurización. Por otro lado, los cangrejos destinados a la producción de productos elaborados o simplemente para la venta de las carnes de cangrejo por separado, se trasladan a la zona de descascarillado tras el enfriamiento con el fin de retirar ambos tipos de carne (la carne blanca de las pinzas y patas y la carne marrón del interior del caparazón). Este proceso se realiza principalmente de manera manual. Finalmente, las carnes de cangrejo son envasadas, de diferentes formas y utilizando diferentes formatos según convenga, antes de aplicarles un tratamiento de pasteurización. Aunque estos pasos son comunes para todos los productores de buey de mar listo para el consumo, existe una gran variabilidad en la calidad entre productos de similares características y formatos. Esta variabilidad se podría atribuir a una falta de estandarización de los procesos debido al pequeño tamaño de las compañías (que generalmente son empresas Chapter 10 244 familiares) y a su reducida inversión en tecnología y optimización de los procesos. Para realizar la optimización de un proceso, se requiere un conocimiento adecuado tanto de la materia prima como de las variables involucradas en el procesado. Además, es también necesario identificar los riesgos y requerimientos de la materia que deben ser cubiertos por el proceso (Stoforos, 1995). En el caso del buey de mar listo para el consumo, muy pocos estudios se han centrado en los microorganismos responsables de su deterioro y ninguno de ellos los ha identificado. La primera parte de esta Tesis Doctoral (Capítulo 2) se centró en la caracterización e identificación de los principales microorganismos presentes en el buey de mar crudo y cocinado listo para el consumo así como la identificación de los microorganismos responsables de su alteración y el estudio de su termorresistencia con el fin de valorar la eficacia de los actuales tratamientos térmicos aplicados a estos productos según la FDA de los EE.UU. Los resultados obtenidos mostraron que los grupos bacterianos presentes en mayor proporción en el buey de mar crudo fueron Pseudomonas spp., Bacillus spp. y Staphylococcus spp. Por otro lado, los grupos bacterianos de mayor importancia en el buey de mar cocinado fueron Bacillus spp. y Staphylococcus spp. aunque sus proporciones variaron dependiendo de la temperatura de almacenamiento. De las bacterias aisladas durante el mismo estudio, se identificaron mediante la secuenciación del ARNr 16S un total de 18 géneros y 31 especies bacterianos diferentes. De las bacterias no formadoras de esporas, Carnobacterium divergens y Shewanella baltica fueron las aisladas con más frecuencia y Bacillus mycoides , Bacillus weihenstephanensis y Psychrobacillus psychrodurans las bacterias formadoras de esporas más frecuentes. Los resultados del estudio de termorresistencia de los microrganismos aislados revelaron a Kocuria atrinae como la bacteria vegetativa más termorresistente de todas las aisladas del buey de mar; sin embargo, el tratamiento térmico recomendado para inactivar Listeria monocytogenes , en Chapter 10 245 productos del mar procesados ( F 70°C7.5°C = 2 min) fue suficiente para conseguir un nivel de inactivación adecuado de este microorganismo. De las bacterias formadoras de esporas, B. weihenstephanensis mostró la mayor tolerancia al calor seguido por P. psychrodurans y finalmente B. mycoides (Capítulo 6). Al comparar los tratamientos térmicos requeridos para alcanzar unos niveles de inactivación adecuados de estas esporas bacterianas con el tratamiento térmico más severo recomendado para inactivar Clostridium botulinum no proteolítico tipo E ( F 90°C8.6°C = 57 min) en estos productos, se detectó que el tratamiento era adecuado para conseguir la inactivación de P. psychrodurans y B. mycoides , pero no para inactivar B. weihenstephanensis . En general, estos resultados demuestran la importancia de Bacillus spp. en el buey de mar tanto por sus niveles de contaminación y prevalencia como por su elevada resistencia a los tratamientos térmicos. Como se ha indicado, en el procesado convencional de buey de mar listo para su consumo, es necesario un proceso de lavado del cangrejo, antes del envasado, para retirar la suciedad del caparazón del cangrejo procedente del ambiente marino así como los exudados proteicos producidos durante el cocinado. Este proceso de lavado puede suponer una recontaminación del cangrejo cocinado lo cual hace necesaria una pasteurización del mismo tras el envasado con el fin de garantizar su seguridad sanitaria así como para alargar la vida útil del producto. Además, este proceso de lavado supone un tiempo considerable en la producción y un elevado consumo de agua. Además, de los riesgos microbiológicos asociados a los pescados y mariscos, en el caso concreto del buey de mar varios estudios han alertado del riesgo de sobrepasar la ingesta semanal tolerable de cadmio, establecida por la EFSA (2009), al consumir este marisco debido a la elevada concentración de este metal pesado, especialmente en la carne marrón del buey de mar (Bolam et al ., 2016; Maulvault, et al ., 2013; Noël et al ., 2011; Wiech et al ., 2017). Chapter 10 246 Una de las soluciones propuestas para afrontar algunos de estos problemas de procesado del buey de mar fue la incorporación de la tecnología de ultrasonidos durante el cocinado. La eficacia de los ultrasonidos para mejorar los procesos de transferencia de calor y de masa, así como para la inactivación microbiana han sido probados extensamente en diferentes procesos utilizando distintas matrices alimentarias (Chandrapala, et al ., 2012; Chemat, et al ., 2011). Es por ello que se evaluaron las posibles ventajas del uso de esta tecnología en el procesado del buey de mar. Los resultados obtenidos en esta Tesis Doctoral prueban que la aplicación de ultrasonidos durante el cocinado del buey de mar mejora la transferencia de calor en el punto frio del animal (abdomen) además de ser útil para reducir el efecto del tamaño y peso de los cangrejos sobre las velocidades de calentamiento (Capítulo 3). Este efecto permitió por un lado reducir el tiempo total de cocinado (hasta un 15%) manteniendo el un valor F actualmente aplicado o por otro lado aumentar entre 2.2 y 3.2 veces el F equivalente total aplicado manteniendo el mismo tiempo de cocinado. Esta tecnología también probó su efectividad para incrementar la salida de compuestos (tales como la suciedad natural, los exudados producidos durante el cocinado y compuestos iónicos) desde el cangrejo al agua de cocción lo cual en un principio podría permitir omitir el lavado de los cangrejos tras el cocinado. Además, la aplicación de ultrasonidos evito la incorporación de sal a la carne de cangrejo durante el cocinado en agua con un 5% p/v de NaCl, al contrario de lo que sucede en el cocinado convencional. Dado el potencial que mostraron los ultrasonidos para eliminar sustancias del buey de mar, también se evalúo su potencial para reducir la concentración de Cd de este crustáceo (Capitulo 4). Para realizar este estudio, se trataron cangrejos hembra a diferentes temperaturas (50, 65 y 80°C) en presencia y ausencia de ultrasonidos, monitorizando la concentración de Cd en el agua de tratamiento. Además, tras el correspondiente tratamiento, se cuantificó la concentración de este metal pesado en las diferentes carnes del buey de mar. La temperatura de tratamiento no mostró una influencia significativa en la Chapter 10 247 velocidad de salida de Cd desde el cangrejo en aquellos tratamientos sin ultrasonidos, pero mostró una gran importancia en los tratamientos en presencia de ultrasonidos. En los tratamientos convencionales (en ausencia de ultrasonidos), la velocidad de salida de Cd también fue independiente de la temperatura determinándose como máximo una reducción del 3% de Cd en el cangrejo. Por otro lado, en los tratamientos combinados, la presencia de ultrasonidos incrementó la salida de Cd de los cangrejos 8,2, 2,1 y 2,7 veces a las temperaturas de tratamiento de 50, 65 y 80°C, respectivamente. La mayor reducción de Cd detectada en los cangrejos fue de un 22,8% tras el tratamiento combinado a 50°C utilizando una energía ultrasónica de 200W. Estos resultados muestran el potencial del uso de la tecnología de ultrasonidos para reducir el contenido de Cd del buey de mar, aunque se requeriría una investigación más en profundidad para optimizar las condiciones ultrasónicas de tratamiento para maximizar la reducción de este metal pesado. En base a los resultados obtenidos en esta Tesis Doctoral, la tecnología de ultrasonidos se podría implementar en la industria del buey de mar durante los procesos de cocción o lavado o incluso se podría proponer un tratamiento previo a la cocción con el objetivo de reducir el contenido en Cd, reduciendo así los riesgos de ingesta asociados con el consumo de este producto. De cualquier manera, en esta Tesis Doctoral se ha demostrado por primera vez que la tecnología de ultrasonidos es un procedimiento físico efectivo para reducir la concentración de Cd durante la cocción del buey de mar. Como ya se ha expuesto, los tratamientos térmicos inducen una serie de modificaciones físico-químicas en los alimentos que alteran sus características. Esta circunstancia también se produce en el buey de mar y sus productos. Por esa razón, en esta Tesis Doctoral, se estudió el efecto de los tratamientos térmicos de pasteurización sobre diferentes parámetros que determinan la calidad del buey de mar y, según los resultados obtenidos, se propusieron diferentes condiciones de pasteurización basadas en ecuaciones matemáticas desarrolladas (Capítulo 5). Chapter 10 248 La temperatura de pasteurización no afectó en el contenido en agua ni en la capacidad de retención de agua de ninguno de los tipos de carne del cangrejo ni tampoco afectó al color de la carne marrón siendo el color de la carne blanca el parámetro más afectado por el tratamiento térmico. Por ello, el color de la carne blanca fue seleccionado como indicador para realizar la optimización del tratamiento de pasteurización del buey de mar. El estudio de la cinética de cambio de color de la carne blanca reveló que la degradación del color de este tipo de carne del buey de mar sigue una cinética de pseudo-primer orden durante el tratamiento térmico. Además, tanto el máximo cambio de color producido por los tratamientos como la velocidad del cambio de color aumentaron exponencialmente con la temperatura de tratamiento. Con el fin de establecer los límites de calidad en los que basar la optimización y antes de llevar a cabo dichos cálculos de optimización, se realizó un “focus group” con industriales del sector para definir la calidad de los productos en base al color de la carne blanca. Gracias a este trabajo, se ha podido establecer por primera vez un ranking de categorías habiéndose definido los siguientes rangos de calidad en base al cambio de color: “Buena calidad” (ΔE* ≤7), “calidad aceptable” (7< ΔE* <9) y “calidad inaceptable” (ΔE* ≥9). En base a las ecuaciones desarrolladas las temperaturas de pasteurización máximas aceptables para el buey de mar entero serían entre 96 y 100°C y entre 104 y 108°C (dependiendo del peso de los cangrejos) para obtener calidades buenas o aceptables, respetivamente. Como se detalla en el Capítulo 5, estas temperaturas de pasteurización se propusieron en base al tratamiento recomendado por la FDA de los EE.UU. para inactivar C. botulinum no proteolítico tipo E ( F 90ºC10ºC= 10 min). Sin embargo y como se ha demostrado en esta Tesis Doctoral, otras especies de bacterias formadoras de esporas, más termotolerantes que C. botulinum tipo E, como B. weihenstephanensis pueden estar presentes en el buey de mar listo para su consumo y sus productos derivados. Chapter 10 249 Por esta razón en esta Tesis Doctoral se evaluó el potencial de tecnologías alternativas al calor en concreto, los ultrasonidos y radiaciones ionizantes para la inactivación microbiana con el objetivo de la pasteurización del buey de mar listo para su consumo y sus productos. En el caso de los ultrasonidos, además de las aplicaciones relacionadas con los procesos de transferencia de masa y de energía, esta tecnología es también considerada como una de las nuevas tecnologías útiles para la inactivación microbiana y ha sido sugerida como alternativa a los tratamientos térmicos convencionales para la pasteurización de alimentos (U.S. FDA. 2000). Como regla general, la resistencia bacteriana a los tratamientos de ultrasonidos disminuye con el tamaño celular siendo mayor en las de forma cocoide (Alliger, 1975; Condón et al ., 2005). Sin embargo, la mayoría de los datos publicados indican que la eficacia bactericida de la tecnología de ultrasonidos es reducida (Lee, et al ., 2013; Meullemiestre et al ., 2017; Jambark, et al ., 2017). Por ello, se ha tratado de mejorar la eficacia bactericida de esta tecnología diseñando procesos combinados (López-Malo et al ., 2005; Lee, et al ., 2013; Raso et al ., 1998a). Algunos de los procesos de ultrasonidos combinados que mejoran la eficacia letal de la tecnología propuestos hasta la fecha son la termosonicacion (TS), manosonicacion (MS) y manotermosonicacion (MTS) (Chemat, et al ., 2011; Piyasena, et al ., 2003; Sala, et al ., 1995). En esta Tesis Doctoral, se evaluó el potencial de los tratamientos combinados de ultrasonidos con presión (manosonicacion) y la combinación de ultrasonidos con temperatura y presión (manotermosonicacion) para inactivar las principales bacterias esporuladas aisladas del buey de mar ( B. mycoides , B. weihenstephanensis y P. psychrodurans ) (Capítulo 6). De los resultados obtenidos, puede deducirse que los perfiles de las curvas de supervivencia, tanto para los tratamientos térmicos como para los tratamientos de ultrasonidos son dependientes de las especies bacterianas. En aquellas especies en las que se observaron hombros en las curvas de inactivación frente a los tratamientos Chapter 10 250 térmicos también se observaron hombros en los tratamientos MS/MTS, aunque la aplicación de ultrasonidos redujo su duración. La eficacia letal de los tratamientos de MTS estuvo directamente relacionada con la termorresistencia de las especies esporuladas. B. mycoides mostró la mayor resistencia frente a los tratamientos de MS y la menor a los tratamientos térmicos y como resultado fue la especie esporulada más sensible a los tratamientos de MTS. El tratamiento combinado de ultrasonidos bajo presión con temperatura manifestó un efecto sinérgico para la inactivación de todas bacterias esporuladas estudiadas. El mayor porcentaje de sinergia se detectó en la especie bacteriana con el mayor valor de z T ( B. mycoides ) mientras que la mayor temperatura a la que se detectó el efecto sinérgico se cuantificó en la especie esporulada más termorresistente ( B. weihenstephanensis ). Los resultados obtenidos demuestran por tanto el potencial de la tecnología de los ultrasonidos para pasteurizar productos líquidos a base de buey de mar (como sopas o caldos) permitiendo reducir los tiempos de tratamiento, entre un 13% y un 85%, dependiendo del microorganismo diana, o las temperaturas del procesado. 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