characterization of pulsed light treatment on the shelf-life and safety of vacuum packaged cold
TRANSCRIPT
CHARACTERIZATION OF PULSED LIGHT TREATMENT ON
THE SHELF-LIFE AND SAFETY OF
VACUUM PACKAGED COLD SMOKED SALMON
By
Allison Maureen Pollock
Department of Food Science & Agricultural Chemistry
Macdonald Campus of McGill University
Montreal, Quebec
February, 2007
A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Master of Science
©Allison Maureen Pollock, 2007
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Suggested short title:
PULSED LIGHT TREATMENT OF SMOKED SALMON
Dedication
In loving memory oftwo Scottish gentlemen who had a significant impact on my life:
Dr. James P. Smith (1946-2006), my thesis supervisor
&
Charles Buchanan (1926-2005), my grandfather
ABSTRACT
Listeria monocytogenes is a common post-processing contaminant in ready-to-eat
vacuum packaged (VP) co Id smoked salmon. Since this psychrotrophic pathogen can
grow at refrigerated temperatures (~4 OC), other safety barriers in addition to temperature
are needed to ensure the continued safety of VP co Id smoked salmon. One such novel
barrier could be the pulsed light (PL) treatment of the product prior to packaging or
treating the product through a transparent package.
Pulsed light destruction kinetics of L. monocytogenes were evaluated while
dispensed into a liquid media, on the surface of a general purpose agar and on the surface
of cold smoked salmon. Results showed that PL technology was an effective surface
sanitation method (a decimal reduction time or D-value of 0.91, 1.37 and 2.25 s exposure
of PL at 800, 700 and 600 V, respectively, and a resulting z value of 500 V) on the agar
plate. However, it had only a limited success when applied to liquid samples as well as
directly on the surface of cold smoked salmon (D-value ranged from 93 s to 24 min).
Sensory quality ofVP cold smoked salmon subjected to selected PL treatments
was monitored during storage for 14 days at 4°C. Both color and odor scores remained
within acceptable limits over the 14 day storage period. Subsequent challenge studies
were carried out with L. monocytogenes applied on VP co Id smoked salmon. An overall
reduction in counts was observed in samples stored at 4°C over 28 days; however, after
PL treatment (day 0), there was no significant reduction in counts. Color and odor scores
maintained acceptable values over 14 days. Additional experiments were carried out to
determine the effects of (1) 1.5% salt, (2) 6% oil, (3) a representative salmon media and
(4) background microflora (lactic acid bacteria) on the PL inactivation of L.
monocytogenes. AU of the se factors significantly affected the destruction of L.
monocytogenes by increasing the D-value (adding resistance to pulsed light destruction).
Overall, these studies have shown that PL treatment in combination with low
temperature storage (4°C) has the potential to extend the shelf-life of VP co Id smoked
salmon products without compromising sensory quality. However further investigation
into higher treatment voltages is necessary in order to achieve a higher target kill of L.
monocytogenes.
RÉSUMÉ
Listeria monocytogenes est un contaminant post-emballage commun du saumon
fumé prêt-à-manger emballé sous vide (ESV). Étant donné que ce pathogène
psychotrope peut croître à température réfrigérée (~4 OC), des barrières additionnelles sont
nécessaires afin d'assurer la continuité de la sécurité du saumon fumé à froid ESV. Une
telle barrière innovatrice pourrait être le traitement préemballage du produit par lumière
pulsée (LP), ou encore de traiter le produit à travers un emballage transparent.
L'évaluation effectuée de la cinétique de la destruction par lumière pulsée de L.
monocytogenes en milieu liquide, à la surface de gélose nutritives tout usage et à la
surface du saumon fumé à froid, révèle l'efficacité du traitement comme méthode
d'assainissement de surface (temps de réduction décimal ou valeur-D de 0.91, 1.37 et
2.25 s exposition à la lumière pulsée de 800, 700 et 600 V, respectivement, et une valeur
z résultante de 500 V) dans les assiettes de gélose. Cependant, l'efficacité du traitement
s'est avérée limitée lorsque appliquée sur échantillons liquides de même que directement
à la surface du saumon fumé à froid (la valeur-D variant de 93 s à 24 min).
La qualité sensorielle du saumon fumé à froid emballé sous vide soumis à une
sélection de traitement à la lumière pulsée fut examinée pour une période d'entreposage
de 14 jours. Les pointages obtenus pour la couleur et l'odeur cette période sont demeurés
à l'intérieur des limites acceptables établies. Des études subséquentes furent exécutées
portant sur l'application de L. monocytogenes sur du saumon fumé à froid emballé sous
vide. Une diminution du dénombrement fut observée dans les échantillons entreposés à
4°C pour une période de 28 jours; cependant après le traitement à la lumière pulsée Gour
0), il n'y eut qu'une diminution légère ou pas de diminution du dénombrement. Les
pointages obtenus pour l'odeur et la couleur demeurèrent acceptables pendant 14 jours.
Des expériences additionnelles furent exécutées pour déterminer l'effet de (1) 1.5% de
sel, (2) de 6% d'huile, (3) d'un milieu représentatif du saumon et (4) de sa microflore
(bactéries acide lactique) sur la croissance de L. monocytogenes. Tous ces facteurs
affectèrent de façon significative la destrucion de L. monocytogenes en augmentant la
valeur-D (ajoutant de la résistance à la destruction par lumière pulsée).
11
En tout et partout, ces études ont démontré que le traitement par lumière pulsée
combiné à un entreposage à température réfrigérée (4°C) a le potentiel d'allonger la durée
de vie sur étagère du saumon fumé à froid emballé sous vide sans compromettre ses
qualités sensorielles. Cependant une investigation plus poussée des traitements à haut
voltage est nécessaire afin d'atteindre des niveaux plus élevés de destruction de L.
monocytogenes.
III
ACKNOWLEDGEMENTS
1 would like to express my sincere thanks to the late Dr. James P. Smith for giving
me the opportunity to study under his supervision. 1 am extremely grateful for his
generous support, constant encouragement and valuable advice. He was definitely a great
inspiration. A special thank you is also extended to his wife, Julie, for her kind
friendship and support.
1 am grateful to Dr. H.S. Ramaswamy and Dr. M.O. Ngadi for gui ding me
through the final phases of my studies. 1 thank them for their incredible patience and
understanding. 1 would especially like to thank them for their assistance in completing
my thesis in the most appropriate way.
My sincere thanks are extended to Mr. Bernard Cayouette for his laboratory
knowledge, his translation skills and also his friendship. 1 thank him for always knowing
how to make me laugh!
1 would like to thank Dr. Andrew Ekins for sharing his microbial wisdom. He
was an incredible resource when developing and troubleshooting microbial experiments
and techniques. 1 also appreciate his help with proofreading posters, abstracts and
manuscripts.
Dr. Miron Teshler is thanked for his help and expertise in performing statistical
analysis. Thank you for your time and patience.
1 would like to acknowledge M. Pierre Fontaine and Mlle. Michèle Tessier from
Fumoir Grizzly, St. Foy, Quebec, for supplying all coho smoked salmon filets and for
their technical assistance. My thanks are extended to Biljana Ushkovska from Cryovac
Sealed Air Corporation for supplying all packaging films. 1 would also like to thank le
Fonds québécois de la recherche sur la nature et les technologies (FQRNT) and the
Canadian Institute of Food Science and Technology (CIFST) for their financial support.
1 owe a special thank you to my parents, grandmother, and two sisters for their
generous love and support throughout my studies. 1 am sincerely grateful for your
encouragement and patience; 1 could not have done it without you. My darling Chris,
thanks for believing in me and supporting me through this work, especially the last phase.
Your love and encouragement means the world to me. 1 love you.
IV
TABLE OF CONTENTS
ABSTRACT ....................................................................................... i
, , RESUME.......................................................................................... ii
ACKNOWLEDGEMENTS... ................................. ............................. ... iv
ABBREVIATIONS.......................................................................... .... xi
LIST OF TABLES.............................................................................. xii
LIST OF FIGURES............. ............................................ ... .............. ... xiii
CHAPTER #1 INTRODUCTION............................................................ 1
CHAPT ER #2 LITERATURE RE"IE~................................................... 4
2.1 Chemical components of fish flesh.............................. ...... ...... ... . . . 4
2.2 Spoilage of fresh fish.................. ...... ... .................. ... . .... . ... . .... .. 6
2.2.1 Microbial spoilage........................................................ 6
2.2.2 Chemistrylbiochemistry offish spoilage............................... 6
2.2.3 Flavor deterioration... ... ... ............... ... ... . .. ...... ... ... . ... .. .. . 8
2.2.4 Changes in texture...... ...... ...... .................. ...... ........ ...... 10
2.3 Traditional preservation methods for fresh fish...... ........... . ... ... ..... . .. .. 10
2.4 Smoking of fish... ... ... ... ... ... ... ................ .. ... ... ...... ... ... ... . .... . ..... Il
2.5 Modified atmosphere packaging (MAP).......................................... 14
2.5.1 Gas packaging... ... ......... ... ... ..... ................ ... ......... ....... 14
2.5.2 Vacuum packaging............... ... ...... ... ... ... ... ......... ...... .... 15
2.6 Microbiological concems about VP smoked fish .......... '" .. ... ... . .. .. . . .... 15
2.6.1 Listeria monocytogenes.................................................. 15
v
2.6.2 Clostridium botulinum... ............ ........................ ... ... ...... 18
2.7 Traditional control measures in ready-to-eat cold smoked salmon........ .... 21
2.7.1 General manufacturing practices (GMPs)... ... .... ........ ...... ...... 23
2.7.2 Storage temperature...................................................... 23
2.7.3 Salt concentration and nitrite............................................ 23
2.7.4 Packaging in films oflower permeability.......................... .... 24
2.7.5 Sorbate/lactate/bacteriocins.............................................. 25
2.8 Pulsed light treatment............... ... ... ..................... ......... ... ...... .... 26
2.8.1 Antimicrobial effects................................................... ... 30
2.8.2 Sterilization ofpackaged products................................... ... 31
2.8.3 Use in foods................................................................ 32
2.8.3.1 Seafoods...................................................... ... 32
2.8.3.2 Meats............................................................ 33
2.8.3.3 Baked goods.................................................... 34
2.8.3.4 Miscellaneous................................................ ... 35
2.8.3.5 Water............................................................ 36
2.8.4 Status and economics..................................................... 36
2.9 Conclusions........................................................................... 37
CHAPTER #3 CHARACTERIZATION OF PULSED LIGHT APPARATUS WITH LISTERIA MONOCYTOGENES.................................................... 38
3.1 Introduction........................................................................... 38
3.2 Materials and methods........................................................... .... 39
3.2.1 Pulsed light equipment.. ................................................ , 39
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3.2.2 Temperature profile....................................................... 39
3.2.3 Bacterial strains......................................................... ... 40
3.2.4 Inoculation and pulsed light treatment................................. 42
3.2.4.1 Relative resistance of different strains of L.
monocytogenes to PL treatment...... ... ... ... ... ... ...... .. . ... . .. .. . 42
3.2.4.2 Spatial power distribution (12 plate experiment)... ........ 42
3.2.5 Analyses................................................................. ... 42
3.3 Results and discussion ............................................................. ,. 45
3.3.1 Temperature profile...................................................... 45
3.3.2 Relative resistance of different strains of L. monocytogenes to PL
treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3.3 Spatial power distribution (12 plate experiment)...................... 47
3.4 Conclusions........................................................................... 55
CHAPTER #4 PULSED LIGHT DESTRUCTION KINETICS OF LISTERIA MONOCYTOGENES (SCOTT A)..................... ....................... ................ 56
4.1 Introduction........................................................................... 56
4.2 Materials and methods........................................................... .... 57
4.2.1 Bacterial strains............................................................ 57
4.2.2 Inoculation and pulsed light treatment................................. 57
4.2.2.1 L. monocytogenes in liquid media in Whirl Pak bags..... 57
4.2.2.2 L. monocytogenes on surface of a solid general purpose
media......... ... ...... ............... ... ... ...... ... ... ...... .... .... ..... 57
4.2.2.3 L. monocytogenes on surface of co Id smoked salmon.... 58
Vll
4.2.3 Analyses.................................................................... 58
4.2.3.1 L. monocytogenes in liquid media in Whirl Pak bags.. ... 58
4.2.3.2 L. monocytogenes on surface of a solid general purpose
media..................................................................... 59
4.2.3.3 L. monocytogenes on surface of co Id smoked salmon.... 59
4.2.3.4 Decimal reduction times and voltage sensitivity.......... 59
4.3 Results and discussion............................................................ ... 60
4.3.1 L. monocytogenes in liquid media in Whirl Pak bags... ...... .... ... 60
4.3.2 L. monocytogenes on surface of a solid general purpose media. ... 61
4.3.3 L. monocytogenes on surface of cold smoked salmon............... 66
4.4 Conclusions........................................................................... 71
CHAPT ER #5 CHALLENGE STUDIES WITH LISTERIA MONOCYTOGENES (SCOTT A)........................................................................................ 72
PART A - Storage and challenge studies on pulsed light treated vacuum packaged cold smoked salmon stored at 4 and 1 rc... ......... ............... ...... ............ ... ... ... ...... 72
5.1 Introduction........................................................................... 72
5.2 Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 73
5.2.1 Sample preparation....................................................... 73
5.2.2 Bacterial strains............................................................ 73
5.2.3 Inoculation.................................................................. 73
5.2.4 Packaging conditions..................................................... 73
5.2.5 Pulsed light treatment.................................................... 74
5.2.5.1 Uninoculated samples......................................... 74
5.2.5.2 Challenge study................................................ 74
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5.2.6 Storage conditions ....................................................... ,. 74
5.2.6.1 Uninoculated samples......................................... 74
5.2.6.2 Challenge study................................................ 75
5.2.7 Analyses........................................................... ...... .... 75
5.2.7.1 Sensory evaluation... ... ..................... ... .... ....... .... 75
5.2.7.2 Microbiological analysis................................... ... 75
5.2.7.3 pH measurement............................................ .... 76
5.3 Results and discussion............................................................ ... 76
5.3.1 Sensory evaluation... ......... ........ ................ ... ...... ...... ..... 77
5.3.2 Microbiological analysis................................................. 79
5.3.3 Changes in pH values.................................................. ... 83
5.4 Conclusions............................................................................ 88
PART B - Effect of fat content, water phase salt content and competing microflora on the growth of Listeria monocytogenes......................................... .................................... 89
5.5 Introduction........................................................................... 89
5.6 Materials and methods........................................................... .... 90
5.6.1 Bacterial strains........................................................ .... 90
5.6.2 Media preparation...................................................... ... 90
5.6.2.1 TSA supplemented with oil or salt....................... .... 90
5.6.2.2 "Salmon" agar............................................... ... 91
5.6.3 Inoculation, PL treatment and storage conditions................. .... 91
5.6.3.1 TSA supplemented with oil or salt and "salmon" agar... 91
5.6.3.2 Destruction kinetics of background microflora............ 91
IX
5.6.3.3 Competition between L. monocytogenes and background
microflora. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.6.4 Analyses.................................................................... 92
5.6.4.l TSA supplemented with oil or salt and "salmon" agar... 92
5.6.4.2 Destruction kinetics of background microflora............ 92
5.6.4.3 Competition between L. monocytogenes and background
microflora... . .. ... ... ... ... ... ... ........................ .. . ... . .... . .... 92
5.7 Results and discussion.. .. .. .. .. .. .. .. . .. . .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .... 93
5.7.1 TSA supplemented with oil or salt.. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. . .. .. 93
5.7.2 "Salmon" agar........................................................... ... 94
5.7.3 Destruction kinetics of background microflora (presumably
LAB)............................................................................... 95
5.7.4 Competition between L. monocytogenes and background
microflora... .. . ... ....... .. ... .. . .. ..... ... .. . .. ... ... ... ... ... . ..... ... ... . ...... 98
5.8 Conclusions...... ...... ... ..................... ... ... ...... ...... ... .... ........ ........ 100
CHAPTER #6 GENERAL SUMMARY & CONCLUSIONS... .................... .... 101
REFERENCES.................................................................................... 104
APPENDIX Application to use biohazard materials.................. ................... 110
x
ATP BFS CFU DMA DNA FAO FDA GMP GRAS HACCP Hx IMP INO LAB MAP MRS OTR PL PUFA RH ROP RNA RTE SOP TMA TMAO TSA UA UV VP WPS Xa
ABBREVIATIONS
Adenosine triphosphate Blow/fill/seal Colony forming unit Dimethylamine Deoxyribonucleic acid Food and Agriculture Organization Food and Drug Administration General manufacturing practices Generally Recognized as Safe Hazard Analysis Critical Control Point Hypoxanthine Inosine monophosphate Inosine Lactic acid bacteria Modified atmosphere packaging De Man, Rogosa, Sharpe agar Oxygen transmission rate Pulsed light Polyunsaturated fatty acids Relative humidity Reduced oxygen packaging Ribonucleic acid Ready -to-eat Standard operating procedures Trimethylamine Trimethylamine oxide Tryptic soy agar Uric acid Ultraviolet Vacuum packaged Water phase salt Xanthine
Xl
LIST OF TABLES
Table 2.1 Chemical composition offish and shellfish per 100g sample......... .... 5
Table 2.2 Bacterial fiora offish caught in clear, unpolluted waters.................. 9
Table 2.3 Reported outbreaks oflisteriosis in RTE seafood products............... 17
Table 2.4 Four major groups of Clostridium botulinum .............................. 19
Table 2.5 Characteristics of the three physiologically distinct clostridia associated with food-borne botulism......................................... 19
Table 2.6 Reported outbreaks of C. botulinum type E in VP smoked fish products........................................................................... 20
Table 2.7 Characteristics of L. monocytogenes and C. botulinum type E........ .... 22
Table 3.1 Qualitative scale to represent growth of L. monocytogenes............... 44
Table 3.2 Growth (on a scale of 1 to 51) of 5 strains and a mixed culture of L. monocytogenes isolated from marine sources and pulsed light treated... 48
Table 3.3 Percent kill of L. monocytogenes at various treatment voltages and distances from the PL source................................................... 51
Table 4.1 Calculation of decimal reduction times (D-values) of L. monocytogenes on the surface of solidified agar plates..................... 65
Table 4.2 Summary oftreatment conditions for destruction kinetics of L. monocytogenes by PL treatment.... .. ... ... ... .................. ... . .. . ....... 70
Table 5.1 Color acceptability scores of PL treated VP cold smoked salmon stored at 4°C..................................................................... 78
Table 5.2 Odor acceptability scores of PL treated VP cold smoked salmon stored at 4°C.......................................................................... .... 78
Xll
Figure 2.1
Figure 2.2
Figure 2.3
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
LIST OF FIGURES
Cold-smoked salmon process - unit operations............................. 13
How pulsed light works. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 28
Comparison of PureBright pulsed light and sunlight... ... ...... ... ... . .... 29
Schematic diagram of a the pulsed light apparatus used in this study. . . 41
Position of Petri plates on PL chamber treatment tray in 12 plate experiment............ ...... ... ......... ... ... ... ........ ....... ......... .......... 43
Temperature profile of pulsed light chamber over a 5 minute treatment period, both on VP cold smoked salmon samples (black lines) and in the chamber itself (gray lines)................................................. 46
Sample representation of growth of 5 strains and a mixed culture of L. monocytogenes isolated from marine sources and pulsed light treated at 600 V and 15 cm distance from the pulsed light source................ 49
Distribution of PL at varying distances from the PL source (600V, 60 s treatment)....................................................................... 52
Distribution of PL at varying distances from the PL source (800V, 60 s treatment)....................................................................... 53
Effect of voltage and distance from the PL source on the percent kill of L. monocytogenes by PL treatment. .. ... ... .. . .. . .. . ... ... ... . .... . .. . . ... 54
Survival curve of L. monocytogenes in liquid media sealed in a 2 oz Whirl Pak bag... ..... . ... ... ... ... ... .. ....... ... . .. ... ... .... ..... ... . .. . .. .. ... 63
Survival curves of L. monocytogenes on solid general purpose media... 64
Decimallogarithm of D-values versus treatment voltages for L. monocytogenes on the surface of general purpose media...... ...... ...... 64
Transmission spectra ofpulsed UV light through Cryovac E300 (OTR = 4,000 cc/m2/day/atm @ 24°C, 0% RH...... ...... ...... ...... ............. 68
Survival curve of L. monocytogenes on the surface of PL treated cold smoked coho salmon, packaged in a clear plastic film..... . ... ... ... .. .... 69
xiii
Figure 4.6 Survival curve of L. monocytogenes on the surface of PL treated co Id smoked coho salmon... ... ... ......... ............. ..... ... ........ . . ..... ...... 69
Figure 5.1 Changes in color scores of control and inoculated VP cold smoked salmon slices, stored at 4°C.................................................... 80
Figure 5.2 Changes in color scores of control and inoculated VP cold smoked salmon slices, stored at 12°C.............................................. ..... 80
Figure 5.3 Changes in odor scores of control and inoculated VP co Id smoked salmon slices, stored at 4°C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Figure 5.4 Changes in odor scores of control and inoculated VP co Id smoked salmon slices, stored at 12°C................................................... 81
Figure 5.5 Changes in L. monocytogenes counts of control and PL treated VP co Id smoked salmon slices, stored at 4°C.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .... 84
Figure 5.6 Changes in L. monocytogenes counts of control and PL treated VP cold smoked salmon slices, stored at 12°C.................................. 84
Figure 5.7 Changes in total aerobic counts of control and PL treated VP co Id smoked salmon slices, stored at 4°C.......................................... 85
Figure 5.8 Changes in total aerobic counts of control and PL treated VP cold smoked salmon slices, stored at 12°C........................................ 85
Figure 5.9 Changes in background microflora counts of control and PL treated VP cold smoked salmon slices, stored at 4°C............................... 86
Figure 5.10 Changes in background microflora counts of control and PL treated VP co Id smoked salmon slices, stored at 12°C......................... ..... 86
Figure 5.11 Changes in pH values of control and PL treated VP cold smoked salmon slices, stored at 4°C.................................................... 87
Figure 5.12 Changes in pH values of control and PL treated VP co Id smoked salmon slices, stored at 12°C................................................ ... 87
Figure 5.13 Survival curves of L. monocytogenes on TSA supplemented with 6% oil and 1.5% salt......... ... ... ... .. . ....... .. ... .. . ... ... ... ...... .. . ... ... .. ... 96
Figure 5.14 Survival curve of L. monocytogenes on "salmon" agar... .. ... . ... . .. .. ... 96
XIV
Figure 5.15 Survival curves of background microflora isolated from cold smoked salmon............................................................................ 97
Figure 5.16 Decimallogarithm ofD-values versus treatment voltages for background microflora on the surface of general purpose media. . . . . . ... 97
Figure 5.17 Effect ofvarying concentrations of background microflora isolated from co Id smoked salmon on the growth of L. monocytogenes. ............. 99
xv
CHAPTERI
INTRODUCTION
A steady increase in global fish consumption has been observed over the past few
decades due to its high nutritive value and the increase in health conscious consumers
aware of the benefits of consuming a di et ri ch in fish. The lean proteins present in fish
are a particularly appealing alternative to red meat, which is receiving much scrutiny for
its undesirable high saturated fat content. The beneficial effects of the polyunsaturated
fatty acids (PUF A) found in fish, particularly omega-3 and omega-6, are well
documented in literature and in the media, thus causing high consumer demand of fish
and fish products.
In addition to being an excellent nutritional source, fish is highly susceptible to
spoilage from the time it is caught. The main cul prit of fish spoilage is a combination of
enzymatic reactions, microbial growth and chemical changes, all of which are accelerated
once fish die. Several processing and preservation techniques have been used to
minimize these undesirable changes in fresh caught fish: refrigeration, freezing, drying,
salting, canning and smoking.
For centuries, fish has been smoked and dried over a wood fire in order to
decrease its water activity, therefore increasing its shelf-life. Currently, fish is smoked
for its desirable flavor and organoleptic properties, not solely as a means of preservation.
However, the process used for cold smoking of fish is not exceptionally rigorous; thus,
there is concern that sorne food-borne pathogens, if present, could survive. Organisms of
primary concern are Listeria monocytogenes and Cloistridium botulinum (psychrotrophic
non-proteolytic type B, E, and F) (Food and Agriculture Organization (FAO), 1999). In
addition to contributing to pathogen survival, the extensive handling of products
following the cold smoking process provides ample opportunities for other food-borne
pathogens (Salmonella spp., Shigella spp., Escherichia coli, Staphylococcus aureus,
Bacil/us cereus, Vibrio parahaemolyticus, and Vibrio cholerae) to contaminate and
survive in the products if insufficient attention is given to Standard Operating Procedures
(SOPs) and Good Manufacturing Practices (GMPs). While C. botulinum has been of
1
concern for many years, L. monocytogenes is a relatively new concern. L.
monocytogenes has been isolated from fishery products on a regular basis (Food and
Agriculture Organization (F AO), 1999); it survives both the salting and the cold smoked
processes and is capable of growth at refrigeration temperatures (Hudson and Mott,
1993). Although this organism is typically present in the raw material, its presence in
ready-to-eat (RTE) foods is primarily caused by contamination during processing (Autio
et al., 1999; Fonnesbech Vogel et al., 2001; Rorvik et al., 1995). The problem is further
complicated by FDA's CUITent policy of "zero-tolerance" for L. monocytogenes on RTE
products.
In order to improve the shelf-life and safety of cold smoked fishery products,
refrigerated storage needs to be supplemented with additional processing and/or
preservation techniques which would aid in preventing the growth of spoilage or
pathogenic bacteria, while maintaining the sensory quality of the final product. One such
technique is pulsed light treatment.
Pulsed light treatment is an innovative technological concept that has great
potential for extending the shelf-life of foods, without a heat treatment step. It is a
method of food preservation that involves the use of intense and short duration pulses of
broad-spectrum "white light", where each pulse, or flash, of light lasts a fraction of a
second and the intensity of each flash is approximately 20,000 times the intensity of
sunlight at sea level. Pulsed light is a sterilization method in applications were light can
access aIl the important volume and surfaces, such as packaging materials, surfaces,
transparent materials and pharmaceutical or medical products. However, due to the non
uniform surfaces and opacity of food stuffs, PL treatment cannot sterilize these products;
however, it can reduce the microbialload of such products. Many studies have been
carried out to evaluate the effects of static UV light treatment of food, however there are
very few studies for the application of pulsed UV (Dunn, 1996; Dunn et al., 1997b; Dunn
et al., 1995; Macgregor et al., 1998; Rowan et al., 1998). These studies have shown the
abi1ity of PL to destroy microogranmisms while maintaining food quality.
Pulsed light technology is still in its infancy and therefore there are a limited
number of studies that have been carried out on the effect of PL in food products and
those performed on fishery products are rare. The purpose of this study was to promo te
2
shelf-life extension ofrefrigerated VP cold smoked salmon by controlling spoilage
bacteria and promote safety by destroying pathogenic bacteria while maintaining the
sensory quality of this RTE product.
The objectives ofthis research were to:
(i) Characterize pulsed light distribution and effectiveness of pulsed light
treatment using various strains of Listeria monocytogenes.
(ii) To evaluate the pulsed light destruction kinetics of Listeria monocytogenes.
(iii) To evaluate shelf-life and quality changes in pulsed light treated cold smoked
salmon stored at different temperatures.
(iv) Finally, to carry out challenge studies on pulsed light treated cold smoked
salmon challenged with Listeria monocytogenes.
3
CHAPTER2
LITERA TURE REVIEW
2.1 Chemical components of fish
Fish and seafood products are an important part of a weIl balanced di et since they
are comprised of four major components: protein, carbohydrate, water and fat (Table
2.1). The high biological value of proteins, unsaturated fatty acids, mineraIs and vitamins
present in fish make them an excellent food choice for inclusion in the human diet
(Sidhu, 2003). However, due to their high water, protein and fat content, fish is an ideal
substrate for microbial growth. As a result, fish have a relatively short shelf-life
(approximately 7 days) even at refrigerated storage conditions.
Fish and seafood product spoilage is dependent on two major factors: the
biological reactions, such as oxidative rancidity, that occur due to the fish's own
enzymes; and the metabolic activities of microorganisms. Fish is usually caught in
waters distant from land and from areas where they are to be consumed. Therefore, a
major problem is the maintenance of fresh quality and prevention of spoilage after
harvesting. There is a growing need for effective preservation methods to prevent the
deteriorative post-mortem changes involved in fresh caught fish, which will thus extend
the shelf-life of the final product. Preservation methods are generally designed to
maintain freshness and quality, beginning from the point ofharvest through storage,
processing, and distribution, until the point of consumption (Ashie et al., 1996).
It has been shown that once fish dies and enters into the stages of rigor mortis, the
microbial flora present in the gills, gut, and skin begin to metabolize the compounds
mentioned above, which leads to deteriorative reactions, resulting in off-flavors, changes
in texture and discoloration (Gram and Huss, 1996).
4
Table 2.1 al., 1991)
Chemical composition of fish and shellfish per 100g sample (Holland et
Fish and shellfish Protein Carbohydrate Water Fat
Cod 17.4 82.1 0.7
Haddock 16.8 1.3 81.3 0.6
Halibut 17.7 1.8 78.1 2.4
Herring 16.8 0.8 63.9 18.5
Mackarel 19.0 0.7 64.0 16.3
Salmon 18.4 1.6 68.0 12.0
Lobster 22.1 2.1 72.4 3.4
Prawnsa 22.6 5.6 70.0 1.8
Shrimpsa 23.8 11.3 62.5 2.4 a - Indicates boiled samples
5
2.2 Spoilage of fresh fish
2.2.1 Microbial spoilage
The flesh ofhealthy live fish is sterile because of the action of the immune system
preventing growth of bacteria on the flesh. At the point of death, the immune system is
no longer capable of protecting the fish, thus bacteria are free to grow.
Gram negative psychotrophic bacteria, which are capable of growing at O°C but
have an optimum growth temperature of ~ 25°C, generally dominate the spoilage offresh
fish. Gram positive bacteria may also be responsible for spoilage in sorne cases (Table
2.2). The bacterialload is an important parameter influencing spoilage. A value of 107
CFU/g ofpsychotrophic bacteria is used as an indicator offish spoilage (International
Commission on Microbial Specification for Foods (ICMSF), 1978).
Although sorne microorganisms are not directly associated with microbial fish
spoilage, they may become a public health concern due to their ability to pro duce
hazardous toxins in fish products. A key example is botulism, an often fatal
neuroparalytic disease caused by toxins produced by Clostridium botulinum type E
(Ashie et al., 1996).
2.2.2 Chemistry/biochemistry of fish spoilage
Enzymes produced by spoilage microorganisms present in the post-mortem tissue
will metabolize fish muscle to produce volatile compounds, resulting in off-flavors and
odors. Trimethylamine oxide (TMAO), which is present in large quantities in marine fish
and shellfish, is broken down to trimethylamine (TMA) by either endogeneous enzymes,
or the bacterial enzyme, trimethylamine oxidase (Phillippy, 1984). This gives fish an
undesirable fishy odor and causes sponginess of flesh during frozen storage. TMAO may
also be decomposed to dimethylamine (DMA) and formaldehyde. The reactions involved
are:
6
AH2 + (CH3)3 NO ~ (CH3)3N + H 20+ A
TMAO TMA
(CH3)3NO ~ (CH3)2NH + HCHO
DMA formaldehyde
where A is an oxidizing substrate and AH2 is the reduced form of substrate.
When fish is alive, adenosine triphosphate (ATP) is generated under aerobic
conditions for muscle contraction. When fish die, anaerobic conditions set in and
glycolysis occurs, with alkaline pyrophosphate and lactic acid being the main products of
post-mortem glycolysis. In addition, any ATP that is produced is degraded to uric acid:
ATP ~ AMP ~ IMP ~ INO ~ Hx ~ Xa ~ UA
where IMP is inosine monophosphate, INO is inosine, Hx is hypoxanthine, Xa is
xanthine and UA is uric acid.
When a certain level of acidity is reached, the proteins become denatured and this
results in stiffness (rigor mortis). As lactic acid increases, the tissues soften and juices
are released. NormaIly, bacteria do not invade the tissue before 3-5 days.
The formation of hydro-peroxides by enzymatic means was discussed earlier;
however there are non-enzymatic reactions that result in the formation of hydro
peroxides, namely oxidative rancidity and non-enzymatic browning.
Oxidative rancidity is weIl known as a major cause of deterioration of seafood
products as weIl as many other foods. The process involves oxidation of unsaturated
fatty acids or triglycerides via the following free radical mechanism:
Initiation:
Propagation:
RH~R*+H*
RH +02 ~ ROO*+H*
R*+02 ~ROO*
ROO*+RH ~ ROOH +R*
7
Termination: ROO * +R* ~ ROOR
R*+R* ~ R-R
ROO*+ROO* ~ ROOR+02
where ROO* is a lipid peroxy radical, R * is a lipid radical, and RH is an unsaturated
lipid.
Once the reaction is initiated, the unstable hydro-peroxides are converted to free
radicals, which are responsible for accelerating the rate of the reaction. The products of
these secondary reactions are responsible for the oxidized flavor in seafood products.
Non-enzymatic browning, which is responsible for the sometimes brown pigment
in fish products, is partly caused by the Maillard reaction, where the sugar ribose and
amino groups react. It has been shown that browning reactions are mostly due to lipid
autoxidation reaction products reacting with proteins. It has been reported that this type
of reaction proceeds through the following three steps: (1) formation of lipid peroxides,
(2) formation of colorless or only slightly colored precursors ofbrown pigments by
interaction of peroxides with active groups of protein and by interaction of carbonylic
peroxide decomposition products with active groups ofprotein, and (3) transformation of
the colorless or light-colored precursors into brown pigments (Khayat and Schwall,
1983). Fresh fish is a highly perishable product that must be rapidly preserved in order to
extend its shelf life. Methods commonly used to extend the shelf life of fresh fish are:
chilling, freezing, canning, salting, drying and smoking.
2.2.3 Flavor deterioration
Most fish species contain a large quantity of long chain lipids with a high
proportion of polyunsaturated fatty acids, which are highly susceptible to oxidation.
Enzymes, such as lipoxygenase and peroxidase, can initiate lipid peroxidation to produce
hydroperoxides. The resulting oxidative off-flavors can be attributed to the breakdown
products of the hydroperoxides; for example, aldehydes, ketones, and alcohols.
8
Table 2.2 1995)
Bacterial flora of fish caught in clear, unpolluted waters (Huss and Gram,
Gram negative
Pseudomonas
Moraxella
Acinetobacter
Gram positive
Bacillus
Clostridium
Micrococcus
Shewanella putrefaciens Lactobacillus
Flavobacterium
Cytophaga
Vibrio
P hotobacterium
Aeromonas
Coryneforms
Comments
Even though S. putrepaciens is sodium
requiring, it has been isolated from
freshwater environments
Vibrio is typical in marine waters
Photobacterium is typical of marine waters
Aeromonas is typical of freshwater
9
2.2.4 Changes in texture
The most important index of quality of fish products is tenderness. Due to the
fact that fish products contain less connective tissue and less collagen than terrestrial
animaIs, they do not require the same amount of time to proceed through rigor mortis.
Therefore, fish and shellfish will soften faster than terrestrial animaIs. Contrary to the
accelerated softening of tissue in terrestrial animaIs, this quality is not desirable in fish
and seafood products.
2.3 Traditional preservation methods for fresh fish
The perishability of fish is notorious, due mainly to the psychrotrophic microflora
associated with the natural fish environment. Chilling the fish above freezing
temperatures (0-4°C) is commonly used to retard the microbial growth and one can
expect a 14-18 day shelf-life. However, sorne detrimental enzymatic reactions and
microbial spoilage causing prote in and fat decomposition may be stimulated at
refrigeration temperatures (Jelen, 1985). The psychrotrophic microorganisms that have
been implicated in the spoilage offish during refrigerated storage include Pseudomonas,
Alteromonas, Flavobacterium, Achromabacter, Shewanella, Acinetobacter and Vibrio
spp (Hubbs, 1991). In addition, pathogens such as Escherichia coli, Listeria
monocytogenes, Campylobacter and Salmonella species can also cause serious food
borne illnesses when introduced during handling and processing (Ingham, 1991).
Freezing is a common method employed to preserve fresh fish, as detrimental
reactions are retarded at sub zero temperatures. A shelf-life of greater than six months
can be achieved, especially when fish is vacuum packaged to prevent oxidative changes.
The quality of the product following the freeze-thaw cycle depends on a key factor: the
speed at which the fish was frozen and subsequently thawed. If the freezing or thawing
process is slow, resulting in the formation of large ice crystals, the final quality of fish
will be inferior to fish that has been rapidly frozen and thawed. Unfortunately, there are
limitations with freezing fish: prote in denaturation and phospholipid hydrolysis occur
10
when temperatures decrease to below -soc (Ashie et al, 1996). In addition, cost for
freezing and storage of fish under ice while in transit is very expensive.
Canning is an efficient way to extend the non-refrigerated shelf life of fish
products. The fish in question is either cooked or raw prior to heat treatment in a metal
can, or more recently, in a metallaminate pouch. This heat treatment renders the fish
"shelf-stable", essentially commercially sterile. Unfortunately, quality offish from a can
does not resemble that of fresh fish, and notable changes in color and texture occur.
However, one major advantage ofthis method is the ability to transport and store fish
under atmospheric (i.e. non-refrigerated) conditions.
Other methods of fish preservation inc1ude salting and drying, both of which
attempt to decrease the water activity, therefore inhibiting microorganisms from
flourishing. Unfortunately, salted and dried fish do not generally appeal to consumers'
taste and can be expensive.
Smoking has been used for centuries to reduce the water activity and extend the
shelf life of fish. This method of fish preservation will be addressed in detail in the next
section.
2.4 Smoking of fish
According to both the Codex Alimentarius Commission (1979) and the
Association of Food and Drug OfficiaIs (AFDO) (1991), cold process smoked fish means
" ... a smoked fish that has been produced by subjecting it to smoke at a temperature
where the product undergoes only incomplete heat coagulation of prote in" (Food and
Drug Administration (FDA), 2001). As a result, cold-smoked fish products undergo a
further heat treatment prior to consumption. A notable exception is cold-smoked salmon,
which is a ready-to-eat (RTE) product, and is consumed uncooked. On the other hand,
hot smoking is "curing fish by smoking at a tempe rature of 70-80°C at sorne stage in the
process in order to cook the flesh" (Bannerman, 2001). This process produces a product
that does not require a further heat treatment prior to consumption (Samuel, 1999). From
this point forward, smoking will refer to the cold smoking process.
Il
For centuries, smoking has been used as a means of extending the shelf life of
fish. Examples of commonly smoked fish include haddock, eel, herring, mackerel, trout
and salmon. This research will focus on the cold smoking process of farmed salmon.
Smoking not only preserves fish but also adds flavor and improves the color and
tenderizes fish flesh. The preservative action is due to the impregnation of the surface
with chemicals such as formaldehyde, a1cohols, phenolic compounds and cresols, which
are found in the smoke produced by burning wood. Liquid smoke, an alternative to
natural smoke, is also used by many large manufacturers of smoked fish products. It is
added to the brine and applied to the product as a dip or sprayed directly onto the product.
Liquid smoke has several advantages over natural smoke: it is possible to eliminate
known carcinogenic compounds that can be found in smoke; it is environmentally more
compatible for processing facilities located near populated areas; it can be incorporated
into the product; and the application (spraying on the surface) is faster (Claus et al.,
1994). The heat and limited drying associated with smoking also aid in the destruction of
microorganisms. The released chemicals are more effective against the vegetative cells
than against spores with their residual effect being directed more against bacteria rather
than molds. The unit operations involved in the co Id smoking process are shown in
Figure 2.1.
There are several safety concerns associated with cold-smoking offish. Firstly,
they are minimally processed, and therefore sorne food-borne pathogens, if present, could
survive. Secondly, they have low salt contents «5% water phase salt, WPS) and aw
>0.97. These conditions are conducive to the growth ofpathogens like Clostridium
botulinum and Listeria monocytogenes. Thirdly, there is the potential for temperature
abuse at all stages of processing, packaging and distribution thereby enhancing the
growth ofthese pathogens. Lastly, cold-smoked fish products are ready-to-eat and do not
receive a heat treatment prior to consumption. Therefore, refrigerated storage alone
cannot be regarded as an adequate safety barrier and additional barriers are needed to
ensure the continued safety of vacuum packaged smoked fish products. Modified
atmosphere packaging (MAP) is one such technique that has the potential to improve the
shelf-life of cold-smoked salmon.
12
Figure 2.1 Cold-smoked salmon process - unit operations (Food and Drug Administration (FDA), 2001)
13
2.5 Modified atmosphere packaging (MAP)
Several processing technologies are currently being used to slow down the
deteriorative reactions in food products. In this thesis, the emphasis is on cold-smoked
fish products which are especially susceptible to microbial, enzymatic and chemical
spoilage, as mentioned in a previous section. Modified atmosphere packaging has been
defined as "the enclosure of a food product in a high gas barrier film in which the
gaseous environment has been changed or modified to slow respiration rate, reduce
microbiological growth, and retard enzymatic spoilage with the intent of extending shelf
life" (Young et al, 1988). The principal goal behind MAP is to reduce headspace
oxygen, which is the major culprit of spoilage reactions.
2.5.1 Gas Packaging
Gas packaging involves removing air from the package and replacing it with a
mixture of gases; the pressure of gas inside the package usually approaches 1 atmosphere
(Smith et al., 1990). Three main gases are commonly used in gas packaging: carbon
dioxide, nitrogen, and oxygen. Nitrogen is used as a filler gas, to prevent package
collapse. Oxygen is normally avoided in gas packaging of high fat products, to prevent
oxidative rancidity. However, when a desirable "bloom" ofred meat is required, oxygen
is used. Carbon dioxide is both bacteriostatic and fungistatic. Several factors influence
the antimicrobial effect of carbon dioxide: microbialload, gas concentration,
temperature, and packaging film permeability (Smith et al., 1990). However, its
bacteriostatic effect is most effective on aerobic microorganisms. In the case of C.
botulinum and L. monocytogenes, which are anaerobic, and facultative anaerobic,
respectively, carbon dioxide has little or no effect and may actually stimulate the growth
of C. botulinum and L. monocytogenes.
14
2.5.2 Vacuum packaging
Vacuum packaging involves packaging a product in a film oflow oxygen
permeability, for example less than 100 cc/m2124hr (i.e. 2 mil polyester) (Seafood NIC,
2002), the removal of air from the package, and the application of a hermetic seal (Smith
et al, 1990). Under good conditions, the resulting oxygen in the package headspace is
reduced to <1 %. Vacuum packaging is widely used in the smoked fish industry as
aerobic spoilage organisms, such as Pseudomonas spp., are inhibited by the reduced
oxygen packaging conditions. However, there is a growing concern that if C. botulinum
and L. monocytogenes were present, ev en in small numbers, they could flourish in an
anaerobic environment, even at refrigeration temperatures. Post-processing
contamination is a major concern with cold-smoked fish products, particularly with L.
monocytogenes. Therefore, in addition to vacuum packaging and refrigeration as
methods of preservation, additional barriers need to be implemented in order to ensure
safety of the final product. This will be discussed in later sections.
2.6 Microbiological concerns about vacuum packed smoked fish
The processes used for co Id smoking of fish are not exceptionally rigorous;
therefore, there is a concern that sorne foodborne pathogens, if present, have the
possibility to survive. Organisms of primary concern are Listeria monocytogenes and
Clostridium botulinum (psychrotrophic, non-proteolytic type B, E, and F) (Food and
Drug Administration (FDA), 2001). These two organisms, in addition to Staphylococcus
aureus and Salmonella spp., have been responsible for a number of food poisoning
outbreaks involving smoked fish products (Gonzalez-Rodriguez et al, 2002).
2.6.1 Listeria monocytogenes
Listeria monocytogenes is a Gram positive non-spore forming rod shaped bacteria
that is both aerobic and facultative anaerobic. It is widespread in nature, occurring in
15
soil, vegetation, water, and many animal and plant products (Lovett and Twetd, 1988). In
addition, it can grow over a wide pH, aw, salt and temperature range.
While C. botulinum has been of concern for many years in cold smoked fish, L.
monocytogenes is a relatively new concern. The Food and Agriculture Organization
(F AO) reported that L. monocytogenes has been isolated from fishery products on a
regular basis since the late 1980s (Food and Drug Administration (FDA), 2001).
Furthermore, this pathogen can survive the cold smoking process and is capable of
growing at refrigerated conditions and under the influence of high salt contents (~1 0%).
There is a diverse range of food products that have been responsible for individual
cases and major outbreaks of listeriosis. Such foods are generaHy highly processed, have
an extended shelf life at refrigeration temperatures, are capable of supporting the growth
of L. monocytogenes, and are consumed without further cooking (Huss et al., 2000). A
typical example is cold-smoked salmon. Examples of outbreaks ofliseriosis from RTE
seafood products are shown in Table 2.3.
Recently, an investigation into the occurrence of L. monocytogenes in ready-to-eat
fish products traded in Osaka City, J apan found that it was in fact isolated from 12 (13 %)
of the 95 products tested (Nakamura et al., 2004). AH positive sampi es were from co Id
smoked fish with 9 being obtained during the summer. Their findings suggest that
persistent strains in each manufacturing facility proliferate during the summer and
contaminate products during manufacturing processes.
Although fresh fish may already be contaminated with L. monocytogenes before
entering the processing stage, there are indications that the raw material is not the
primary source of contamination of the final product (Rorvik et al., 1995). In addition,
the prevalence of L. monocytogenes in cold-smoked fish is highly variable. It has been
found to range from <1.4% to 100% in cold-smoked salmon samples from three different
production sites (Jorgensen and Huss, 1998).
16
Table 2.3 Reported outbreaks oflisteriosis in RTE seafood products (Food and Drug Administration (FDA), 2001)
Year Country Food Numberof
Cases Deaths
1980 New Zealand Fish and 29 9
molluscan
shellfish
USA Contaminated 9
(Connecticut) shrimp
Tasmania Smoked 3
mussels
Sweden Cold-smoked 9
rainbow trout
17
2.6.2 Clostridium botulinum
Clostridium botulinum is generally gram positive, at least at the early stages of
growth. Most strains are strictly anaerobic and are capable of forming spores.
Clostridium botulinum is divided into 4 major groups that are based principally on their
ability/inability to metabolize complex proteins (Table 2.4). In addition, clostridia can be
separated into three physiologicaUy distinct groups that have been shown to be associated
with food-borne botulism (Table 2.5). The specific type that is a major concem for cold
smoked, vacuum packaged fish is the non-proteolytic type E. However, there is an
additional concem for types A and B if the fish originates from temperate waters.
Due to the high post-mortem pH ofmost fish (>6.0), germination and growth of
C. botulinum type E is not inhibited (Gram and Huss, 1996). Type E is capable of
growing and producing toxin at temperatures as low as 3°e, i.e. refrigerated temperatures.
Fortunately, the toxin is thermolabile and a 30 min heating at 800e will inactivate aU
toxins; however, the cold-smoke process will not pro vide an adequate temperature and
time combination to inactivate the toxin.
There have been several reported cases of foodbome botulism. Outbreaks of C.
botulinum type E in vacuum packaged smoked fish products are represented in Table 2.6.
In order for an foodbome outbreak to occur, several conditions must be met: (1) presence
of spores, (2) suitable substrate for germination and growth, (3) survival of spores during
processing, (4) environmental conditions favorable after processing, (5) insufficient
cooking time in the home, and (6) ingestion of the food in question.
18
Table 2.4 Four major groups of Clostridium botulinum
Group Classification
I C. botulinum A, B, F (proteolytic)
II C. botulinum B, E, F (non-proteolytic)
III C. botulinum C, D
IV C. botulinum G
Table 2.5 Characteristics of the three physiologically distinct clostridia associated with food-borne botulism
Characteristic
Neurotoxins formed
Minimum growth temperature tC)
Minimum growth pH
Spore heat resistance
(D JOo°c)(min)
Foods involved in botulism outbreaks
Potential food problems
Proteolytic C. botulinum
A,B,F
10-12
4.6
>15
Home-canned foods, faulty commercial processing
Canned foods
N on-proteolytic C. botulinum
B,E,F
3
5.0
<0.1
F ermented marine products, dried fish, vacuum packed fish
Refrigerated processed foods
with a long shelf life
N eurotoxigenic C. butyricum
E
10-15
4.0-5.2
<1-5
Vegetable-based foods in Asia
Bakery products (Sevu) in lndia
19
Table 2.6 Reported outbreaks of C. botulinum type E in VP smoked fish products (Advisory Committee on the Microbiological Safety of Food (ACMSF), 1992)
Number of
Year Country Food Cases Deaths
1960 USA VP smoked ciscoes 3 2
1963 USA VP smoked whitefish chubs 17 5
1970 Germany VP smoked trout 3
1991 Sweden VP smoked salmon 2 o
20
Psychotrophic, non-proteolytic C. botulinum is found both in freshwater and
saltwater species of fish; hence its prevalence is widespread but its incidence is low
(Food and Drug Administration (FDA), 2001). The packaging environment and the
storage temperature are critical factors influencing the growth of C. botulinum. Since C.
botulinum is an anaerobe, the potential for botulism to develop exists when reduced
oxygen packaging (ROP) or vacuum packaging is used when treatments do not destroy
the spores of C. botulinum. Mild heat treatments, such as cold-smoking, in combination
with vacuum packaging, may actually promote growth of C. botulinum by inhibiting
aerobic competitors (Seafood NIC, 2002). Since cold-smoking does not pro vide
commercial sterility, the product must be carefully refrigerated to prevent spoilage and
ensure product safety. Control of C. botulinum can be established by maintaining the
product temperature between 3.0 and 3.3°C throughout distribution and storage; however
this is not a realistic option. For this reason, a combination of low temperature storage
and salting (3.5% salt) (Food and Drug Administration (FDA), 2001) are used to prevent
growth of C. botulinum type E and enhance safety of the product. Commercially
produced cold-smoked fish has rarely been responsible for any outbreaks of botulism.
Therefore, it can be conc1uded that the cold storage temperature in combination with a
high salt (>3.5%) keeps the hazard under adequate control (Food and Drug
Administration (FDA), 2001). A summary of characteristics for L. monocytogenes and
C. botulinum type E are represented in Table 2.7. Based on the se characteristics, several
control measures, both traditional and novel, must be applied in order to ensure a safe end
product.
2.7 Traditional control measures in ready-to-eat cold smoked salmon
Fish is a highly perishable food. The cold smoking process in combination with
vacuum packaging is designed to extend the shelf-life offish at refrigerated temperatures.
However, the potential for temperature abuse ofthese products as weIl as the
characteristics of L. monocytogenes and C. botulinum type E merit the use of additional
control measures to ensure safety of the final product. Sorne of the se are described
below:
21
Table 2.7 2002)
Characteristics of L. monocytogenes and C. botulinum type E (Smith et al.,
L. monocytogenes C. botulinum type E
General Gram positive, non spore Gram positive, spore forming, aerobic and forming, anaerobic facultative anaerobic
Symptoms of disease Mild flu-like symptoms, Nausea, vomiting, blurred septicemia, meningitis, or double vision, fatigue,
encephalitis, and abortion in paralysis of muscles, and pregnant women death
Causative agent Infection T oxin in food - type E produces a deadly
neurotoxin at temperatures as low as 3°C
Onset of illness 2-35 days 12-72 ho urs
Duration of illness 1-90 days 1-10 days
Associated fish products Has been isolated from Indigenous organism of fishery products on a aquatic environments; 1-2
regular basis since 1980s spores to a few hundred per kg offish
Challenges in co Id smoked Capable of surviving cold ROP and cold smoking may salmon smoking process, can grow select for growth by
at refrigeration conditions inhibiting aerobic and under the influence of competitors
NaCl (~10%)
Preventative measures Strict temperature of Strict temperature of finished product «2°C), fini shed product «4°C), additional barriers (pH, additional barri ers (pH, water activity reduction, water activity reduction, chemical preservatives), chemical preservatives),
strict personal strict personal hygiene/sanitation, and hygiene/sanitation, and
good manufacturing good manufacturing practices (GMPs) practices (GMPs)
22
2.7.1 Good manufacturing practices (GMPs)
It is considered impossible to completely eliminate L. monocytogenes from
processing environments in which RTE foods are produced (Food and Drug
Administration (FDA), 2001). The high prevalence ofthis organism is largely due to
post-processing contamination. It is essential that the manufacturing facility adhere to, at
the minimum, GMPs or a Hazard Analysis Critical Control Point (HACCP) program.
Three areas of utmost importance in the environmental control of L. monocytogenes are:
training of staff, reduction or elimination of the organism in niches, and lastly,
monitoring of the contamination.
2.7.2 Storage temperature
Both L. monocytogenes and C. botulinum type E are capable of growing at
refrigeration temperatures. It has been generally recommended that VP cold smoked fish
be stored at temperatures lower than 2°C to prevent the growth of L. moncytogenes.
However, it has also been shown that this organism is capable of growth at these
temperatures. To prevent growth and germination of C. botulinum type E spores, and
neurotoxin formation, it is recommended the storage temperature of the final product be
below 4°C. The lowest established temperature limit for growth and toxin production by
strains ofpsychrotrophic C. botulinum is 3.3°C (Advisory Committee on the
Microbiological Safety of Food (ACMSF), 1992).
Unfortunately, these guidelines do not provide a great deal of flexibility in terms
oftemperature abuse, as the majority ofrefrigerators are not kept at temperatures below
2°C. Therefore, refrigeration alone is not an adequate control measure to ensure a safe
product.
2.7.3 Salt concentration and nitrite
L. monocytogenes is a halotolerant bacterium; therefore salting is not likely to
inhibit its growth. Water phase salt concentrations in the final VP smoked fish product
23
range from 3 to 12%, in a few cases, but levels typically range from 3.5-5% (Food and
Drug Administration (FDA), 2001). The level of salt required to have an inhibitory effect
on L. monocytogenes is greater than 10%, however, this level of salt is generally too high
organoleptically. On the other hand, under optimal conditions, C. botulinum will grow in
up to 5% salt. However, at reduced temperatures, less salt is tolerated by the organism.
Therefore, the recommended level of salt, in combination with low temperature storage,
required to prevent the growth of C. botulinum is 2.5-5%.
Nitrite has also been used in VP meats and fish due to its anti-botulinogenic
properties. Nitrite (at 200 ppm) has an inhibitory effect against L.monocytogenes at 5°C,
if used in combination with salt. The addition of nitrite may reduce the amounts of salt
required to inhibit C. botu/inum toxin formation; a nitrite level of 200 ppm is appropriate
in this case (Food and Drug Administration (FDA), 2001). The use of nitrites is not
permitted in Canada as there are concems about the potential carcinogenic effects of
nitrosamines.
2.7.4 Packaging in films of lower permeability
Many studies have documented that oxygen removal enhances toxin formation.
This information led the FDA to recommend a film permeability of at least 10,000
cc/m2/day for VP smoked fish products. However, it is now known that the growth of C.
botu/inum in foods does not depend upon the total exclusion of oxygen, nor does the
inclusion of oxygen as a packaging gas ensure its suppression (Dufrense et al., 2000).
There is a major concem that VP products may promote the growth of C. botu/inum by
inhibiting aerobic spoilage microorganisms, which ifunder temperature abuse conditions,
could lead to toxigenesis before spoilage. The new recommendation by the FDA would
ensure that the product would spoil, due to the presence of oxygen for aerobic spoilage
organisms, before toxigenesis occurred.
24
2.7.5 Sorbate/lactate/bacteriocins
Sorbate is Generally Recognized as Safe (GRAS) substance and a preservative,
and may therefore be used as a food additive. It is effective against C. botulinum at pH
values below 5.5. It has been shown to result in a marked reduction in growth rate of L.
monocytogenes; however no studies on cold-smoked fish have been performed. Sorbate
is not permitted in countries such as the UK; and from a technological point of view, it
may be difficult to apply it on cold smoked salmon.
Lactate is inhibitory to psychrotrophic C. botulinum and studies have shown that a
concentration of 2% can completely inhibit L. monocytogenes at 5°C. Lactate is often
used as a flavor enhancer; however, it is not known what the sensory effect of lactate
would be on smoked fish. In addition, it may be difficult to absorb 2% lactate into the
water phase of the fish (Advisory Committee on the Microbiological Safety of Food
(ACMSF), 1992; Food and Drug Administration (FDA), 2001).
Lately, numerous studies have been conducted regarding the effect ofbacteriocins
on various food products, including fish. For example, the addition of 1000 ppm ofnisin
to co Id smoked salmon caused an initial reduction in numbers of L. monocytogenes, but
after 2 weeks, growth in vacuum packs at 5°C resumed as in the control (Nilsson et al.,
1999). Unfortunately the practical application of bacteriocins is hampered by several
aspects: only sorne countries permit the use ofbacteriocins; their stability and activity in
food products is unpredictable; and resistance to bacteriocins by L. monocytogenes has
been shown to occur quite readily (Food and Drug Administration (FDA), 2001).
Due to the potential of temperature abuse at aIl stages of storage and the limited
efficiency of traditional control methods, it is essential to investigate novel control
measures of food preservation to ensure the safety of VP cold smoked salmon. Although
it is possible to utilize heat treatments for fish preservation, such as microwave or ohmic
heating, the emphasis of a non-thermal technology was paramount to maintain the
structural integrity of the co Id smoked salmon product. One such non-thermal
technology is pulsed light.
25
2.8 Pulsed light treatment
A novel processing method that could be used as a post-packaging barrier is
pulsed light. Cold-smoked fish presents a serious dilemma. It has been a part of our diet
for centuries, and only now are questions of safety being raised. Is the product safe? If
yes, then why? If no, then why? Is it possible to modify the process to reduce the
potential risk of pathogens? Will that modified food be acceptable to the consumer?
There have been major advances in cold-smoked fish preservation methods, one ofwhich
is pulsed light technology. Pulsed light technology has great potential to be used on a
commercial scale, not only to increase shelf-life but to also ensure the microbial safety of
the final product.
Pulsed light technology is a relatively new method of food preservation that was
introduced by Pure Pulse Technologies in San Diego California in the 1990s. The
process involves the use of intense and short duration pulses of broad-spectrum "white
light". The spectrum of light for pulsed light treatment includes non-ionizing
wavelengths in the ultraviolet (UV) region (comprising 25%) and the near infrared (NIR)
region (Barbosa-Canovas et al., 2000; Dunn et al., 1997b). Pulsed light kills high levels
of different microorganisms exposed to the light, including bacteria, fungi, spores,
viruses, protozoa, and cysts (Pure Pulse, 1997b). Pulsed white light is applicable as a
sterilization method or as a method to reduce the microbial population on the surface of
packaging materials, transparent pharrnaceutical products, surfaces, and transmissive
materials, including water, air, and many solutions. Many foods, which are opaque with
complex surfaces, cannot be sterilized by exposure to pulsed light; however pulsed light
is very effective for reducing microbialloads on the surface and extending the shelf life
of such products (Dunn et al., 1997b). The pulsed light method has been approved by the
U.S. Food and Drug Administration (FDA) after being evaluated for both safety and
effectiveness (Dunn et al., 1997b).
Pulsed light is produced using engineering technologies that multiply power
several fold. Power is magnified by accurnulating electrical energy in an energy storage
capacitor over relatively long times (fractions of a second) and releasing this stored
energy to do work in much shorter times (millionths or thousandths of a second). The
26
result is very high power during the dut y cycle with the expenditure of only mode st
average power consumption (Figure 2.2) (Dunn et al., 1995). As a result, the process is
economical. An in depth look at pulsed light treatment at 4 J/cm2 showed usage cost
estimates of approximately 1-4 cents/m2 of treated surface area (including amortization of
the capital expenditure, lamp replacement, maintenance, and electrical costs) (Dunn et
al., 1997b).
The process can treat products on-line at high throughput (approximately 50
packages per minute). This is due to the fact that a single lamp can produce multiple
flashes per second and only a few are required to be effective in reducing microbial
populations. The effectiveness ofthe white flash is due to its rich broad-spectrum UV
content, short duration, and very high power (Dunn et al., 1997b).
Although a range of pulsed spectra, durations, and intensities have been found to
be useful for the treatment of foods and packaging, pulsed light development focuses on a
broad-spectrum "white" light flash containing wavelengths from 200 nm in the uv to
about 1 mm in the NIR (Dunn et al., 1995). This spectral resolution is similar to that of
sunlight, having peak emission between 400 and 500 nm (Figure 2.3). However, each
pulsed light flash is approximately 20,000 times the intensity of sunlight at sea level
(Barbosa-Canovas et al., 2000; Dunn et al., 1997b; Dunn et al., 1995; Pure Pulse, 1997a).
Light energy on the surface is measured as fluence, or incident light energy per
unit area, in Joules per square centimeter. A Joule is less than V4 calorie, and therefore
more than 4 Joules of energy are required to raise the temperature of 1 g of water 1°C
(Dunn et al., 1995).
27
WALL POWER
(AC)
DC POWER SUPPLY
'" '" 2:i 2:i crJ crJ
CAPACITOR
~ 2 2 l\ VERAGE ~ ............................................................. ·· .. · ........ POWER o ~ CHARGING
TIME
SWITCH
HIGH POWER PULSE
POWER = ENERGY TIME
POWERIS MAGNIFIED MANY-FOLD
Figure 2.2 How pulsed light works (Dunn et al., 1995; Pure Pulse, 1997a)
28
1.2 ---~-------'--l
E 1 s:::::
~ fi) 0.8 s:::::
S -+- PureBright s::::: 0.6 "C ___ Sunlight CI)
~----~--- ._.
N 0.4 .-ëU 1
E 1 ... 0.2
- ~ TT .--c-,: 0 z
0
R:>C) flI
C)C) ~
~C) ~
C)C) '\
~C) Oj
Wavelength (nm)
Figure 2.3 Comparison of PureBright pulsed light and sunlight (Pure Pulse, 1997a)
29
2.8.1 Antimicrobial effects
Dunn et al. (1997b) reported that a single pulsed light flash at 1-2 J/cm2 will ki1l6
log colony-forming units (CFU) of bacterial spores per square centimeter of inoculated
surface. They also showed that treatment with a few flashes to total fluence of about 4-6
J/cm2 will kill more than 7-8 log CFU/cm2 ofbacterial or mold spores and more than 9
log CFU/cm2 of vegetative cells. A previous study involving a micro drop assay for a
gram-positive pathogen (L. monocytogenes), a gram-negative pathogen (E. coli
0157:H7), aerobic bacterial spore (B. pumilus), and fungal conidiospores (A. niger)
showed no surviving organisms at concentrations of approximately 105 /cm2 using pulsed
light treatment of a single flash at 0.5-1 J/cm2, and 7-9 logs reduction per cm2 using a few
flashes at 1 J/cm2 per flash (Dunn et al., 1995). In addition, Rowan et al. (1998)
conc1uded that light pulses of low or high UV content were capable of reducing counts of
L. monocytogenes, S. enteritidis, P. aeroginosa, B. cereus, and S. aureus by up to 2 or 6
logs orders, respectively.
More recently, a study conducted by the XENON Corporation showed the ability
ofXENON's SteriPulse-XL 3000 equipment to kill bacterial endospores of Bacillus
subtilis. They concluded that the killing was rapid (1 second or less) and reduced
viability by a significant factor when starting with spore suspensions at 108 or 107 spores
per mL; it was possible to completely eliminate viability with three pulses of UV light. It
must be noted that the equipment used in this study is reported to be 50,000 to 100,000
times stronger than sunlight at sea level (XENON Corporation, 2003).
The antimicrobial effects of UV wavelengths in the pulsed light spectrum are
primarily mediated through absorption by highly conjugated carbon-to-carbon double
bond systems in proteins and nuc1eic acids (Barbosa-Canovas et al., 2000; Dunn et al.,
1995). "The mode of action of the pulsed light process is attributed to unique effects of
the high peak power and the broad-spectrum of the flash" (Barbosa-Canovas et al., 2000).
Nucleic acids are a primary cellular target. Chemical modifications and cleavage of
DNA are two of the several mechanisms of inactivation. It is presumed that the impact of
pulsed light on proteins, membranes, and other cellular material occurs concurrently with
nucleic acid destruction. The combination of high energy and intensity of pulsed light
30
amplify the known mechanisms of destruction of cellular components caused by
individual wavelengths of light. Therefore, the sum of the damage caused by the broad
spectrum light is thought to produce extensive irreversible damage to DNA, proteins, and
other macromolecules (Barbosa-Canovas et al., 2000). The proposed mechanisms of
action of pulsed UV light in macromolecules mentioned above are as follows:
• DNA: demonstration of strand breaks and di mer formation in vivo and vitro
• RNA: single stranded breaks and formation of dimmers
• Proteins: peptide bonds not broken; inactivation of enzyme activity controlled
or minimized by controlling the delivery of critical parameters (XENON
Corporation, 2003).
The antimicrobial effect of pulsed light is significantly greater than that of
conventional UV sources or high-intensity mercury vapor lamps. A few pulsed-light
flashes kills high levels of all exposed organisms. For example, it was reported that when
A. niger, a common bread mold that is relatively resistant to conventional UV was
sprayed onto packaged material, conventional UV exposure produced about 2.5-4.5 log
reduction in CFU during the first 3-10 seconds oftreatment (Cerny, 1977). However,
longer exposure times did not significantly increase the effect of UV light. On the other
hand, a few flashes ofpulsed light applied in a fraction of a second resulted in 7 log
CFU/cm2 reduction of A. niger in the packaged bread. This study confirmed previous
results which showed that pulsed light resulted in greater reduction in microbialload in a
much shorter time compared to conventional static UV methods (Dunn et al., 1997b).
2.8.2 Sterilization ofpackaged products
Many plastics transmit light efficiently and the final packaged product may be
subjected to treatments through the se materials. Examples of such plastics are:
polyethylene, polypropylene, nylon, ethylene vinyl acetate and ethylene vinyl alcohol,
and many other non-benzene based plastics. Polyaromatic hydrocarbon-rich plastics, for
example polyethylene terephthalate, polycarbonate, polystyrene, and polyvinyl chloride,
do not generally transmit pulsed light well enough to allow the treatment of products
through such materials (Dunn et al., 1997b).
31
Another useful application of pulsed light in packaged products is for
blowlfiU/seal (BFS) packaging systems that produce asepticaUy filled containers for
dispensing a variety of products. Dunn et al. (1997a) investigated the effect of pulsed
light on BFS packages inoculated with organisms highly resistant to traditional
sterilization methods (Bacillus pumilus spores, B. subtilis strain niger variety globigii
spores, B. stearothermophilus spores and A. niger spores). They concluded that no viable
organisms were recovered from the treated samples. These results demonstrated that
pulsed light was more effective than traditional sterilization methods to inactivate
resistant microorganisms treated through the package.
2.8.3 Use in foods
As was mentioned previously (Section 2.8), pulsed light will not sterilize foods,
but it is effective in reducing the microbial population, particularly on the surface of
baked goods, seafood and meats, fruits and vegetables, and many other foods have shown
significant reduction in their microbialload, enhanced shelf-life, added safety, with no
change in their nutritional properties after pulsed treatment (Dunn et al., 1995). A
considerable amount of work on the effects of pulsed light on various food products has
been done and the results of these studies are summarized below (Dunn et al., 1997b;
Dunn et al., 1995). However, there is still a lack of research on pulsed light as a post
packaging barrier.
2.8.3.1 Seafoods
Shrimp that was treated with pulsed light and stored under refrigeration
temperatures for 7 days remained edible, while untreated shrimp showed extensive
microbial spoilage, were discolored and fouI smelling, and were no longer edible. Pulsed
light also resulted in a significant extension of both microbial and sensory shelf life of
fresh fish fiUets.
32
A recent study has shown the ability of PL to inactivate 1 log of E. coli 0157:H7
and L. monocytogenes Scott after 60 s treatment at 8 cm from the PL source, without
affecting the quality (Ozer and Demirci, 2004).
However, there is lack of research on pulsed light as a post packaging technology
to control pathogens, such as L. monocytogenes and C. botulinum, in smoked fish
products. Inactivation of L. monocytogenes by pulsed light will be the primary focus of
this work.
2.8.3.2 Meats
Extensive studies conducted on a variety of meats have shown that pulsed light
can be used to enhance product shelf-life and safety. It was concluded that pulsed light
reduced the counts ofall microbial types by 1-310gs on a variety ofmeats. The main
disadvantage with meat is that its uneven surface allows sorne microorganisms to avoid
exposure to pulsed light resulting in higher counts than on media, packaging materials, or
relatively simple surfaces.
When frankfurters were inoculated with Listeria spp. (3 and 5 CFU/g), the
microbialload was reduced by ~ 2 logs using pulsed light. Nutrient analysis (prote in,
riboflavin, nitrosamine, benzpyrene, and vitamin C) performed on samples showed no
differences between pulsed light and untreated samples, even when over-treated samples
(30 J/cm2 total treatment) were analyzed. Ofparticular concem is riboflavin, which is
extremely sensitive to degradation by light, heat and oxidation. Furthermore, a study of
beef, chicken and fish showed that even excessive pulsed light treatment did not
influence riboflavin concentration.
Pulsed light also has a significant bene fit in terms of microbial and sensory shelf
life on retail beef. Total aerobic, lactic, enteric, and Pseudomonas counts were decreased
by about 1-3 logs using pulsed light treatment.
33
2.8.3.3 Baked goods
Essentially, baked goods normally come from the oyen mold-free. Mold spoilage,
however, occurs as a result of post-baking contamination from the bakery atmosphere
and equipment, such as slicing machines, and bakery personnel.
A study was performed on commercially prepared off-the-shelf bread loaves
divided into two and placed in polyethylene bags. Samples were treated through the bag
(3 pulsed light flashes at 1 J/cm2/flash). Mold appeared on untreated samples after 5-7
days at room temperature, and the samples very moldy by day Il. Treated samples,
packaged and stored under similar conditions were mold-free for the duration of storage.
Preservative-free breadsticks (i.e. without calcium propionate and potassium
sorbate) packaged in low-density polyethylene bags were treated with eight flashes of
pulsed light at 0.5 J/cm2 at three positions along the long axis of each breadstick with
1200 radial rotation in between treatment positions. Approximately 17% of the control
breadsticks showed visible molding by day 6 while 100% were moldy by day 7.
However, less than 4% of the treated breadsticks showed visible signs ofmold by day 13,
and no further mold growth occurred throughout the 26 days of storage.
Cream-filled chocolate cupcakes frosted with chocolate and a white frosting stripe
were used. The cake portion was reformulated to be preservative-free, however the
normal preservative-containing cream filling was used because it is assumed that the
cream filling would be unaffected by the pulsed light treatment, due to the depth of
penetration. Cupcakes were not directly inoculated, but the location where they were
cooled and packaged resulted in natural contamination by mold spores. Samples were
treated with eight flashes at 0.5 J/cm2/flash approximately 14 cm below the unit.
Cupcakes were treated at various angles to the incident light, i.e. similar to breadsticks.
Approximately 6% of control samples had mold growth by day 8, 53% by day 12,82%
by day 15, and 94% by day 19. A different trend was observed for pulse treated samples,
with 8% showing mold growth by day 15,18% by day 19, and 58% by day 23. The test
showed that treatment with pulsed light provided an approximate 10-day extension in
mold-free shelf-life of cupcakes.
34
Commercially pre-baked pizza crusts are often gas packaged in a high gas barrier
pouch. This is a very effective way of preserving the crusts as aerobic molds cannot
survive in an environment that is oxygen free. However, this means of packaging is very
expensive. Therefore, the application of pulsed light has the potential to substantially
decrease packaging costs. Samples were removed from their commercial packaging and
placed in polyethylene pouches under atmospheric conditions. Samples treated with
pulsed light, both on top and bottom to provide complete exposure, showed no mold
growth, while mold growth was evident in untreated samples. This suggests that
integrating pulsed light treatment in the packaging process may allow the use of less
expensive packaging systems.
A similar test was performed on pre-baked pizzas, with approximate composition
of 60% dough, 18% tomato sauce, and 18% cheese by weight. Pizzas were inoculated by
environmental exposure for 15 minutes on either side. The pizzas were sealed in
polypropylene pouches and divided. The treated samples were processed through the
pouch with an exposure of 12 flashes at 0.5 J/cm2. By day 30, greater than 80% of the
untreated samples showed visible signs of mold growth. However, mold growth was
detected visually on only one of the pulsed light treated samples after 30 days storage at
7°C.
2.8.3.4 Miscellaneous
Pulsed light treatment has been used in other food stuffs, such as milk, honey and
cornmeal. A logJO reduction obtained in milk varied from 0.16 to 8.55 10gJO CFU/mL
demonstrating the ability of pulsed UV -light to inactivate Staphylococcus aureus
(Krishnamurthy et al., 2004b). In another study, PL treatment enhanced the inactivation
of Clostridium sporogenes up to 90% in honey, however it failed to inactivate the spores
completely due to insufficient penetration ofUV-light (Hillegas and Demirci, 2003).
Lastly, a maximum of 4logJO reduction of Aspergillus niger was achieved in corn me al
after a 100 s treatment time at 3800 V and 8 cm from the PL source (Jun et al., 2003).
However, treatment was limited because sample temperature increased beyond 120°C,
leading to changes in food properties and quality.
35
2.8.3.5 Water
Pulsed light is also an effective treatment for water destined for use in the food
industry. Laboratory tests performed to model the potential effectiveness of a pulsed
light water treatment system show high inactivation levels of Klebsiella terrigena,
Cryptosporidium parvum oocysts, and other microorganisms in water suspensions. These
organisms were chosen for testing because oftheir relative importance for water
treatment. Two pulsed light flashes at less than 0.5 J/cm2 per flash resulted in no viable
counts of K terrigena from a suspension initially containing 6 or 7 10gs/mL.
Suspensions also containing 6 or 7 logs/mL of C. parvum oocysts were rendered
noninfectious by one pulsed light flash at 1 J/cm2.
2.8.4 Status and Economics
As discussed previously, pulsed light technology is very effective at eliminating,
or reducing the microbial population on surfaces and in transparent media, such as water.
Although its effect on opaque and irregular surfaces is not as evident, a 1-3 log reduction
in total microbial counts could result in significant shelf life extension of a product and
may also enhance its safety.
Pulsed light has been approved by the FDA. The agency conducts studies to
consider changes in chemical composition of the food that may be induced by the
proposed treatment, including any potential changes in nutrient levels. The agency
concluded that the treatment is effective in reducing the numbers of microorganisms on
the surface of treated foods and that treated foods will be at least as safe, from a
microbiological standpoint, as untreated foods that are currently on the market (Dunn et
al., 1997b).
Pulsed light treatment costs are also very favorable. Conservative estimates for
pulsed light used at 4 J/cm2, including equipment costs, electricity and maintenance, yield
costs of less than a few tenths of a cent per square food of treated area (Dunn et al.,
1995).
36
2.9 Conclusions
Pulsed light treatment is an effective, economical, safe, FDA-approved food
preservation technology capable of being used in food processes at high online
throughput rates. It can also be used to treat packaging materials, bulk products, water,
air, and surfaces. The results suggest that pulsed light processing can also be an effective
preservation method for new products and packaging systems.
L. monocytogenes continues to be a problem as a post packaging contaminant of
cold smoked salmon, primarily due to post processing contamination in the
manufacturing facility. There have been no reported outbreaks ofbotulism from RTE
cold smoked salmon, but C. botulinum type E continues to be a regulatory concern in
these products.
With respect to cold smoked salmon, high intensity pulsed light has the potential
to: (i) be an effective post packaging barrier to reduce the levels of L. monocytogenes in
cold smoked salmon; (ii) enhance the safety of cold smoked salmon while minimizing
changes in organoleptic characteristics; (iii) further extend market areas and open up new
markets for cold smoked salmon; (iv) satisfy consumers' and regulatory agencies
concerns for safe, convenient minimally processed seafood products; and (v) keep
process costs low (~1-4 cents/m2 ofproduct).
37
CHAPTER3
CHARACTERIZATION OF PULSED LIGHT APPARA TUS WITH LISTERIA MONOCYTOGENES
3.1 Introduction
Pulsed light (PL) treatment is a new and emerging technology that involves the
use of intense and short duration pulses ofbroad-spectrum "white light". The spectrum
of light for pulsed light treatment includes wavelengths in the ultraviolet (UV) to the near
infrared region. This technology is applicable mainly in sterilizing or reducing microbial
populations on the surface of packaging materials, transparent pharmaceutical products,
or other surfaces (Dunn et al., 1997b). However, pulsed light may also be used to extend
the shelf-life or improve the quality of produce.
Before embarking on destruction kinetics and challenge studies with Listeria
monocytogenes, a common post-processing contaminant in vacuum packaged (VP) cold
smoked salmon, it was found desirable to characterize the pulsed light system and
treatment conditions. The effectiveness of high intensity pulsed light treatment depends
on several factors related to the equipment, process, product and target microorganism.
Prior to its use, several of these factors need to be standardized. The intensity of a light
and its effect depends on its initial power and distance of the target from the light source.
This chapter deals with evaluation/characterization of the spatial power distribution in the
pulsed light apparatus using severity of destruction of Listeria monocytogenes subjected
to the PL treatment.
The specific objectives ofthis study were to:
1) Evaluate the temperature profile for the pulsed light system over a five minute
treatment period (to make sure the treatment did not result in heat damage to the
product);
2) Calibrate the pulsed light unit with 5 strains (from marine sources) and a mixed
culture of L. monocytogenes (to select the most resistant strain);
38
3) Investigate the effect of depth, intensity and treatment time on the distribution of
pulsed light in the treatment chamber (to optimize the treatment).
3.2 Materials and methods
3.2.1 Pulsed light equipment
A bench-top pulsed UV system fitted with a low-pressure xenon flash lamp
(Model PUV-OI, Magnavolt Technologies Inc., Plattsburgh, NY, USA) was used to
subject samples to pulsed light treatment (Figure 3.1). In principle, the electric power is
magnified by accumulating electrical energy in an energy storage capacitor over
relatively long times (for example, a decimal fraction of a second) and releasing this
stored energy to do work in much shorter times (milliseconds or nanoseconds). This
results in very high power during the short dut y cycle with the expenditure of only
modest average power consumption.
PL treatments were achieved by varying four of the PL parameters: treatment
time (0-60 s), voltage intensity (0-1000 volts), pulse frequency (0.1-1 pulse per second
(Pps)), and distance from the PL source (5-35 cm). Temperature at different locations
was monitored with Reversible Temperature Labels placed directly beside treated
samples (Multi Temp Liquid Crystal Strips, models RLC series 60, OMEGA Engineering
Inc., Stamford, CT, USA). Once the temperature in the unit reached 5°C above ambient
temperature, the unit was flushed with cool air supplied by a fan until ambient conditions
were re-established.
3.2.2 Temperature profile
Pre-sliced frozen cold smoked coho salmon (Oncorhynchus kisutch) filets
(Fumoir Grizzly, St. Foy, Quebec) stored at _20°C were defrosted in a refrigerator
overnight. Slices were placed into 210 x 210 mm moderate barrier bags (OTR = 4,000
cc/m2/day/atm @ 24°C, 0% RH) (Cryovac Sealed Air Corporation, Mississauga, Ontario)
39
and vacuum packaged using a Multivac chamber-type, heat seal packaging machine
(Model 300A/42).
A Reversible Temperature Label (Multi Temp Liquid Crystal Strips, models RLC
series 60, OMEGA Engineering Inc., Stamford, CT, USA) was placed directly on top of
refrigerated packaged samples before PL treatment. Duplicate samples were evaluated
for temperature increases every 15 seconds over a 5 minute period at 600 and 800 V, 5
cm from the PL source, at 1 pps. The temperature inside the chamber was also evaluated
at 600 and 800V.
3 .2.3 Bacterial strains
Five strains of Listeria monocytogenes; HPB strains Scott A (smoked salmon
isolate), 323 (shrimp iso1ate), 392 (lobster isolate), 439 (crab isolate) and 976 (smoked
salmon isolate) were obtained from the Microbial Hazards Bureau (Health Protection
Branch (HPB» Health Canada, Ottawa, Canada). The cultures were maintained frozen at
-20°C (700 ilL of a 24 h culture grown in TSB (tryptic soy broth) + YE (yeast extract)
(Difco) with 300 ilL of a 50% (v/v) glycerol solution).
A loopful of the above culture was streaked onto tryptic soy agar (TSA, Difco)
plates and incubated at 3YC for 48 h. Isolated colonies were then transferred to a tube
containing 9 mL oftryptic soy broth (TSB, Difco) supplemented with 0.6% yeast extract
(TSB/YE) and incubated for approximately 12 h at 35°C to give a suspension of
approximately 1x109 CFU/mL. Inoculums were prepared using this culture which was
further diluted with the appropriate volume of 0.1 % peptone water to achieve the desired
inoculum concentration (10'-109 CFU/mL).
AlI sample preparations and inoculation were carried out in a Purifier™ Class II
Safety Cabinet (Labconco, Model #36205-04, Labconco, Kansas, MI, USA) equipped
with a HEP A filter to ensure minimal contamination of the sampI es and the surrounding
environment as well as the safety of the research personnel.
40
Discharge cable
Pulse generator
Treatment cham ber
Figure 3.1 Schematic diagram of the pulsed light apparatus used in this study
41
3.2.4 Inoculation and pulsed light treatment
3.2.4.1 Relative resistance of different strains of L. monocytogenes to PL
treatment
Room temperature solid tryptic soy agar (TSA, Difco) Petri plates were
inoculated by spreading 0.1 mL ofa 1x107 CFU/mL suspension of L. monocytogenes
(Scott A, 323, 392, 439, 976 and a mix of all five (in equal volumes)) evenly onto each
plate using a sterile hockey stick, to give a final inoculum level of 1x1 06 CFU/mL.
Control samples were inoculated in a similar fashion with sterile 0.1 % peptone water.
The plates were then subjected to the following PL treatments under the center of the
flash lamp, with their lids removed: 600 or 800 V; 5, 10 or 15 cm distance from the PL
source, for 20, 40 or 60 s at 1 pps. It must be noted that previous results (not shown)
concluded that 1 pps was the ideal and most efficient pulse frequency (rather than 0.1 or
10 pps).
3.2.4.2 Spatial power distribution (12 plate experiment)
Petri plates were inoculated as above (3.2.4.1), however only L. monocytogenes
Scott A was used. Twelve inoculated plates were placed on the treatment tray as shown
in Figure 3.2 and subjected to the following PL treatments, with their lids removed: 600
or 800 V, 5, 10 or 15 cm distance from the PL source, for 60 s at 1 pps.
3.2.5 Analyses
Following PL treatment (sections 3.2.4.1-3.2.4.2), plates were incubated at 35°C
for 24 h. In order to facilitate these initial studies, a qualitative scale (Table 3.1) was
used to represent growth.
42
Figure 3.2 Position of Petri plates on PL chamber treatment tray in 12 plate experiment
43
Table 3.1 Qualitative scale to represent growth of L. monocytogenes
Growth Description of growth Approximate number of colonies
5 Lawn of growth TNTC 1
4 Extensive growth (distinct colonies) >300
3 Heavy growth (countable) > 300
2 Medium growth (countable) ~30
1 Light growth ~ 10
o No growth 0
ITNTC = too numerous to count
44
3.3 Results and discussion
3.3.1 Temperature profile
Figure 3.3 shows the temperature rise observed in the PL chamber over a 5
minute treatment period. The gray lines represent the increase in temperature observed
inside the treatment chamber (600 and 800V). In both cases, the chamber started at
ambient temperature, in this case 22°C. After the 5 minute treatment time, the
temperature inside the chamber increased slightly to 24 and 26°C, respectively.
For VP cold smoked salmon sarnples (black lines), the initial temperature
observed was 14-15°C. Over the 5 minute treatment period, the temperature increased to
26 and 28°C for 600 and 800 V treatment, respectively. Since cold smoked salmon is
typically smoked at temperatures below 30°C (Samuel, 1999) and it has been reported
that L. monocytogenes is capable of surviving the smoking process and is capable of
surviving at refrigeration temperature (~4°C) (Food and Drug Administration (FDA),
2001). The increase to 26 and 28°C observed during 5 minutes of PL treatment is
acceptable as L. monocytogenes will not be inactivated by thermal means.
It is important to investigate the increase in temperature in PL units, because as a
non-thermal process, PL is generally used to maintain the integrity ofvalue-added
products, such as co Id smoked salmon, while decreasing the possible risk associated with
consumption. A recent report showed that the temperature observed following PL
treatment ofraw salmon filets increased to as much as 85°C, for 60 s treatment (Ozer and
Demirci, 2004). At this temperature, both quality changes and microbial degradation will
occur in cold smoked salmon, which results in detrimental effect on the quality and hence
would compromise the integrity ofthis value-added product.
45
29
27
25
Ô 23 'L I!! :::s Ë 21 CIl Co E ~ 19
17
15
13 ---
o 2 3
Treatment time (minutes)
1
:--~-------~ 800V chamber
-40"' 600V chamber
..... 800V salmon
1
l ...... 600V salmon ---~--_._-----------_._-
4 5
Figure 3.3 Temperature profile of pulsed light chamber over a 5 minute treatment period, both on VP cold smoked salmon samples (black lines) and in the chamber itself (gray lines)
46
3.3.2 Relative resistance of different strains of L. monocytogenes to PL treatment
The qualitative growth of 5 strains and a mixed culture of L. monocytogenes
following PL treatment are shown in Table 3.2 and a representative figure (treatment at
600 V and 15 cm from the PL source) is shown in Figure 3.4. It was important to
investigate the possible differences in resistance of 5 strains of L. monocytogenes, aIl
from marine sources, to pulsed light treatment. From these results, if appropriate, a
representative/high resistance strain would be chosen to complete aIl experiments.
It can be seen in Table 3.2 and Figure 3.4 that strain Scott Ais generally equally
or more resistant to the other 4 marine-source strains tested in aU situations. Therefore
this strain was used in aIl experiments to foIlow.
These findings have also shown that in general, as the number of pulses is
increased (i.e. increasing the treatment time), microbial destruction is increased (Figure
3.4). Recently, pulsed UV inactivation of Staphylococcus aureus in milk showed an
increase of ~ 7 10gIO reduction when treatment time was increased from 30 to 180 s, while
holding distance from the PL source and volume constant (Krishnamurthy, 2004b).
3.3.3 Spatial power distribution (12 plate experiment)
Because the distribution of PL in the chamber varies at different distances from
the PL source, it was important to look at the exposed treatment area at 5, 10 and 15 cm
distance from the PL source. Figures 3.5 and 3.6 are photographs of the distribution of
PL at varying distances from the PL source at 600 and 800 V, respectively. The percent
surface area exposed increased as the distance from the PL source is increased. In order
to establish the inactivation of L. monocytogenes at each distance from the PL source, the
average kill of L. monocytgenes was calculated for plates inside the exposed area at the
respective distances from the PL source (Table 3.3). Figure 3.7 represents a model of the
effect of voltage (600 and 800 V) and distance (5, 10 and 15 cm) from the PL source on
the percent kill of L. monocytogenes by PL treatment.
47
Table 3.2 Growth (on a scale of 1 to 51) of 5 strains and a mixed culture of L. monocytogenes isolated from marine sources and pulsed light treated
Distance from pulsed light source = Sem
Voltage Time(s) Growth
Scott A 976 323 439 392 MIX 600 20 1 0.5 0.5 0.5 0.5 0.5
40 1 0 1.5 0 0.5 0.5 60 1 0.5 0.5 0 0 0.5
800 20 0.5 0.5 1 0.5 0 0.5 40 0.5 0 0 0 0 0 60 0 0 0 0 0 0
Distance from pulsed light source = 10cm
Voltage Time(s) Growth
Scott A 976 323 439 392 MIX 600 20 2 1 1 1 1 2
40 1 0 0 0.5 0 1 60 0 0 0 0.5 0.5 1
800 20 0.5 0.5 0 0.5 0 0.5 40 0 0 0.5 0 0 0 60 0 0 0 0 0 0
Distance from pulsed light source = IScm
Voltage Time(s) Growth
Scott A 976 323 439 392 MIX 600 20 5 4 3 4 4 5
40 1 1 0 1 1 1 60 1 1 0.5 0.5 1 1
800 20 1 1 1 0.5 1 1 40 1 1 0 0.5 0 0.5 60 0 0.5 0 0.5 0 0
1 Where 0 represents no growth and 5 represents extensive growth (see Table 2.1)
48
5
4.5
4
3.5
3 .s::. l o 2.5 ... (!)
2
1.5
1
0.5
0 20 40
Time (sec)
• Scott A .976 11323 11439 0392 OMIX
60
Figure 3.4 Sample representation of growth of 5 strains and a mixed culture of L. monocytogenes isolated from marine sources and pulsed light treated at 600 V and 15 cm distance from the pulsed light source
49
The regression equation which has been fitted to the data is shown below:
% Kill = 132.333 - 0.0183333V - 14.4D + 0.014VD
R2 = 94.8456
where V is the voltage and D is the distance with the values of the variables specified in
their original units.
From the se results, optimum maximum and minimum values were determined for
percent kill of L. monocytogenes. A maximum value (100% kill) would be achieved at
800 V and 5 cm distance from the PL source. A minimum value (31.3%) would be
achieved at 600 V and 15 cm distance from the PL source. These finding justify that the
lethality of pulsed light increases with increasing light intensity or fluence (Pure Pulse,
1999).
Serious consideration of sample surface area must be taken into account before
voltage and distance from the PL source are selected. If the surface area of a sample fits
into the exposed treatment area of the pulse co ne at 5 cm, a lower voltage may be used;
compared with a large sample that would require treatment at 15 cm and a higher voltage.
50
Table 3.3 Percent kill of L. monocytogenes at various treatment voltages and distances from the PL source
Treatment voltage (V)
600
800
Distance from PL source (cm)
5
10
15
5
10
15
Surface area exposed to PL treatment (%)
33
75
92
33
75
92
Average kill of L. monocytogenes in the
exposed treatment area (%)
93
58
33
100
89
68
51
5cm 10 cm 15 cm
Figure 3.5 Distribution of PL at varying distances from the PL source (600V, 60 s treatment)
5em 10cm 15 cm
Figure 3.6 Distribution of PL at varying distances from the PL source (800V, 60 s treatment)
~111 ~ .~
~ 91
~ (l) 71 u È) 51 ~
31 60~0~64~0~~~~--~ 680 720 760 800
1315 9 11
5 7 · D1stance Voltage
Figure 3.7 Effect ofvoltage and distance from the PL source on the percent kill of L monocytogenes by PL treatment
54
3.4 Conclusions
This chapter focused on characterization of the pulsed light apparatus with L.
monocytogenes. Results found that of 5 strains of L. monocytogenes isolated from
marine sources, strain Scott A was equally, if not more resistant than the other strains to
PL and was therefore used in the remaining studies. In addition, an increase in treatment
time (also the number of pulses received, at 1 pps) increased the microbial destruction.
The findings from the effect of voltage and distance from the PL source on the
inactivation of L. monocytogenes showed that as voltage increases from 600 to 800 V,
microbial inactivation is increased. As the distance from the PL source is increased
(from 5 to 15 cm), the percent kill of L. monocytogenes is decreased as the intensity of
the PL is decreased (as was seen in the photographs). Therefore, it is imperative when
choosing PL parameters (for the Magnavolt PUY -01 in this case), to consider the sample
surface area.
From the results ofthis chapter, a combination of 800 V, 5 cm distance from the
PL source for 60 s at 1 pps was recommended, whenever possible, for future
experiments, to maximize microbial destruction.
55
CHAPTER4
PULSED LIGHT DESTRUCTION KINETICS OF LISTERIA MONOCYTOGENES (SCOTT A)
4.1 Introduction
Fresh fish is a highly perishable commodity and its shelf-life is limited by
microbial spoilage and/or enzyme activity. The smoking process reduces the water
activity and impregnates preservatives into the flesh offish, both ofwhich reduce the
degree ofmicrobial spoilage. However, vacuum packaged (VP) cold smoked fish is a
ready-to-eat product that is not heated prior to consumption and is susceptible to spoilage
by L. monocytogenes, which is capable of surviving the cold smoking process and
growing at refrigeration temperatures (~4°C) (Cortesi et al., 1997; Rocourt et al., 2000;
Rorvik, 2000; Rorvik et al., 1995). Therefore, additional safety measures, such as pulsed
light (PL) treatment, are needed to ensure the continued safety ofVP co Id smoked
salmon.
The previous chapter focused on the characterization and optimization of the
pulsed light apparatus for treatments used in this study. These results were used as a
guideline to determine the proper treatment procedure for evaluating the destruction
kinetics of L. monocytogenes.
Unfortunately, very little research has been conducted using PL treatment in food
stuffs, as this treatment method is still in its infancy. In terms of PL treatment offish,
Dunn et al. (1997b) reported that PL treatment offish fiUets yields a significant extension
ofboth microbiological and sensory shelf-life. More recently, a study demonstrated that
one log reduction of E. coli 0157:H7 and L. monocytogenes Scott A could be achieved
after 60 s treatment at 8 cm distance from the PL source without affecting the quality of
fresh salmon fiUets (Ozer et al., 2004). However, there is a lack ofresearch of PL
treatment of cold smoked salmon. The objectives ofthis study were to evaluate the
pulsed light destruction kinetics of L. monocytogenes:
1) in a liquid medium treated through a packaging film;
56
2) inoculated on the surface of general purpose agar plate; and
3) inoculated directly on the surface of cold smoked salmon.
4.2 Materials and methods
4.2.1 Bacterial strains
The bacterial cultures were prepared as described in section 2.2.3.
4.2.2 Inoculation and pulsed light treatment
4.2.2.1 L. monocytogenes in liquid media in Whirl Pak bags
A 20 mL aliquot of a 0.1 % peptone water solution containing 1 x 1 08 CFU/mL L.
monocytogenes Scott A was aseptically transferred to a 2 oz transparent polyethylene
sampling bag (Whirl Pak, Fisher Scientific, Ottawa, ON, Canada) and heat sealed.
Control samples were packaged in a similar fashion with 0.1 % peptone water. The 20
mL aliquot represented a sample thickness of approximately 7 mm in the sealed Whirl
Pak bags. AH samples, in duplicate, were PL treated in the center of the sample tray
(directly under the flash lamp) at 5 cm from the flash lamp, 800 V. Samples were
subjected to treatment times from 0 to 120 seconds at 1 pulse per second (pps).
4.2.2.2 L. monocytogenes on surface of a solid general purpose media
Room tempe rature solidified tryptic soy agar (TSA, Difco) Petri plates were
inoculated by spreading 0.1 mL ofvarying concentrations of L. monocytogenes Scott A
evenly onto each plate using a sterile hockey stick. Control samples were inoculated in a
similar fashion with sterile 0.1 % peptone water. The plates, with their lids removed,
were then subjected to the following PL treatments under the center of the flash lamp:
600, 700 or 800 V, 5 cm distance from the PL source, 1 - 10 s treatment time at 1 pps.
57
4.2.2.3 L. monocytogenes on surface of cold smoked salmon
Pre-sliced frozen cold smoked coho salmon (Oncorhynchus kisutch) filets
(Fumoir Grizzly, St. Foy, Quebec) stored at -20°C were defrosted ovemight in a
refrigerator. Slices were trimmed to 70 x 70 mm squares and placed into 210 x 210 mm
moderate barrier bags (OTR = 4,000 cc/m2/day/atm @ 24°C, 0% RH) (Cryovac Sealed
Air Corporation, Mississauga, Ontario). Samples were inoculated by spreading 1 mL of
culture suspension evenly onto each cold smoked salmon sample using a sterile hockey
stick to give a final inoculum level of approximately 104 CFU/50 cm2 (exactly 6. 9x 1 04
CFU/50 cm2). Control smoked salmon samples were inoculated in a similar manner with
the same volume of 0.1 % sterile peptone water.
Samples were vacuum packaged and heat sealed using a Multivac chamber-type,
vacuum packaging machine (Model300A/42) and subjected to pulsed light treatments at
800V,5 cm distance from the PL source for 1-5 min per side at 1 pps.
The above experiment was repeated without the vacuum packaging step. Instead,
70 x 70 mm cold smoked salmon samples were placed on sterile sheets of aluminum foil
and inoculated in the same way with 0.1 mL of culture suspension to give a final
inoculum level ofexactly 3.06x104 CFU/50 cm2• As before, control samples were
inoculated in a similar fashion with 0.1 mL of 0.1 % sterile peptone water. Samples were
performed in duplicate. These unpackaged samples were PL treated the same way as in
the above experiment but treated for 5 and 10 min.
4.2.3 Analyses
4.2.3.1 L. monocytogenes in liquid media in Whirl Pak bags
Following PL treatment, Whirl Pak bags were aseptically opened and 1 mL
aliquots were transferred to tubes containing 9 mL of 0.1 % sterile peptone water and
further dilutions were then made, again using 0.1 % sterile peptone water. The samples
were enumerated for L. monocytogenes by plating 0.1 mL of the appropriate dilutions on
tryptic soy agar (TSA, Difco Laboratories, Detroit, MI) by the spread plate method. TSA
58
plates were incubated at 35°C for 24 h. AU plates were do ne in duplicate. Countable
plates (30-300 colonies) were reported as IOglO Colony Forming Units per mL of sample
(lOglO CFU/mL).
4.2.3.2 L. monocytogenes on surface of solid general purpose media
FoUowing PL treatment, plates were incubated at 35°C for 24 h. Countable plates
(30-300 colonies) were reported as IOglO CFU/mL.
4.2.3.3 L. monocytogenes on surface of co Id smoked salmon
FoUowing PL treatment, packaged samples were asepticaUy opened. From this
point forward, aU packaged and unpackaged samples were analyzed in the same way.
The smoked salmon piece was placed in a sterile stomacher bag and blended with twice
its weight of 0.1 % sterile peptone water in a Stomacher (Model 400, A.J. Seeward,
London, UK) for 1 min. One mL ofthis slurry was then added to tubes containing 9 mL
of 0.1 % sterile peptone water and further dilutions were then made, again using 0.1 %
sterile peptone water. AU samples were performed in duplicate.
The inoculated samples were enumerated for L. monocytogenes by plating 0.1 mL
of the appropriate dilutions on Oxford agar (Oxoid) supplemented with Listeria Selective
Supplement (SRO 140E). Control samples were enumerated in a similar fashion. AU
plates were incubated at 35°C for 24-48 h. Countable plates were reported as 10glO
CFU/50 cm2).
4.2.3.4 Decimal reduction times and voltage sensitivity
The pulsed light destruction kinetics of L. monocytogenes were analyzed based on
a first-order reaction indicating a logarithmic order of death, and expressed as:
59
where N is the number of organisms that survived the PL treatment for time t (min), No is
the initial number of microorganisms present before PL treatment and k is the reaction
rate constant (min-1). The treatment time at any given voltage resulting in 90%
destruction of the existing microbial population, is referred to as the decimal reduction
time (D-value). This was obtained as the negative reciprocal si ope of the log (N1N0) vs.
time, expressed as:
where NI and N2 represent survivor counts at time tl and t2, respectively. Since the
survivor curve can have a lag period, this must be taken into account while using the data
for practical applications.
The voltage dependence of the kinetic parameters D-value was analyzed by
voltage z-value (zv). The voltage sensitivity parameter ofD-values (the voltage z-value,
Zy) is determined by plotting the decimallogarithm ofD-values vs. treatment voltage.
From the regression slope oflog (D-values) vs. voltage, Zy was determined as the
negative reciprocal slope:
where DI and D2 represent decimal reduction times at voltages VI and V2, respectively.
The Zy value of the treatment represents the voltage range that results in a 10 fold
change in D-value.
4.3 Results and discussion
4.3.1 L. monocytogenes in liquid media in Whirl Pak bags
The survival curve for L. monocytogenes in liquid media is shown in Figure 4.1,
which represents a small reduction in counts after 120 s oftreatment at 800 V. The figure
60
demonstrates a good fit of data for the first order model (R2 = 0.97) which suggests that
the destruction of L. monocytogenes follows the logarithmic destruction.
A decimal reduction time of 93 s resulted from the PL treatment. It must be noted
that due to the limited penetration of PL treatment, the samples, which had a thickness of
approximately 7 mm, were too thick to achieve a reasonable rate of destruction.
Secondly, the penetration power of the light gets attenuated and as in most cases drops in
exponential form. Although there could have been substantial reduction in counts at the
surface, the survivor in the deeper layers would dominate the total counts when mixed.
For example, even if 80% of the cells in the light's path get destroyed by severallog
cycles, leaving the other 20% in the central region unaffected, the overall count will still
be in the same logarithmic scale as in the beginning. This means the overall reduction
would be less than one log cycle. Obviously, it would be desirable to use the liquid in
much thinner profiles so that the destruction occurs throughout.
Difficulty with PL treatment in thick and opaque samples has been well
documented by Dunn et al. (1995,1996, 1997a, 1997b). More recently, a study
monitoring the ability of PL to inactivate Staphylococcus aureus in milk showed that the
10glO reduction decreased as the sample volume increased from 12 to 48 mL
(Krishnamurthy,2004b). They also observed that at lower volumes, there is a rapid
increase in 10glO reduction for increase in treatment time, when compared to higher
volumes due to poor penetration capacity of UV -light. This group also observed that the
opacity of the sample plays a vital role in activation. They reported that a 7 to 8 log
reduction of S. aureus in O.1M phosphate buffer was achieved after 5 s treatment,
whereas a similar log reduction in milk required 3 min of PL treatment (Krishnamurthy,
2004a).
4.3.2 L. monocytogenes on surface of solid general purpose media
The survival curves for L. monocytogenes on the surface of the solidified agar
plates at 600, 700 and 800 V are shown in Figure 4.2, which indicates that the destruction
was significantly influenced (P < 0.05, by comparison ofmeans) by the voltage used and
the treatment time. The survival curves at higher voltages (800 V) were steeper than at
61
lower voltages (600 V) which illustrate that the destruction rate of L. monocytogenes is
higher at higher voltages.
The decimal reduction times for 800, 700 and 600 V were 0.91, 1.37 and 2.25 s,
respectively. It must be noted that D-values were computed by not taking into account an
initiallag period (between 4 and 7 sec), only after which a first order rate of destruction
was observed. The associated lag periods must be used along with the respective D
values in order to have a meaningful prediction of the pulsed light destruction power.
The lag times and associated D values are summarized in Table 4.1. For example, at
800V, the associated D value is 0.91 s and the lag is 4 s. So for achieving one decimal
reduction in counts, the treatment time would be the D value of 0.91 s plus the las of 4 s
for a total of 4.91 s. However, if 5 decimal reduction are required, the exposure time
would be (0.91 x 5) + 4 = 8.55 s.
Compared with a D-value of 93 s in liquid media (3.3.1), the D-values in this
experiment show that PL has an incredible potential as a surface sanitation method.
Surface inactivation of L. monocytogenes was monitored in a similar study that reported a
7 10glO reduction in L. monocytogenes, on the surface of a general purpose media,
following PL treatment for 512 s at 30 kV and 1 pps (MacGregor et al., 1998). The
longer treatment time required for the reduction in counts in MacGregor's study was due
to the lower stored energy in the pulse generator of3 J/pulse, versus 12.8 J/pulse utilized
in section 4.3.2.
Although the surface of the general purpose media used in this study was uniform
and dry, it does not necessarily represent the surface of co Id smoked salmon both in
texture and composition. Therefore, it was important to investigate the effect of PL on
the surface of co Id smoked salmon, which is not uniform and dry (discussed later in
section 4.3.3).
Lower D-values are associated with higher voltages demonstrating a higher
destruction rate. Figure 4.3 demonstrates the curve oflog (D-value) vs. treatment voltage
to compute the voltage sensitivity parameter (zv value). The Zv value of L.
monocytogenes was ca1culated to be 500 V, meaning that for every 500 V increase in
treatment voltage, the D-value would be reduced by 1 log cycle. A reference D-value
and z fully describes the pulsed destruction kinetics over the range of voltages studies.
62
6
3
2
o
Figure 4.1 pak bag
o
R2 = 0.9749 o value = 93 sec
20 40 60 80 100 120 Treatment time (seconds)
survival curve of L. monocylogenes in liquid media sealed in a 2
oz Whirl
63
~
~ t 7.00 u o 5 6.00 E::ï 'E
:::; 5.00 Ou. cO o 0
~ c5 4.00 bO c...J QI-g 3.00 o (,,)
2.00
1.00
, 1 1 1 , 1 1 1 , 1 1 1 1
1 1
" 1
i>eOOv
11111700V
A600V
x eoo after lag
:K 700 after lag
- 600 after lag
0.00 - -----~' --+_~-_'---_-,--------~---_.--, ------------ ----r--
o 5 10 15 20
Treatment time (sec)
Figure 4.2 Survival curves of L. monocytogenes on solid general purpose media
0.40
0.35
0.30
0.25 R2 = 0.9969 z value = 500V
œ 0.20 :l iii ~ 0.15 c
ë:5' 0
0.10 ...J
0.05
0.00
5 0 600 650 700 750 -0.05
-0.10
Treatment voltage (volts)
Figure 4.3 Decimallogarithm of D-values versus treatment voltages for L. monocytogenes on the surface of general purpose media
25
850
64
Table 4.1 Calculation of decirnal reduction tirnes (D-values) of L. monocytogenes on the surface of solidified agar plates
Treatment voltage D-value following Lag time (s)
(V) lag period (s)
800 0.91 4
700 1.37 5
600 2.25 7
1 PL exposure tirne for n decirnal reductions = (D x n) + lag tirne
Total time for one
D-value (S)l
4.91
6.37
9.25
65
4.3.3 L. monocytogenes on surface of cold smoked salmon
It must be noted that the packaging film used in this study, Cryovac film type
E300 (OTR = 4,000 cc/m2/day/atm @ 24°C, 0% RH), was tested with a pure culture of L.
monocytogenes to ensure that the packaging film did not absorb the UV light. Initial
testing showed that a 6 log reduction in L. monocytogenes resulted when a pure culture
was surface streaked onto TSA media, covered with a layer of E300 film and PL treated
at 800 V for 60 seconds at 1 pps. The transmission spectra of PL through said packaging
film is shown in Figure 4.4 (Xenon Corporation, Wilmington, MA, USA).
Figures 4.5 and 4.6 indicate very minimal destruction of L. monocytogenes during
the pulsed light treatment. Meaningful D values could not be obtained from these
figures. Based on averages over a five min period, approximate D-values of 19-24 min
for packaged and unpackaged salmon samples, respectively, were computed from the
curves for comparison purposes only. Unfortunately, aD-value ofthis magnitude (19-24
min) pales in comparison to a D-value of 4.91 s (inclusive oflag), as was seen in section
4.3.2 when L. monocytogenes was surface inoculated on the general purpose agar media.
In terms of industrial application of PL technology, it seems unrealistic to apply a PL
treatment of 19-24 min and then only get a 1 log reduction in L. monocytogenes, when a
2-3 log reduction would most probably be necessary to prolong the shelf-life of the co Id
smoked salmon.
The lack of destruction of PL treatment on the surface of co Id smoked salmon
may be explained for a variety of reasons. Firstly, the reduced water activity of co Id
smoked salmon may quickly absorb the liquid culture under the surface, and therefore
render the cells impenetrable by PL treatment. PL treatment is incapable of penetrating
opaque products, such as co Id smoked salmon, but it has been shown to reduce the
microbialload of such products in sorne previous studies (Dunn et al., 1997b). Secondly,
the chemical composition of cold smoked salmon may protect the culture from the effects
of PL treatment. The high fat content (6.5%), moderate water phase salt (1.5%) and
lactic acid bacteria may all protect L. monocytogenes. It has been reported that the
natural microflora of cold smoked salmon display a capacity to inhibit spoilage as well as
several strains ofpathogenic microorganisms, such as L. monocytogenes (Tomé et al.,
66
2006). Inhibition results from the production of natural preservatives such as organic
acids, hydrogen peroxide and diacetyl, and antimicrobials, such as bacteriocins (Nilsson
et al., 1999). Lastly, since the destruction of microorganisms are described on
logarithmic scales, as pointed out before, even if the PL treatment eliminated 50% of the
total amount of L. monocytogenes on the surface of the sample, presence of even small
amounts of residual volumes (of full concentration) in the sample will overly undermine
the total destruction. This is an important factor to take into consideration when
evaluating a processing technology.
67
Percent Transmission McGi11 University - Sample # 3 -- E300 Cryovac -OTR = 4000 cc/mA2/day
100
1 90
80
70
60)'
50
40t t
301
20+
101 i
k O~[ --__ ~----~--~--L_~ ____ _
200 300 400
Wavelength (nm)
500
Figure 4.4 Transmission spectra ofpulsed UV light through Cryovac E300 (OTR = 4,000 cc/m2/day/atm @ 24°C, 0% RH
600
4.90
4.80
4.70
4.60 ...... '" CI 4.50 -..
0 Z 4.40 -Z ~ 4.30 0 ...1
4.20
4.10
4.00
3.90
0 2 3 4 5
Treatment time (min)
Figure 4.5 Survival curve of L. monocytogenes on the surface of PL treated co Id smoked co ho salmon, packaged in a clear plastic film
4.6
4.55
4.5
","""
CI :r 4.45 o
Z -Z ~ 4.4 o ...1
4.35
4.3
4.25
o 5
Treatment time (min)
10
Figure 4.6 Survival curve of L. monocytogenes on the surface of PL treated co Id smoked coho salmon
69
Table 4.2 Summary oftreatment conditions for destruction kinetics of L. monocytogenes by PL treatment
Liquid media in Whirl Pak bag (20 mL)
Surface of general purpose media
Surface of packaged cold smoked salmon
Surface of unpackaged cold smoked salmon
Treatment voltage (V)
800
600
700
800
800
800
Lag time (s)
4
5
7
Decimal reduction time
(D-value)
93 s
0.91 s
1.37 s
2.25 s
19 min
24 min
Voltage sensitivity parameter (zv value)
500V
4.4 Conclusions
Destruction kinetics for L. monocytogenes were evaluated in a liquid media, on
the surface of a general purpose media and directly on the surface of co Id smoked salmon
following various PL treatments were studied. Table 4.2 shows a summary of results
from the various conditions and PL treatments. PL treatment showed incredible promise
to sanitize smooth and dry surfaces (a D-value of 0.91 sec at 800V, and a resulting Zv
value of 500 V) however, its penetration to thick and opaque samples was poor, as was
the case in the liquid media and on the surface of co Id smoked salmon (93 sec and 19-24
min, respectively).
It is now important to evaluate the effect of PL on the sens ory characteristics of
cold smoked salmon, as it is a value added product. In addition, the effect of PL on shelf
life at refrigeration temperature and moderate temperature abuse conditions is equally
important. These will be addressed in the next chapter.
71
CHAPTERS
CHALLENGE STUDIES WITH LISTERIA MONOCYTOGENES (SCOTT A)
PART A - Storage and challenge studies on pulsed light treated vacuum packaged cold smoked salmon stored at 4°C and l]OC
5.1 Introduction
It has been reported that the incidence of Listeria monocytogenes contamination
in both imported and domestic seafood in the United States is between 5 and 6%
(McCarthy, 1997). The Center for Science in the Public Interest (CSPI, USA) reported
that between 1987 to 1998, there were 112 Class 1 recalls for domestic or imported
ready-to-eat seafood products contaminated with L. monocytogenes. These observations
are important to the food industry and safety of public health as L. monocytogenes has
been found to survive, and even grow, at refrigeration temperatures.
This is especially of major concem with ready-to-eat products, such as cold
smoked salmon. The process of smoking fish imparts a degree of microbial stability, but
it is a function of several factors: the salt level obtained after brining, the amount of heat
applied, the inhibitory action of smoke, and the dehydrating effect of the smoking process
(Dodds et al., 1992). Consumer demands for a more delicate and palatable product have
led to reduced salting/drying and cold smoking of raw material, thus increasing the
microbiological risk (Rorvik et al., 1995).
Pulsed light treatment, a non-thermal processing technique, may be an attractive
option for smoked fish producers to reduce the number of outbreaks of RTE smoked fish
products. However, there is a lack of research on PL treatment as a post-packaging
technique for microbial pathogen control.
The objectives ofthis study were to monitor the effect of PL treatment on:
1) the change in color and odor in uninoculated VP cold smoked coho salmon stored
at refrigeration temperature (4°C);
72
2) the physical, chemical, sensorial and microbial changes in VP co Id smoked coho
salmon sarnples challenged with L. monocytogenes and stored at refrigeration
(4°C) and moderate temperature abuse conditions (12°C).
5.2 Materials and methods
5.2.1 Sample preparation
Pre-sliced frozen cold smoked coho salmon (Oncorhynchus kisutch) filets
(Fumoir Grizzly, St. Foy, Quebec) stored at -20°C were defrosted in a refrigerator
overnight. On sampling day, packages were aseptically opened and individual slices
(~20-30 g each) were separated for use.
5.2.2 Bacterial strains
The bacterial cultures were prepared as described in section 4.2.2.
5.2.3 Inoculation
In challenged samples, inoculation was do ne by spreading 0.1 mL of culture
suspension evenly onto one side of each cold smoked salmon slice using a sterile hockey
stick to give a final inoculum level of approximately 105 CFU/slice. In this study, control
smoked salmon samples were also inoculated with L. monocytogenes, but were not
subjected to PL treatments. AlI experiments were carried out in duplicate.
In the case of samples used for the salmon discoloration trial (unchallenged
samples), samples were not inoculated with L. monocytogenes.
5.2.4 Packaging conditions
Inoculated slices, with the uninoculated side down, were packaged in 210 x 210
mm moderate gas barrier bags (OTR = 4,000 cc/m2/day/atm @ 24°C, 0% RH) (Cryovac
73
Sealed Air Corporation, Mississauga, ON) on a commercial backboard. These
backboards are used in the smoked fish industry to pro vide structural integrity to the final
product. This backboard was made of a cardboard material coated with a foillaminate on
one side (the side that cornes into contact with the smoked salmon). AlI samples were
vacuum packaged using a Multivac heat seal packaging machine (Model300A/42).
5.2.5 Pulsed light treatment
5.2.5.1 Uninoculated samples
VP samples were PL treated individually in the center of the treatment tray placed
under the flash lamp. Treatment voltage, pulse frequency and distance from the PL
source were kept constant, at 800V, 1 pps and 5 cm, respectively; and samples were
treated for 20, 40 or 60 s. Control samples were not subjected to PL treatment. AlI
control and PL treatments were carried out in duplicate.
5.2.5.2 Challenge study
AlI samples were subjected to PL treatment, as above, at 800 V, 5 cm from light
source at 1 pps. AlI samples were treated for the same amount of time, 60 sec. Control
samples (which were challenged with L. monocytogenes) were not subjected to PL
treatment.
5.2.6 Storage conditions
5.2.6.1 Uninoculated samples
Following PL treatment, aIl samples were stored at 4°C. Duplicate samples were
used for each sampling day; analysis was performed on day 0, 2,5, 7, 9, 12 and 14.
74
5.2.6.2 Challenge study
Following PL treatment, one half of samples were stored at refrigeration
temperature (4°C), while the other halfwere stored at moderate temperature abuse
conditions (12°C). Duplicate samples were used for each sampling day; analysis was
performed at day 0, 7, 14,21 and 28.
5.2.7 Analyses
5.2.7.1 Sensory evaluation
On each sampling day, sensory analysis was performed under fluorescent light by
an untrained panel consisting of 3 panelists. Samples were coded and presented in
random order. Panelists were asked to evaluate cold smoked salmon color through the
unopened primary package.
Panelists were asked to use a five-point hedonic scale (5 = extremely desirable, 1
= extremely undesirable) to evaluate color. A score of 3 was regarded as borderline for
acceptability. Samples were then aseptically opened and panelists used the same five
point hedonic scale to rate the acceptability, in terms of odor. In aIl cases, fresh co Id
smoked salmon samples were provided as a control on each sampling day.
5.2.7.2 Microbiological analysis
On each sampling day, packaged samples were aseptically opened. The smoked
salmon piece (~20-30g) was placed in a sterile stomacher bag and blended with twice its
weight ofO.l% sterile peptone water in a Stomacher (ModeI400, A.l. Seeward, London,
UK) for 1 minute. One mL ofthis slurry was then added to tubes containing 9 mL of
0.1 % sterile peptone water and further dilutions were then made, again using 0.1 % sterile
peptone water.
On each sampling day, the inoculated samples were enumerated for L.
monocytogenes by plating 0.1 mL of the appropriate dilutions on Oxford agar (Oxoid)
75
supplemented with Listeria Selective Supplement (SR0140E). Control samples were
checked for the presence of 1. monocytogenes on each sampling day, using the method
described previously. AH samples (inoculated and controls) were monitored on each
sampling day for total aerobic counts as weH as for lactic acid bacteria counts. Total
aerobic counts were determined by plating appropriate dilutions on Tryptic Soy Agar
(TSA, Difco Laboratories, Detroit, MI) using a spread plate method. Background
microflora were enumerated using Lactobacillus MRS Agar (Difco Laboratories, Detroit,
MI) using the spread plate technique of the appropriate dilution. AlI counts were done in
duplicate. Oxford and TSA plates were incubated aerobically at 35°C for 24-48 h, while
MRS plates were incubated anaerobically at 35°C for 48 h in anaerobic jars (BBL
Microbiology Systems, Cockeysville, MD). Countable plates were reported as IOglO
Colony Forming Units per slice (IOglO CFU/slice).
5.2.7.3 pH measurement
The pH of each homogenized sample was measured in duplicate after
microbiological analysis using previously calibrated (buffer solution ofpH 4 and 7,
Fisher Scientific, Nepean, Ontario) Coming pH meter (ModeI220, Coming Glass Works,
Coming, NY).
5.3 Results and discussion
In the challenge study, values for color score, odor score, pH, and aIl
microbiological counts were analyzed for statistical significance by a one-way ANOV A
test (to compare the means of control versus PL treated samples). If the means were
statistically significant, a Duncan's test (pairwise multiple comparison) was performed.
A probability (P) of < 0.05 was considered to be significantly different.
76
5.3.1 Sensory evaluation
The color and odor scores were evaluated using a hedonic scale, ranging from 1 to
5. A score of 3 was considered to be the upper limit of acceptability, implying that the
sensory shelf-life was terminated when this score was reached.
The purpose behind color and odor evaluation of uninoculated co Id smoked
salmon slices was to determine if PL treatment alone had a significant effect on the
sensory quality. The color acceptability scores for PL treated uninoculated VP cold
smoked salmon are shown in Table 5.1. Control (untreated) and samples PL treated for
20 sand 40 s maintained a perfect color score of 5 during the 14 day storage period.
Samples treated for 60 s showed a graduaI decrease in color scores after 7 days.
However, at the end of the storage period, samples treated for 60 s had a color score of 4,
which was still above the upper limit for acceptability.
The odor acceptability scores are shown in Table 5.2. Both control and samples
PL treated for 20 s maintained a perfect score of 5 throughout the 14 day storage period.
Samples treated for 40 and 60 s showed a graduaI decrease in odor scores. However, as
was the case with the color scores, the odor scores were above the upper limit of
acceptability after the 14 day storage period. Therefore, PL treatment alone does not
significantly affect the sensory quality ofVP cold smoked salmon stored for 14 days at
4°C.
Changes in the general appearance (color and odor) of inoculated PL treated
samples and inoculated controls (which were not PL treated) (challenge study) stored at
4°C and 12°C are shown in Figures 5.1-5.4. Both at 4 and 12°C storage temperature,
there was no significant difference (P > 0.05) between color and odor scores for control
and PL treated samples.
At 4°C, color and odor scores remained within acceptable limits for the duration
of the 28 day storage period. At 12°C, a graduaI decrease was observed in color and odor
scores after 7 days of storage and the sensory-shelf life was terminated between days 21
and 28 for inoculated PL treated samples.
77
Table 5.1 4°C
Table 5.2 4°C
Color acceptability scores of PL treated VP cold smoked salmon stored at
Treatment time Days of storage at 4°C
(seconds) 0 2 5 7 9 12 14
0 5 5 5 5 5 5 5
20 5 5 5 5 5 5 5
40 5 5 5 5 5 5 5
60 5 5 5 5 4.5 4.5 4
Odor acceptability scores of PL treated VP cold smoked salmon stored at
Treatment time Days of storage at 4°C
(seconds) 0 2 5 7 9 12 14
0 5 5 5 5 5 5 5
20 5 5 5 5 5 5 5
40 5 5 5 5 4.5 4.5 4.5
60 5 5 5 5 4.5 4 4
78
The effect of PL treatment (at 3800 V and 3 pps) on color of fresh salmon fillets
was monitored by Ozer et al. (2004). They noticed that although decreasing the distance
between the fish fillets and PL source to 5 cm increased the 10glO reduction, samples
treated for more than 30 s exhibited visible color and quality changes. It was therefore
recommended that samples be refrigerated prior to and after PL treatment in order to
compensate for the temperature increase, which is favorable for product autolysis. It
must be noted that the PL treatment used in Ozer's study was considerably stronger than
that used in this study, and therefore color changes in this study remained within
acceptable limits. However, since PL has the potential to adversely affect quality,
samples should be exposed to the minimum level of PL required for pathogen control.
5.3.2 Microbiological analysis
L. monocytogenes, enumerated on Oxford agar, supplemented with Listeria
selective supplement are shown in Figures 5.5-5.6. L. monocytogenes was not detected in
the co Id smoked coho salmon used in this study (data not shown). There was no
significant difference between control (inoculated but not PL treated) and inoculated PL
treated samples (P> 0.05). However an overall decrease in counts was observed, from an
initiallevel of ~ 104 CFU/slice to ~ 10' CFU/slice, after 7 days of storage; followed by a
graduaI increase in counts over the next 21 days to a finallevel of ~ 103 for control
samples and 102 for inoculated PL treated samples. One possible explanation for the
initial decrease in L. monocytogenes counts may be the production of bacteriocins by the
competing flora (lactic acid bacteria) that prevented the growth of L. monocytogenes.
RecentIy, the time required for a 4 log reduction of L. monocytogenes counts in Serbian
fermented sausages was significantIy decreased by the addition of Lactobacillus casei (a
bacteriocinogenic lactic acid bacteria) (Drosinos et al., 2006).
79
5*":--------~ ~-------*--------~
4
l!! o u ~3t---------------------o "0 ()
2
1 ---
o 7 14 21 28
Days of storage at 4°C
Figure 5.1 Changes in color scores of control and inoculated VP co Id smoked salmon slices, stored at 4°C
5·
Control
4 ""*"" PL treated
l!! 0 U 1/1 3 -~
0
1 "0 ()
2-
o 7 14 21 28
Days of storage at 12°C
Figure 5.2 Changes in color scores of control and inoculated VP co Id smoked salmon slices, stored at 12°C
80
5'~------~
4 * CI) ... 0 (.) !II 3 ... 0 ------'C 0 ~"Contorl
2 ""'*"" PL treated
o 7 14 21 28
Days of storage at 4°C
Figure 5.3 Changes in odor scores of control and inoculated VP cold smoked salmon slices, stored at 4°C
f o (.)
5 tIii< ,------31<:
4· ""*"" PL treated
~ 3"----------------------------------------~~--~)· o 'C o
2
o 7
- -- --1
14 21 28
Days of storage at 12°C
Figure 5.4 Changes in odor scores of control and inoculated VP cold smoked salmon slices, stored at 12 oC
81
At 12°C, counts of L. monocytogenes increased from ~104 to > 106 CFU/slice over
the 28 storage period, with no significant difference (P > 0.05) between inoculated
control and inoculated PL treated samples. However, there was a significant difference
(P = 0.003) between counts of L. monocytogenes in inoculated PL treated samples stored
at 4 and 12°C. These results are in agreement with a challenge study that showed
significant increases in growth rate of L. monocytogenes in cold smoked salmon samples
(similar in composition to samples used in this trial) stored at 4 and 8°C, with 8°C having
a higher growth rate than 4°C (Cornu, 2006). The final counts of> 106 CFU/slice at 12°C
seen in this study confirm the importance of strict temperature control in conjunction
with VP to inhibit the growth of L. monocytogenes.
Total aerobic counts and background microflora counts are shown in Figures 5.7-
5.10. There was no significant difference in counts (P > 0.05) between inoculated
controls (not PL treated) and inoculated PL treated samples for both aerobic and
background microflora counts at both storage temperatures. In addition, there was no
significant difference between total aerobic counts for inoculated PL treated samples
stored at 4 and 12°C. However, at both storage temperatures, the final aerobic counts
(day 28) exceeded the maximum microbiologicallimit for fresh fish (International
Commission on Microbial Specification for Foods (lCMSF), 1978) of 107 CFU/g.
Initial counts for the background microflora were ~ 102 CFU/slice. For inoculated
PL treated samples stored at 4°C, counts increased steadily to 107 CFU/slice at the end of
storage. At 12°C, they increased to ~ 103 _104 CFU/slice by the end of storage, which was
significantly different (P = 0.03) from inoculated PL treated samples at 4°C. The slower
increase in background counts observed at 12°C may be attributed to the fact that at
12°C, L. monocytogenes grows at a much faster rate than at 4°C (shown above), therefore
competing considerably more with the background microflora for nutrients.
It has been widely reported that the dominant background microflora present in
cold smoked salmon is lactic acid bacteria (LAB) (Gonzalex-Rodrigues et al., 2002;
Gram and Huss, 1996; Hansen and Huss, 1998; Leroi et al., 1998). In order to confirm
this observation, representative colonies isolated from the co Id smoked salmon used in
this study and grown on Lactobacillus MRS agar were Gram stained and observed under
82
a microscope. Analysis showed that an colonies were Gram positive cocci, being
conducive with characteristics of lactic acid bacteria.
5.3.3 Changes in pH values
The changes in pH values for control (inoculated) and inoculated PL treated VP
cold smoked salmon slices, stored at 4°C and 12°C are shown in Figures 5.11-5.12. At
4°C there was a significant difference (P = 0.03) between control and PL treated samples,
the latter with a decrease from a value of 6 to ~5.4 at the end of the 28 day storage period.
Although there was no significant difference for aIl microbiological counts between
control and PL treated samples (section 5.3.2), PL treatment may have reduced the ability
of the lactic acid bacteria naturally present in the cold smoked salmon to produce acid
and lower the pH; therefore resulting in the significant difference between the pH values
of control (untreated) and PL treated samples.
At 12°C, the difference in pH values for control and PL treated samples was not
significant over the 28 day period (P > 0.05). However, there was a significant difference
(P = 0.03) between PL treated sample stored at 4 and 12°C. This is attributed to the fact
that at moderate temperature abuse conditions (12°C), microbial growth and activity
occurs at a faster rate than at refrigeration temperature (4°C), thus decreasing the pH
more quickly.
83
5
4
-0 3 Z -z 5' o ..J 2
o o
------t----~-------· ----.-,---~-.------- -
7 14
Days of storage at 4°C
~~Control
-*"" PL treated
---1-- ---------- ------
21 28
Figure 5.5 Changes in L. monocytogenes counts of control and PL treated VP cold smoked salmon slices, stored at 4°C
ô z
7
6
~ 5 (!) o ..J
4
3 +----
o 7
~Ak- Control
-*"" PL treated
I----·-----------,------~_·"
14 21 28
Days of storage at 12°C
Figure 5.6 Changes in L. monocytogenes counts of control and PL treated VP co Id smoked salmon slices, stored at 12°C
84
-o Z Z c;-O ..J
11
10
9
8
7
6
5+------,
o 7
[---~-----~ ---~~
-+~Control
-r PL treated ~-----
14 21 28
Days of storage at 4°C
Figure 5.7 Changes in total aerobic counts of control and PL treated VP cold smoked salmon slices, stored at 4°C
10
1 9.5
9
8.5
'ô 8 z Z _ 7.5 C) o ...J 7
6
5·:I~_ o 7
1--------1 Control
1
-r PL treated 1
______ . ___ .~ _______________ ~ ____ J
14 21 28
Days of storage at 12°C
Figure 5.8 Changes in total aerobic counts of control and PL treated VP co Id smoked salmon slices, stored at 12°C
85
8
7
6
~5 Z 5 9 4
3
2
1 ;:;~ 1
1 L---------.--------r-------------,------------, o 7 14 21 28
Days of storage at 4°C
Figure 5.9 Changes in background microflora counts of control and PL treated VP cold smoked salmon slices, stored at 4°C
5.5
5
4.5
4
"ô ~ 3.5 Z 5 9 3
2.5
2
1.5
1 "--' --------.----------"-~-.- -- - .. -l' - ----~ .------- ----- -
o 7 14
Days of storage at 12°C
21 28
Figure 5.10 Changes in background microflora counts of control and PL treated VP cold smoked salmon slices, stored at 12°C
86
6r --___ ___ 5.9
:Il 5.8 :::::1
""iii > =a 5.7
5.6
5.5 -
5.4
o -----,------------r-
5 10 15 20
Days of storage at 4°C
[-- --
.-Control
..... PL trea~~ ___ _
25 30
Figure 5.11 Changes in pH values of control and PL treated VP cold smoked salmon slices, stored at 4°C
6.1
6
[------
"*4\>$ Control
~PL treated ._-----._--~~-~~--
5.9
5.8
:« :s ~ 5.7
:I: Q,
5.6
5.5
5.4
5.3 +----------,---------------, ---~------~----.~---____r_---------,
o 5 10 15 20 25 30
Days of storage at 12°C
Figure 5.12 Changes in pH values of control and PL treated VP cold smoked salmon slices, stored at 12°C
87
5.4 Conclusions
With decreased use of smoke, salt and bacteriocins in the smoking process and
continued risk of contamination with L. monocytogenes, which is capable of growing at
refrigeration temperature, acidic pH and salt concentrations up to 10%, there is a need for
a processing technology that will ensure the safety of the final product without
jeopardizing the sens ory quality. PL treatment was evaluated for this purpose.
In this study, there was little or no effect of PL treatment on the sensory quality of
uninoculated VP cold smoked salmon stored at 4°C for 14 days. Co Id smoked salmon
slices challenged with L. monocytogenes, vacuumed package d, PL treated and stored at 4
and 12°C showed no significant difference in counts, when compared to controls that
were inoculated but not PL treated. However, at 4°C, there was an overall decrease in L.
monocytogenes counts from day 0 (104 CFU/slice) to day 7 (~101 CFU/slice), followed
by a more graduaI increase in counts over the 28 day storage period. At 12°C, there was
a steady increase in counts of L. monocytogenes to a finallevel of ~ 107 CFU/slice after
28 days. Therefore, moderate temperature abuse of fish products contaminated with L.
monocytogenes poses a considerable problem as the growth ofthis pathogen may be
enhanced.
88
PART B - EfJect of fat content, salt content and competing microflora on the growth of Listeria monocytogenes
5.5 Introduction
In the previous study (Part A) it was shown that PL treatment had no significant
effect on L. monocytogenes in VP cold smoked salmon. This was also shown in Chapter
#3, where PL treatment showed little to no effect on L. monocytogenes on VP and
unpackaged cold smoked salmon. However, Chapter #3 also showed that PL treatment
was able to substantially decrease counts of L. monocytogenes on the surface of a general
purpose agar medium. This brought us to more carefully look at the difference between
the se two cases, one in which the bacteria was inoculated on a nutrient agar and the other
on the surface of smoked fish. The latter case obviously had less nutrients, and possibly
more competitors for the growth of bacteria, notably presence of salt and fat and the
presence of other competing microflora which could potentially inhibit the growth and
activity of L. monocytogenes. It has been reported that factors, such as the type of tissue
(% fat), pH of the tissue and nature of the competing flora affect the growth of L.
monocytogenes (Sheridan et al., 1995).
Therefore, in addition to the inability of PL to penetrate below the surface of co Id
smoked salmon, it was found useful to investigate the effect of additional factors (such as
fat and salt content) on the growth of L. monocytogenes so that the reason for the
observed results could be postulated. Since these factors could not be removed from cold
smoked salmon, we decided to do the other possibility, adding salt and fat to the agar
medium to see any possible inhibitory effects.
The objectives ofthis study were therefore to monitor the effect of the following
factors on the growth of PL treated L. monocytogenes, Scott A on agar medium enriched
with:
1) Fat and salt;
2) Competing microflora (presumably lactic acid bacteria isolated from co Id smoked
coho salmon).
89
5.6 Materials and methods
5.6.1 Bacterial strains
L. monocytogenes Scott A was prepared as described in 4.2.2.
The lactic acid bacteria (LAB) culture was prepared by swabbing the surface of a
slice of co Id smoked salmon, then streaking onto Lactobacillus MRS Agar (Difco
Laboratories, Detroit, MI) and incubating them anaerobically at 35°C for 24-48 h.
Distinct colonies were then re-streaked onto MRS agar for purity and incubated again at
35°C for 24-48 h. Distinct colonies were then transferred to a tube containing 9 mL of
tryptic soy broth (TSB, Difco) supplemented with 0.6% yeast extract (TSB/YE) and
incubated for approximately 12 h at 3 Y C to give a suspension of approximately 109
CFU/mL. Inocula were prepared using this culture which was further diluted with the
appropriate volume of 0.1 % sterile peptone water to achieve the desired inoculum
concentration (101_109 CFU/mL). The LAB cultures were maintained frozen at -20°C
(700 I-lL of a 24 h culture grown in TSB (tryptic soy broth) + YE (yeast extract) (Difco)
with 300 I-lL of a 50% (v/v) glycerol solution).
A loopful of the above culture was streaked onto MRS plates and incubated
anaerobically at 35°C for 48 h. Distinct colonies were then transferred to a tube
containing 9 mL of tryptic soy broth (TSB, Difco) supplemented with 0.6% yeast extract
(TSB/YE) and incubated anaerobically for approximately 12 h at 35°C to give a
suspension of approximately 109 CFU/mL. Inoculums were prepared using this culture
which was further diluted with the appropriate volume of 0.1 % peptone water to achieve
the desired inoculum concentration (10 1-109 CFU/mL).
5.6.2 Media preparation
5.6.2.1 TSA supplemented with oil or salt
Tryptic soy agar (TSA, Difco) was supplemented with 6% olive oil (w/w basis) to
represent the highly unsaturated fat content of the cold smoked coho salmon from Chile,
90
which was used in the challenge study. Separately, TSA was supplemented with 1.5%
salt (w/w basis) to represent the water phase salt (WPS) content of the same cold smoked
salmon. Both ofthese supplemented media were poured, separately, into Petri plates and
allowed to dry overnight.
5.6.2.2 "Salmon" agar
A 50:50 ratio (w/w basis) of co Id smoked salmon to distilled water was
homogenized in a Waring blender for 1 minute. This mixture was autoclaved and re
homogenized before being combined with double strength Oxford media (Difco) in a
ratio of 1: 1 and poured into Petri plates (as above). The resulting concentration of
salmon in the media was 25%.
5.6.3 Inoculation, PL treatment and storage conditions
5.6.3.1 TSA supplemented with oil or salt and "salmon" agar
Dried Petri plates were surface inoculated with L. monocytogenes as in section
4.2.2.2. The plates were then subjected to the following PL treatments under the center
of the PL source, with their lids removed: 800 V, 5 cm distance from the PL source for 1
- 9 s at 1 pps. AlI plates were incubated at 35°C for 24-48 h.
5.6.3.2 Destruction kinetics of background microflora
Dried Petri plates were surface inoculated with a culture of background
microflora isolated from co Id smoked salmon, as in section 4.2.2.2. The plates were then
subjected to the following PL treatments under the center of the PL source, with their lids
removed: 600, 700 or 800 V, 5 cm distance from the PL source for 1 - 21 s at 1 pps. AlI
plates were incubated at 35°C for 24-48 h.
91
5.6.3.3 Competition between L. monocytogenes and background microflora
Appropriate volumes of diluted cultures of L. monocytogenes and isolated
background microflora (in 0.1 % peptone water) were aseptically transferred,
consecutively, to a 2 oz transparent polyethylene sampling bag (Whirl Pak, Fisher
Scientific, Ottawa, ON, Canada) to give a final volume of25 mL, then heat sealed. The
concentration of L. monocytogenes was held constant in aIl samples (103 CFU/mL) while
the concentration of the background microflora varied from 101 - 105 CFU/mL. Control
samples were 25 mL of the appropriate dilution of either L. monocytogenes, at 103
CFU/mL, or varying concentrations of the background microflora, from 101 - 105
CFU/mL. Samples were not PL treated, but incubated at 35°C for 24 h.
5.6.4 Analyses
5.6.4.1 TSA supplemented with oil or salt and "salmon" agar
Following incubation, countable plates (30-300 colonies) were reported as 10gIO
Colony Forrning Units per mL of sample (lOglO CFU/mL).
5.6.4.2 Destruction kinetics of background microflora
Following incubation, countable plates (30-300 colonies) were reported as 10gIO
Colony Forming Units per mL of sample (lOglO CFU/mL).
5.6.4.3 Competition between L. monocytogenes and background microflora
Following incubation, Whirl Pak bags were aseptically opened and a 1 mL aliquot
was transferred to tubes containing 9 mL of 0.1 % sterile peptone water and further
dilutions were then made, again using 0.1 % sterile peptone water. The samples were
enumerated for L. monocytogenes by plating 0.1 mL of the appropriate dilutions on
Oxford agar (Oxoid) supplemented with Listeria Selective Supplement (SR0140E).
92
Background microflora were enumerated using Lactobacillus MRS AGAR (Difco) using
the spread plate technique. Oxford and MRS plates were incubated aerobically and
anaerobically, respectively, at 35°C for 24 and 48 h, respectively. Countable plates (30-
300 colonies) were reported as 10gIO Colony Forming Units per mL of sample (lOglO
CFU/mL).
5.7 ResuUs and discussion
5.7.1 TSA supplemented with oil or salt
The purpose behind the supplementation oftryptic soy agar with 6% oil or 1.5%
salt was to separately mimic the fat and water phase salt content of the cold smoked
salmon used in Part A ofthis chapter, and evaluate their respective effect on the growth
of L. monocytogenes. The two survival curves for L. monocytogenes on TSA
supplemented with 6% olive oil and 1.5% salt are shown in Figure 5.13 along with the
survival curve for L. monocytogenes on unsupplemented TSA (from Chapter #4), which
indicates that the destruction was influenced by the treatment time, as in Chapter #4. The
decimal reduction times for L. monocytogenes on TSA supplemented with 6% oil and
1.5% salt were 2.07 and 1.03 s, respectively. However, it must be noted that D-values
were calculated by not taking into account an initiallag period of 3 seconds, only after
which a first order rate of destruction was observed. These lag periods must be used in
conjunction with the respective D values in order to have a meaningful prediction of the
PL destruction power.
In order to determine if the effect of salt and oil content were significant on the D
value of L. monocytogenes, the slopes of the survival curves for 1.5% salt and 6% oil
were compared to that of L. monocytogenes on regular unsupplemented TSA (from
Chapter #4). A probability CP) of< 0.05 was considered to be significantly different.
The D-value for L. monocytogenes plated on regular TSA and PL treated at 800 V
was 0.91 s. Therefore, by comparison, 1.5% salt and 6% oil significantly (both P < 0.01)
increased the D-value, making L. monocytogenes more resistant to PL treatment. In
terms of the effect of salt content, the results are in agreement with a challenge study that
93
showed a decrease in growth rate (higher D-value) when the salt content of co Id smoked
salmon was increased (Cornu et al., 2006). However, it must be noted that the phenolic
content (smoke compounds) was not equal in the compared samples in Cornu's study and
it has been reported that phenolic compounds also have an effect on the growth rate of L.
monocytogenes (Sunen et al., 2001; Thurette et al., 1998). In terms ofthe increase in
resistance of L. monocytogenes seen with the addition of 6% oil, this may be due to a
protective effect. For example, a high pressure processing study with Listeria innocula
noticed that an increase of fat content of milk resulted in a progressive protection against
inactivation (Gervilla et al., 2000). In order to investigate the combined effect of the
physiochemical characteristics on the growth of L. monocytogenes, a representative
salmon agar was used.
5.7.2 "Salmon" agar
The purpose behind this study was to evaluate the effects of the physiochemical
characteristics of cold smoked salmon on the destruction kinetics of L. monocytogenes.
The survival curve at 800 V is shown in Figure 5.14, where a D-value of 1.53 s resulted.
A significant difference (P < 0.01) between the growth of L. monocytognes on "salmon"
agar versus regular TSA was observed when a comparison for slopes was performed (as
in 5.7.1). This is in agreement with the conclusions of Cornu et al. (2006) that the
physiochemical characteristics (pH, salt content, water-phase salt content and phenolic
content) significantly affect the growth rate of L. monocytogenes. The addition of25%
cold smoked salmon to the Oxford media also had a protective effect on the PL
destruction of L. monocytogenes.
In general, each of the above mentioned treatments resulted in D values being
increased by 25% to 125%. Assuming that aU these components being there
simultaneously on the surface of the smoked fish, and assuming each one to be
consecutive, a significantly larger D value of about 4-5 s could be expected for the L.
monocytogenes on cold smoked salmon. Ifthere is sorne synergy a further greater effect
could be expected. Still the D-value may be short of the value found for smoked salmon.
The only other explanation then would be that a fairly large fraction (for example 1110)
94
of the bacterial culture was protected from the PL exposure. In this case, because of the
logarithrnic nature of the destruction, the residual count would remain at 90% level ev en
after long exposure times.
5.7.3 Destruction kinetics of background microflora (presumably LAB)
It was important to evaluate the destruction kinetics for the background
microflora present in cold smoked salmon and compare then to the destruction kinetics
for L. monocytogenes, as was done in Chapter #4.
The survival curves for the background microflora at 600, 700 and 800 V are
shown in Figure 5.15, which indicates that the destruction was influenced by the voltage
used and the treatment time. The survival curves at higher voltages (800 V) were steeper
than at lower voltages (600 V), which illustrates that the destruction rate of the
background microflora is higher at higher voltages.
The decimal reduction times for 800, 700 and 600 V were 0.93, 1.34 and 2.31 s,
respectively. It must be noted that D-values were computed by not taking into account an
initiallag period (between 3 and 5 sec), only after which a first order rate of destruction
was observed. This lag period is required along with the respective D-values in order to
have a meaningful prediction of the PL destruction power.
Lower D-values are associated with higher voltages demonstrating a higher
destruction rate. Figure 5.16 demonstrates the curve oflog(D-value) vs. treatment
voltage to compute the voltage sensitivity parameter (Zy value). The Zy value of the
background microflora was calculated to be 500 V, meaning that for every 500 V
increase in treatment voltage, the D-value would be reduced by 1 log cycle. The Zv value
for L. monocytogenes was also 500 V (computed in Chapter #4), which means that the
destruction rate of L. monocytogenes is equally sensitive to changes in voltage than the
background microflora.
95
9.00
~ & 8.00
t (,) 7.00 o c:: e :::J 6.00
.,J.§ '0 ~ 5.00 cO o 0
:;:::l cf 4.00 ]:0 C...J ~ -- 3.00 c o (J
~ :::l
"C
~
2.00
0.00 o 2 4 6 8 10
Treatment time (sec)
+ Regular
+1.5% salt
+6%oil
- Regular after lag
x +1.5% salt after lag
l"'_+6% oil aft=~_I:g
12
Figure 5.13 Survival curves of L. monocytogenes on TSA supplernented with 6% oil and 1.5% salt
9.00 -
~ & 8.00
~ ~ 7.00· o ~ e ::J 6.00 .,J.§ '0 ~ 5.00 . cO o 0
~ cf 4.00 bO C...J ~ -- 3.00 c o (J 2.00 ~ :J
&
R2
= 0.9934 D value = 1.53
~ 1.001 0.00 ------~---,------------,---------_r-----~-~,_--.--- .. - - ....
o 2 4 6 8 10
Treatment time (sec)
Figure 5.14 Survival curve of L. monocytogenes on "salmon" agar
96
C "C 0 C E i5iO
9.00 Î
8.00 -------------llC ... 1/)
Cl"C ~ CI) (.)~ lU 0
7.00
..c E CI) 1/) _ 6.00 oC ...J +-"CE
'0 '8:3 5.00 cEu.2
00
rti ... 0 ...... c5 4.00 'E:"CO CI).2!...J
g -E - 3.00 8 .!!!
2.00 -
1.00
1 1 1 1 1 1 1 1 1
0.00 ---~..............!-___t____"____~--~~- r~------------
o 5 10 15
Treatment time (sec)
800V
700V
600V
- 800V after lag
1 X 700V after lag
l_~_6_0_0V __ aft_e_rlag
20 25
Figure 5.15 Survival curves of background microflora isolated from co Id smoked salmon
-Q) :l ëU ~ c -(!) 0 ...J
0.40
0.30
0.20
0.10
0.00 --6 0
-0.10
620 640
R2 = 0.9872 z value = 500 V
660 680 700 720 740 760
Treatment voltage (volts)
Figure 5.16 Decimallogarithm ofD-values versus treatment voltages for background microflora on the surface of general purpose media
97
5.7.4 Competition between L. monocytogenes and background microflora
There have been numerous reports of inhibition of L. monocytogenes by lactic
acid bacteria in a variety of vacuum packaged, ready-to-eat products: Italian salami (de
Carvalho et al., 2006), frankfurters and cooked ham (Amezquita and Brashears, 2002),
and cold smoked salmon (Tome et al., 2006; Vescovo et al., 2006). It was therefore
important to investigate the effect of the background microflora, presumably LAB, on the
growth of L. monocytogenes. Figure 5.17 shows the effect of varying concentrations of
background microflora isolated from co Id smoked salmon on the growth of L.
monocytogenes at 3S oC for 24 h. After 24 h, aIl control samples had reached 109
CFU/mL (results not shown). With the concentration of L. monocytogenes held constant
in aH samples, the graduaI increase in concentration of background flora began to have an
effect on the growth of L. monocytogenes. This trend continued until the concentration of
both cultures in the final sample was approximately equal after the 24 h storage period;
this occurred when the initial concentration of background microflora was 105 CFU/mL.
The growth of Listeria innocula has been shown to be controIled by or reduced by the
addition of Lactobacillus casei, Lactobacillus plantarum and Carnobacteria piscicola
(which aIl grow at SOC and pro duce antimicrobial substances) singly, or in association to
vacuum packaged cold smoked salmon (Vesvoco et al., 2006). Therefore, the
background microflora of cold smoked coho salmon used in this study appears to have an
effect on the growth of L. monocytogenes.
98
10
9
8
::J' 7
E 6 -~ LI.
2- 5 ... c5 4 0 ...J
3
2
1
0 101\3
LM:101\1 BKG
101\3 LM:101\2
BKG
101\3 LM:101\3
BKG
101\3 LM:101\4
BKG
I~ ~ta~ae:biC coun'
DL. monocytogenes ------ _ ... _-------- --- -
101\3 LM:101\5
BKG
Concentration of L. monocytogenes (LM): background microflora (BKG) (CFUlmL)
Figure 5.17 Effect of varying concentrations of background microflora isolated from cold smoked salmon on the growth of L. monocytogenes
99
5.8 Conclusions
This study has shown that a low salt content of 1.5% and a fat content of 6%
considerably retarded the destruction of L. monocytogenes, most probably by offering the
cells protection from the PL treatment. In addition, the presence of background
microflora naturally present on cold smoked salmon, presumably lactic acid bacteria, had
an inhibitory effect on the growth of L. monocytogenes at 35°C. At refrigeration
temperature, it is reasonable to expect that the background microflora would have a
greater effect on the growth of L. monocytogenes as the latter grows at a slower rate at
low temperatures, as was confirmed in Chapter #4.
100
CHAPTER#6
GENERAL SUMMARY & CONCLUSIONS
The seafood processing industry is an extremely important component of the
Canadian food processing industry, with 1 billion dollars ofrevenue generated annually.
The fish smoking industry is currently struggling with cold smoked fish as the low
temperature used for cold smoking provides an ideal environment for L. monocytogenes,
which is capable of surviving the cold smoking process and growing at refrigeration
temperature (4°C). Therefore, the industry is constantlY looking for new preservation
technologies to ensure the safety of ready-to-eat smoked products, while maintaining
their organoleptic properties. One such approach is pulsed light (PL) treatment.
Preliminary studies showed that voltage, treatment time (equivalent to number of
pulses, due to 1 pulse per second frequency), distance from the PL source, and position in
the PL treatment chamber play a significant role in the destruction of L. monocytogenes.
Maximum destruction was achieved at high voltage and minimum distance from the PL
source. However, due to the increase in treatment area as the distance from the PL source
increases, sample surface area must be considered before choosing the appropriate
combination of PL factors.
Destruction kinetic studies were performed on L. monocytogenes in liquid media,
on the surface of general purpose agar and directly on cold smoked salmon (both with
and without vacuum packaging). It became apparent that PL technology is effective in
sanitizing smooth and dry surfaces (a D-value of 0.91, 1.37 and 2.25 sec at 800, 700 and
600 V, respectively, and a resulting z value of 500 V), however, it struggles to penetrate
thick and opaque samples, as was the case in the liquid media and on the surface of cold
smoked salmon (93 sec and 19-24 min, respectively).
Color and odor acceptability were monitored in uninoculated PL treated VP cold
smoked salmon samples over a 14 day period. A graduaI decrease in color and odor
scores was observed in samples treated for 60 s. However, after 14 days, aIl samples
were within acceptable limits.
101
Challenge studies were performed on inoculated vacuum packaged cold smoked
salmon over a 28 day period at refrigerated (4°C) and moderate temperature abuse (12°C)
conditions. Although PL treatment did not have a significant effect on L. monocytogenes
counts (on treatment day 0), there was an overall reduction in counts (from day 0 to day
28) with no observed change in color or odor scores for 14 days at 4°C. However, at
moderate temperature abuse conditions (l2°C) a dramatic increase in L. monocytogenes
counts (to ~lxl07 CFU) was observed with a decrease in color and odor scores.
Due to limited kill of L. monocytogenes in the challenge studies, experiments
were carried out to determine the effect of (l) 1.5% salt content, (2) 6% oil content, (3) a
representative salmon media and (4) background microflora (lactic acid bacteria) on the
growth of L. monocytogenes. It was shown that aIl of the above conditions significantly
effect the PL destruction of L. monocytogenes by increasing the D value (adding
resistance to pulsed light destruction).
Although PL technology is limited by its inability to penetrate opaque and non
smooth surfaces, there is an application for such a technology in the Canadian smoked
fish industry. Since the majority of L. monocytogenes contamination occurs in post
smoking processing (for example in the slicing machines) a PL unit could be placed
above the slicing unit to sterilize the blades between slices. Contamination is usually
low; therefore a few flashes of exposure would dramatically reduce the microbialload.
However, the slicer would need to be completely enclosed, to prevent UV exposure of
personnel, thus an automatic slicer would be imperative, and possibly an added expense
to the processor.
These studies have shown that PL treatment in combination with low temperature
storage (4°C) has the potential to extend the shelf-life of VP cold smoked salmon
products without compromising sens ory quality.
Future Recommendations
The voltages used in this study did not significantly reduce the population of L.
monocytogenes in cold smoked salmon; therefore, in order to improve the microbialload
reduction by PL treatment, further studies utilizing higher voltages (1000-3500 V) seem
102
to be necessary. In addition, the advantage to using higher voltages is a decrease in the
treatment time, which is desirable for commercial purposes.
These studies have focused on the PL destruction of L. monocytogenes as it is the
primary pathogen of concern in RTE cold smoked salmon. However, it would be
advantageous to obatin PL destruction kinetics for Clostridium botulinum type E as it
continues to be a regulatory concern in RTE VP fishery products.
103
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109
.1:..1Œ,')EARCH PERSONNFL' (attach additiol~,l shecls ifl'referred) . «m, ,,,,,, ___ •
NlIIlle Departmenl
""'''
James P . Smith Fd. Science & Ag. Chem 'M' ... ,·,"" __
BernardC ayouette Fd. Science & Ag, Chom
lIock Alison Po Fd. Science & Ag, Cham -----_ .. ~ .... _._-_.~,--"
~ .... "-,,. -----------
6. Briefly describe:
Jo1> Tit1e1Classification
"-Professorllnvestigator
_HH'H'
TechnÎcian
Masler's student
Attendcd Seminar on Safe Useoflliological Ctlbiools? Sa let y
, __ lf:Œ5, indlCiIIC • .< .!ate of atwud,mce.
I~;;L.) t"'''~ .. ·----,--, -,--,,-...
cÂ
Yes fcY~ Y ~s (cr:::~~~ c
,---Ji No
i) the biohazardotls material involved (e.g. bacleria, viruses, human tissues) & designated biosafety risk group
Bacteria:
At McGiII: Listeria monocytogenes (Risk Group 2) Bacillus cerous (Risk Group 2)
At Health Canada, Ottawa: Protoelytic and non-proteolytic strains of Clostrdium bo!ulinum
ii) the procedures învolving bîohazards
Ail stock cultures are prepared trom frozen cultures in glycerol and grown overnlght at 30 to 370C by ML Bernard Cayouette. Preparation of ail cultures, dilutions and plates, as weil as inoculation of ail food samples, are done under aseptic conditîons in a biological
csafety cabinet Ali staff/studenls were face masks, lab coats and rubber gloves during
ail microbiok>gical procedures, No pipetting is done by mouth, Benches are routinely washed with a 1 % hypochlorite solulîon at the end of each day while the safety cabinet is swabbed with 70% ethanol and the UV Iight left on overnight. Ali conlaminatêd containers, plates, dilutiOnbottles, etc., are sterilized by autoclaving priar to discarding and are clearly identified as "Autoclaved and Sterilized' before pick:up by janltorial staff.
iii) the protocol for decont'\lllillutillg spills
The protocol for decontaminating spills of type 2 microorganisms ts as oullined in both the McGiIl and Department laboratory sarety manuals. Spill are wiped up and trealed with 1 % hypochlorite for -30 minutes, and then wiped (anar drying) with papertowels soaked with 70% ethanol. If the spill occurs in the UV. cabinets, the light is letton for-1 h aftar cleaning, Studentststaff do no! enter into the lab for at least 1 hour afler a spil!. Ali towelS, contaminated paper cloths, etc., lab coats, masks are placed in autoclavable bags and decontarninatêd by sterilization, Ali bags are clearly labeled 'Autoclaved and Sterilized", If a spill oceurs on the body, the clothing is removed and sterilized, Splashes to the face are washed with gerrnicidal soap and hot water. Ali spins are reporte<! ta the laboratory supervisor and a follow up session is do ne with the students to go over the cause{s) of the spill and to re-enforco preventive measures,
7. Does the protoco! present conditions (e,gc handling of large volumes or high concentrations of pathogens) that could incre<1~e the ha7~lf(!s of the illfediolls agent(s)?
No. Only srnall volumes of each microorganislll are used.
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8. Do the specifu; procedures to be employed involving genetically engineered orgallÎSms have a hislory of safe use'!
1'\10 geneti(',3l1y rnodified microorganisms are used in t.his stucly.
9, What precautions will be taken to reduce production of infcctiolls droplets and aerosols?
Students are trained in proper hanoling and inoculation of cultures, Haming of loops etc. Ali work is done in the safety cabinet and students wear gloves. masks and lab coats at ail Umes. These micoorganisms do not cause a problem as aerosols but through their growth in food and consumptÎOn of the contaminated food.
Copies of the McGiIl and departmental safety protocols are in the lab at ail Umes for perusal by the sludents, Fu rthermore , most of the work done in our laboratory Is under the constant supervisÎOn of Mr. Bernard Cayouette.
10. Will the biohaz,m!ot!s materials in this project expose memhers of the research lcam to ully risks that might require special training, vaccination or other protective mensures'? If yes, plcase explain.
None for pathogens used at McGilI. For work wilh Clostridium bolulinum at Health Canada, studenls will be vaccinated and receive training on the handling of this pathogen,
LI. Will this project produce combined ha:r.ardous waste - i.e. radioactive biohazardous waste., biohazardous animal carcasses contaminated with texic chemicals, etc.? Ifyes, please explain how disposai will be handled.
NIA
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12. List the biolotùcal safetv cabinets to be used. -- _<o<"h_.,,",~ ..
Building RoomNo. Manufacturer ModelNo. Seriai No. Date Certifted ---, .... _------_.-
Macdonald Stewart 1·055 labconco 36205-04 247196 21/11/03 _., ~,~''''' ..
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