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INFORMAnON TO USERS
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SHELF-LIFE AND SAFETY STUDIES
ON RAINBOW TROUT FlLLETS PACKAGED
UNDER MODIFIED ATMOSPHERES
By
IsabeUe Dufresne
Department of Food Science
" Agricultural Chemistry
Macdonald Campui
of
MeGill University
Montrea., Quebee
A thesls submitted to the Faculty of Gnduate Studles and Resean:b iD partial
rulftllment of the requirements for the degree ofMaster ofScience
November 1999
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ABSTRAcr
SHELF-LIFE AND SAFETY STUDIES ON RAINBOW TROUY FlLLETS
PACKAGED UNDER MODIFIED ATMOSPDERES
The combined effect ofvarious gas packaging atmospheres (air, vacuum and gas
packaging), films ofdifferent oxygen transmission rate (OTR) and storage temperature (4
and 12°C) were investigated on the shelf-life and safety offtesh rainbow trout flUets.
Preliminary studies were done to determine the optimum packaging atmospheres
to maintain the bright pink color of trout packaged in a higb gas barrier film. Both
vacuum and gas packaging (85% C~:15%N2) resulted in the longest shelf-life (-28
days) in terms ofcolor at 4°C. Based on these optimum gas atmospheres for color, shelf
life studies were performed at bath refrigerated and temperature abuse conditions (12°C).
A 3-4 day extension in the shelf-life of ftesh trout fillets was possible through gas
packaging (85% CÛ2:15% N2). At 12°C, the shelf-life oftrout fillets was tenninated after
-2 days in all packaged trout
Challenges studies were also done with Listeria monocytogenes and C!ostridium
botulinum type E, two psychrotrophic patbogens of concem in modified atmosphere
packaged (MAP) fish. While gas packaging had a slight inhibitory effect on the growth
ofL. monocytogenes. it grew well in both air and vacuum packaged trout filIets stored al
4°C and inall gas atmosphcres at 12°C.
ln challenge studies with C. hotu/inu", type E spores (l~ sporesIg), toxin was not
detected in trout fillets packaged in air, vacuum or in a C~:N2 (85:15) las mixture and
stored at 4°C. However, alI fish were toxic by day 5 at 12°C and spoilage preceded
toxigenesis.
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Subsequent studies were donc 10 determinc the effect of various levels of
headspace oxygen (o-l000At, balance CÜ2) or film OTR on the time to 10xicity in trout
stored at 12°C. In all cases, trout were toxic within 5 days , irrespective of the initial
levels of oxygen or film OTR. Similar results were obtained in cballenge studies with
vacuum packaged cold and hot smoked trout in films ofdifferent OTR and stored al 4, 8
and 12°C. While no samples were toxic at 4°C, 500A. of the cold smoked trout fiIlets and
75% ofthe hot smoked trout fillets were toxic at 8°C, and all products were toxie after 28
days at 12°C. In some cases, spoilage preœded toxigenesis, while in other cases,
toxigenesis preceded spoilage.
In conclusion, these studies have shown that storage temperature plays a more
critical raIe on spoilage and time to toxigenesis in both fresh and smoked trout tillets than
either the inclusion of elevated levels of oxygen within the MA products or OTR of the
packaging film. Additional barriers, other than oxygen, need to be considered to ensure
the public healtb safety ofthese products, particularlyal abuse storage conditioDS.
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tTUDE SUR LA CONSERVATION ET LA SÉcVRITt
MICROBIOLOGIQUE DE LA TRUITE ARC-EN-CIEL EMBALLÉE SOUS
ATMOSPHERE MODIFIÉE
Les effets combinés de différentes atmosphères (air, sous vide et gaz) ainsi que
diverses pellicules d 7 emballage et températures d 7 entreposage (4 et 12°C) ont constitué le
sujet d 7étude sur la durée de la conservation et de la sécurité microbiologique de la truite
arc-en-ciel.
Des études préliminaires ont permis de déterminer les abnosphères optimales
d 7emballage afin de maintenir la couleur rose/orangée de la truite emballée dans une
pellicule de faible perméabilité. L 7emballage sous vide et l'emballage sous atmosphère
gazeuse (85% CO2:15% N2) de la truite ont démontré la plus longue durée de vie en
terme de couleur à 4°C. Suite à ces résultats calorimétriques, une étude sur la durée de
conservation de la truite a été réalisée à des températures de réfrigération (4°C) et sous
des conditions abusives de températures d'entreposage (12°C). Une extension de 3 à 4
jours a été possible pour la truite emballée sous atmosphère gazeuse (85% CÛ2: 15% N2).
A 12°C, la longévité microbiologique des truites a été réduite à -2 jours.
Des études de cas ont été menées avec la Lister;a monocytogenes ainsi qu'avec le
C/ostridium botu/inum de type E, deux pathogènes psychrotrophs d'inquiétude pour les
poissons emballés sous atmosphères modifiées. Un certain effet inhibiteur sur le
développement de la Listeria monocyrogenes a été démontré lorsque les truites ont été
emballées sous atmosphère gazeuse (85% C(h: 1S% N2) à 4°C. Lorsque emballées sous
air et sous vide à 4°C ainsi que sous toutes conditions d'emballage à 12°C, la croissance
de la Listeria monocytogenes a été observée.
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Aucune toxine botulinique n'a été détectée pour les filets de truites emballés sous
air, sous vide ou sous atmosphère gazeuse (85% C(h:15% N2) à 4°C lors d'études de cas
sur le C/ostridium botu/;num de type E. Par contre, à 12°C, tous les filets de truite étaient
toxiques tout en étant altérés sensorieUement à l'intérieur de 5 joUIS.
Des études subséquentes ont été réalisées afin de déterminer les effets de
différentes concentrations d'oxygène ainsi que de l'utilisation de diverses pellicules
d'emballage sur le temps de toxicité pour la truite entreposée à 12°C. IndéPendamment
des conditions d'emballage, les truites étaient toxiques dans l'espace de 5 jours. Des
résultats similaires ont été obtenus lors d'études de cas utilisant des filets de truites
fumées à froid et à chaud emballés dans différentes pellicules à des températures
d'entreposage de 4, 8 et 12°C. Aucun échantillon n'était toxique à 4°C, 500AJ des truites
fumées à froid et 75% des truites fumées à chaud étaient toxiques à 8°C, et tous les
produits étaient toxiques à 12°C après 28 jours. Dans certains cas, les échantillons étaient
détériorés avant l'apparition de la toxine botulinique, dans d'autres cas, cette toxine était
présente avant le rejet sensoriel des produits.
En conclusion, ces études ont démontré que la température d'entreposage joue un
rôle plus que détenninant sur la qualité et la sécurité microbiologiques de la truite fraîche
et fumée. Ainsi, des paramètres additionnels autres que la présence d'oxygène dans
l'emballage, doivent être considérés afin d'assurer la sécurité alimentaire de la truite
emballée sous atmosphère modifiée, _ spécialement lors d'abus de température
d'entreposage.
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ACKNOWLEDGMENTS
My tbanks goes to my supervisor Dr. J.P. Smith for bis patience, understanding
and continuous encouragement in completing this study. 1 will also like to acknowledge
bis academic guidance as weU as bis persona! support throughout the yean. He was
definitely a great inspiration. Jbese tbanks are further extended to bis family for their
tiiendship.
1 am very grateful to Mrs. Dsemarie Tarte for her technical assistance and for ber
generosity. 1would also like to thank Dr. John W. Austin and Mr. Burke Blanchfield for
giving me the opportunity to work al the Bureau of Microbial Hazards, Health Canada 1
really appreciated their advice and assistance in the later stages ofmy research.
1 would a1so like to acknowledge my mends, many of whom participated in my
research as sensory panelists and who all provided me with great memories both in and
out of the classroom. These include Fabienne Crumïère, Ndeye Dioum, Robert
Cocciardi, Andreij Wnorowslci, Xavier Penaud, Galen BagdaD, Daphne Phillips, Sameer
Al..Zenki, Caroline Simard, Anis EI-Khoury, Sam Choucha and Charif Geara. 1 would
also like to mention special thanks to my part-time roommate, and friend, Jiunni Liu with
whom 1developed a strong bond through helping each other with our respective projects.
1 would a1so like to thank Mr. Steven Thibault, Mrs. Lucie Labbé and Mn.
Jennifer Caron from Via-Mer, St-Hyacinthe, Quebec for supplying the ftesh rainbow trout
fillets as weU as Mr. Murray Brookman and Mr. Lome Brookman from Levitts, Montreal,
Quebec for supplYing the smoked rainbow trout fillets. My thanks are extended to Mrs.
Jennifer Morris ftom Cryovac Sea1ed Air Corporation for supplies ofthe packaging films.
1would a1so Iike to thank NSERC (National Sciences and Engineering Research Councü
ofCanada) for their financial support.
FinaUy, 1 owe special thaoks 10 my parents and boytiiend Nicolas for their love
and support tbroughout my studies.
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TABLE OF CONTENTS
J\IJ~1rFlJ\<:1r••.••.•.••••.••••.•••.•••••••••••••••••••••.••.••••••••••••••.•.••.•••.••••••••••••.••••••iiiIll:~~••.........•....................•...........•......•..•.•.••.•..••..•.......•................."~(:~()~I)(]J:~~..•...•••..•..•••...••.....•.........••..•..•...••......................'fÏi~I~1r ()j: 1rJ\ll~~•...•.•...•...•.........•....•.........•..•..••.•..••.••..•.................•..•.~~~1r ()j: j:I(J~~ ~
1. INTR()DUCTI()N AND LITERATURE lŒVIEW 1. .•....................................11.1. IntrCNi\lctÏOIl••••••••••••••••••••••••••••••••••••.•••••••••••••••••••.••••••••••••••••••••••••••••• 11.2. (:hemiœl compositioll offish 2
1.2.1. Fish proteins.. . . . . . .. . . . . . . . . . . . . . .. . . .. . . . . . .. . . .•. . . . . . . . 21.2.2. Fish lipids...................•..............•......•..........•.....................•...21.2.3. Fish~hyCbnltes 31.2.4. Fish ~tamjns and mineraIs.............................•............................3
1.3. Quality offish 61.3.1. Extemal appearal1ce of fish 61.3.2. Text\lre offish ~ 6
1.3.2.1. Rigor mortis 71.3.2.2. j:~tors adBf~tU1~texttun= 7
1.3.3. Taste and aroma offish 81.3.4. Color offish 8
1.3.4.1. C>riginofthe red color ÎI1 salmol1Îds...............................•....91.3.4.2. Asthaxmthîn md CaJ1thaxanthin..........................•............101.3.4.3. Depositioll ofpigDleJ1ts 111.3.4.4. Absorption ofpigDleJ1ts 121.3.4.5. Discoloration ofpigmœts 12
1.4. Spoilage offish 151.4.1. (Jelleral spoilage pattern offish 151.4.2. Chemical deterioration offish 151.4.3. Microbial degradation offish 17
1.5. Presenration offish.............................................•..............................231.5.1. Modified atmosphere packaging of fish..........•..............................24
1.5.1.1. Methods ofgas packaging 241.5.1.1.1. Packaging films 261.S.1.1.2. Gas compositioll..................•..........................271.5.1.1.3. Antimicrobial effects 27
I.S.l.2. Concems associated with modified atmosphere packaging 301.S.1.2.1. Concems with Listeria monocytogenes 30I.S.1.2.2. (:ODCems with Clostridium botulinum 32
1.6. (:ODClusioD...................•.•.••.• ,. ......•.......•...•..••••.•••..•••..••..•.•.......•.•••..•391.7. Ile~hobj~"es ...............•...........................................................~
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2. THE COLOR SHELF-LIFE STUDIES Of TROUT FlLLETS PACKAGEDUNDER VARIOUS GAS ATMOSPHERES AND STORED AT 4°C 41
2.1.In~on......................................................•..............................412.2. Materials and methods.........................................•......••......................43
2.2.1. Sample preparation................................•................................432.2.2. Packaging and storage conditions........•.......................................432.2.3. A.nalyses•••.•••••••••.••••••.••.••.....••.•..••.•..••••.••.••••••••.•.....••...•.••.••44
2.2.3.1. Subjective color measurements 442.2.3.1.1. Sensorial color acceptability 442.2.3.1.2. Sensorial color detennioation 44
2.2.3.2. Objective color measurements 442.2.3.3. Statistical analysis 48
2.3. Results and discussion 492.3.1. Subjective color measurements...........•........................................49
2.3.1.1. Changes in color acceptability scores•••••.•••••••••••...•..••.•.•••••492.3.1.2. Changes in Roche color chart scores 49
2.3.2. Objective color measurements 532.3.2.1. CIE-LAS values 53
2.3.2.1.1. Changes in L* (lightness) values 532.3.2.1.2. Changes in a· (redness) values 542.3.2.1.3. Changes in b· (yellowness) values 542.3.2.1.4. Changes in C· (chroma) values 552.3.2.1.5. Changes in h (hue angle) values•............................55
2.3.2.2. Comparison ofobjective color measurements to other studies 562.3.3. Relationship between subjective and objective measurements ofcolor 60
2.4. Conclusion 64
3. SHELF-LIFE STUDIES ON TROUT FILLETS 653.1. Introduction 653.2. Materials and methods.....•..................................................................66
3.2.1. SauDœplepre~on 663.2.2. Packaging and storage conditions 663.2.3. Analyses 67
3.2.3.1. Headspace gas analysis 673.2.3.2. Instrumental color measurements 67
3.2.3.3. Sensory evaluation 683.2.3.4. Measurement ofdrip loss 683.2.3.5. Microbiologjca1 analyses....................•...........................683.2.3.6.})11ID~ent 693.2.3.7. Thiobarbituric &Cid test••..•••.•••••••.•••••••.••••••••••••••••••••..•••693.2.3.8. Statistica18D8lysis 70
3.3. Results and discussion.....•.............................•....................................723.3.1. Changes in headspace gas composition...........•..•...........................723.3.2. Color measurements (CIE-LAB values).........••..•...........................72
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3.3.2.1. Changes in L* (ligbtness) values........•..............................753.3.2.2. Changes in a* (redness) values..........•......•.......................753.3.2.3. Changes in b* (yeUowness) values......•..............................753.3.2.4. Changes in C* (chroma) values 763.3.2.5. Changes in h (hue angle) values 76
3.3.3. Sensory evaluation 793.3.3.1. Changes in color acceptability scores..•..............................793.3.3.2. Changes in Roche color chart scores 793.3.3.3. Changes in texture scores 823.3.3.4. Changes in general appearance scores 823.3.3.5. Changes in odor scores 85
3.3.4. Changes in drip loss 853.3.5. Microbiological analyses 88
3.3.5.1. Changes in mesophilic counts 883.3.5.2. Changes in psychrotropbic counts 883.3.5.3. Changes in lactic acid baeteria counts 893.3.5.4. Changes in aerobic and anaerobic spore forming bacteria.....•...89
3.3.6. Changes in pH values 893.3.7. TBA anaIysis 933.3.8. Overall shelf-life studies 96
3.4 COl1clltSioll ~
4. CHALLENGE STUDIES WITH USTERlA MONOCYTOGENES 1004.1. lotr()dhœctiOIl 1004.2. Materials and methods 101
4.2.1. Sample preparation. 1014.2.2. Bacterial strains•....•.•..••..••.•.......•.•••.••••..••........••........•••.••••..•1014.2.3. Inoculation 1024.2.4. Packaging aJld stora.ge conditioQS 102
4.2.5. J\Jtal~••••.••••••••••••••••••••.•••••••••••••.•••••••••••••••••••••••...•••••••••• 1034.2.5.1. Microbiologica1 analyses 103
4.2.6. Statistical analysis 1044.3. Results and discussion 1OS
4.3.1. Changes in headspace gas composition 1OS4.3.2. Sensory evaluation....•...........................................................1OS4.3.3. Changes in pH values 1074.3.4. Microbiological8D8lyses 112
4.4. COl1clltSÎon.....................................................................•..............118
S. CHALLENGE STUDœS WITH CLOSTRIDIUMBOTULINUMTYPE E..•.......119PART A: Challenge studies on MAP of ftesh trout fillets stored at 4 and 12°C....1195.1. Introduction....................•......................................•...•..................1195.2. Materials and methods....•........................................•........................120
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5.2.1. Sample preparation.........................•.................................... 1205.2.2. Bacterial strains and inœu1ation 120
5.2.3. Packaging and storage conditioDS 1205.2.4. Analyses 121
5.2.4.1. Headspace gas analysis 1215.2.4.2. Toxin assay 121
5.2.5. Statistical analysis 1225.3. Results and discussion 123
5.3.1. Changes in headspace gas composition 1235.3.2. Sensory evaluation 123
5.3.2.1. Changes in general appearance scores 1235.3.2.2. Changes in odor scores 125
5.3.3. Changes in pH values 1285.3.4. Toxin assay 128
5.4. Conclusion 134
PART B: Challenge. studies on MAP offtesh trout fillets stored at 12°C in anenvironment ofdiffereni oxygen concentratioDS..............•............................... 135s.s. Introduction 1355.6. Materials and methods 136
5.6.1. Sample preparation and inœu1ation 1365.6.2. Packaging and storage conditioDS............•.................................1365.6.3. Analyses..............................................•....•....................... 1365.6.4. Statistical analysis 136
5.7. Results and discussion 1385.7.1. Changes in headspace gas composition 1385.7.2. Sensory evaluation 138
5.7.2.1. Changes in color acceptability scores...•.......................... 1385.7.2.2. Changes in texture scores 1405.7.2.3. Changes in odor scores................•.............................. 140
5.7.3. Changes in pH values 1405.7.4. Toxin assay 143
5.8. Conclusion 146
PART C: ChaUenge studies on MAP fresh trout fillets stored at 12°C in films ofdifferent oxygen transmission rate 1475.9. IntreMiuction 1475.10. Materials and methods.....................................•............................... 148
5.10.1. Sample preparation and inœu1ation...•..............•....................... I485.10.2. Packaging and storage conditioDS.................•........................... 148
5.10.2.1. Film permeability: Oxygen transmission rate (OTR) I485.10.3. Analyses.......................................................•...................1495.10.4. Statistical anaIysis..............................•................................ 149
5.11. Results an.d discussion 1515.11.1. Changes in headspace gas composition.....•............•...............•....15 1
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5.11.2. SeDSOry eva1uatioD•........•.•.........•...................•.....•..... ..........151S.11.2.1. Changes in color acceptability scores•............................1S3S.11.2.2. Changes in texture scores 1S35.11.2.3. Changes in odor scores 1S3
S.11.3. Changes in pH values 1SS5.11.4. Toxin assay......................•............................ .................... ISS
S.12. Conclusion 1S8
6. CHALLENGE STUDIES ON COLD AND HOT SMOKED TROUT~~TS 1S~
6.1. Introduction l S~6.2. Materials and methods 161
6.2.1. Samples preparatioD.........................................•......•..............1616.2.2. Bacterial strains and inoculation 1616.2.3. Packaging and storage conditioDS....•....................•..................... 1616.2.4. Analyses 161
6.2.4.1. W&ter activity determination 1626.2.4.2. Salt concentration 162
6.2.5. Statistical analysis................•.........•................•......•.............. 1626.3. Results and discussion 1M
6.3.1. Sensory evaiuation...............•................................................ 1M6.3.1.1. Changes in color acceptability scores 1M6..3.1.2. Changes in texture scores 1686.3.1.3. Changes in ooor scores 169
6.3.2. Changes in pH values 1706.3.3. Toxin assay 170
6.4. Conclusion.................................•..•........................•...................... 175
GENERAL CONCLUSION 176~~E~C~S....................................•............................................... 17~
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USTOFTABLES
Table 1: Average cbemical composition ofrainbow trout 4
Table 2: Differences in the lipid content offish species 5
Table 3: Suggested fasting period based on water temperature.......•......•.........14
Table 4: Bacterial tlora offish caught in clear, unpolluted waters 21
Table 5: Intrinsic factors affecting spoilage rate offish species 22
Table 6: Sensory acceptability scale 45
Table 7: Standard Roche color chart 46
Table 8: Estimated subjective color shelf-life (days) oftrout fillets packaged under
ditTerent gas atmospheres and stored al 4°C based on the sensory acceptability scale and
the sensory Roche color chart 52
Table 9: Estimated objective shelf-life (days) extrapolated ftom seosory color
acceptability for trout filIets packaged under difTerent gas atmospheres and stored al
4°C 62
Table 10: Estimated objective shelf-life (days) extrapolated from sensory Roche
color chut for trout flUets packaged under different gas atmospheres and stored at
4°C 63
Table Il: Outline of microbiological analysis performed on rainbow trout
fillets '71
Table 1%: Changes in color coordinates of trout fillets packaged onder different gas
atlnospheres and stored al 4°C........................................................•...........7'7
Table 13: Changes in color coordinates of trout fillets packaged onder different gas
atlnospheres and stored al 12°C........................•...................•.....................78
Table 14: Estimated shelf-life (days) of air, vacuum and gas packaged trout fillets
stored al 4 and 12°C................................................................•...............97
Table 15: Changes in headspace Ch and CCh of control and inoculated trout fillets
packaged under ditferent gas atmospheres and stored al 4 and 12°C 106
Table 16: Changes in headspace Ch and 'CÛ2 of control and inoclliated trout fillets
packaged under different gas atmospheres and stored al 4°C...............•..............•124
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Table 17: Changes in headspace Ü2 and C02 of control and inoculated trout fillets
packaged under different gas atmospheres and stored al 12°C 124
Table 11: Estimated shelf-life (days) based on sensory analysis of control and
inoculated trout fiIIets packaged under different gas atmospheres and stored at
~oC................................•....................•............•.........................•.....• 126
Table 19: Estimated shelf-Iife (days) based on sensory analysis of control and
inoculated trout fiIIets packaged under different gas atmospheres and stored at
12°C.................................................. .............................•..................12ir
Table 20: Time to toxigenesis in trout fillets packaged under different gas
atmospheres and stored at ~oC........................................•.......................... 131
Table 21: Time to toxigenesis in trout fiIlets packaged under different gas
atmospheres and stored at 12°<:; 132
Table 22: Packaging treatments of control and inoculated trout fillets stored at
12°C 137
Table 23: Changes in headspace Û2 and CÛ2 of control trout fiIIets packaged under
ditTerent gas atmospheres and stored at 12°C 139
Table 24: Changes in headspace Ch and CÛ2 of inoculated trout fillets packaged
under different gas atmospheres and stored al 12°C 139
Table 25: Estimated shelf-life (days) based on sensory analysis ofcontrol trout fillets
packaged under ditTerent gas atmospheres and stored al 12°C 141
Table 26: Estimated shelf-life (days) based on sensory anaIysis of inoculated trout
fillets packaged under different gas atmospheres and stored at 12°C ~ ..•........ 1~1
Table 27: Time to toxigenesis in trout fillets packaged under different gas
~osph~and storedat 12°(: I~S
Table 28: Oxygen ttansmission rate (OTR) of films used to package control and
inoculated trout fillets stored at 12°C : .....................•.......•..............1SO
Table 29: Changes in headspace Û2 and CÛ2 levels of control and inoculated trout
fillets packaged under different gas atmospheres in films of ditTerent. OTR and stored at
12°C............................................................•........•..............••...........152
•
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xv
Table 30: Estimated shelf-life (days) based on sensory analysis and pH anaIysis of
control and inoculated trout fi1Iets packaged under different gas atmospheres in films of
different OTR and stored at 12°C 1S4
Table 31: Time to toxigenesis in trout fi1Iets challenged with C. botulinum type E
(1fil ceUslg) and packaged under different gas atmospheres in films ofdifTerent OTR and
stored al 12°C 1S6
Table 32: Oxygen ttansmission rate of films used for vacuum packaged control and
inoculated cold and hot smoked trout fillets stored al 4, 8 and 12°C 163
Table 33: Estimated shelf-life (days) based on sensory analysis and pH anaIysis of
control and inoculated smoked trout fillets vacuum packaged in films ofdifferent oxygen
transmission rate and stored al 4°C 165
Table 34: Estimated shelf-life (days) based on sensory analysis and pH analysis of
control and inoculated smoked trout fillets vacuum packaged in films ofdifferent oxygen
transmission rate and stored al 8°C 166
Table 35: Estimated shelf-life (days) based on sensory analysis and pH analysis of
control and inoculated smoked trout fillets vacuum packaged in films ofdifferent oxygen
transmission rate and stored al 12°C 167
Table 36: Time to toxigenesis in cold smoked trout tillets vacuum packaged in films
ofdifferent OTR and stored at 4, 8 and 12°C 171
Table 37: Time to toxigenesis in hot smoked trout fillets vacuum packaged in films
ofdifferent OTR and stored at 4, 8 and 12°C 172
••
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xvi
LIST OF FIGURES
Figure 1: Major changes due to autolysis and baeterial activity 18
Figure 2: General schematic offish deterioration 19
Figure 3: Autoxidation reaction sequence 20
Figure 4: Examples of food currendy heing packaged under MAP in North
~eri~ 25
Figure 5: Oxygen requirements ofmicroorganisms 29
Figure 6: Color acceptability scores of trout fillets packaged under different gas
atnlospheres and stored al 4°C 51
Figure 7: Roche color chart scores oftrout fillets stored packaged under different gas
atmospheres and stored at 4°C 51
Figure 8: L* values of trout fillets packaged under different gas atmospheres and
sto~ at4°C 57
Figure 9: a* values of trout flUets packaged under different gas atmospheres and
sto~ at4°C 57
Figure 10: b* values of trout fillets packaged under different gas atmospheres and
st()~at4°C 58
Figure Il: C* values of trout flUets packaged under different gas atmospheres and
st()~ at4°C 58
Figure 12: h values of trout fillets packaged under different gas atmospheres and
st()~at4°C 59
Figure 13: Changes in headspace <h of trout fillets packaged under different gas
atmospheres and stored at 4°C.. : 73
Fipre 14: Changes in headspace <h of trout fillets packaged under ditTerent gas
atmospheres and stored at 12°C 73
Figure 15: Changes in headspace C(h of trout fillets packaged under different gas
atm~heresandstoredat4°C...•................................................................74
Flpre 16: Changes in headspaœ C(h of trout fillets packaged under dift"erent gas
atmospheres .and stored at 12°C 74
•
•
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xvii
Figure 17: Changes in color acceptability scores of trout fillets packaged under
different gas atmospberes and stored at 4°C 80
Figure 18: Changes in color acceptability scores of trout fillets packaged under
different gas atmospberes and stored at 12°C 80
Figure 19: Changes in Roche color chart scores of trout fillets packaged under
difIerent gas atmospheres and stored at 4°C 81
Figure 20: Changes in Roche color chart scores of trout tillets packaged under
different gas atmospheres and stored at 12°C 81
Figure 21: Changes in texture scores of trout fillets packaged undcr different gas
atlnospheres and stored at 4°C 83
Figure 22: Changes in texture scores of trout fillets packaged undcr different gas
abDospheres and stored at 12°C.........................................•.........................83
Figure 23: Changes in general appearance scores of trout tillets packaged under
different gas atmospheres and stored at 4°C 84
Figure 24: Changes in general appearance scores of ttout tillets packaged under
different gas atmospheres and stored at 12°C 84
Figure 25: Changes in odor scores of trout fillets packaged under different gas
atIllospheres and stored at 4°C 86
Figure 26: Changes in odor scores of trout fillets packaged under different gas
atmospheres and stored at 12°C.........................................................•.........86
Figure 27: Changes in drip 10ss (%w/w) of trout fillets packaged under different gas
atulospheres and stored at 4°C 87
Figure 28: Changes in drip 105s (%w/w) of trout fillets packaged under different gas
atmospheres and stored at 12°C 87
Figure 29: Changes in mesophilic counts of trout fillets packaged under different gas
~tulospheres and storedat4°C ~ ~
Figure 30: Changes in mesophilic counts of ttout fillets packaged under different gas
~tulospheres id1d storedat 12°C..............•....................................................~
Figure 31: Changes in psychrotrophic counts of trout fillets packaged under different
gas atmospheres and stored al 4°C...........................•....................................91
•
•
•
xvüi
Figure 32: Changes in psychrotrophic counts of trout fiIlets packaged under different
gas atrnospheœs and stored at 12°C............................................................•..91
Figure 33: Changes in Iactic acid bacteria COUDts of trout fillets packaged under
different gas atmospheres and stored al 4°C...............•...................................•.92
Figure 34: Changes in lactic acid bacteria cOUDts of trout fillets packaged under
different gas atmospheres and stored al 12°C 92
Figure 35: Changes in pH values of trout fillets packaged under different gas
atmospheres and stored al 4°C.................•...............•...................................94
Figure 36: Changes in pH values of trout flUets packaged under different gas
atmospll~and storedat 12°C 94
Figure 37: Changes in TBA numbers of trout fiUets packaged under different gas
atmospll~andstored at ~oC 95
Figure 38: Changes in TBA numbers of trout fiUets packaged under different gas
. atmosplleresandstoredat 12°C 95
Figure 39: Changes in general appearance scores of control trout fillets packaged
under different gas atmospheres and stored al 4°C 108
Figure 40: Changes in general appearance scores of inoculated trout fiUets packaged
under different gas atmospheres and stored al 4°C 108
Figure 41: Changes in general appearance scores of control trout fillets packaged
under different gas atmospheres and stored at 12°C 109
Figure 42: Changes in general appearance scores of inoculated trout flUets packaged
under different gas atmospheres and stored at 12°C l 09
Figure 43: Changes in pH values of control trout fillets packaged under different gas
atmospheres and stored al 4°C = 110
Figure 44: Changes in pH values of inoculated trout fillets packaged under different
gas atmospberes and stored al ~oC : ..................•................................110
Figure 45: Changes in pH values of control trout fiUets packaged under different gas
atInospheres and stored al 12°C 111
Figure 46: Changes in pH values of inoculated trout fiIlets packaged under different
gas atmospheres and stored at 12°C.......................•....•............................... 111
•
•
•
xix
Figure 47: Changes in counts ofL. monocylogenes ofinoculated trout fillets packaged
under ditTerent gas atmospheres and stored at 4°C 114
Figure 48: Changes in counts ofL. monocytogenes of inoculated trout fillets packaged
onder different gas atmospheres and stored at 12°C 114
Figure 49: Changes in mesophilic counts of control and inoculated trout fiIlets
packaged under different gas atmospheres and stored at 4°C 115
Figure 50: Changes in mesophilic counts of control and inoculated trout fiIlets
packaged onder different gas atmospheres and stored at 12°C 115
Figure 51: Changes in psychrotrophic counts of control and inoculated trout fiIlets
packaged onder different gas atmospheres and stored at 4°C 116
Figure 5%: Changes in psychrotrophic counts of control and inoculated trout fiIlets
packaged under different gas atmospheres and-stored at 12°C 116
Figure 53: Changes in lactic acid bacteria counts ofcontrol and inoculated trout fillets
packaged under ditTerent gas atmospheres and stored at 4°C 117
Figure 54: Changes in lactic acid bacteria counts ofcontrol and inoculated trout fillets
packaged under different gas atmospheres and stored at 12°C 117
Figure 55: Changes in pH values of control trout fillets packaged under ditTerent gas
atmospheres and stored at 4°C ,; 129
Figure 56: Changes in pH values of inoculated trout filIets packaged under different
gas atmospheres and stored at 4°C 129
Figure 57: Changes in pH values of control trout fillets packaged under different gas
atmospheres and stored al 12°C 130
Figure 58: Changes in pH values of inoculated trout fillets packaged under different
gas atmospheres and stored at 12°C 130
Figure 59: Changes in pH values ofcontrol trout fillets packaged onder various levels
ofheadspace Û2 and stored at 12°C 142
Figure 60: Changes in pH values of inoculated trout fillets packaged under various
levels ofheadspace Ch and stored at 12°C....................•................................142
•
•
•
1
CBAPfERl
INTRODUcnON AND LITERATURE REVIEW
1.1. Introduction
Approximately 700A. of the total world's area i.e. approximately 360 million
square kilometers of the earth's surface is covered by seas (Riedel, 1961). Fish are
aquatic animais and are the MOst numerous of the vertebrates with more than 20, 000
known spccies (Thurman and Webber, 1984). The various fish speçies cao he sub
divided as foUows: 58% from the marine environment and 42% from fteshwater
(Thunnan and Webber, 1984). Due to the increasing world population and shortage of
land, more emphasis bas been placed on fish as a protein source. Over the past few years,
there bas been a growing interest in aquaculture to meel these needs. Aquaculture
involves the cultivation and harvesting of fresh and marine aquatic animais and is more
comparable to agriculture than to commercial fishing. Aquaculture provides lOto15% of
the seafood consumed worldwide. The most popular aquacultured products are salmon
with -30010 of aU salmon consumed in the world coming from fish farms (Rumsey, 1988;
Bjorndahl, 1990). Trout is another product which is farmed worldwide with France being
the main producer (Gabriel, 1990). In 1987, more than 250, 000 tons were PrOduced with
16 million lb. produced annually in the United States alone (Simpson et a/., 1981).
Rainbow trout, also known as Sa/mo gairdneri, and sometimes Sa/mo irideus, which bas
recently changed its name to Oncorhynchus mylciss (Billard and Nado, 1989) is the fish
species exploited in aquaculture that bas heen the Most studied in laboratorics.
•
•
•
2
1.2. Cbe_ut composition of fis.
The chemical composition offish varies greatly from species to species and within
individual tish of the same species according to age, sex, environment and season (Russ
and Borresen, 19958). The average chemical composition of rainbow trout is shown in
Table 1.
1.2.1. Fish proteins
Fish proteins are divided into 3 groups: (i) structural proteins: e.g., actin, myosin,
topomyosin and actomyosin (70 to SOOAt of the total protein) (ü) sarcoplasmic proteins:
e.g., myoalbumin, globulin and enzymes (25-300A. ofthe total protein) and (iü) connective
tissue proteins: e.g., collagen « 1% of total protein). Fish proteins contain aU the
essential amino acids, such as lysine, tryptophan, histidine, phenylalanine, leucine,
isoleucine, threonine, methionine-cystine and valine (Huss and Borressen, 1995a).
1.2.1. Fish lipids
Fish lipids are mainly found in the abdominal and dorsal subcutaneous adipose
tissue and in the intermuscular adipose tissue of tish. A small amount may a1so be
deposited in the red muscle fiber (Fauconneau et a/., 1990). The composition of tish
lipids varies according to species, diet, temperature, salinity, selective mobilization,
selective distribution ete. (Lovern, 1950). In generai, tish lipids comprise of -40010 long
chain, high1y unsaturated, fatty acids (Stansby and HaU, 1967). Haard (1 992a), showed a
relationship between saturation and temperature with fatty 8Cids becoming progressively
more unsaturated as temperature decreased. Fish fats are consequently more or less liquid
at low temperature, depending on the proportion of polyunsaturated fatty acids.
Depending on the amount and the location of lipid, fish C8D be categorized as either lean
or fatty. If the lipids are stored in the liver, the fish is considered lean, wbüe ü the Iipids
are stored in the fat cells and distributed in other body tissue, it is coDSidered as a fatty
•
•
•
3
fish (Huss and Borresen, 19958). Based on this criteria, rainbow trout is regarded as a
fatty fish (Table 2).
1.2.3. Fish carbohydrates
The carbohydrate content of fish muscle is <0.5%. It is comprised of glycogen
which is a component of nucleotides (Huss and Borressen, 19958). Although the
carbohydrate content is relatively smaIl, it varies according to seasonal changes
associated with diet, spawning and water-temperature (Ang and Hsard, 1985; Black and
Love, 1986). As expected, the level of Cree sugars is not higb enough to impart a sweet
taste to fish but it contributes to non-enzymatic browning during fish processing (Haard
and Arcins, 1985).
1.1.4. Fish vitamins and minerais
Fish is a good source of vitamins, especially vitarnins of the B complexe Sînce
salmonids are considered fatty fish, they are a1so a good source of vitamins A and D
(Murray and Burt, 1969). The minerai content of fish is directly proportional to the
minerai content present in the water habitat. Fish therefore is regarded as an important
source of minerais, specifically calcium, phosphorus, iron, copper and selenium.
Minerais in11uence the nutritive value and taste offish (Love, 1988).
•
Table 1: Average chemical composition ofrainbow trout (Kiessling et a/., 1989)
•
•
Constituents
Water
Protein
Lipid
Onchorhynchus mylciss
70-800.!ct
15-22%
2-90/'0
•
Table Z: Differences in the lipid content offish species (Jacquot, 1961)
Fatty Semifatty Lean
Rening Barracuda Coalfish
• Mackerel Bass Cod
Pompano Mullet Haddock
Pilee Perch Hake
Salmon Sbark Plaice
Shad Smelt
Trout
Tuna
•
•
•
•
6
1.3. Quality of fish
The world demand for good quality ftesh fish is increasing. Consumers'
satisfaction of fishery produets depends on various traits such as safety, nutrition, tlavor,
taste, texture, colof, appearance and suitability of the raw material for processing and
preservation (Haard, 1992a), as weil as the aesthetic appearance and fteshness of tish
(Huss and Nielsen, 1995a). Each of these quality parameters will be discussed in the
following sections.
1.3.1. Extemal appearance offish
Healthy fish should have an intact skin of uniform color, free of abnormalities
(e.g., fungi infection). It should he covered by a transparent mucus layer; small black or
white spots on the fish body may he an indication of parasites. Hea1thy fish also have
transparent eyes with black pupils. If the eyes are white or iDflated, the fish MaY be
underfed. The gills should be bright red and non-swollen. If they are light pink, it is an
indication ofanemia or a Iack ofoxygen (Hansen and Landry, 1982).
1.3.%. Texture offish
Prior to death, fish should remain in clean, oxygenated, cold water never
exceeding 12°C in order to obtain a firm flesh. Theyalso need to fast prior to slaughter in
order to remove ail the food that might be present in their digestive tract (Table 3). The
fasting enhances the product quality, especially when evisceration is not performed
correctly.
Once fish have been harvestecl, it is essential 10 keep them as fresh as possible.
This is achieved through good temperature control (1°C). If the temperature fluctuates, it
is necessary to eviscerate fish within the 30 minutes of harvesting (Hansen and Landry,
1982).
• 1.3.2.1. Rigor mortis
7
•
Evisceration (removal of the viscera, gills and kidney) must be performed as soon
as possible after death. At the point ofdeath, there is no more cardiac aetivity and so the
brain and tissues are no longer provided with oxygen. Immediately after death, the
muscle is totally relaxed and the elastic texture persists for severa! hours using up reserve
oxygen and nutrients. After death, glycogen or fat is oxidized or bumed by tissue
enzymes producing C02, water and ATP (Huss, 1995). As a consequence of these
changes, 6sh pH decreases and ATP will start to breakdown causing muscle stitTening
due to contraction of the transverse skeletal muscle (Amlacher, 1961). The whole body
becomes inflexible and fish enter rigor mortis for a day or more until resolution.
Resolution of rigor renders the muscle soft again but it will not recover its initial
elasticity. Resolution of rigor morlis varies according to fish species, temperature,
handling conditions, size and physical condition of the fish. The resolution of rigor is
thought to be associated with the activation of one or more muscle enzymes assimilating
some components of the rigor morlis complex (Huss, 1995). It is relevant to know the
state of the biochemical changes proceeding for transformation. In filleting for example,
the operation will yjeld a poor product and may even lead to gaping if it is performed
during rigor morlis. If the procedure is carried out pre-rigor, the muscle will contract
fteelyand shorten following rigor. For thermal processing, the texture will be soft and
pasly if cooked pre-rigor, tough if cooked in rigor and firm, elastic and delicious if
cooked post-rigor (Huss and Nielsen, 1995b).
1.3.2.2. Factors affecting texture
•Texture of fish is affeded by severa! factors including the rate and extent of rigor
mortis, the amount and type of fatty aciels, distribution of muscle fat and the amount of
exercise (Mobr, 1986). Swimming, in fact, improves the texture of fish by retarding
postbarvest softening and, depending on the velocity, it may increase the quantity of red
muscle (moderate velocity) or white muscle (greater velocity) (Davison and Goldspink,
•
•
•
8
1977). Since the exercise pattern of fann-raised fish is different, it results in fish with a
softer texture. Another factor that influences texture is the size of the tish. The number
and size of muscle cells is directly proportional to the size of the tish. Therefore, larger
fisb tend to have a firmer texture compared to smaller fish within the same species (Love,
1988).
1.3.3. Taste and aroma offish
Flavor compounds include Cree amino acids, minerais, organic &eids and
quatemary ammonium bases (Konosu, 1979; Love, 1980; Fine, 1992). The ftee amino
&Cid content increases during growth 50 mature fish have a stronger taste (Suzuki and
Suyama, 1980). Since farmed fisb tend to have less ftee amino &Ciels than wild fisb, their
taste appears weaker or insipid to some people (Haard, 1992a). The taste of fish may be
atTected by its diet. For example, a study showed that farmed salmon fed shrimp waste as
a source of carotenoid pigment resulted in fish· with a better fish t1avor than when
synthetic carotenoids were used (Haard, 1992b). The amount of fat in the diet, as weil as
the type of lipids, may influence fish taste. For example, high levels of soybean oil in
salmonid feed was responsible for the off-flavor termed the "hatchery f1avor" (lnoue et
al., 1988).
The aroma from fish is mainly due to enzyme eatalyzed reactions of
polyunsaturated fatty acids, also, the environment in which the fish is raised may
contribute to its odor c.g., smalt concentrations of algae Metabolites in water (Josephson
and Lindsay, 1986).
1.3.4. Color of fish
''We eat first with our eyes". This expression explains the importance of color as
a quality parameter. In fact, the chroma, hue, shade and tint of fish will almost instandy
determine its acceptability as a food. Consumers will reject a food that bas an unusual
•9
color since this will be perceived as a sigtl of jmmaturity, spoilage, inadequate proces5iDg
or adulteration (Klaui and Bauemfeind, 1981). Therefore, it is essential for a product to
maintain proper and consistent color.
1.3.4.1. Origin ofthe red color in salmonids
•
Salmonids have a reddisb color wbich does not originate from myoglobin but
trom carotenoids (Huss and Borresen, 1995b). Carotenoids are a group of fat-soluble,
natural pigments that provide yellow, orange and red cololS in plants and animaIs
(Matsuno and Hirao, 1989) and are the pigments responsible for the pinklred muscle
coloration of salmonids (No and Storebak:ken, 1992). In the wildemess, carotenoids are
synthesized by primary producers (bacteria, a1gae, yeast and higher plants (Liaaen-Jensen,
1991». They are passed up the food chain until reaching salmonids where they are
assimilated and stored in their flesb imparting a distinctive pinklred color (Youngson et
a/., 1997). AnimaIs, including salmonids, are in fact incapable of de novo synthesis of
carotenoids. Farm raised trout are fed a diet including carotenoid pigments, astaxanthiD
and canthaxanthin, to impart the desired muscle color to the fish.
Canthaxantin bas heen trequendy used for pigmentation of the salmonid flesh
(Simpson et al., 1981).
o
•o
Canthaxanthin or Diceto 4,4'p-carotene
•10
However, this pigment is now being replaced by astaxanthin, the main pigment of wild
salmon and crustaceaos (Simpson et a/., 1981).
o
o
Astaxanthin or 3,3'-dihydroxy..JJ,p.carotene 4,4' dione
1.3.4.2. Astaxanthin and canthaxanthin
•
•
Astaxanthin imparts a pink color approaching the color of wild fisb while
canthaxanthin imparts a rich orange color to the tlesh. This deep orange color is
perceived as added colorant by Most consumers (Blockhus, 1986; Mac, 1990).
Astaxanthin is an oxycarotenoid (formula C.wHS20..) with a molecular weigbt of 596.86.
Il bas been shown to be deposited more efficiendy than canthaxanthin but a synergistic
effect was observed (bigber deposition of total muscle pigment ) when both pigments
were ingested in combination rather than individually. The pigments are absorbed ftom
the diet which usually contain ~7S mg astaxanthin and/or canthaxanthin per kg
(Torrissen et al., 1989). They are distributed throughout the fish muscle where they bind
to actomyosin (Henmi et a/., 1987). The choice and concentration ofcarotenoids utilized
is made on the basis of the desired color and legal status. Astaxanthin is listed as a color
additive in salmonid feed to pigment their flesh by the United States Food and Drug
Administration while canthaxantbin is listed in the CFR (Code ofFederal Regulations) as
a food color additive (Turujman et a/., 1997). The addition of carotenoid pigments bas
been shown to be saCe without any toxicity effect on muscle cells~ 1993). However,
in order to obtain consistency in pïnk..red coloration of salmonids, feeding procedures
must he established. Genera1ly, the diet will be supplemented when fish have reached
•11
50010 of their final weight (Hansen and Landry, 1982). Most salmonids, in fact
accumuJate astaxanthin in their muscle when they have reached a certain body size.
1.3.4.3. Deposition ofpigments
•
•
There are many factors intluencing the amount ofcarotenoid present in salmonid
flesh. For example, there are variations in the distrIbution of the pigment in the tish
body; 10ngitudina1ly, there is 30-4()0/O more carotenoid al the caudal (mid..section) part
while radially there is an increase in pigmentation towards the backbone (March and
MacMillan, 1996). Different species may also show various responses 10 dietary
supplementation. Muscle of Atlantic salmon was distinctly less pigmented than rainbow
trout for a similar intake of dietary astaxanthin. However, the pigmentation pattern was
similar for both species (March and MacMillan, 1996). There are also ditrerences
between sexes and ~ong individuals within the same sex of similar strains of salmonid
species (Choubert et a/., 1992). Out of the breeding period, female muscles contain more
carotenoids and consequently are more colored than males but, al sexual maturation,
carotenoids are relocated to the ova and the level of carotenoids in the tissue decreases
(Torrissen and Torrissen, 1985). Other important changes occur during sexual maturation
due to self imposed starvation. Such modifications result in a decrease in long chain
highly UDSaturated fatty acids in muscle plus a shift in pH (Haard, 1992a).
Carotenoids are deposited mainly in tish muscle but also in the skin, eggs, gonads,
milt, liver and eyes (Simpson et a/., 1981). A level of 3 to 4 mw'kg or greater of
astaxanthin results in good fish flesh coloration. However, during bandling it may fade or
isomerize, so levels of >4 mWJcg should he aimed when marketing salmonids (Tonissen
et a/., 1989). Astaxanthin is stored in the skin as esters and in the tlesh and eggs as fiee
astaxanthin (non..esteritied). Canthaxanthin is deposited to color salmonid muscle
(Simpson et a/., 1981). It bas been demonstrated that astaxantbin is morc efticiently
utiljud than canthaxanthin (Torrissco et a/., 1989) but astaxantbin is depleted at a faster
rate as it is chemically less stable than cantbaxanthin in salmon (SigurgisladoUir et al.,
•12
1994). However, it is important to note that the absorption of carotenoids is generally
poor i.e., <1 ()oA» of carotenoid in the feed. This low level of absorption may he due to
oxidation in the alimentary canal (Johnson and An, 1991). However, the metabolism of
carotenoid ingestion, transportation to deposition in the muscle bas not been elucidated
(Withler, 1986).
1.3.4.4. Absorption ofpigments
•
Several factors are known to influence pigmentation including species, strain,
size, age, diet, carotenoid source and supplementation rate (Tonissen et al., 1989).
Several studies have been done in this area to maximize the effect of fish nutrition on
tlesh coloration. A positive correlation in a relatively short period oftime (2 months) bas
been reported between the increase in dietary lipids and the fixation of canthaxanthin.
Moreover, no correlation was observed with an increase in the protein or calorie content
This phenomenon was explained on the basis of liposolubility of the pigment. The
Iipophilic nature of fat content had a positive effect on the dispersibility ofcanthaxanthin
and therefore its absorption and transfer within the flesh (Malak, 1975). Diets
supplemented with a-tocopherol increased the levels of pigment deposited in the tlesh.
Vitamin E is therefore regarded as an essential nutrient in the diet of salmonids. In
addition to enhancing retention of astaxanthin, a-tocopherol protected the pigments from
oxidation in the salmonid muscle (Sigurgisladottir el al., 1994). However, an excess of
vitamin A in the diet bas a1so been shown to have an antagonistic effect on the fixation of
canthaxanthin (Malak, 1975).
1.3.4.5. Discoloration ofpigments
•Even though astaxanthin and canthaxanthin impart a desirable red-pink coloration
to the flesh of salmonids, undesirable changes in color with time, due to various factors,
accor. First, discoloration O18y result ftom rough handling during harvesting. Physical
mishandling in the net (large catches, long trawling time) or on the dcck, may cause
•
•
•
13
marks and tearing ofblood vessels (Huss and Borresen, 199Sb). Discoloration may also
occur as a result of Iipoxygenase 8Ctivity (Simpson et a/., 1981). Browning of the flesh
muscle O18y aIso he encountered from oxidation of myoglobin and hemoglobin.
However, this phenomenon varies in intensity since usually the fish muscle is properly
bled (Sainclivier, 1983). Contrary to Meat pigments, which require oxygen for blooming,
seafood coloration is not a problem if oxygen is limited unless it is a higbly pigmented
sPeCies (Stammen et a/., lm). Red muscle pigmentation is dynamic i.e. it will intensify
when the fish becomes more active and fade when it is at rest (Love et al., 1977). Lipids
may he oxidized and modify the tlesh color rendering it yeUowish (Gendron et al., 1993).
Astaxanthin bas a role as a scavenger of lipid oxidation and so bleaching of carotenoids,
as a result of complex autoxidation reactions, will occur (Andersen et al., 1990). Since
the chemical structure of carotenoids contains long conjugated double bonds, light, heat,
acids, oxygen and enzymes have a detrimental effeet and cause the destruction of the
pigment through oxidation (Johnson and An, 1991). Light, air circulation and heat will
also enhance or accelerate the oxidation process and result in the loss of natural color
(fading, darkening or change in hue) in both natura! and synthetic carotenoids
(Bauemfeind et af., 1977). Discoloration occurs mainlyal the abdominal wall of fish
(Daewood et af., 1986; Teskeredzic and Ptiefer, 1987). Therefore, fillets are more likely
to he discolored. Processes used in the preparation or preservation may aIso alter
pigments chemically and physically and consequently their color (KIaui and Bauemfeind,
1981). For example, if packaging stimulates the accumulation of mebnyoglobin in red
fleshed fish, il will result in darkening of the fish muscle. Also, in gas packaging, if the
carbon dioxide concentration is too high, the color of the beUy flaps, comea and skin may
he modified (Haard, 1992c).
•
•
•
IS
1.4. SpoUage of filh
1.4.1. General spoilage pattern offish
Spoilage of fish occurs mainly as a result of autolysis (self- digestion) and
microbial growth (Figure 1). Spoilage is initiated by tissue enzymes (autolysis) foUowed
by microbial growth. Microbial spoilage occurs either direcdy through production ofotT
odors or indirectly by excreting enzymes that cause tissue decomposition. Tissues with a
high metabolic rate are more susceptible to spoilage than ones with a low metabolic rate.
Microbial spoilage depends on the level of contamination and type of spoilage bacteria.
Microorganisms come from water, through cross-contamination on ship and through
handling and processing (Huss and Gram, 1995a). The characteristic pattern of fish
deterioration is shown in Figure 2.
In the case of sensory quality of fish, four stages are encountered during fish
deterioration: (1) fish is fresh with a sweet, seaweedy delieate taste (2) disappearance of
distinctive odor and taste (neutral) (3) signs of spoilage accompanied by undesirable
malodorous substances varying in intensity depending on the fish species and type of
spoilage; texture becomes either soft and watery or tough and dry and (4) tish is putrid
and overtly spoiled (Huss and Nielsen, 199Sb).
1.4.2. Chemical deterioration offish
Two distinct reactions occur to fish lipids: hydrolysis and oxidation. 80th
reactions result in the production of a rancid taste and smell and may also affect the
texturai properties oftish. The re&etions may either be enzymatic (microbial, intraeeUular
or digestive enzymes) or non-enzymatic and depend on fish species and storage
temperature (Huss and Jorgensen, 1995). Fatty fish are more prone 10 Iipid degradation
than lean fish. Rancidity is also more pronounced directly under the skin where most fat
is located (Bramstedt and Auerbach, 1961). Hydrolysis resu1ts in the formation of free
•
•
•
16
fatty 8Cids from triglycerides cleaved by lipases (Huss and Jorgensen, 1995). The process
ofautoxidation is shown in Figure 3.
During the initiation step, two criteria must he met to start autoxidation. Firsdy, a
hydrogen atom must he removed and secondly, molecular oxygen must he present (from
the atmosphere). The removal ofhydrogen cao he achieved by the interaction with light,
heat or enzymatically Oipoxygenase which is present in various amounts in fish tissue).
From the schematic, R-H =- Re + He, a ftee radical is formed which is extremely
reactive. This combines with another ftee radical or another hydrogen atom. However, if
oxygen is present, it will react with this as its tirst choice (almost zero activation energy)
provoking a degradative reaction in the product. The propagation step involves the
reaction ofoxygen with a free radical to form a peroxy radical, R- +~ =- R-O-O-. The
peroxy radical is unstable and reacts with an other molecule to remove its hydrogen R-H
+ R-Q-o- ~ R·O-O-H + R-. Hydroperoxides are formed and another ftee radical is
Iiberated which will undergo a similar cycle causing an accumulation of hydroperoxides
with time. Another reaction (less likely to occur in comparison to propagation) cao
terminate autoxidation depending on the reactive spccies present. In fact, the peroxy
radical cao react with a free hydrogen, Re + H- =- R-H, with a ftee radical, R-Q-o- + R
~ R-O-O-R, or with another peroxy radical R-O-O- + R-O-O- => R·O-Q-R + 02 causing
the formation of neutral species (Gordon, 1990). The hydroperoxides produced in large
amounts are tasteless, but their breakdown leads to the formation of secondary
autoxidation products, such as aldehydes, ketones, alcohol, small carboxilic acids and
alkanes. lbese are responsible for malodorous substances and, in some cases, to a
yellowish discoloration of fish flesh (Russ and Jorgensen, 1995). However, fish possess
certain mechanisms to proteet against oxidation e.g., the enzyme glutathione peroxidase
which reduces the production of hydroperoxides. Fish aIso contains vitamin E (a
tocopherol) .which is an important natural antioxidant (Russ and Jorgensen, 1995).
Inhibition of Iipid oxidation is possible through limiting oxygen availability and tbrough
low temperature. The latter effcct influences both enzyme activity and microbial growth
(Barnett et al., 1982).
•
•
•
17
1.4.3. Microbial degradation offish
The tlesh ofhea1thy live tish is sterile because of the action of the immune system
preventing growth of bacteria in the flesh. At the point of death there is no more
protection ftom the immune system and bacteria are Cree to grow. Bacteria tirst attack the
fish surface and tlesh by moving between the muscle fibers. The early stages of fish
spoilage occur in the extemal part of fish as a result of baeterial enzymes diflbsing into
the flesh (Russ and Gram, 1995a).
The spoilage microflora is dominated by Gram negative, psychrotrophic bacteria
which are capable of growth at O°C but have an optimum growth temperature of - 25°C.
Gram positive bacteria may also be found but generally the spoilage microOora of fish is
dominated by Gram negative bacteria (Table 4). The bacterial load is an important
parameter influencing spoilage. A value of 101 cfulg ofpsychrotrophic bacteria is used as
a standard offish spoilage (lCMFS, 1978). However, values of 108_109 cfulg bave been
proposed before overt spoilage 0CCUfS. (Stier et a/., 1981). Several factors determine if
fish is likely to undergo rapid spoilage or slow spoilage onder similar storage conditions
(Table 5). Microbial degradation of protein is responsible for most of the undesirable
changes in tlavor and odor (Brady, 1989). Distinctive amino 8Cids are precursors of
malodorous substances. For example, lysine can he converted to cadaverine and
putrescine while arginine can be converted to 3-aminovaleric &Cid or &.aminovaleric
aldehyde (Oba~ 1952-1953).
•
10,---------------------.
141210
Bacterial activity
8642
8
O"'--.....:.~-__+--_+--__+_--.........-..:..-._+_-__t
o•Figure 1:1976)
Major changes due to autolysis and bacterial activity (Adapted ftom Buss,
•
•
•
Catch
UDeath
UIntenuption ofblood circulation
UCessation ofoxygen supply
UDrop in redox potential
UInterruption ofcellular respiration
uDropinATP
uRigor morlis
uStorage ofhypoxanthin
UBacterial growth
UDegradation compounds
Glycogen => Iactic &Cid
DecreaseinpH
Cathepsins activation
protein ~ amino &Cid
•Figure 2: General schematic of fish deterioration (Jacober and Rand, 1982)
•
•
Initiation: R-H => R- + H-
Propagation: R- + Û2 => R-O-Oe
R-H + R-Q-O- => R-O-O-H + R
Tennination: R- + H- => R-H
R-o-O- + R- => R-o-O-R
R-o-O- + R-o-Oe => R-o-o-R + Ch
•
Figure 3: Autoxidation reaction sequence
•
Table 4: Bacterial flora of tish caught in clear, unpolluted waters. (Huss and Gram,1995a)
Gram Relative Gram positive
Pseudomonas Bacil/us
Moraxel/a Clostridium
Acinetobacter Micrococcus
• Shewanella putrefaciens Lactohacillus
CommeRas
Even thougb S. putrefaciens is sodium
requiring it bas been isolated from
freshwater environment
•
F/avohacterium
Cytophaga
Vibrio
Photohacterium
Aeromonas
Coryneforms
Vibrio is typical in marine waters
Photobacterium is typica1 ofmarine waters
Aeromonas is typical of freshwater
•
TableS: Intrinsic factors atTccting spoilage rate offish 5peCies (Huss & Gram,1995b)
•
•
Factors affecting spoUage Fast relative spoUage rate Slow relative spoU_le rate
rate
Size small fisb larger fish
Post mortem pH highpH lowpH
Fat content fatty species lean 5peCie5
SIdn properties thin sm thickskin
•
•
•
23
15. Preservation offis"
Fish is an unstable, bighly perishable food (Brody, 1989). Several preservation
techniques cao be used today to market fish in various forms; live, fresh, frozen, boneless,
filet, canned, marinated, pre-eooked, stuffed, dried (Hansen and Landry, 1982). Smoking
is also a Mean of preserving fish that bas been known for many centuries. The vast
majority of smoked fish products are cold smoked, and therefore treated as raw fish and
require to be cooked at home. However, smoked salmon and trout are notable exceptions
in that they are cold smoked that are consumed uncooked. Another method is hot
smoking where the products are normally consumed cold. In hot smoking, the core
temperature rcaches 70°C, therefore, the heat of the process will destroy aImost all the
vegetative microbial ceUs present in the raw 6sb, and this alone will extend the shelf-life
by severa! days if the temperature during distribution and retail display is maintained
below 4°C. Part of the preservative mechanism, however, still depends upon the brining
and subsequent chilled holding process allowing a surface brine-soluble protein pellicle
to form. This damp skin takes up most of the antioxidant and bacteriostatic substances
ftom the smoke hefore hardening to complete a banier against further bacterial invasion
which in tom delays rancidity. However, once this barrier is broken, the fish flesh is an
ideal medium for the growth ofmost spoilage organisms present in the air or on the bands
of operatives and can rapid1y become a health bazard Cold smoking provides a similar
antioxidativelbacteriostatic/physical pellicle, but the process does not increase the internai
temperature of the fish to kill the internai microtlora nor lowers the water 8Ctivity
sufficiently to inhtoit post-processing growth. Even with the ovemight salting of fish
sides prior to smoking, the product's sub-surface water activity remains higher tban 0.96,
which is insufficient to prevent growth of many spoilage and Pathogenic organislDS
(Homer, 1992). However, consumers prefer ftesh fish. Several methods bave been
developed to prolong the shelf-life of ftesh fish ftom chilling with ice to modified
atmosphere packaging. Modified atmosphere packaging (MAP) bas been defined as "the
enclosure of food produets in a high gas barrier film in which the gaseous environment
•
•
24
bas been cbanged or modified to slow respiration rates, reduce microbial growth and
retard enzymatic spoilage with the intent ofextending shelf Iife" (Youog et al., 1988).
1.5.1. Modified atmosphere packaging offish
MAP can he used for shelf-life extension of fresh fish in conjunction with
refrigerated storage (Reddy et al., 1992). lbere is an increased cOJ1S!1lDer demand for
fresb and chilled preservative free products. With portion control and the decrease in
sales of canned and frozen food, MAP is rapidly becoming the food
processinglpreservation technology ofthe future (Farber, 1991). In Europe, sales ofMAP
fish, produced from 1986 to 1990, increased five fold to 250 X 106 packages (Davis,
1995). However, the use of MAP in North America is still in its infancy as the
distribution chain is longer and consumers are not as weU edueated conceming MAP
storage requirements (Stammen el al., 1990). Examples of products packaged under
MAP conditions in North America are summarized in Figure 4. However, MAP fish is
only permitted in Canada
A recent Canadian survey showed that consumers had a positive PerCeption of
MAP technology as a Mean of saving money and reducing waste (Smith et al., 19908).
MAP is not a relatively new preservation technique. The effect of increased carbon
dioxide and reduced oxygen levels on shelf-life stability of fish under controlled
temperature was noted in the 1930's (Smith et al., 1990b). MAP can extend the shelf-life
of ftesh fish, but it must he used with low temperature storage (Brody, 1989).
1.5.1.1. Methods ofgas packaging
•There are 3 main metbods to package fish under modified atmospberes; bulle
container, master pack and individual packs. Master packaging consists of fisb packaged
in expanded POlystyrene trays (EPS) with a polyvinyl chloride (PVC) ovcrwrap. They are
placed in a master pack made of high barrier matcrial which is then tlusbcd with the
•
•
A. Fresil ra", mea"
e.g. sliced bacon
steak
heefhearts
pork kidneys
oxtails
B. Cooked meats
e.g. hamburgers
beefjerky
sausage rolls
sliced meats
C. Poultry
e.g. whole carcasses
nuggets
cbicken parts
peeled bard cooked eggs
D. Fisll (Canada only)
E. Clleese
F. Prepared saladl
(mainly al restaurant level )
G. Puta
H. Various types of sandwlclles
•
Figure 4: Examples of food currently being packaged under MAP in North America(Farber, 1991)
26
appropriate gas mixture. This creates a gaseous equilibrium within the master package
and tish will only be removed from the master pack in the supermarket (Brody, 1989).
Strict temperature control during transport and distribution is essential (Stammen et al.,
1990). Barnett et al. (1982) found that salmon was organoleptically acceptable after 2
weeks storage in l000A. carbon dioxide. The advantage of master packaging is that the
tish is ready for retail display. However, once removed from the master pack. normal
spoilage indicators (Gram negative bacteria) will predominate (E1dund, 1982).
Consumers also perceive this product as fresh and do not suspect it to have an extended
shelf-Iife (Stammen et al., 1990). However, there are several disadvantages to master
packing. Fish must be arranged properly in the primary pack for retail display and the
film used must he strong to withstand changes in pressure occurring during removal ofair
and tlushing of the appropriate gas mixture. The master pack film should therefore be
robust to avoid puncturing and tearing (Lindsay, 1981).
•
• 1.5.1.1.1• Packaging films
•
Packaging films are essential for the success ofMAP foods. According to the Sea
Fish Industry Authority, the quality of packaging materials include strength, clarity,
thickness, transparency, thermoformability, ability to form a seal and seal strength, anti
fogging properties, permeability, ease of displaying priee sticker and stability during
storage. (Sea Fish Industry Authority, 1985) Polymers which are used for gas packaging
include polyamide (nylon), polypropylene (PP), polyvinylidene chloride (PVDC),
ethylenevinyl a1cohol (EVOH) and polyethylene (PE). &ch polymer bas distinct
characteristics and lamination of polymers cm be achieved to take advantage of their
individual properties. Common combinations are nylon-PVDC-PE and nylon-EVOH...PE;
where nylon provides strength to the externallayer, PVDCIEVOH provides gas barrier
and PE is the heat sealant layer (Smith et al., 199Ob).
• 1.5.1.1.2. Gas composition
27
•
Normally carbon dioxide, nitrogen and oxygen are used in gas packaging of tish.
Smce these gases are the ones we breathe, therefore theyare not considered dangerous
nor are they considered as food additives (Smith et al., 199Oa). Nitrogen is an mert gas
and bas no microbiological effect on food. It is mainly used as a filIer gas to prevent
package collapse due to carbon dioxide absorption from the product (Farber, 1991). It is
also used to replace oxygen in low water activity (aw) products and therefore prevent
oxidative rancidity. Oxygen is generally avoided in MAP foods with the exception ofred
Meats and to continue respiration in certain commodities, such as fruits and vegetables
(Smith et al., 1990b). In fact, oxygen is omitted in fatty fish due to oxidative rancidity
(Sea Fish Industry Authority, 1985). Carbon dioxide is the Most important gas in MAP
foods. CÛ2 is bacteriostatic and fungistatic. At optimal concentrations of 600" CÜ2
(balance N2), it retards growth of spoilage microorganisIDS and reduces oxidative
rancidity offat (Brody, 1989). Higher concentrations ofCÛ2 provide no further extension
in shelf life or increase antimicrobial effect and it mayeven cause some problems, such as
discoloration and drip loss in muscle foods (Coyne, 1933a). Several factors influence the
antimicrobial effect of carbon dioxide specifically; microbial load, gas concentration,
temPerature and packaging film permeability (Smith et al., 1990b).
1.5.1.1.3. Antimicrobial effects
•
Carbon dioxide is highly soluble in water and will form carbonic &Cid as shown:
CÜ2 + H20 ~ H2CO] <=>~ + HCO]-. It cao consequently reduce the pH of fish, ftom
6.3 to 5.7-5.8 (Brody, 1989). This dissolution phenomenon is dePendent on temperature,
moi5ture content and concentration of carbon dioxide; as temperature increases, the
solubility of CÜ2 decreases and microbial growth increases (Ogrydziak and Brown,
1982). Carbon dioxide bas been proven more effective against Gram-negatïve bacteria
than Gram-positive bacteria (Yasuda et al., 1992). The specifie manner in which carbon
•
•
•
28
dioxide exerts its bacteriostatic etTect is unknown but several theories have been
postulated (Smith et a/., 199Oa). These are:
1. Displacement of oxygene However, this is not the sole reason since aerobic spoilage
microorganisms grew weil in 1()()oAt nitrogen and not in 1()()OAt carbon dioxide.
(Coyne, 1933b).
2. Reduction of extracellular pH. (Valley and Reuger, 1927). However lowering pH to
the same extent by HCI did not inhibit bacterial or mold growth.
3. Reduction ofintracellular pH and therefore atTecting cell metabolism (Wolfe, 1980).
4. Altering cell membrane permeability (Sears and Eisenber, 1961; Enfors and Molin,
1978).
The overall efIect of carbon.dioxide, together with the appropriate temperature
control, is to increase both the lag phase and generation time ofspoilage -microorganisms
(Smith et al., 19908)
The microbial sensitivity to carbon dioxide varies considerably dePending on the
oxygen requirement of the microorganisms (Figure 5). The predominant Gram-negative,
psychrotrophic bacteria in cold water fish are Moraxella, Acinetobacter, Pseudomonas,
Flavobacterium and Vibrio (Ward and Baj, 1988). Carbon dioxide is most effective
against these aerobic spoilage microorganisms. However, it has Iittle or no eiTeet on
facultative organisms, sucb as Enterobacteriaceae, Broncothrix thermophacta or
microaerophilic lactic &Cid bacteria which are capable of growing at concentrations of
l000At C(h (Brody, 1989). The Clostridium genera are not intluenced by carbon dioxide
(Smith et al., 19908) and gas packaging conditions O18yeven he favorable to their growth.
The numbers, types and age of the microbial population greatly affect the
antimicrobial properties of carbon dioxide (Brody, 1989). The earlier the time of
packaging i.e., bacteria which are still in the lag phase, the greater the effect of carbon
•
•
•
Figure s:
Aerobes-reguire atmospheric oxygen for growthPseudomonos
Acinetobaeter/MoraxellaMierococcusFilmyeasts
Molds
MicroaeroDhiles... reguire low levels ofoXYlenCampylobacterLactobacilJus
Facultative organisms...growing in the presence or absence ofoxygenBrochothrix thermosphacto
Staphylococcus speciesBoeillus species
EnterobacteriaceaeVibrio
Fermentative yeasts
Anaerobes- inhibited (or killedl by oxysenClostridium botu/inum
Clostridium perfringens
Oxygen requirements ofmicroorganisms (Smith et al., 19908)
•30
dioxide. MAP does not improve the quality of a product but helps retard its further
degradation.
1.5.1.2. Concems associated with modified atmosphere packaging
The main concems associated with MAP is that since normal indicators of
spoilage are inlubited, the growth of pathogens may occur or even he stimulated. The
main organism of public health concem is the growth ot: and toxin production by
C/ostridium botu/inum type E in MAP fish. There is also concem about psychrotrophic
pathogens such as Listeria monocytogenes, Aeromonas hydrophi/a and Yersina
entera/inca (Farber, 1991).
Listeria monocytogenes is a small, gram-positive, non-spore forming, motile,
hemolytic, rod-sbaped bacterium (Bahk and Martb, 1990). This organism can grow either
in aerobic as well as in anaerobic conditions (pelroy et a/., 1994). L. monocytogenes was
reported to growat temperatures between .().4 and 45°C, a pH between 4.39 and 9.4 and
at a minimum water sctivity (aw) of 0.92 (lCMSF, 1996). This bacterium is very salt
tolerant, as it cao survive for 4 months in a solution of 25.5% NaCI held at 4°C.
Carbobydrates are essential for the growth of L. monocytogenes. In fact, glucose serves
as a source of carbon and energy (Bahk and Marth, 1990). L. monocytogenes is
psychrotrophic. Although it grows best al 3~3-r»C, the organism thrives at remgeration
temperatures. It is heat sensitive with a D value (lime for 9()OA. destruction) at 71.-r»C of
-1 second (Bahk and Marth, 1990).
•1.5.1.2.1. Concems with Listeria monocytogenes
•Since L. monocytogenes is ubiquitous in nature, fish and seafood barvested fiom
naturaI environments are regarded as potential sourœs of Listeria in the human diet
(Ryser and Marth, 1991). In fact, in the United States, the organism bas been found in a
variety ofboth raw and ready-to-eat fishery products (pelroy el al., 1994). The high level
••
•
•
31
of contamination may he due to the use of river water tlowing· tbrougb agricultural land,
rearing fish in earth ponds instead of concrete ponds or raceways, no stalVation of fish
prior to slaughter and total Iack of regular mecbanical and chemical cleaning in fish
farms. Hygienic defaults during packaging cao also lead ta cootarninated ready-to-eat
products (Jemmi and Keush, 1994). It was reported that raw fish were more frequently
contaminated than finished produets (McCarhty, (996). McAdams (1996) isolated L.
monocytogenes Iike colonies (0 to 51 CFU/t00g) in both whole fish and fiUets of
aquacultured rainbow trout. Eldund et al. (1995) reported L. monocytogenes as a
common cODtaminant of raw, eviscerated salmon supplied to smoked salmon processor
with the organism found in 4/19 samples of slime, 30/46 skins, 8/t7 heads, 6/9 tails and
1/15 belly C8vity and beUy tlap trimmjngs. The situation may he further complieated
since seafood processing plants are an ideal environment for survival and growth of
certain microorganisms such as Listeria monocytogenes (McCarthy, 1996).
L. monocytogenes is a widely rec::ognized enteroinvasive pathogen (Ryser and
Marth, 1991). L. monocytogenes is the causative agent of listeriosis. In spite of the
relatively low incidence of this disease, Iisteriosis is a serious illness and this is reflected
by the apparent high mortality rate in many cases with fatalities averaging -300» (Newton
et al., 1992). In Canada, approximately 40-60 cases are reported annually (Farber and
Harwig, 1996). In addition, 2-6% ofbealthy people are reported to he asymptomatic fecal
carriers ofL. monocytogenes (Rocourt, 1996). Individuals al greatest risk from listeriosis
are pregnant women and their fetuses, the elderly and immunosuppressed patients. The
clinica1 symptoms include central nervous system infections and primary bacteraemia, but
it cau also include endocarditis. .In pregnant women, sPQntaneous abortion, stillbirth or
birth of a severely ill baby due to infection of the fetus can occur (McLauchlin, 1993).
The Food and Drug Administration bas detennined that there is a zero tolerance « one
organism per 25g ofsample) for Listeria species in food (Farber and Peterkin, 1991).
Modified atmosphere PaCkaging (MM) can extend the shelf-Iife of many
perishable products including tish. The use of reduced oxygen (<h) and increased carbon
•32
dioxide (C(h) concentratioDS can increase the shelf-Iife by inhibiting the growth of
aerobic spoilage bacteria. Under such conditioDS, the growth of psychrotrophic bacteria,
including such species as L. monocytogenes, may Dot be inhtbited. In fact, it bas been
reported tbat MAP does not completely inhibit the growth ofL. monocytogenes but may
exteDd its Iag phase and generation time (ICMfS, 1996). L. monocytogenes bas been
found to outgrow spoilage bacteria on cooked chicken (Marshal1 et QI., 1991). Therefore,
legitimate concems have been expressed regarding the microbiological safety of MAP
food contaminated with this pathogen (Farber, 1991). The chiefconcems are the growth
and multiplication of this psychrotrophic pathogen to an undesirable level on fresh fish
during a normal refrigerated storage period and the possible cross-contarnination of ftesh
tish with cooked ready-to-eat products with L. monocytogenes during market handling or
in the home refrigerator. (Fernandes et al., 1998).
Members of the genus Clostridium are obligately anaerobic, gram-positive, rod
shaped, sporulating bacteria. Those that produce a neurotoxic protein, which elicits
botulism, are all placed in the one species, C. botulinum. The resulting species is
composed of strains of diverse cultural properties. The toxin itself cao he serologically
different in that the toxicity of one toxin is neutralized ooly by the antitoxin for that
particular antigenic type. There are presently seven neurotoxic types (A, D, C, D, E, F
and G) (Sugiyama, 1990). Types A, B, E and F have caused the majority of human
botulism (Eklund, 1982). Types A, D, E and F cao he further divided ioto two groups
based upon their biochemical and physiological characteristics. Group 1 consists of the
proteolytic types A, B and F.and group fi cODSists of the non-proteolytic types S, E and F.
Members ofgroup 1 attaek complex proteins, and their growth is usually, but Dot a1ways,
accompanied by off-odors. They have a mjnimum growth temperature of lOOC (however,
their optimal growth temperature is 3t»C) and are inhtbited in foods below pH 4.6 and
above 8.S (Blocher and Busta, 1983; Sperber, 1982). The strains of these types are the
most heat resistant and will grow and produce toxin in foods containing 8-90" water
•
•
1.S.1.2.2• Concems with Clostridium botulinum·
•
•
•
33
phase sodium chloride. Foods with water activity below 0.93 are iDlubitory to thcir
growth (Riemann, 1969). Members of group n on the other band, are the Most heat
sensitive types and are inlubited by 5-6% water phase NaCI. They will grow in foods
with a water activity of 0.96 or higher and are inhtbited by pH below 5.0 (Riemann,
1969). They have the unique properties of being non-proteolytic and growing al
temPel'8tures as low as 3.3°C, however, their optimal growth temperature is 30°C.
Because of tbeir non-proteolYtic characteristics, their growth in foods cannot he deteeted
by off-odors.and off-flavors (Schmidt et al., 1961).
Reat resistance is commonly expressed as the decimal reduction time or D- value.
D-value is the time required to inactivate 9QO/'o of the population al a specified
temPel'8ture. D-values vary considerably among C. botulinum strains even within the
same group. At the reference temperature ofthe food industry, DI210C or ~OoF for spores
of group 1 strains, the mast resistant form of C. botulinum is 0.21 minute. Spores of
group n strains have lower D-values. At 180~, DIBOoF is 2 minute while type E bas a
D82.2oCofO.l..Q.3 minute (Lynt et al., 1982; Simunovic et al., 1985).
Oxidation potential a1so influences growth since the organism is an obligate
anaerobe and therefore requires reducing conditions. Il is more likely to grow in foods
with low oxidation-reduction (redox) potentials, a pH dependent value expressed as Eb
(Brown and Emberger, 1980), but a negative redox potential is not necessary. Growth of
C. botulinum type E spores occurred atpH 7.0 in a bacteriological medium ofEb of+126
mV and in sterilized milk of Eb +144 mV (Lund and Wyatt, 1984). The potential of a
food with a low redox poising capacity cau be lowered by growth of otber organisms.
Consequently, oxygen may inhibit C. botu/inum by increasing the redox potential. C.
botulinum would not grow on the surface of a product exposed to air but could do 50
below its surface if there is a sufficient concentration ofredox-reducing constituents, such
as thiol compounds e.g. in meat and fish (Sugiyama, 1990).
•
•
•
34
Increasing levels of microbial contamination significandy decreases the risk of
toxigenesis since C. botulinum is not generally a good microbial competitor. However,
c. botulinum growth, under certain conditions, may he enhanced rather than inhibited by
other bacteria Extensive growth of surface bacteria reduces the redox potential or
oxidize 8Cids and increase the pH to values permitting growth and toxin production by C.
botulinum (Garcia and Genigeorgis, 1987).
Botulism bas heen recognized as a foodbome disease for more tban 1, 000 years
(Eklun~ 1982). Botulism is a neuroparalytic disease caused by toxigenic strains of C.
botulinum. Today, botulism stiU remains a significant public health bazard. Botulism in
man occurs in four distinct forms: food poisoning botulism, wound botulism, infant
botulism, and possibly a similar fonn in adults (unclassified botulism). The kind that bas
heen longest known is the food poisoning which results ftom ingestion of preserved food
in which the causative organism had grown and fonned toxin (Smith and Sugiyama,
1988). Therefore, foodbome botulism is an intoxication caused by the ingestion of food
with preformed toxine There are four fondamental prerequisites for foodbome botulism
to exist: (1) the presence of the organism i.e., the food must he contaminated with C.
botulinum spores or vegetative cells ftom the environment; (2) inadequate processing i.e.,
the processing treatment must be inadequate to inactivate the C. botulinum spores, or the
product must be contaminated. after processing; (3) food cao support toxin formation i.e.,
the food must support the growth and toxin production of C. botulinum when storage
temperature exceeds 3.3°C. Most botulism outbreaks have been traced to foods that were
poorly processed and temperature abused. (4) the food must be consumed i.e., the food
must he acceptable to the consumer and consumed without cooking or after insufficient
beating to inactivate the botuliDal toxin (Eldun~ 1982).
Food poisoning botulism bas an incubation period of 12..36 bours or less ifmore
toxin is consumed. The iUness may start with gastrointestinal problems such as nausea,
vomiting and dianbea, but these effects may not he caused by the toxin since they are not
seen in infant or wound botulism. Constipation is a common symptom when typical
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3S
botulism signs develop. Fatigue and muscular weakness are the tirst indications of
botulisme They are soon followed by ocuIar effects, such as droopy eyelids, sluggish
response of pupils to light, blurred and double vision. Effects in the mouth include
dryness with difficulty in speech and swallowing. The muscles controlling the limbs and
respiration become progressively paralyzed, with death occurring within 3-5 days ftom
respiratory failure (Sugiyama, 1990).
In fact, botulinum neurotoxin acts by blocking the release of acetylcholine al the
neuromuscular junction in the three-step process: (1) the toxin· molecule binds 10
receptors on the nerve ending; (2) the toxin molecule, or a portion of il, is intemalized;
and (3) within the nerve ceU, the toxin interferes with the release of acetylcholine by an
unknown mecbanism. Botulinum neurotoxin prevents the passage of stimuli ftom the
motor nerves to the muscles (~t effecting the neuromuscular junctions of the head and
neck) and, as the disease progresses, more and more muscles fait to respond to their
sPecific stimuli until the muscles needed for breathing, or the cardiac muscles, fait thus,
resulting in death (Smith and Sugiyama, 1988).
Botulism in Canada is primarily foodbome with the great majority of the
outbreaks (890,4.) involving C. botu/inum type E among Inuit and American coastal
Indians with mortality rates as high as 17.7% from 1971 to 1984 (Hauschild and
Gauvreau, 1985). C. botu/inum type E strains are more likely 10 be present in ftesh water
and marine environments 50 that botulism from foods made of fish and aquatic mammaJs
is predominantly type E (Sugiyama, 1990). Since aquatic environments often show fairly
high levels of contamination, it is not surprising that fish have the highest level of
contamination with C. botulinum type E. The Baltic Sea bas the highest level of C.
botulinum type E contamination in the world (Huss, 1980). The high mortality rate from
type E botulism in Alaska bas resulted in surveys on the incidence of C. botulinum in the
environment. A high incidence of C. botu/inum type E spores (74%) were found on the
beaches in northwestem AIMka (Miller el a/., 1972). Fantasia and Duran (1969) showed
that 6 to SOOAa of the intestines of fish from different parts of the Great Lakes were
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36
contarninated with C. botulinum type E. Laycok and Loring (1972) round that 18% ofthe
sediment ftom the St Lawrence River were aU positive with C. botu/inum type E spores.
Botulism is an important problem where salmonids are raised artificially. ft is
known as the '~ankrupt disease" because of the serious economic consequences to
commercial fish farmers. In Denmark, rainbow trout losses where as high as 2, 200 kg
daily (Huss and Eskildsen, 1974). In outbreaks in Oregon and Washington ftom 1979 to
1984, severa! millions of fish were lost (Eklund et al, 1984). Sîmilar outbreaks among
rainbow trout have been reported in Britain (Caon and Taylor, 1982). The conditions
leading to these outbreaks have been described in detail. Type E bas always been
involved because fish are extremely susceph"ble to toxin of this type (Eklund et al., 1984).
The source C. botulinum type E is usually water tlowing into the ponds in which the fish
are mised or organisms growing in the sediments of the tlowing streams. The organisms
can grow in ponds, in the sediments of food, fish excreta and dead fish. The organisms
picked up by farmed fish do not harm the fish nor do they produce toxine However, when
fish die, C. botulinum type E in the intestine multiply and invade the flesh. Toxin is
acquired by live fish eating the tlesh of dead fish, which is common in farm raised fish
(Smith and Sugiyama, 1988). Botulism outbreaks usuallyoccurs during the summer and
fall when the water temperatures are higher. As the water temperature decrease, the
growth rate of C. botu/inum type E a1so decreases and less toxin is PrOduced in the dead
fish carcasses (Eldund et al., 1984).
In the light of this evidence, natural contamination of farm raised fish is beyond
our control. AlI fish should be assumed to be caniers of C. botulinum type E spores and
therefore classified as high hazard food products (Baker and Genigeorgis, 1990).
As stated earlier, the desire of the fishery industry to extend. the shelf-Iife of ftesh
fish and increase market demand bas led to the adoption of packaging techniques which
have been successful in the preservation of other foods. One such approach is the use of
modified atmosphere (MA) packaging. However, commercial use of MA to extend the
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shelf-Iife offishery products bas been limited by the potential ofç. botulinum growth and
toxin production in refrigerated, MA packed fish, without overt sensory evidence of
spoilage. The hypothesis is that in this environment, the microorganism may have a
competitive advantage over the normal psychrotrophic bacteria that are not able to grow
under the reduced oxygen environment inside the package. If this occurs, noticeable
spoilage characteristics may not he noticeable and the consumer may inadvertently ingest
fish containing botulinum toxin (Garcia and Genigeol'gÎS, 1987). Despite these concems,
fillets of ftesh fish packaged under MA and stored continuously at temperatures below
3°C have appeared in European supennarkets. No cases ofbotulism have been associated
with the consumption ofsuch products this far (Baker and Genigeorgis, 1990).
Severa! investigators reported that spoilage preceded toxigenesis in MA raw fish
product held below 10°C. In fact, Garren et al. (1995) reported that botulinum toxin was
detccted after 6 days al 10°C in packaged trout; however, fish was noticeably spoiled
before that tÎlDe. The Torry Fish Research Station (Caon et al., 1983; Caon et al., 1984)
indicated that spoilage ofwhole trout , salmon fillets and cod fiUets stored onder 4O-6()OA.
C~ MA and vacuum al 10GC preceded toxigenesis by non-proteolytjc B and E strains
(100 spores/g). However, other investigators have shown that toxin production by c.botu/inum may precede organoleptic spoilage in fish samples tbat have been packaged
under MA. Garcia et al. (1987) showed that the earliest lime C. botulinum was detccted
in salmon fillets, irrespective ofMA, al 30, 12 and 8°C was after l, 3-9 and 6-12 days of
storage. Toxin detcction coincided with spoilage al 30°C, but preceded spoilage al 8 and
12°C. TItese observations ÎDdicate that a hazardous situation may arise as a result of
storing salmon filIets al mild abusive temperatures. Eklund (1982) bas shown that in 60
and 9()OAt C(h, spoilage was not obvious after 10 clays storage ofsalmon st 100e but that a
number ofsamples, inoculated with ~ I~ type E sporeslg fish, were toxie before 10 days.
Lindsay (1983) also concluded tbat C. botulinum tyPe E toxigenesis cao occur in fish
without overt signs ofspoilage.
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With processed fish, sucb as smoked fish, the rate of C. hotu/inum toxin
production is slowed down, primarily because of the salt concentration. Nevertbeless,
since spoilage is also slowed down, toxin cao appear in such products before they become
spoiled (Robbs, 1976). Commercial smoked curing of gutted fish as currendy practiced
does not eliminate the numbers of C. hotu/inum type E spores present. Further
tempera~ control and salt concentration are therefore necessary (Christiansen et a/.,
1968). From a public health point of view, all smoked fish should be assumed to be
carrier of C. hotu/inum (Huss et a/., 1974). Industrially processed vacuum packaged hot
smoked salmonids have been responsible for numerous recent outbreaks in northern
Europe. The consumer's demand for reduced use of sodium salts and the vacuum
packaging used to prolong shelf-life bas created high-risk products that are largely
dependent on refiigeration for safety (Eklund, 1992). In the United Kingdom 5 out of a
total of 646 vacuum packed smoked fish products were found to contain C. hotu/inum
type E (Hobbs et a/., 1965; Caun et a/., 1966). In Denmark, an incidence rate of 1.68% of
C. hotulinum type E was reported in smoked salmon purchased from retail oudets
,(Nielsen ~d Pedersen, 1967). Caon et a/. (1980) reported toxin production by C.
botulinum type E in vacuum packed cold smoked salmon after 13 days at 10°C.
It is generally agreed that fish cannot be protected from natura! contamination
with C. botulinum. Therefore, control of botulism must be achieved by adequate process
and storage control which requires a thorough understanding of all the factors affecting
survival, growth and toxin production (Hobbs, 1976).
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1.6. ConclasioD
The current consumer trend is towards refrigerated fresh food produc:ts. In an
effort to enlarge market shares ofhigh quality fish products, research bas been condueted
at extending the shelf-life of tish. Chemical changes take place within fish and adversely
affect freshness, color, flavor and texture. Microbial changes are also responsible for
spoilage of tish. One approach to retard these undesirable changes is through modified
atmosphere packaging which involves a knowledge ofraw products, packaging materials,
production and refrigeration to ensure safety ofproduct.
The product of interest in this study is rainbow trout. Two major concerns will be
addressed: the effcct of MAP on discoloration and the safety of modified atmosphere
packaged fish with respect to growth of Listeria monocytogenes and Clostridium
botu/inum.
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1.7. Research objeetives
To date, few groups bave concentrated their research on the role ofpackaging and
storage conditions on color changes of fresh rainbow trout fillets. Furthermore, the effect
of MAP on the overall quality of fresh trout is of main concerne Consequently, it is
essential to optjmize the various storage and packaging environments, focusing on the
overa11 effect of MAP to ensure optimum product color without compromising safety.
The specifie objectives of this research are:
(1) To study the effect ofvarious abnospheres in MAP rainbow trout fillets on the color
stability offi'esh fillets.
(2) To determine the physicai, chemical, microbiological and sensory changes of MAP
fresh rainbow trout fillets.
(3) To determine the public hea1th safety of MAP rainbow trout fillets in challenge
studies with Lialeria monocytogenes and C/ostridium botu/inum type E.
(4) To determine the levels of additional barriers (if any) to ensure the public health
safety of MAP fresh and smoked rainbow trout fillets with regards to the growth of
C/ostridium botu/inum type E.
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41
CBAPl'ER2
THE COLOR SRELF...LIFE STUDIES OF TROUT FlLLETS PACKAGEDUNDER VARIOUS GAS ATMOSPHERES AND STORED AT 4°C
2.1. Introduction
Fresh trout filIet is an expensive, highly perishable and biologiœlly unstable food
product. Flesh color of fresh trout is an important quality parameter for consumer
acceptance. In fact, one of the major signs of ftesh quality degradation of trout fillets is
loss of color. Consumer acceptance of foods is based primarily upon appearance which,
in tom, influences purchase, use and repurchase of that particu1ar food.
Pigments, principally the carotenoids astaxanthin and/or canthaxanthin, are
responsible for the appealing pink...arange color of trout tlesh. The oxidation)state of/
these flesh pigments determines the apPearaDce of the fish tissue. In the ~complete
absence of oxygen, these carotenoid pigments are less Iikely to deteriorate. Therefore,
packaging of trout fillets in a gaseous environment of various levels of carbon dioxide
(balance nitrogen) mayhave a beneficial effect on trout color.
RandeU et al. (1995) showed that the optimum color of fresh trout was obtained
by packagjng in an atmosphere of C02:N2 (60:40). However, other studies bave shown
that the inclusion of oxygen in the gas mixture (e.g., CÛ2:N2:02 (40:30:30» was
necessary for optimum color retention during remgerated storage (Via-Mer, Persona!
Communication, 1998). Chen et al. (1984), showed tbat air packaged trout fillets had
better color retention al refrigeration temperature over a 14 day period compared to trout
tillets packaged under vacuum or in 10001'0 C(h. In view of the conflicting results
concern.ing the optimum gaseous atmosphere for color stability and retention of color in
ftesh trout, the objectives of this study were: (i) to monitor the pigment chaDges of trout
fillets packaged under various atmospheres usÎDg objective and subjective methods (ü) 10
determine the optimum gaseous conditions for color retention in trout tilIets stored at
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42
refrigerated temperature (4°C) and (ili) to determine ifthere was a signific:ant correlation
between subjective and objective measurements offish color.
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%.2 Materl'" aad Methods
%.2.1. Sample preparation
Freshly caught and eviscerated rainbow trout (Onchorynchus mylciss), obtained
ftom a local seafood producer (Via-Mer, St-Hyacinthe, Quebec), were cut into fillets of
approximately 250g cach. They were placed on ice in expanded polystyrene
(Styrofoam(l) containers and transferred to our laboratory.
%.l.%. Packaging and storage conditions
Tbree types ofpackaging conditions (air , vacuum and gas packaging) were used.
Trout fiUets were placed in expanded polystyrene (Styrofoam (1) trays (140 mm X 265
mm) containing a moisture absorbing pad and wrapped with a layer ofpolyvinyl chloride
low banier film (PVC) with an oxygen transmission rate (OTR) of 17, 050 cc/m2/day/atm
@ 20°C, OOAtRH as the primary package. A high barrier film, with an om of 12
cclm2/day/atm @ 24°C, OOAtRH) (Cryovac Sealed Air Corporation, Mississauga, Ontario)
was used as the secondary packaging material. Air packaged samples were heat sealed
directly without any further modification to the package atmosphere. Vacuum packaging
was achieved, foUowed by an instantaneous sealing of the secondary package, using a
gas/vacuum packaging machine (Model A300/42, Multivac, Germany). The same
machine was used to gas package trout fillets with 96.3% CÛ2, 85.1% CÛ2 and 590At C02
respectively (balance nitrogen). A Smith's proportional gas mixer (Model 299-028,
Tescom, Corp., Minneapolis, Minnesota) was used to give the desired proportions ofCÛ2
and N2 in the package headspace. AlI five packaging conditions were done in triplieate.
AIl packaged products were stored al a remgeration temperature of 4°C and examined
after 3, 7, 14,21 and 28 days.
•44
%.2.3. Analyses
On each sampling day, samples were removed from the refrigerator for subjective
and objective color assessment using a destructive sampling pIan.
%.%.3.1. Subjective color measurements
Sensory analyses were performed by six untrained panelists on pooled samples.
Panetists were Dot given any prior information about the samples. Sensory evaluation
was carried out as foUows: panelists were asked 10 evaluate trout Met color through the
unopened primary package which had been removed ftom its secoDdary package.
To evaluate coJor, a seven-point hedonic scale, descnDed by Greer (1993) (Table
6) was used. A score of 3.S was regarded as borderline for acceptability. The shelf-Iife
ofpackaged trout fiUets was determined by the time (days) a score of3.S was reached.•
%.2.3.1.1.
%.2.3.1.2.
Sensorial color acceptability
Sensorial color determinatioD
The color of trout flUets was also compared subjectively by panelists using a standard
Roche color chart with values ranging tram Il (Iight orange) to 18 (clark red) (Table 7).
This chart reflects the color specification of salmonid products during storage. The
Roche color chart is used by the fish industry to monitor the color of fresh trout and was
therefore used byour panel to grade color changes subjectively on each sampling day.
Trout color was measured objectively using a Minolta spectrophotometer CM
S08d (Minolta Co., Osaka, Japan). This instrument possesses an integrating sphere and•2.2.3.%. Objective color measurements
•
7
6
S
4
3
2
1
Table 6:
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Sensory acceptability scale (Greer, 1993)
Order ofdeslrabllty
Extremely Desirable
Desirable
Slightly Desirable
Neither Desirable Nor Undesirable
Slightly Undesirable
Undesirable
Extremely Undesirable
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47
bas a measuring area of8 mm in diameter and an illumination arca of Il mm in diameter.
Doring measurements, the instrument was placed directly on the surface of the trout
tillets overwrapped with the primary polyvinyl chloride (pVC) packaging film (OTR =17, OSO cclm2/day/atm at 20°C, ()oAt RH). Five readings were taken al five different
locations on each sample surface. A light source~ corresponding to a tungsten filament
lamp, operated at color temperature of 2845°1{, was diffused into the integrating sphere.
Retlectance measurements were collected using a 10° field ofview. The instrument was
adjusted to have the specular component excluded (SPE) which includes only üght
retlected diffusely from the specimen surface; such measurements provide results which
closely match those of the visual evaluation by a trained observer. In fact, the ügbt
retlected specularly from the specimen surface passes through the hole in the integrating
sphere uncovered by the door and enters a ligbt trap, which prevents the light from re
entering the integrating sphere. Before data collection, the instrument was calibrated
(zero and white calibrations) to account for anyeffect of stray light due to luminous
signais of the spectrophotometer's optical system (Minolt&, 1994). Zero calibration was
achieved by aiming the spectrophotometer in the air where there was no object within 1
meter distance. White cah"bration was perfonned using a standard-white retlector that
was covered with a PVC film (primary packaging material) to obtain base reading of the
instnlment.
Data were collected in CIBLAB (Commission Internationale de l'Eclairage) color
space values as an indicator ofproduct L* (lightness), a* (red-green chromaticity) and b*
(yellow-blue chromaticity) values. Other color space values were also used 10 evaluate
the color oftrout tillets; L* C* h, where the L* values corresponds to ligbtness (as in the
L*a*b* color space values), C* values to the chroma and h to the hue angle. C* values,
also known as color saturation or vividness, descn"bes the purity or lack ofgrayness ofan
object. Il is calculated as foUow: C* = .../(a*)2 + (b*)2. The hue angle (h) in the world of
color is used for the classification of red, yellow, blue, etc. The hue angle (h) ranges
between pure red (hue angle =0°), pure yellow ( hue angle = 90°), pure green (hue angle
•48
= 180°) and pure blue (hue angle =270°). The hue angle is calculated using the
following formula: h =tan·} (b·/a·) [degrees].
Statistical analysis
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A 3 X 5 X 6 factorial design was used as the experimental design throughout this
study. Each fish sample (in triplicate) was subjected to 5 packaging treatments and tested
on 6 ditTerent test days as descnDed previously. A general Linear Model Procedure was
used to statistically analyze the color measurement data and a Duncan's multiple range
test was used for comparison of the means, utilizing the Statistical Analysis System
(SAS, 1988). A probability (P) of less than 0.05 was considered 10 he significandy
different. The Pearson test was performed to determine correlation coefficients (R
values).
•49
2.3. Results and Diseassioa
2.3.1. Subjective color measurements
2.3.1.1. Changes in color acceptability scores
•
The color acceptability scores for the various packaging treatments are shown in
Figure 6. Trout flUets were regarded as unacceptable when a score of 3.5, corresponding
to the mid..point between "Neither Desirable Nor Undesirable" and 6'Slightly
Uodesirable", on a subjective scale of7 was reached (Greer, 1993). As expccted, all trout
flUets had the highest color acceptability scores during the initial days of storage (Figure
6). However, as storage progres~ed, color acceptability decreased. The estimated shelf..
Iife oftrout fiUets packaged under different conditions, based on the time in days to reach
a score of 3.5 on the sensory acceptability scale, is shown in Table 8. The best results
under the conditions ofthe test were obtained by vacuum packagiDg and by gas packaging
in CO2:N2 (85.1:14.9). Trout flUets packaged under these conditions had acceptable color
scores of 3.9 and 3.5 respectively after 28 days al 4°C. The lowest sensory color
acceptability scores were found in air packaged trout fillets which reached an
unacceptable score of 3.5 after -18 days. The rapid discoloration ofair packaged trout is
probably due to oxidation of fish pigments (astaxantin and canthaxantbin) which are
responsible for the pink-orange color of trout. An increase in microbial growth,
particularly 00 the fish surface, may also influence the discoloratioo of air packaged trout
tiUets. Statistically, the color acceptability scores al day 0 and day 3 were significantly
ditTerent (P < 0.05) from those at day 7 and day 14 for alI treatments, while the latter
scores were also significantly different from clay 21 and clay 28 scores.
2.3.1.2. Changes in Roche color cbart scores
• The results for the color scores of trout fillets stored al 4°C under different
packaging conditions using the standard Roche color cbart are shown in Figure 7. Each
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so
untrained panelist matched the color of trout fillets with correspondïng color values on
the standard Roche color cbart. Ideally, tiesh trout fillets should bave values around 14
15 on this chart. Trout flllets are regarded as unaccepiable wben values are below 13
(Via Mer, Personal Communication, 1998). The initial scores for trout fillets were 13.6.
In general, ail trout fillets became paler with lime i.e., decreasing standard Roche color
chart values, irrespective of packaging treatment. However, vacuum and gas (CÛ2:N2
(85.1: 14.9» packaged trout flllets had an overall redder/darker color, i.e., higber color
chart values (scores between 14.1-13.4 and 14.0-13.1 respectively) of all the packaging
treatments after 28 days (Figure 7). Air packaged trout fillets had the lowest color score
(12.2) after 21 days at 4°C. After 28 days ofstorage, the lowest scores were observed for
trout flUets packaged in CÛ2:N2 (96.3:3.7) and CÛ2:N2 (59:41) with scores of 11.7 and
12.1 respedively (Figure 7).
The estimated shelf-life of trout fillets packaged under different treatments based
on the Roche sensory color cbart are shown in Table 8. The shortest shelf-life was
observed ftom trout fillets packaged in air with an estimated shelf-life of -18 days.
Vacuum and gas packaged trout fillets in C(h:N2 (85.1:14.9) had the longest shelf-life as
their color remained appealing tbroughout 28 days.
StatisticaUy, the sensory shelf-life of vacuum and gas packaged trout fillets
(C02:N2 (85.1: 14.9» did not show any significant difference (P > 0.05) between
treatments based on both subjective color measurements (sensorial color acceptability and
sensorial color determination frOID the Roche color cbart). However, the latter treatments
were significantly different (P < 0.05) from air, CÛ2:N2 (96.3:3.7) and CÛ2:N2 (59:41)
packaged samples (Table 8).
•7
1:1·1
3
U 2
1
0 5 10 15 20 25 30
0.,.
-+-Nr___Vacuum
-'-96.3" C02~85.1"C02___sn.C02
•Figure 6: Color acceptability scores of trout fillets package<! under difJerent gasatmospheres and stored at 4°C
18
17
116
115
114
t 13II:
12
110 5 10 15 20 25 30
0.,.
~,..
"",,-V8QUft
......98.3"C02-M-85.1" C02....-sn.C02
•Figure 7: Roche color chart scores of trout fillets packaged under different gasatmospheres and stored at 4°C
• • •Table 1: Estimated subjective color shelf·life (days) oftrout flUets packaged onder difTerent gas atmospheres and stored at 4°C
based on the sensory acceptability seale and the sensory Roche color chart(R=O.9381)
'acbglnl treatalentl
Air
Vacuum
C02:N2 (96.3:3.7)
CÛ2:N2 (85.1:14.9)
C02:N2 (59:41)
Color acceptablUty
Shelf·llfe·
(day.)
....18
>28
-19
....28
....18
Roche color chart
Shelf.life b
(day.)
....18
>28
-22
>28
....22
• Based on the time (days) necessary for the eolor oftrout flUets to reach a score of3.5 on the sensory aceeptability scaleb Based on the lime (days) necessary for the color oftrout flUets to reach a score of 13 on the sensory Roche color chart scale
•53
2.3.2. Objective color measurements
2.3.2.1. CIE-LAB values
Changes in five variables were monitored spectrophotometrically: L* values
Oightness), a* values (redness), b* values (yellowness), C* values (chroma) and h values
(hue angle). These are shown in Figures 8 to 12 respectively.
2.3.2.1.1. Changes in L* (lightness) values
•
•
The initial brightness values (L*) ofall trout fillets was 45.54. For most packaged
trout fillets stored at 4°C, L* values decreased during the first few days of storage as
shown in Figure 8. In fset, trout fillets resehed their darkest color after three days with
trout fillets packaged in air, in C02:N2 (85.1:14.9) and C(h:N2 (59:41) having L* values
of 43.14, 44.42 and 41.71 respectively. For vacuum packaged trout fillets, the darkest
color was observed after 7 days of storage with an L* value of 43.99. However, this
initial decrease in L* values was foUowed by a graduai increase, i.e., color- was darker
during the tirst days of storage and then became paler with tÎDle. Only trout packaged in
CO2:N2 (96.3:3.7) did not follow this trend and its L* values increased progressively from
day 1. An increase in paleness with time can he attributed to a combiDation of bacterial,
enzymatic and chemical spoilage. In Cact, breakdown of tissue over an extended time
period will eventually lead to a leakage ofpigments that will further undergo autoxidation
and hence discoloration (paIeness) offish tissues.
Interestingly, three separate distinct trends were observed in the color curve fit,
i.e., day 0-3, day 7-14 and day 21-28. Each storage period had significantly difTerent (P <
0.05) L* values indicating that L· values of trout fiUets were significantly different (P <
0.05) from one week to another. However, the L* values for each day, within the same
interval, were not significantly different (P > 0.05).
• 2.3.2.1.2. Changes in a· (redness) values
•
Changes in a* values with storage for trout under various packaging conditions
are shown in Figure 9. For most packaging treatments, a* values increased gradually
with time indicating that trout was becoming redder in color. Fluctuations in a * values
were observed mainly within the first few days ofstorage. The a* values obtained at day
o were significantly ditTerent (P < O.OS) than those at day 3 which in tom were all
markedly different (P < O.OS) to the a· values from days 7 to 28. Trout fillets packaged in
air, and in C02:N2 (85.1:14.9) had significantly difTerent (P < 0.05) a· values. Trout
packaged in air appeared to be less red in color than otber packaging conditions reaching
minimum a* values of 15.09 after 21 days. Conversely, maximum redness was observed
after 14 days of storage (a· values of 21.66) for trout flUets packaged in CÛ2:N2
(85.1:14.9). Trout fillets packaged under vacuum and in CÛ2:N2 (96.3:3.7) showed a
graduai increase in redness i.e., trom a· values of 12.98 to 19.97 and 19.54 respectively
after 21 days. However, during the Iast 7 days of storage, vacuum and CÛ2:N2 (96.3:3.7)
packaged trout flUets decreased slightly in redness.
2.3.2.1.3. Changes in b* (yeUowness) values
•
Fluctuations in b* values over time for trout fiUets packaged under difTerent
packaging treatments are shown in Figure 10. For ail packaging conditions, the b* values
of trout fillets were highest al day O. The MOst dramatic changes in yellowness were
observed during the first three days storage. The b· values decreased espeçially for trout
fillets packaged onder vacuum, in CÛ2:N2 (59:41) and in air reaching values of 16.01,
16.48 and 17.3 respectively. Thereafter, all b· values increased gradua1ly for these
packaging treatments until the end ofstorage. However, the final b· values never reached
initial values of 24.87. The decrease in b· values indicated that trout fillets were
becoming less yellow with lime. Statistically, b* values obtaincd al day 0 were
significantly difTerent (P < O.OS) than the b* values obtained al day 3. From day 7 10 day
•55
28 however, the b· values were not very different (P > 0.05) from each other. AIl trout
flllets, packaged under C02:N2 (85.1:14.9) had higher b· values than air packaged trout
fillets. Furthennore, trout flUets packaged in a mixture of C(h:N2 (85.1: 14.9) were
signiflcandy different (P < 0.05) tram air packaged trout flUets with respect ta yellowness
at the end ofstorage.
2.3.2.1.4. Changes in C· (chroma) values
•
The changes in C· values for trout flUets stored at 4°C are shown in Figure Il.
This attribute measures the variation in color saturation. The C· values increased with
lime compared ta the initial value of 19.51 observed al day O. In fact, for a11 packaging
conditions, trout fillets appeared more vivid in color as storage time increased. Less
bright colors were observed for air packaged trout flUets (minimum C· value of 22.35
after 21 days of storage) while more vivid colors were observed for trout packaged onder
a mixture of C(h:N2.(SS.I: 14.9) (maximum C· value of 33.11 after 14 days of storage).
In terms of contrast in C· values, the most signiflcant changes were re<:orded during the
first week with c· values obtained al day 0 being significantly ditTerent (P < 0.05) from
those obtained at day 3. The latter was also marked1y different (P < 0.05) from the C·
values observed throughout storage. However, the C· values trom day 7 to day 28 were
not signiflcantly different (P > O.OS) trom one another. Similarly, non-signiflcant
differences (P > 0.05) in terms of C· values were observed for trout fillets packaged
onder vacuum, in CÛ2:N2 (96.3:3.7) and in C(h:N2 (59:41). However, notable
ditrerences (P < 0.05) were recorded for ail trout fillets packaged either in air or in
CCh:N2 (85.1:14.9).
2.3.%.1.5. Changes in h (hue angle) values
•Changes in h (hue angle) values for trout fillets stored al 4°C and packagcd under
various atmospheres are shown in Figure 12. From day 0 to day 3, trout fillets could be
divided into two groups, one group with increasing b values and another with decreasing
56
h values, compared to the initial h value of 48.22 observed at clay O. The tirst group
included trout fillets packaged onder vacuum, in COz:N2 (96.3:3.7) and in C(h:N2
(85.1: 14.9) with h values of 48.96, 48.54 and 49.28 respectively. The other group
included trout fillets packaged in air and in CO2:N2 (S9:41) with h values of 47.11 and
47.27 respectively. A general increase in h values was observed &om day 3 to day 7 with
the exception of trout fillets packaged onder vacuum where its h value continued to
decrease to 48.33. From day 7 until the end of the storage, trout fillets packaged under
various gaseous conditions sbowed a decrease in hue angle, with the exception of trout
flUets packaged under the gaseous mixture ofCÜ2:N2(96.3:3.7) which bad an b value of
51.63 after 28 days of storage. Statistical analysis sbowed that trout fillets packaged
under CÜ2:N2 (96.3:3.7) were significandy different (P < 0.05) ftom trout packaged
under CÛ2:N2 (85.1:14.9). Furthermore, both treatments were significandy different (P <
O.OS) trom vacuum, air and gas packed CÜ2:N2 (59:41) packaged trout fillets.
•
• 2.3.2.%• Comparison ofobjective color measurements to other studies
•
The initial bright pink/orange color of trout fillets is the Most important indicator
of quality. Skrede et al. (1989) studied the objective color characteristics of raw trout.
They reported initial L* values between 43.5 and 52.9, a* values of4.3 to 14.7, b· values
of IS.5 to 23.4 and h values of 57.9 to 74.5. Their initial b· values were lower and their
initial h values were higher than the objective measurements found in our laboratory, i.e.,
initial b· value of 24.87 and h value of 48.22. However, L* values and a· values were
similar. In another study, Gobantes et al. (1998) showed that the L· values of vacuum
packaged trout increased from 43 to 51.8 after 15 days at 4°C. They a1so monitored
changes in C* and h values ofvacuum packaged trout al 4°C and reported tbat C· values
reached 16.7 after 15 days of storage i.e., slighdy brighter than the initial value of 15.3
while h values decreased ftom 61.3 ta 55.1 at the end of storage. These results compare
favorably to our studies with vacuum paclœged trout.
•53
51
49
1 47
J:'45
43
41
390 5 10 15 20 30
0.,.
~A;
-e-Vacum-'-96.3% 002~85.1%oo2
-11-59%002
•Figure 8: L· vaiues of trout fillets packaged under ditrerent gas atmospheres andstored at 4°C
22"T--------~...._--------~
20
1 18
1•• 16
14
~A;
~V8QUl'l
-'-96.3% C02-M-85.1% C02-11-59%002
3025201S
0.,.
10S12 .f----+----+----+----+----+----I
o
Figure 9: a* values of trout fillets packaged under difJerent gas atmospheres andstored al 4°C•
•26,.-----------------_22
11 18
•14
-,-AiI~Vacuum
-'-96.3% C02-M-85.1% C02-'-59%C02
30252015
0.,.
10510 ~--_+_--..._--..._--_+_--_+_-___f
o
•Figure 10: b* values of trout fillets packaged onder different gas atmospheres andstored al 4°C
35-r--------------------,30
11 25
Û
20
-'-AlI~VIICUUI'ft
-'-96.3% C02-M-85.1% 002-11-59% 002
30252015...,.1015 +---........--_+_--+----+---......----f
o
Figure Il: C· values of trout fillets packaged under different gas atm~beres andstored at 4°C•
•52
51
50
149~
48
47
460 5 10 15 20 25 30
0.,.
~AII
~V8QUII
-"'-96.3% 002-M-85.1%oo2___sn.C02
•
•
Figure Il: h values of trout filIets packaged onder different gas atmospheres andstored at 4°C
•
•
•
60
2.3.3. Relationship between subjective and objective measurements ofcolor
Two subjective methods were used to compare color: sensory color aceeptability
and the Roche color chatt. A sensory color aceeptability value was assigned at each
sampling day by six panelists from a hedonic scale (range 1-7, Table 6) ta trout fillets
packaged under various gas atmospheres. When the sensory acceptability value reached a
score of 3.5, corresponding to the mid-point between 6Weither Desirable Nor
Undesirable" and 6'Sligbtly Undesirable, the shelf-life of trout fillets was considered
terminated (Greer, 1993). A value of 13 on the standard Roche color chart (Table 7) was
considered to be the cut-otT point for color of trout fillets (Via Mer, Personal
Communication, 1998). Based on these two subjective measurements, the sensory color
shelf-life of trout tillets packaged under different treatments was estimated as shown in
Table 8. The R-value (0.9381) indicated a significant correlation between these two
subjective methods of color detennination. Furthermore, it showed that either method
could be used to determine the color acceptability or estimated shelf-life based solely on
calor.
Subjective color measurements were a1so correlated to objective measurements.
Air packaged trout fillets had the shortest shelf-life i.e., -18 days based on subjective
color observations (Table 8). Th~refore, the L·,a·,b·,C· and h values, corresponding to
this shelf-life were chosen as the eut-offpoint CIE values and hence, termination ofshelf
life, for the other packaging treatments. For example, air packaged trout fillets had a
color acceptability shelf-life of 17.5 days. Therefore, the shelf-life based on L· value was
detennined by drawing a line of acceptability/unacceptability at 18 days to give an L·
value of49.2. Thereafter, for the other packaging treatment, trout fillets were regarded as
unacceptable, and hence shelf-life tenninated, when an L· value of 49.2 was reached.
The upper limit ofacceptability based on a, b, C and h values was detennined in a similar
manner. The estimated shelf-Iife of trout fillets based on these standards are shown in
Tables 9 and 10 respectively.
•
•
•
61
It is evident form Tables 9 and 10 that there W8S a poor correlation between
objective and subjective measurements. Tbese observations are in agreement with Setser
(984) who reported that it was impossible to correlate a subjective scale of color to
instrumental readings of lightness, redness, yeUowness, chroma or hue angle. In fact,
color preference for a specifie product varies from one individual to an other. lberefore,
on a subjective scale, different values would be givm by different panelists. However, by
using an objective method, only one physical measurement would be given.
• • •Table 9: Estimatcd objective shelf-life (days) extrapoJatcd from sensory coJor acceptability for trout flUets packaged under
different gas atmospheres and stored at 4°C
L* value' b* valuel d h value''.caglnl Color a* value e C* value'treatmentl acceptabliiti
Air .....18 .....18 .....10 .....3 -9 -3
Vacuum >28 .....27 .....5 .....2 .....5 ....16
CO2:N2 -19 ....20 ....3 >28 -3 >28(96.3:3.7)
CO2:N2 .....28 -6 -2 >28 -3 >28(85.1:14.9)
C~:N2 -18 -17 -4 ....3 -4 -26(59:41)
R=-O.l219 R=-O.4202 R=O.2008 R=-0.3365 R=O.l905• Based on the ûme (days) necessary for the color oftrout flllets to reach a score of15 on the sensory acccptability scalcb Bascd on the time (days) necessary for the color oftrout fillet to reach aL· value of49.2C Bascd on the lime (days) necessary for the color oftrout fillet ta reach an a· value of 16.2cl Bascd on the lime (days) necessary for the coJor oftrout fillet to reach a b· value of 17.6e Bascd on the time (days) necessary for the color oftrout fillet to reach a C· value of24.0r Bascd on the lime (days) necessary for the coJor oftrout fillet to reach an h· value of47.2
• • •Table 10: Estimated objective shelf-life (days) extrapolated from sensory Roche color chart for trout fiUets packaged under
different gas atmospheres and stored at 4°C
'aeu"nltreatmenll
Rocheeolorehart
L* value b a* vllue C b* vllues r C* vllue' --h vllue'
Air
Vacuum
C02:N2(96.3:3.7)
C02:N2(85.1:14.9)
C02:N2(59:41)
....18
>28
....22
>28
-22
....18
....27
-20
-6
-17
....10
-s....3
....2
-4
....3
....2
>28
>28
....3
-9
-s
-3
-3
-4
....3
....16
>28
>28
-26
R=-0.1l27 R=-O.6739 R=O.2725 R=-O.6113 R=O.48IS• Based on the time (days) necessary for the color oftrout fillets to reach a score of 13 on the sensory Roche color chartb Based on the time (days) necessary for the color oftrout fillet to reach a L* value of49.2C Based on the lime (days) necessary for the color oftrout fillet to reach an a* value of 16.2d Based on the time (clays) necessary for the color oftrout fillet to reach a b* value of 17.6e Based on the time (days) necessary for the color oftrout fillet to reach a C* value of24.0f Based on the time (days) necessary for the color oftrout fillet to reach an h* value of47.2
•
•
•
64
2Â. Conclusion
Color is probably the most important factor consumers associate with fish quality
and freshness. In fact, ifa food is visually unappetizing, it will certainly not be purcbased
or consumed. This study bas shown that the color ofpackaged trout tillets at refrigeration
temperature (4°C) was dependent on the gas atmosphere within the package. Five
packaging treatments were investigated and a significant increase in color shelf-life of
fresh trout fillets could be achieved using modified atmosphere packaging. The most
noticeable color changes were observed within the tirst week as noted for ail objective
color values such as L·, a·, b·, C· and h. Both subjective and objective results showed
that, based on color attributes only, trout fillets packaged onder vacuum had the longest
expected shelf..life of > 28 days. The second most successful packaging treatment for
trout fillets was a gas mixture of CÛ2:N2 (85.1:14.9). The shortest shelf-Iife for trout
fillets (days) was obtained by packaging in air or in a mixture of C(h:N2 (59:41).
However, since shelf-life was determined solely on the basis ofcolor, further studies need
ta be done to determine the shelf..life of trout based on physical, chemical and bacterial
changes oftrout fillets packaged under optimum gaseous conditions for color Le., vacuum
and CO2:N2 (85.1:14.9).
•
•
•
6S
CBAl'TER3
SHELF-LIFE STUDIES ON TROUY FILLETS
3.1. Introduction
Rainbow trout (Onchorhynchus my/dss) is a very popular fish species worldwide.
However, its shelf-life is limited by physica1, chemical and microbiological spoilage.
Extending the shelf-life of commercial ftesh trout beyond the S-7 days (Bamett et a/.,
1987) would he ofgreat significance to the food industry. Recently, modified atmosphere
packaging (MAP), involving a mixture ofcarbon dioxide (85% CÛ2) and nitrogen (15%
N2), bas been used to increase the color shelf-life of fish. By packaging trout fillets under
such gaseous conditions, using a high gas barrier film, the calor shelf-life of ftesh trout
could he extended by -10 days if strict temperature control was maintained. However,
little is known about the influence ofthe gas atmosphere on overall quality of trout fiIlets.
Therefore, the objective of this study was to determine the physical, chemical, sensorial
and microbiological quality ofrainbow trout fillets packaged in air, under vacuum and in
gas (CÛ2:N2 (85:15» conditions and stored at refiigeration (4°C) and mild temperature
abuse conditions (12°C).
•
•
•
66
3.2. Materials and Methods
3.2.1. Sample preparation
Freshly harvested rainbow trout filIets (Oncorhynchus mylciss) were obtained ftom a local
fish processor (Via-Mer, St-Hyacinthe, Quebec) and used throughout this study. Trout
filIets were transported in expanded polystyrene (Styrofoam<l» containers to our
Iaboratory.
3.2.%. Packaging and storage conditions
The trout fillets were packaged under three gaseous conditions: air, vacuum and gas
packaging C(h:N2 (87.2: 12.8). Trout flUets were placed in 140 mm X 26S mm expanded
polystyrene (Styrofoam @) trays containing a moisture absorbing pad and wrapped with a
layer of polyvinyl chloride (PVC) breatbable film with an oxygen transmission rate
(OTR) of 17, OSO cc/m2/day/atm @ 20°C, ()oA. RH) as primary package. Bach primary
package was than placed in a 210 mm X 42S mm high gas barrier Cryovac bag (W.R.
Grace" CO. of Canada Ltci. Cryovac division, Mississauga, Ontario) with an OTR of 12
cc/m2/day/atm @ 24°C, OOA. RH serving as secondary package. Air packaged trout fiUets
were introduced into the secondary packaging material and then heat sealed. Vacuum and
gas packaBing was done using a Multivac Chamber type vacuumlgas packaging machine
with an heat sealer (Model A 300/42, Multivac, D8941 Wolfertschwenden, Germany). In
gas packaging, the bags were tirst evacuated and then back tlushed with a mixture of
C(h:N2 (87.2:12.8). A Smith's proportional gas mixer (ModeI299-028, Tescom, Corp.,
Minneapolis, Minnesota) was used to gjve the desired proportions of C(h and N2 in the
package headspac:e. Duplieate samples per treatment were stored al refiigeration
temperature (4°C) for 21 days and mild abuse temperature conditions (12°C) for 7 days.
AlI packaged trout fillets were monitored after 2, S, 7, 14 and 21 days.
•67
3.2.3. Analyses
On each sampling day, two packages from the 4 and 12°C storage and corresponding to
each packaging treatment were analyzed for headspaœ gas composition, color, sensory,
drip loss, microbiological, pH and oxidative rancidity changes.
3.2.3.1. Headspace gas analysis
•
Changes in headspace gas composition were measured usÏDg a gas chromatograph
(Model 3300, Varian, Canada) at day 0 and on each sampling day. At each sampling, 0.5
ml of gas was withdrawn from each bag usÏDg a gas-tight pressure-Iock syringe and
needle (Precision Sampling Corp., Baton Rouge, La) inserted througli an adhesive
silicone septum attached to the outside of each secondary package. The gas sample was
then injected ioto the gas chromatograph fitted with a thermal conductivity detector.
Separation of the gas mixtures was achieved by Porapak Q (80-100 mesh) and Molecular
Sieve SA (80-100 mesh) columns using helium as the carrier gas (flow rate of 20
ml/min). The column temperature was set at 60°C while the injector and detector were
set at 100°C. Resolution of the gas peaks was done with a Hewlett-Packard recorder
integrator (ModeI3390A, Hewlett-Packard Co., Avondale, PA).
3.2.3.2. Instrumental color measurements
•
Color coordinates and reflectance measurements were calculated using the CIE
LAB system. The values were recorded for a CIE standard illuminant A (2856°K) and
the CIE 10° standard observer. Instrumental color measurements were canied out with a
Minolta Spectrophotometer CM-S08d (Minolta Co., Osaka, Japan) equipped with an
integrating sphere and with the optio~ of specular comPOnent excluded as outlined in
section 2.2.3.2. of Cbapter 2. During the measurements, the instrument was placed
direcdy on top of the fish tillet surface over-wrapped with the primary packaging film.
Measurements were taken at five different locations on each sample surface. Color
•68
variables recorded were L*, a*, b*, C* and h descnbing lightness, red-green chromaticity,
yellow-blue chromaticity, chroma «8*2 + b*2)~ and hue angle (arctan b*/a*) respectively
(Minolta, 1994).
3.2.3.3. Sensory evaluation
Sensory analysis was perfonned under fluorescent Hght by an untrained panel consisting
of 6 panelists. Samples were coded and presented in a random order. Color, texture,
overall acceptability and odor of raw trout fillets were evaluated using a hedonic scale
(7 Extremely desirable, 1=Extremely undesirable as descnbed in Table 6). The
appearance ofunopened packages was compared against a standardized Roche color chart
(Table 7).
Drip loss was measured using a Mettler Toledo PB3001 balance (Switzerland).
The percentage drip loss was caIcuJated al each storage day by ditTerences in the initial
and the final weight of trout tiUets. Packaged samples from each treatment were weighed
initially. On subsequent sampling days, packages were opened, drip poured into a sterile
measuring cylinder and the samples re-weigbed. Drip loss was expressed as a percentage
ofthe initial weight oftrout fiIlets (%w/w).
•3.2.3.4•
3.2.3.5.
Measurement ofdrip loss
Microbiologica1 analyses
•
Microbiological analysis was conducted on duplieate samples of trout fillets.
Tests performed included total plate count (bath 35°C and 4°C incubation), I&ctic acid
bacteria count and bath aerobic and anaerobic spore counts.
Ten grams oftrout fiIlet was weighed aseptically into a stomacher bag and 90 ml
of0.1% (w/v) sterile peptone water (Difco Laboratories, Detroit, Michigan, USA) added.
•69
The bags were stomached (Lab Blender 400, BA6021, Seward Medical, London) for 1
minute. This 10-1 dilution was then used for subsequent dilutions again using peptone
water.
The media and the methodology used for enumerating the various types of
microorganisms are summarized in Table Il. For all counts, 0.1 ml of the appropriate
dilution was plated in duplicates using a spread plate technique. Plates were incubated
aerobically with the exception of lactic acid bacteria and anaerobic spore counts whicb
were incubated anaerobically using anaerobic jars (BBL Microbiology Systems, Becton
and Dickenson St Co., Cockeysville, Maryland, USA). Spores, both aerobic and
anaerobic, were determined by tirst heat shocking the appropriate dilutions at 75°C for 20
minutes. Countable plates (30-300 colonies) were reported as IOS10 Colony Forming
Units per gram of sample (log CFU/g).
• 3.2.3.6• pH measurement
The pH of each homogenized sample was measured in duplicate after
microbiological analysis using a previously calibrated (butTer solutions of pH 4 and 7,
Fisher Scientific, Nepean, Ontario) Coming pH meter (Model 2220, Coming Glass
Works, Coming, NY).
3.2.3.7. Thiobarbituric acid test
•
TBA analysis was performed according to the method of Tarladgis et a/., (1960).
A lOg portion of rainbow trout fillets was transferred to a blender jar witb 50 ml of
distilled water and then blended (Osterizer, Sunbeam) at higb speed for 2 minutes. The
homogenate was transferred to a round bottom Oask by washing with an additional47.S
ml of distiUed water. To this mixture, 2.5 ml of4N HCI was added. A small amount of
antifoaming agent (Antifoam ~'B" BDR IDc., Toronto, Ontario) was placed onta the lower
neck of the tlask and a few glass beads were added to prevent bumping. The Oask wu
•70
then connected to a distillation unit and heated at 100°C until 50 ml of distillate was
coUected in a volumetrie flask A 5 ml aliquot of this distillate was transferred to screw
cap test tubes and 5 ml TBARS (thiobarbituric &Cid reactive substances) reagent added.
After mixing, ail test tubes, covered with aluminum foil, were heated for 3S minutes at
I()()OC in a water bath (Fisher Scientific Isotemp® waterbath modeI15-45S-20, Niles, a)
and then cooled under running tap water. A distilled water-TBA reagent blank was also
prepared in a similar manner. A portion of each samples was transferred to cuvettes
(Fisher Scientitic, 4.5 ml macrocuvettes, model 14-385-985). The optica1 density orthe
sample was read against the blank al a wavelength of 538 Dm using a spectrophotometer
(phannacia Biotech U1trospec® 1000). Multiplication of the readings by a factor of 7.8
was ~quired to convert to mg of malonaldehyde per l000g of sample and hence obtain
the corresponding TBA nomber.
Two factorial designs, a 2 X 3 X 3 and a 2 X 3 X 5 were used throughout this
study. Each tish sample (in duplicate) was subjected to 3 packaging treatments for 3
(storage al 12°C) or S (stomge at 4°C) test days as described previously. A Duncan's test
was used for separation of the means that had significant treatment variation using
Statistical Analysis System (SAS, 1988). A probability (P) of < 0.05 was considered to
he signiticantly different.
•
•
3.2.3.8• Statistica1 analysis
•
Table 11: Outline ofmicrobiological anaIysis performed on rainbow trout fillets
Organis. Medial SuppUer Metbod IncubatloD Incubation
temperature tbnel
rC)Mesophiles TSA Oifco Spread 35 48h
• Psychrotrophs TSA Oifco Spread 4 7d
Lactic &Cid bacteria MRS Oifco Spread 35 48h
Aerobic spores TSA Oifco Spread 35 48h
Anaerobic spores TSA Oifco Spread 35 48h
i TSA: Tryptic Soy Agar; MRS: De Man, Rogosa and Sbarp agar2 h=hours; d=days
•
•
•
•
72
3.3 Results and DiKussion
3.3.1. Changes in headspace gas composition
Changes in headspace Û2 and CO2 levels of trout fillets packaged onder difTerent
gas atmospheres and stored at 4 and 12°C are shown in Figures 13-16 respectively. For
trout fillets packaged in air and stored at 4°C, headspace Û2 decreased to < 5% within 7
days (Figure 13) while headspace CÛ2 increased to 17% within the same time period
(Figure 15). In air packaged trout flUets stored at 12°C, headspace Ch decreased ta < 2%
after 5 days of storage (Figure 14) while headspace C(h increased to 61.1% after 7 days
of storage (Figure 16). A reduction in headspace O2and increase in CÛ2 is mainly due ta
the growth of aerobic and facultatively anaerobic bacteria. However, some endogenous
enzymes may also contribute to a reduction in headspace Û2. In gas packaged trout flUets
(C02:N2 (87.2:12.8» stored at 4°C, Û2 ranged between 2-3% during the first 7 days of
storage (Figure (3) while gas packaged trout fillets stored at 12°C had headspace Û2
levels of< 1% after 5 days of storage (Figure 14). Headspace C02 decreased to 77-80010
in gas packaged trout flUets CÛ2:N2 (87.2:12.8) at 4 and 12°C (Figures IS and 16). This
decrease in C02 cao he attributed to the dissolution of C02 in the aqueous phase of the
fish. Thereafter, headspace C02 increased slightly (less than 3%) towards the end of
storage mainly due to the growth, and metabolism, ofboth aerobic and anaerobic spoilage
bacteria of fish.
3.3.2. Colol' measurements (CIE-LAB values)
Changes in the L* (lightness), a* (redness), b* (yeUowness), C* (chroma) and h
(hue angle) are shown in Tables 12-13, respectively.
•25
20
15
~eS -'-G8s#10
5
Figure 13: Changes in headspace Û2 of trout tillets packaged under different gasatmospheres and stored at 4°C
•25
20
15
~eS# -'-GM
10
5
Figure 14: Changes in headspace Û2 of trout fillets packaged under different gasatmospheres and stored at 12°C
•
•100
80
tS 60
~u -'-G.-.,.40
20
00 5 10 15 20 25
o.,s
•Figure 15: Changes in headspace CÛ2 oftrout flUets packaged under different gasatmospheres and stored al 4°C
100
80
tS60
~u -'-Ges.,.40
20
00 1 2 3 4 5 8 7
~
•Figure 16: Changes in headspace CÛ2 oftrout fillets packaged onder different gasatmospheres and stored al 12°C
• 3.3.2.1. Changes in L- (lightness) values
75
The changes in lightness (L* values) for trout fillets packaged under different gas
atmospheres and stored al 4 and 12°C are shown in Tables 12-13 respectively. It is
evident from these tables that the lightness (L- values) increased as storage lime
increased. This may he attributed to the bleaching of pigments (astaxanthin and
canthaxanthin) with time. These pigments are responsible for the color oftrout, so any
loss will lead to an increase in paleness. In fact, the L* values for trout tillets stored al
4°C increased from 45.30 to 55.11 for air packaged trout, to 52.60 for vacuum packaged
trout and to 55.59 for gas packaged trout. The L* values also increased with lime for
trout fillets stored al 12°C with final L* values of56.85, 50.62 and 61.10 for air, Vaalum
and gas packaged trout respectively.
The changes in redness (a* values) for trout fillets packaged under ditTerent gas
atmospheres conditions and stored al 4 and 12°C are shown in Tables 12-13. For all air
packaged trout fillets, irrespective of storage temperature, a* values increased steadily
throughout storage. Vacuum packaged trout flUets showed the largest increase in redness
al 4°C with final a* values of 23.38. At 12°C, the final a* values were of 20.60 after 7
days for vacuum packaged trout fillets. The a* values ofgas packaged trout fillets stored
al 4 and 12°C tluctuated throughout storage. However, the final a* values for gas
packaged trout flUets stored at 4 and 12°C, 19.70 and 24.20 respectively, were higher
than the initial a* value of 16.47.
•3.3.2.2•
3.3.2.3.
Changes in a* (redness) values
Changes in b* (yeUowness) values
•Changes in the yeUowness (b* values) for trout ti1lets packaged under different
atmospheric conditions and stored at 4 and 12°C arc shown in Tables 12-13. The b*
values fluctuated througbout storage irrespcctive of packaging and storage conditions.
•76
However, the final b* values for ail packaging atmospheres stored at 4 or 12°C were ail
higher than the initial b* value of 20.00. Carbon dioxide is known to bleach color and
react with oxymyoglobin to form metmyoglobin increasing a* values and b* values 10
produce a brown color (Brown et al., 1980). The effect is, however, limited since
myoglobin is not the main precursor of color for rainbow trout. Free radical oxidative
rancidity bas also been reported to increase b* values (Przybyrski et al., 1989).
Changes in C* (chroma) values for trout fillets packaged onder different
atmospheric conditions and stored al 4 and 12°C are shown in Tables 12-13 respectively.
In ail cases, C* values increased with lime with final chroma values always exceeding the
initial value of25.91. For trout filIets packaged at 4 and 12°C, there was no significant
difference (P > 0.05) between packaging treatments.
•
3.3.2.4.
3.3.2.5.
Changes in C· (chroma) values
Changes in h (hue angle) values
•
Changes in h (hue angle) values for trout flUets packaged onder various
atmospheres and stored al 4 and 12°C are shown in Tables 12-13. At 4°C, air and
vacuum packaged trout fillets showed no significant difference (P > 0.05) in terms ofhue
angle pattern over time. In both cases, h values decreased to 49.53 and 49.58 respectively
after 21 days. Tbere was also DO signiticant difference (P > 0.05) for air and vacuum
packaged trout flUets stored al 12°C. The h values remained fairly constant during the
tirst 2 days of storage (data not shown) and then decreased to 46.88 and 48.24
respectively by the end ofstorage. The final h values for air and vacuum packaged trout
fiUets were a1ways lower than the initial h value of 50.46, irrespective of storage
temperature. Gas packaged trout filIets had higber final h values, 52.64 and 50.52
respectively, for fillets stored al both 4 and 12°C.
• • •
Table 11: Changes in color coordinales of trout flUets packaged under difTerent gas atmospheres and stored at 4°C
'ackagiDI Color coordlnatntreatment
L* values .* values b*'values C* values h valunInldal Final Initiai Final Initiai Final Initiai Final Initiai Fini.
(DIY O)_(DIY 1IL(DIY O)--!Day 11) (Oay 0UOIY 11)--!DIY O)_(Day 11UOay O)_(OIY 11l
Air 45.30 55.11 16.47 21.20 20.00 24.80 25.91 32.62 50.46 49.53
Vacuum 45.30 52.60 16.47 23.38 20.00 27.40 25.91 36.03 50.46 49.58
CO2:N2 (87.2:12.8) 45.30 55.59 16.47 19.70 20.00 25.80 25.91 32.46 50.46 52.64
• • •
Table 13: Changes in color coordinates of trout flUets packaged onder different gas atmospheres and stored at 12°C
'acopng Color coordlnateatreatment
L* values .* values b* values C* values h valuesInitiai Final Initiai Final Initiai Final Initiai Final Initiai Final(Da, OUO., 7)_(DI' O'_(DI' Lcoa, O).-!Day 7}_(Da, 0LCOlY 7) (Day OL(Day 7)_
Air 45.30 56.85 16.47 25.39 20.00 27.04 2S.91 37.11 SO.46 46.88
Vacuum 45.30 50.62 16.47 20.60 20.00 23.10 25.91 30.96 50.46 48.24•
C(h:N2 (81.2:12.8) 45.30 61.10 16.47 24.20 20.00 29.38 25.91 38.06 50.46 50.52
•79
3.3.3. Sensory evaluation
Changes in the sensory properties for color acceptability, Roche color chart,
texture, general appearance and odor scores of trout fillets packaged under difl'erent
atmospheres and stored at 4 and l~oC are shown in Figures 17..26 respectively. Trout
fillets were regarded as unacceptable when a score of 3.5 on a subjective scale of 7 was
reached (Greer, 1993). However, for the sensorial detennination of color based on the
Roche color chatt, values of 13 or less on a scale of Il 10 18 were regarded as
unacceptable (Via Mer, Personal Communication, 1998).
3.3.3.1. Changes in color acceptability scores
•At 4°C, optimum color acceptability was observed in vacuum packaged trout
fillets. In fact, sensory color scores never reached 3.5 or less throughout 21 days of
storage (Figure 17). The lowest sensory color acceptability scores were obtained for trout
fillets packaged in CÛ2:N2 (87.2:12.8) with a score of 3.5 after -15 days. The sensory
shelf-Iife ofair packaged trout fillets, based on color acceptability scores, was tenninated
after -17 days. At 12°C, color acceptability scores for ail packaging treatments were not
significantly different (P > 0.05). Air, vacuum and gas packaged trout flUets bad sensory
scores of3.5 after -4 days respectively (Figure 18).
3.3.3.2. Changes in Roche color chart scores
•
The initial scores for trout tilIets were 13.64. In general, trout filIets tended to
become paler with tilDe, irrespective of storage temperature. This trend was observed for
aU air and gas packaged trout fillets which had final Roche color chart values of 12.42
and 11.67 after 21 clays at 4°C and 13.07 and 11.44 after 7 days al 12°C (Figures 19..20).
However, as reported in section 2.3.1.2., vacuum packaged trout fillets were redder and
darker in color at the end of storage with Roche color cbart scores of 14.25 after 21 days
at 4°C and 13.82 ailer 7 days al 12°C. At both stomp temperatures, gas
•7.,...-------------------_
1:~:::a.r
'4~~~~=___+f
2
2520151051+-----+---~+__---+__---+_--_.f
O,
•Figure 17: Changes in color acceptability scores of trout fillets packaged underdifferent gas atmospheres and stored at 4°C
7.,...--------------------.
I:~~~~i{41
3J---------~~~-~~__+
S2
7853211+----......-~~-_+--_+--_+_O-- ........----f
o
•Figure 18: Changes in color acceptability scores oftrout filIets packaged underdifferent gas atmospheres and stored al 12°C
•18,.----------------------,
17
1,8
115
t 14--=.....~_....t 13-+-------::~~~:::::::----=~~--___I_~
12
201510511 ......---+-----+-----+-----+----~
o
•Figure 19: Changes in Roche color chart scores oftrout fillets packaged underdifferent gas atmospheres and stored at 4°C
18 -------------------.,
17
1,8
c.1 15u
114;-====~~J13f--------=::~~~;;::::;:~~=f
12
7653211 +---+------l~-____+-- ......--........--........--...
o
•Figure 20: Changes in Roche color chart scores of trout fillets paclœged underdifferent sas atmospheres and stored at 12°C
•82
packaged trout fillets bad a score of 13 after -4 and -3 days al 4 and 12°C respectively
(Figures 19-20). The longest color shelf-life (> 21 days) was obtained al 4°C with
vacuum packaged trout, continning previous studies.
Texture values increased from an initial score of5.29 to 5.83 and 5.92 after 5 days
at 4°C for air and vacuum packaged trout fillets respectively. Texture scores then
decreased with time but never reached the unacceptable score of 3.5 by day 21 (Figure
21). However, a gradual decrease in texture scores was observed for gas packaged trout
flUets at 4°C, with shelf-life being terminated after day 14. At 12°C, ail packaged trout
flUets followed a similar trend i.e., a decrease in texture scores with lime (Figure 22),
although, all packaged trout flUets still bad an acceptable texture after 7 days.
•
3.3.3.3.
3.3.3.4.
Changes in texture scores
Changes in general appearance scores
•
The general appearance scores are influenced primarily by the amount of
discoloration present on the surface of the trout flUets which may be caused by a
combination of residual oxygen in the package and microbial growth. At 4°C, vacuum
packaged trout flUets had the highest general appearance scores. Based solely on this
parameter, the shelf..life ofvacuum packaged trout fillets was > 21 days (Figure 23). Air
packaged trout flllets had an estimated shelf-life of -17 days foUowed by gas packaged
trout flUets which bad a shelf-life of -12 days. At 12°C, the general apPe8l'8llce scores
decreased significandy within the first 2 days. Shelf-life, based on general appearance
scores, was terminated after only -4 days for trout fillets stored al 12°C and packaged in
air, vacuum and CÛ2:N2 (87.2:12.8) respectively (Figure 24).
•7
6
r 8~Vce 4-'-Ges
J32
10 5 10 15 20 25
---
•Figure 21: Changes in texture scores of trout fillets packaged under different gasatmospheres and stored al 4°C
7
6
15
8~VcI! 4
!3 -'-Ges
2
10 1 2 3 4 5 6 7
o.,s
•Figure 22: Changes in texture scores oftrout fillets packaged onder ditTerent gasatmospheres and stored al 12°C
•7.,......--------------------.
25201510. 51+----+-----+----_~--_~----4
o
•Figure 23: Changes in general appearance scores oftrout flUets packaged onderdifferent gas atmospheres and stored at 4°C
7
16
1 5
~c
14 ~V8C
-'-Gea•J:1
0 1 2 3 4 5 6 7
0.,.
•Figure 24: Changes in general appearance scores of trout fillets packaged underditTerent gas atmospheres and stored at 12°C
• 3.3.3.5. Changes in odor scores
8S
•
•
OtT-odors were characterized as rancid (probably due to oxidative rancidity) or
putrid Newton and Rigg (1979) reported that the growth of lactic &Cid bacteria and other
fermentative bacteria could cause off--odors by depleting glucose on the meat surface and
enhancing amino acid breakdown and the production ofputrefactive odors.
Changes in odor scores were significandy ditTerent (P < O.OS) for all storage
temperatures and packaging treatments on each sampling day. Air packaged trout fillets
had signiflcantly (P < O.OS) lower odor scores throughout storage at 4 and 12°C and their
shelf-life was terminated after -10 and -3 days respectively (Figures 25-26). Vacuum
and gas packaged trout flUets showed significandy different (P < O.OS) odor scores al 4°C
and their shelf-life was terminated after -13 and >21 days respectively. However, at
12°C, shelf-life was terminated after -4 days.
3.3.4. Changes in drip loss
Drip loss occurs due to the degradation ofmuscle tissue which results in a 10ss of
water holding capacity. Changes in drip loss for trout flUets packaged under ditTerent gas
atmospheres and stored al 4 and 12°C are shown in Figures 27-28 respectively. As
expected, the weight loss of trout fillets stored at both temperatures increased steadily
throughout storage and the percentage drip loss was consistendy higher at 12°C than at
4°C. This can he explained by the accelerated rate ofboth chemical and microbiological
spoilage al higher temperatures and hence greater proteolysis and breakdown of muscle
structure. At 4°C, drip loss was greatest in vacuum packaged trout fillets (3.78% w/w)
after 21 days of storage while at 12°C, the greatest drip Joss was observed for air
packaged trout flUets (S.75% w/w) after 7 days. At 4°C, the lowest drip loss (1.71% w/w)
was observed in gas packaged trout fillets (CÛ2:N2 (87.2:12.8» after 21 days while at
12°C, vacuum packaged trout fillets bad the lowest percentage drip loss (2.28% w/w)
after 7 days.
•7?"-------------------.......
2520151051+----........j~--___+---_+---__+-----f
o
6
2
1 5
! 41
j 3 t-------~-~~.::;;;;;.....----I
•Figure 25: Changes in odor scores of trout fillets packaged onder different gasatmospheres and stored al 4°C
7~----------------------.
785321
6
2
1:j 3 t------~----..;:~-----I
•Figure 26: Changes in OOor scores oftrout fillets packaged onder different gasatmospheres and stored al 12°C
•6
5
14
~
J 3
Î21
00 2 5 7 14 21
o.w-
Figure 27: Changes in drip 10ss (%w/w) of trout fillets packaged under different gasatmospheres and stored at 4°C
•6
5
141
~- .VecJ 3 DGesILa 2
1
00 2 5 7
•Figure 21: Changes in drip 1088 (%w/~) oftrout flUets packaged underdifferent gasatmospberes and stored st 12°C
•88
The amount of drip loss may be directly interrelated 10 the pH of fish tissue. The
pH of trout fillets decreased with time for ail packaging conditions. When pH decreases,
the isoelectric point of flsh proteins will be reached and proteins will become closer due
ta less electrostatic repulsion and hence, squeeze out more moisture.
3.35. Microbiological analyses
Changes in microbiological counts ofmesophilic, psychrotrophic and lactic &Cid
bacteria are shawn in Figures 29-34 respectively.
3.35.1. Changes in mesophilic counts
•Total aerobic plate count (APC) results for trout tillets s10red at 4 and 12°C are
shown in Figures 29..30 respectively. Ali packaged trout tillets had an initial plate count
of 6.4 X 104 CFU/g. In other studies, the initial bacterial Joad (APC) of rainbow trout
fillets bas been reported to range ftom 1~-1<r CFU/g (Yasuda et al., 1992) to lOS CFU/g
(Bamett et al., 1987). At 4°C, APCs increased in ail packaged trout fillets to > 108, 107
,
and 106 CFU/g for air, vacuum and gas packaged trout fi1Iets by day 7 respectively
(Figure 29). At 12°C, air packaged trout flUets reached an APC ofS X 108 CFU/g by clay
5 i.e., the highest count obtained for ail filIets. Vacuum and gas packaged trout flUets bad
APC counts of 1.26 and 3.23 X 108 CFU/g respectively by the end ofstorage (Figure 30).
3.3.5.2. Changes in psychrotrophic counts
•
Changes in psychrotrophic counts for trout tillets packaged in difTerent gas
atmospheres and stored at 4 and 12°C are shown in Figures 31-32. The time to reach a
count of 107 CFU/g was regarded as the standard to terminate shelf-life (lCM8f, 1978).
The initial psychrotrophic COUDts for ail samples were -106 CFU/g, indicating poor
manufacturing practices at the process plant. At 4°C, air paclœged trout fillets reached
counts of 107 CFU/g in -2 days while in vacuum paclœged trout fillets, tbis limit was
•89
reached after -3 days. Gas packaged trout fillets had the longest shelf-life of -6 days.
AlI packaged trout tillets had psychrotrophic counts of>108 CFU/g by the end of storage.
Shelf-life was tenninated after -2 days for air, vacuum and gas packaged trout fillets
stored at 12°C (Figure 32).
3.35.3. Changes in laetic acid bacteria counts
•
Changes in lactic acid bacteria (LAB) counts for difTerent packaging treatments of
trout tillets stored at 4 and 12°C are shown in Figures 33-34. Initial counts ofLAB were
_104 CFU/g which increased to -107 CFU/g by day 7 at 4°C, irrespective of packaging
treatment (Figure 33). At 12°C, ail packaged trout fillets bad LAD counts of _107_108
CFU/g by day S and remained at these levels until the end of storage (Figure 34). These
results are in agreement with sensory odor PerCeption. In fact, upon opening packages, a
distinct acidic odor was noticeable, which was probably due to the inc:rease in LAS
numbers throughout storage.
3.35.4. Changes in aerobic and anaerobic spore forming bacteria
•
Growth of aerobic or anaerobic spore forming bacteria was not observed for any
packaged trout fiUets al storage of 4 or 12°C (data nor shown). These results are
surprising since fish can be contarninated with Clostridium spp., particularly C. botulinum
type E.
3.3.6. Changes in pH values
Changes in pH values for trout fillets packaged under different abnospheres and
stored at 4 and 12°C are shown in Figures 35-36. The pH values did not change
significandy (P < 0.05) in any of the packaging treatments for trout fillets stored al 4°C.
For trout fillets packaged in air, pH decreased sligbdy ûom 6.55 10 pH 6.32 (clay 2) and
•9 __------------------..,
8
5
252015105.. ~--~~----+-----+---- .........---""'"
o
•Figure 29: Changes in mesophilic counts oftrout flUets packaged under different gasatmospheres and stored al 4°C
9
8
l7U
J6
5
..0 2 3 .. 5 6 7
o.wa
•Figure 30: Changes in mesophilic counts of trout fillets packaged onder different gasatmospheres and stored at 12°C
•9,---------------------,8
~ 7 +-~~-_+--~-----~.",_e:.~--I~u
Je
5
2015105.-+-----+----+-----+----+----~
o
•Figure 31: Changes in psychrotrophic counts oftrout-fillets packaged under differentgas atmospheres and stored at 4°C
•
9
8
i 7~u
Je
5
.-0 1 2 3 .- 5 8 7
0.,.
Figure 32: Changes in psychrotrophic counts oftrout flUets packaged under differentgas atmospheres and stored al 12°C
•9-poo----------------------.8
5
252015105.+-------I~--_+---__+_---_+_----f
o
•Figure 33: Changes in lactic acid bacteria counts oftrout fillets packaged underdifferent gas atmospheres and stored at 4°C
•
9
8
~7lA.u
Js
5
•0 1 2 3 4 5 6 7
---Figure 34: Changes in lactic &cid bacteria counts oftrout fillets packaged underdifferent gas atmospheres and stored at 12°C
•
•
•
93
then subsequendy increased to pH 6.46 alter 14 days. A sirnilar trend was observed for
ail vacuum packaged trout fillets. For trout fillets packaged under a gaseoua atmosphere
ofCÛ2:N2 (87.2:12.8), pH values decreased from 6.55 to 6.18 after 21 days (Figure 35).
The slight reduction in pH (0.37 units) in these samples may be explained by the
dissolution of carbon dioxide from the package headspace at refrigerated teD1Pel'8tures
and the growth of lactic 3Cid bacteria.
Trout fiUets stored al 12°C showed similar pH changes for a1l packaging
conditioDS. The pH values decreased initially and then increased. However, only air
packaged trout had pH values greater than the initial value of 6.55 (Figure 36). The
subsequent increase in pH during stomge may he explained by the buffering effect of
muscle proteins and release of amino acids, probably as a result of proteolytic activity of
facultativelyanaerobic spoilage bacteria.
3.3.7. TBA aoalysis
When fish are caught and tissues damaged, certain enzymes, such as lipoxygenase
of fish gilI and skin, may initiate lipid peroxidation (Hsieh et al., 1988). The higb degree
of unsaturated fish Iipids makes tbem suscepbble to oxidation producing unstable
hydroperoxides and therefore leading to quality deterioratioD. In fact, the breakdown
products of hydroperoxides (carbonyls) are a sourœ of oxidative off-tlavors that
adverselyaffect the taste and smeU of fish (Josephson et al., 1984). Pigments present in
trout however, have shown to have an effective protection against oxidation of lipids.
Asthaxanthin (2 hydroxy groups) provides a good contact to hydroperoxides (Andersen et
al., 1990).
In this study, trout flUets stored al 4 and 12°C under various gas atmospheres were
analyzed for TBA values throughout stonge (Figures 37-38). A TBA value of 3 was
considered to he the upper limit ofacceptability in terms of fish lipid oxidation based on
the rePOrt of SînDhuber and Yu (1958). At 4°C, air and vacuum packaged trout fiUets
•7
6.8
1 6.6
"1i. 6.4
6.2
60 2 5 7 14 21
•Figure 35: Changes in pH values oftrout fillets packaged onder di1ferent gasatmospheres and stored al 4°C
7
6.8
1 6.6 5Jl .Vec
i. 6."aGas
6.2
60 2 5 7
•Figure 36: Changes in pH values oftrout fillets packaged under difTerent gasatmosphercs and stored at 12°C
•3.2 -y--------------------...,
0.8
201510
o.wa5
0-+-----+----.-...01-------+------1o
•Figure 37: Changes in TBA numbers of trout fiUets packaged under difTerent gasatmospheres and stored al 4°C
3.2~------------------__.
2.4
0.8
-- -141210862
0-+---~---+--_+_--_+_--+---~---4
o
• Figure 38: Changes in TBA numbers oftrout fillets packaged under different gasatmospheres and stored al 12°C
•
•
•
96
showed a graduai increase in TBA values, ftom 0.32 to 0.49 and 0.39 respectively aftcr
20 days. However, gas packaged trout fillets had the largest increased in mA values to
3.05 throughout storage i.e., the only packaging condition to reach the upper limit of
acceptability of 3 for lipid oxidation. The shelf-life of gas packaged trout fillets was
estimated to he of-20 days.
At 12°C, air and vacuum packaged samples foUowed a similar trend, with their
final TBA values heing 0.42 and 0.33 respectively after 13 days. The final mA value of
gas packaged trout fillets was again higher st 12°C (1.35) but not as higb as st 4°C.
Bamett et al. (1987) reported a significant increase in TBA values in MAP trout between
day 10-18 at 3S~ (l.~C).
3.3.8. Overall shelf-Iife studies
The estimated shelf-life (days) for ail packaged trout fillets stored st 4 and 12°C is
summarized in Table 14. Sensory evaluation scores for color, texture, odor and general
appearance are the four most important factors consumers relate to fish quality and
freshness. The time to reach a score of 3.5 (rejection point in acceptability scale) or 13
(rejection point in Roche color chart scale) from either color, texture, odor or general
appearance was used as an indicator of sensory shelf-life. The most limiting sensory
parameter was the Roche color scores for air and gas packaged trout fillets stored al 4°C
and vacuum and gas packaged trout fiUets stored at 12°C, with sensory shelf-life of7, -4,
-3 and -3 days respectively. Sensory odor scores were also used as standards of the
overall sensory shelf-Iife. For example, vacuum packaged trout fiUets stored al 4°C and
air packaged trout fillets stored al 12°C had an overaU sensory shelf-life of -13 and -3
days respectively and were rejected due to strong off-odors. It is interesting 10 note that in
most cases, color and odor were the determining factors for rejection.
• •
Table 14: Estimated shelf·life (days) ofair, vacuum and gas packaged trout flUets stored at 4 and 12°C
•
'aetall·1conditions
Air
TempenturerC)
4
Sensory Sensory Sen10ry Sensorycolor Roche color teltare- general
acceptablUaya chanb appeannce-16.7 7 > 21 16.6
Senlory000'"
9.8
Mlerobla.t
(APe)
1.6
TRAd
>20
Vacuum
CÛ2:N2(87.2:12.8)
Air
Vacuum
4
4
12
12
> 21
14.7
4.2
4
>21
3.6
3.6
3.4
> 21
14
>7
>7
>21
12.1
4
4.2
13.1
> 21
2.6
4.1
2.7
6.4
1.8
1.7
>20
19.7
> 13
> 13
C02:N2 12 4.3 2.9 > 7(87.2~ 12.8)
aRejcction point: lime (daya) to rcach a score of3.5Jtaejection point: time (days) to reach a score of 13CRejection point: time (days) to reach psychrophilic counts of 107 CFU/gdRejcction point: time (days) to reach a TBA number of3.0
4.3 4.0 1.6 > 13
•
•
•
98
The microbiological shelf-life of trout fillets was based on the time necessary for
the psychrotrophic counts 10 he> 107 CFU/g. Fresh trout flUets had a shelf-life of -2
days at 12°C irrespective of packaging treatment. However, at 4°C, a slightly longer
microbiological shelf-life of -2...3 days was obtained for air and vacuum packaged trout
flUets respectively while a longer extension in shelf...life (-6 days) was possible by gas
packaging and remgeration.
The shelf-life oftrout fillets based on oxidative rancidity levels was detennined by
the tinte (days) to reach a TBA nomber of 3. Based on this criteria, TBA shelf-life was
generally Dot terminated by the end ofstorage. However, gas packaged trout fillets stored
at 4°C had a TBA shelf...life of-20 days.
For most trout fillets, product rejection was based on microbiological shelf-life
rather than sensorial or oxidative rancidity (TBA) shelf...life
•
•
•
99
3.4. CODd_loD
This study bas shown that a slight extension in shelf-üfe of fresh trout tillets can
he achieved using gas packaging. These results are in agreement with previous studies
using MAP to extend the quality of ftesh fish (Bamett et al., 1987). However, shelf-life
is dependent on the initial quality of fish, which in this study was relatively poor. At
12°C, no differences were observed in shelf-life between packaging treatments. This
study again shows the importance of strict temPerature control and good manufacturing
practices (OMPs) if MAP is to he etTective to extend both the sensory and
microbiological shelf-life oftrout fillets.
•
•
•
100
CllAPTER4
CHALLENGE STUDIES WITB LlSTERIA MONOCYTOGENES
4.1. Introduction
The incidence ofListeria monocytogenes CODtamination in imported and domestic
seafood in the li.S. bas been reported to he between S and 6% (McCarthy, 1997).
Recently, L. monocytogenes was isolated from both whole fish and tillets of aquacultured
rainbow trout (McAdams, 1996). Thèse observations are of importance to the food
industry since L. monocytogenes bas been found to survive, or even grow, al refrigeration
temperatures, i.e., temperatures previously considered to prevent the growth of food
pathogens. Another concem is the possibility ofcross-eontamination of ftesh tillets with
cooked ready-to-eat products with this psychrotrophic pathogen during market handIing
or in the home.
Modified atmosphere packaging bas been slmwn to extend the shelf-life of fish
products by inhibiting the growth of aerobic spoilage bacteria (Chapter 3). However,
under MAP conditions, the growth of psychrotrophic pathogenic bacteria, such as L.
monocytogenes, may not be inhibited. Therefore, concems have heen raised regarding the
microbiological safety ofMAP food.
The objectives of this study were to monitor the physical, chemical, sensorial and
microbiological changes in rainbow trout fillets inoculated with L. monocylogenes
packaged in air and two modified atmospheres, and stored al retiigeration (4°C) as well as
temperature abuse conditions (12°C).
•
•
•
101
4.2. Materials and Metbods
4.2.1. Sample preparation
Rainbow trout fillets (Oncorhynchus my/ciss) were obtained &am a local seafood
producer, Via-Mer, St-Hyacinthe, Quebec. Fillets were immediately stored on ice and
transported to our laboratory in expanded polystyrene (Styrofoam~ container. Prior to
inoculation and packaging, the fresh fillets were cut into pieces each weighing
approximately l00g.
4.2.2. Bacterial strains
Five strains ofL. monocytogenes; Strain Scott A (human isolate), and HPB strains
323 (shrimp isolate), 392 (Iobster isolate), 439 (crab isolate) and 976 (salmon isolate)
were obtained from the Microbial Hazards Bureau (Health Protection Branch (HPB),
Health Canada, Ottawa, Canada) from the culture coUectioD of Dr. J. Farber. The
cultures were maintained frozen at -24°C (700 J.L1 of a 24 hours culture grown in TSB
(tryptic soy broth) + YE (yeast extraet) (Difco) with 300 JJl of a 500A. (v/v) glycerol
solution).
A loopful of the above culture was streaked ooto Tryptic Soy Agar (TS~ Difco)
plates and incubated at 35°C for 48 hours. Isolated colonies of each strain were then
transferred to separate tubes containing 5 ml of Tryptic Soy Broth (TSB, Difco)
supplemented with 0.6% yeast extract (TSBIYE) and incubated for 13 hours at 35°C to
give a suspension of approximately 2 X 109 CFU/ml. The inoculum was prepared using
these cultures which were then further diluted with 0.1% peptone water to 2 Xl06
CFU/ml. Appropriate volumes of each strain (5 X 200J.tL) were then diluted again in 9
ml of0.1% peptone water (Difco) to yield a mixed-strain suspension ofL. monocytogenes
ofapproximately 2 X lOS ceUs/mi.
•
•
•
102
4.%.3. Inoculation
Fillets were inoculated by spreading 1 ml of culture suspension evenly onto each
rainbow trout fillet using a sterile hockey stick to give a final inocu1um level of
approximately 103 CFU/g (exactly 1.185 X 10" CFU/g). Control trout fillets were
inoculated in a similar manner with the same volume of 0.1% sterile peptone water.
Sample preparation and inoculation was canied out in a Purifier1M Class II Safety Cabinet
(Labconco, Model # 36205-04, Labconco, Kansas, Missouri, USA) equipped with a
HEPA filter to ensure minimal contamination of the samples and the surrounding
enviromnent as weil as the safety ofthe research personnel.
4.2.4. Packaging and stomge conditions
Control and inoculated trout fillets (- l00g) were- packaged individually in 210 X
425 mm high gas barrier bags (OTR = 12 cclm2/day/atm @ 24°C, OO/oRH) (Cryovac
Sealed Air Corporation, Mississauga, Ontario), and thm subjccted to the foUowing three
packaging treatments: air, vacuum and gas (C02:N2 (88.8: 11.2». Air packaged samples
were sealed dircctly without further modification to the package headspace. Vacuum and
gas packaging of trout fillets were done using a Multivac chamber-type, heat seal
packaging machine (Model 300A/42). A Smith's proportional gas mixer (Model 299
028, Tescom Corp., Minneapolis, Minnesota) was used to give the desired proportions of
C(h and N2 in the package headspace. Samples at refrigeration temPerature (4°C) were
stored for 21 days while flUets at mild temperature abuse (12°C) were only stored for 7
days. Duplicate samples were used for each condition and for each sampling day.
Analysis was performed al day 0,2,5,7,14 and 21 .
•103
4.2.5. Analyses
Headspace gas anaIysis, sensory analysis (general appearance only) and pH
measurements were carried out as descnDed in sections 3.2.3.1., 3.2.3.3. and 3.2.3.6.
respectively.
4.2.5.1. Microbiological analyses
•
•
On each sampling day, bags corresponding to each treatment were aseptically
opened. The trout fillet (-1OOg) was placed in a sterile stomacher bag and blended with
twice its weight of 0.1% Peptone water in a Stomacher (Model 400, AJ. Seeward,
London, UK) for 2 minutes. One ml of this slurry was then added to tubes containing 9
ml ofO.lo/oSterile Peptone water (l0-1) and fiJrther dilutions were then made, again using
0.1% sterile PePtone water.
On each analysis day, the inoculated samples were enumerated for L.
monocytogenes by plating lOOpL of the appropriate dilutions on PALCAM agar (Oxoid)
supplemented with Palcam Selective Supplement (SRlSOE). Control samples were
checked for the presence ofL. monocytogenes on the first and last day of the storage trial
using the method described previously. AlI samples (inoculated and control trout tillets)
were monitored at the beginning and end of the storage trial (day 7 for storage at 12°C
and day 21 for storage at 4°C) for total aerobic counts (mesophiles and psychrotrophs) as
weil 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. Lactic 8Cid bacteria were enumerated using Lactobaci//us MRS
Agar (Difco Laboratories, Detroit, MI) using a spread plate technique of the appropriate
dilutions. AIl counts were done in duplicate. PALCAM plates, as weil as TSA plates,
were incubated aerobically at 3SoC for 48 hours. TSA plates for psychrotrophic counts
were incubated aerobically at 4°C for 7 days, while lactic &Cid bacteria wcre incubated
anaerobically at 3SoC for 48 hours (Anaerobie Jars, BBL Microbiology Systems,
•
•
•
104
Cockeysville, MD). Countable plates (30-300 colonies) were reported as 10810 Colony
Forming Units per gram ofsample (lOglo CFU/g).
4.%.6. Statistical analysis
Two factorial designs, a 2 X 3 X 3 and a 2 X 3 X 5 were used tbroughout this
study. Bach fish sample (in duplieate) was subjected to 3 packaging treatment during 3
(storage at 12°C) or 5 (storage at 4°C) test days as descnDed previously. Duncan's
multiple range test was used for comparison of the means, utilizing Statistical Analysis
System (SAS, 1988). A probability (P) of less than 0.05 was considered to be
significantly different
•
•
•
lOS
4.3. Results and Discussion
4.3.1. Changes in headspace gas composition
Changes in headspace gas composition (Ü2 and CÛ2) of control and inocuJated
trout samples stored at 4°C and 12°C are shown in Table IS. In control and inoculated
trout tillets stored al 4°C, Û2 decreased while C02 increased throughout storage as a
result ofmicrobial activity. For inoculated gas packaged trout fillets, Û2 levels remained
low (below 1.5%) throughout storage. The CÛ2 concentration in air packaged inoculated
trout increased to -26% after 21 days white CÛ2 increased slighdy in gas packaged trout
from 88.8% to -9S% during the first week and then retumed to ils originallevel towards
the end ofstorage, probably due to its absorption in the aqueous phase ofthe filleL
At 12°C, changes in headspace Û2 in non..inoculated and inoculated samples were
similar. Headspace 02 decreased in air packaged trout, from 20.7% to < 1% in control
samples and to 3.6% in inoculated samples. Headspace 02 remained fairly constant in gas
packaged trout (control and inoculated) with 02 levels < 1.6S% tbroughout storage. For
ail packaging conditions, headspace C02 increased to similar levels as control samples
throughout storage (Table 1S).
4.3.2. Sensory evaluation
Changes in the genera1 appearance (color and texture scores) of inoculated and
control trout tillets stored at 4°C and 12°C are shown in Figures 39-42. The appearance
of the fish was evaluated using a hedonic scale, ranging ftom 1 to 7 (Greer, 1993). A
score of 3.S was consideree! to be the upper limit of acceptability, implying tbat the
sensory shelf..life was terminated when this score was reached.
Control trout fillets stored at 4°C (Figure 39) showed a graduai decrease in
general appearance alter 21 clays of storage. The sensory shelf..life of air paclœged
• • •Table 15: Changes in headspace 02 and C02 ofcontrol and inoculated trout flUets packaged under different gas atmospheres and
stored at 4 and 12°C
Pack.glnlareatalent
Inoculation Stor.getemperature
Headspace .as composition (%v/v)
Inldal Final02 COI QL C02
20.7 0 9.2 16.0
1.6 88.8 0.7 92.7
Air
C02:N2
(88.8: II.2) 4
Air +
C02:N2 +(88.8:11.2)
20.7
1.6
o88.8
1.0
1.1
25.9
88.0
Air .. 20.7 0 0 44.8
C(h:N2 .. 1.6 88.8 0.6 99.8
(88.8: 11.2) 12
Air + 20.7 0 3.6 44.3
CO2:N2 + 1.6 88.8 0 95.9
(88.8: Il.2)
•
•
•
107
control trout fillets was <21 days al 4°C. Even though the sensory shelf-life for vacuum
and gas packaged samples was >21 days, gas packaged samples had higher acceptance
scores (6 versus 4) at the end ofstorage compared to vacuum packaged trout. Differences
were observed in the sensory evaluation of inoculated trout fillets stored at 4°C under the
various packaging conditions (Figure 40). Vacuum packaged inoculated trout maintained
a perfeet score of 7 during the tirst week of storage, then, their general appearance
decreased progressively, althougb, by the end of storage, the sensory shelf-life was still
acceptable. No specific trends were observed for air and gas packaged inoculated trout,
however, their sensory shelf life was -II and -14 days respectively (Figure 40).
Statistically, the latter treatments were not significantly different (P>O.05).
At 12°C, a constant decrease was observed in the general appearance ofaU control
packaged trout (Figure 41). Sensory shelf-life was <7 days for air and vacuum packaged
trout and >7 days for gas packaged trout. A similar trend was observed for aU inocuJated
packaged trout stored at 12°C (Figure 42). Values during the tirst 2 clays of storage were
significantly higher (P<O.05) than during the remainder of storage. Sensory shelf-life,
based solely on appearance, was estimated to he -4, -6 and -4 days for inoculated air,
vacuum and gas packaged trout respectively stored at 12°C.
4.3.3. Changes in pH values
The changes in pH values for cODuol and inoculated trout tillets stored al 4°C and
12°C are shown in Figures 43-46. For ail conditions, pH remained fairly constant
(between pH unit of 6 and 7) througbout tbis study. At 4°C, pH decreased for aU 3
packaging conditions from an initial value of 6.8 to -6.4-6.6 for control and inoculated
trout respectively (Figures 43-44). At 12°C, pH decreased ftom an initial value of 6.8 ta
-6.5-6.7 for control and inocu1ated trout samples respectively (Figures 45-46). The pH
values for air and vacuum inoculated packaged trout were significantly dift"erent &am gas
inoculated packaged samples (p<O.OS). This slipt decrease difference can be attributed
•7~::=======-16
1
rt----~~IJ3
2
2520151051+------4'-----+---...-+----.......-----4
o
•Figure 39: Changes in general appearance scores ofcontrol trout fillets packagedunder ditTerent gas atmospheres and stored al 4°C
7 ...._.1----1.........---------------.
5 10 15 20 25
•Figure 40: Changes in general appearance scores of inoculated trout filIets packagedunder different gas atmosphcres and stored al 4°C
•7
6
1J 5
1 EJI!~Vecuum
14-A-G-.
)32
10 1 2 3 4 5 6 7
0.,.
•Figure 41: Changes in general appearance scores ofcontrol trout fillets packagedunder ditTerent gas atmospheres and stored al 12°C
Figure 42: Changes in general appearance scores ofinoculated trout fillets packagedunder ditferent gas atmospheres and stored at 12°C•
7
16
ii> 5
1
14
r2
10 1 2 3 4 5 6 7
0.,.
•6.8..,....----------------------.
6.7
6.6
1 6.5
1i. 6."
6.3
6.2
6.1
E]AIraveaun.0..
o 21
•Figure 43: Changes in pH values ofcontrol trout fillets packaged under different gasatmospheres and stored al 4°C
Figure 44: Changes in pH values of inoculated trout Mets packaged under differentgas atmospheres and stored at 4°C•
7
6.8
1 6.6
1i. 6."
6.2
60 2 5 7 14 21
E]AIraveaun.0..
•7
6.8
1 6.6 5jJ aVecuum
i. 6.4.Ges
6.2
60 7
•Figure 45: Changes in pH values ofcontrol trout fillets packaged under different gasatmospheres and stored at 12°C
7-y--------------------_6.8
16.6
1i. 6.4
6.2
6
Figure 46: Changes in pH values of inoculated trout fillets packaged under difTerentgas atmospheres and stored al 12°C•
o 2 5 7
•
•
•
112
to the growth of lactic acid bacteria and dissolution of beadspaœ C(h in gas packaged
trout.
4.3.4. Microbiological analyses
Counts of L. monocytogenes, enumerated on PALCAM agar, for the various
packaging treatments of fresh trout are shown in Figures 47-48. L. monocytogenes was
not detected in any control samples stored at bath 4°C and 12°C (data not shown). In the
inoculated study, L. monocytogenes counts increased from an initial level of 1.2 X 10"
CFU/g to 3.2 X las CFU/g and 9.9 X las CFU/g for air and vacuum paclœged trout fillets
stored al 4°C (Figure 47). Vacuum packaging may have enhanced the growth of L.
monocytogenes since it is a microaerophilic microorganism. However, gas paclœged
samples showed a graduaI decrease (- 1 Log) in counts of L. monocytogenes during
storage. In fact, counts varied from 1.2 X 10" CFU/g to 2.S X loJ CFU/g after 21 days.
These results showed that not ooly L. monocytogenes was inhibited in a higb CÛ2
atmosphere but it also decreased in numbers. The gaseous mixture used also reduced the
L. monocytogenes counts by 2 orders of magnitude compared to air and more compared
to vacuum packaged trout fillets stored at 4°C al 21 days (Figure 47). Similar results
were reported by Sheridan et al. (1995) who showed an inhibitory effect of l000A. CÛ2 on
the growth ofL. monocytogenes in gas packaged lamb. Furthermore, Avery et al. (1994)
reported inhibition of L. monocytogenes in beef striploin steaks packaged under a
saturated CO2 controUed atmosphere and stored al SoC and 10°C.
At 12°C, counts ofL. monocytogenes increased to >107 CFU/g over 7 days for all
packaging treatments (Figure 48). lbese results confirm the importance of strict
temperature control in conjunction with MAP 10 inlubit the growtb of this patbogen.
Other factors which bave been reported to atTect the growth ofL. monocytogenes include
the type of tissue (% fat), pH of the tissue and nature of the competing Oora (Sheridan et
al., 1995).
•
•
•
113
Mesophilic, psychrotrophic and lactic &Cid bacteria counts are shown in Figures
49-54. At 4°C, mesophilic counts reached 107 CFU/g for Most packaging conditions of
both control and inoculated trout after 21 days, with the exception of air inoculated and
gas control packaged trout (Figure 49). In these packaging treatments, counts increased
to 108 CFU/g and 106 CFU/g after 21 days respectively. At 12°C, mesophilic counts
reached >IOs CFU/g for both control and inoculated trout after 7 days, irrespective of the
packaging conditions (Figure 50).
The initial psychrotrophic counts of fresh trout fillets were of 3.8 X lOS CFU/g.
At 4°C, psychrotrophic counts increased to 108 CFU/g by day 21 in both air and vacuum
packaged trout. However, for gas packaged trout, these levels were not reached until the
end ofstorage (Figure SI). At 12°C, psychrotrophic counts reached lOS CFU/g by the end
of storage for ail packaging conditions (Figure 52). At both storage temperatures, the
final psychrotrophic counts (after 21 clays al 4°C and 7 days al 12°C) exceeded the
maximum microbiological limit for fresh fish recommended by the Intematioaal
Commission on Microbiological Specifications for Foods (lCMSF, 1978) of 107 CFU/g.
Initial lactic acid bacteria counts were -loJ CFU/g. At 4°C, LAD counts
increased to 107 CFU/g in ail packaging conditions throughout storage. (Figure 53). At
12°C, LAS counts increased to lOS CFU/g in air packaged trout while counts increased 10
107 CFU/g in vacuum and gas packaged trout by the end of storage (Figure 54).
Therefore, no particular packaging treatment appeared to have a major etrect on the
growth oflactic acid bacteria.
•8-r--------------------.,7
2520151053+---~~-=--_+---_+_---_+_-----f
o
•Figure 47: Changes in counts ofL. monocytogenes of inoculated trout fillets packagedunder different gas abnospheres and stored at 4°C
8~------------__::Jl_---- ...7
7654323+-----t----+---......--......-~~-__+--......
o
• Figure 48: Changes in counts ofL. monocytogenes ofinoculated trout fiUets packagedunder different gas atmospheres and stored al 12°C
•9-w-----------------.
~Car*aI (Ak)
~Inoalllflld (Air)
-6-Car*aI (V*'U'n)
~1nrxU1C1~ (V1ICWm)
~Ccri'aI (c;.)
~Inoa"""(o-)
20151053 ~--+__--......--_+_--_+_--_4
o
7
8
•Figure 49: Changes in mesophilic counts of control and inoculated trout fiIIetspackaged under ditTerent gas atmospheres and stored at 4°C
Figure 50: Cbanges in mesopbilic counts of control and inoculated trout fiIIetspackaged onder different gas atmospheres and stored at 12°C•
9
8
7"='..
~6J
5
4
30 2 4 6 8
---
~CCnIraI (AIr)
-e-Inoadrt d (Ar)
-'-CCnIraI (V8QUIt)
-*-1nocUIIted (Veaun)___ConIraI (Gel)
~Inoalileed(Gea)
•9.,..------------------.8
6
-'-Cor*oI (Ar)
~tnoalIIIted(AW)
-'-Cor*oI (V8QUft)-)E-tnoa.... (Vecuum)___Cor*oI (G8a)
~1~(G8s)
2520151055+---.-.+----+----+--~~--..f
o
•Figure 51: Changes in psychrotrophic counts of control and inoculated trout filletspackaged under different gas atmospheres and stored al 4°C
9 -r---------------....--.,
Figure 52: ChaDges in psychrotrophic counts of control and inoculated trout filletspackaged under different gas atmospheres and stored al 12°C
-'-ConraI (Ar)
~ Incallllted (Ajr)
--'-ConraI (V8QUft)
~ Inonlleted (Vacuum)___CcnroI (G8a)
..-lma..... (Gas)
862
8
6
5+-----+----+-----+------1o
":'..~
l)7
J
•
•9 __--------------_
8
7
..
-+-ConIraI (Mo)
-e-1noCI1IBI8d (AIr)
-'-ConIraI (V1IClUft)~InX'IIBI8d(V8QUII)
-ll-ConIraI (c;.)
-.-1noCI1IBI8d (c;.)
2520151053+----+----+----+--~--........
o
•Figure 53: Changes in lactic acid bacteria counts ofcontrol and inoculated trout filletspackaged under difTerent gas atmospheres and stored al 4°C
Figure 54: Changes in lactic acid bac:teria counts ofcontral and inoculated trout filletspackaged under difTerent sas atmospheres and stored al 12°C•
9
8
7"':'.~
~6
J5
.-3
0 2 .. 6 8
o.r-
-+-ConIraI (Mo)___1noa1lBl8d (~)
ConIraI (V1IClUft)
~1noCI1IBI8d (V8QUII)
-Jl-ConIraI (Gaa)
~InX'!fIIted(c;.)
•
•
•
118
4.4. ConclusloD
Fresh fish is a highly perishable commodity and its shelf-life is limited by
microbiological spoilage. In this study, trout fillets were inocu1ated with L.
monocytogenes, packaged under three different atmospheres and stored al normal
refrigeration (4°C) and slighdy abusive temperatures (12°C). It bas been shown that L.
monocytogenes grew well in trout (a nutrient rich substrate) with a pH near or above 6,
which is in agreement with other studies done on muscle foods (Glass and Doyle, 1989).
Therefore, L. monocytogenes, if present in fish, could pose a potential health threat to
consumers as it could easily increase to undesirable levels during normal refrigerated
storage and mild temperature abuse conditions. Packaging bad an important effect on the
growth ofL. monocytogenes. White gas packaging had more than an inhibitory effect on
the growth ofL. monocytogenes, it grew weU in air and in vacuum packaged trout al 4°C.
However, counts of L. monocytogenes increased to > 107 CFU/g in a11 packaging
treatments at 12°C. Therefore, moderate temperature abuse offish products contarninated
with L. monocytogenes may significandy enhance the growth of this pathogen. Survïval
and growth ofL. monocytogenes in fish is dependent on atmospheric conditions, storage
lime, temperature and the nature ofthe food product.
•
•
•
119
CllAPl'ERS
CHALLENGE STUDIES WITH CLOSTRIDIUMBDTULINUMTYPE E
PART A: CHALLENGE STUDIES ON MAP OF FREsa moUT FILLETSSTORED AT 4 AND 11°C
S.l. Introduction
A major factor limiting the wider application of modified atmosphere packaging
(MAP) of fresh fish is the concem about the potential growth of Clostridium botulinum.
This concern is justified since (a) the prevalence of C. botulinum spores in ftesh and salt
water fish is very high (Eklund et al., 1984) (h) nonproteolytic C. botulinum type E cm
grow and produce toxin al temperatures as low as 3.3 to 4°C (Smith and Sugiyama, 1988)
and (c) the inhibition of the normal aerobic spoilage bacteria of ftesh tish by reduced~
and increasedC~ levels may retard spoilage and enhance the probability ofC. botulinum
growth during prolonged storage, particularly al mild temperature abuse conditions
(Garcia et al., 1987). Several studies have indicated that C. botulinum can produce toxin
in MAP seafood products (Stier et al., 1981; Post et al., 1985; Rhodehamel et al., 1991
and Reddy et al., 1996). More recendy, Lyver et al. (1998) reported toxin production in
commercially sterile value-added seafood products packaged onder a modified
atmosphere. Therefore, the objective of this study was to determine the public hea1th
safety ofMAP fresh rainbow trout fiIlets in challenge studies with C. botulinum type E.
•
•
•
120
5.2. Materia" and Metllods
5.2.1. Sample preparation
Rainbow trout fiUets (Onchorhynchus mylciss) were obtained from a local seafood
producer, Via-Mer, St-Hyacinthe, Quebec. Fillets were stored on ice and transported to
our laboratory in expanded polystyrene (Styrofoam~ boxes. The fillets were cut into
uniform pieces each weighing -100g prior to inoculation and packaging.
5.2.2. Baeterial strains and inoculation
Four strains of C. botulinum type E: Russ, Gorden, Bennett and 8550 were
obtained from the Microbial Hazards Bureau (Health Protection Branch (HPB), Health
Canada, Ottawa, Canada) fram the culture collection of Dr. J. Austin. Cultures were
grown in tl'ypticase peptone glucose yeast-extract (TPGY) broth at 35°C for 10 days in an
atmosphere of 1()OA, H2, 1(}GAt C(h and 8oo" N2 in an anaerobic chamber (Coy Laboratory
Products Inc., ADn Arbor, Michigan, USA). Spores were harvested in sterile distilled
water, centrifuged al 17t SOO X g for 20 minutes at 4°C and washed three times. The
spores were frozen al -SO°C in gelatin phosphate butTer at pH 6.6 until use. Equal
oumbers of spores of each strain were mixed to form a single suspension of 1.1 X 105
spores per ml. The spore mixture was heat shocked at 60°C for 20 minutes prior to
inoculation of trout fillets. Trout was inoculated in three different locations (each with
30J,tl) 00 the fillet, to give a final inoculum level of 1~ CFU/g. Control trout fillets were
inoculated with a similar volume ofsterile gelatin phosphate buffer at pH 6.6.
5.2.3. Packaging and storage conditions
Control and inoculated trout fiIlets (-1OOg) were packaged individually in
duplieate in 210 X 210 mm high gas banier bags (Om 12 cc/m2/day/atm @ 24°C,
OOAtRH) (Cryovac Sealed Air Corporation, Mississauga, Ontario). Packages were then
•121
subjected to the following three packaging treatDlents: air, vacuum and gas (CÜ2:N2
(85:15». Air packaged samples were sealed using an Impulse heat sealer. Vacuum and
gas packaging of trout fillets was done using a Multivac Chamber type, heat seal
packaging machine (Model 300A). A Smith's proportional gas mixer was used to give
the desired proportions ofC(h and N2 in the package headspace. Samples were stored at
4°C for 56 days and at 12°C for 7 days. AlI trout fillets were monitored for physical,
chemical, microbiological and sensory changes at weeldy intervals (4°C) and daily
intervals (12°C) or until shelf-life was tenninated.
5.%.4. Analyses
Sensory analysis and pH measurements were carried out as descnbed in section
3.2.3.3. and 3.2.3.6. respectively.
• 5.1.4.1• Headspace gas analysis
Packages were anaIyzed for headspace gas composition by withdrawing gas
samples using a 10 cc gas-tight pressure-Lock syringe (Model 790-002, Macon,
Minneapolis, Minnesota, U.S.A.) through silicon sea1s attached to the outside of each
package. Headspace gas composition was analyzed with an Ü2 and CÜ2 analyzers
(Models HS-750 and PG-lOO respectively, Mocon, Minneapolis, Minnesota, U.S.A.).
5.2.4.2. Toxinassay
•
At each sampling time, trout fi1lets were weighed into stomacher bags and twice
the weight ofsterile peptone water was added. The mixture was stomached for 4 minutes
(400 LabBlender A.J. Seeward, London, DK) and the homogenate was centrifuged at 11,
000 rpm for 20 minutes at 4°C (Induction drive centrifuge, Model J2-21M, Beclanan,
Mississauga, Ontario). The clear supematant was filter sterilized (Lot #SLHA02510,
Millipore, Mississauga, ON) and trypsinized to activate toxin prior to mouse toxicity
•
•
•
122
tests. Duplicate mice (20-2Sg) were injected intraperitonea11y with O.SSml sample. Micc
were observed for 3 clays for symptoms of botulism (pincbed waist, Iabored breathing
and/or death). Mice showing severe distress were eutbanized immediately. Two
additional mice were injected if only one mice died. Samples were considered positive
for toxin if 212 or ~4 mice died (Hauscbild et a/., 1975). Control samples were
prepared and injected in a similar manner. To ·contirm the presence of C. botu/inum
toxin, toxin neutralization was carried out on the earliest samples to he considere<!
positive for toxin (Lot #3015-11 trivalent ~,E: type A (7 500 IU), type B (5 500 lU)
and type E (8 500 IU), Pasteur Mérieux Connaught Canada, Toronto, ON).
5.%.5. Statistical analysis
For trout fillets stored at 4°C, a 2 X 3 X 4 factorial design was used tbroughout
this study. Each fish sample (in duplicate) was subjected ta 3 packaging treatments
during 4 test days (7, 14,28 and 56). For trout fillets stored at 12°C, a 2 X 3 X7 factorial
design was used. Each fish sample (in duplicate) was subjected to 3 packaging treatments
during 7 test days (1, 2, 3,4, S, 6 and 7). A Duncan's multiple range test was used for
comparison of the means, utilizing Statistical Analysis System (SAS, 1988). A
probability of less than 0.05 was considered to be significantly differenL
•
•
123
5.3. Results and DlseulSloB
5.3.1. Changes in headspace gas composition
Changes in headspace gas composition of ooly control and inoculated trout fillets
packaged in air or in gas (CCh:N2 (85:15» and stored at 4 and 12°C are shown in Tables
16 and 17. Headspace gas for vacuum packaged samples was not performed due to
limited headspace in the packages and driploss. At 4°C, changes in headspace (h of air
packaged control trout tillets decreased from 20.70At to 1.6% by day 56 while, CCh
increased ta 4S.90A.. For gas packaged (C(h:N2 (84.9:15.1» control trout fillets,
headspace (h remained al <1% while, C(h decreased graduaUy tbrougbout storage to
78.2%. In air packaged inoculated trout fiUets, headspace Û2 decreased to <1% while,
headspace C(h increased ta 43.9OAt by the end of storage. In gas packaged inoculated
trout fillets, headspace 02 remained al <1% throughout storage while headspace CO2
decreased to SO.OOAt probably due to its dissolution into the fish tissue. SimïIar trends
were observed for changes in headspace gas composition for aU packaged trout flUets
stored al 12°C (Table 17).
5.3.2. Sensory evaluation
Changes in the sensory scores for general appearance and odor of trout fillets
packaged under different gas atmospheres and stored al 4 and 12°C are shown in Tables
18 and 19. Trout flUets were regarded as unacceptable when a score of 3.5 on a
subjective scale of7 was reached (Greer, 1993).
5.3.2.1. Changes in general appearance scores
•Changes in sensory general appearailce scores of control and inocu1ated trout
fiUets packaged in air, vacuum or gas (C02:N2 (84.9:15.1» and 5tOred al 4°C are shown
in Table IS. The general appearance scores decreased gradually during storage.
•Table 16: Changes in headspace~ and CÛ2 ofcontrol and inoculated trout fillets
packaged under different gas atmospheres and stored at 4°C
PackaginltreatmeDt
Inoculation Headspace gas composition (%v/v)
•
Inidal Flaal______________......(D--...Y......O) (D.YS6) _
Oa cOa Oa COa
Air 20.7 <1%1 1.6 48.9
CÛ2:N2 (85:15) < 1%1 84.9 < 1%1 78.2
Air + 20.7 < 1%1 < 1%1 43.9
CÛ2:N2 (85: 15) + < 1%1 84.9 < 1%1 80.01 Below the detection limit ofthe apparatus
Table 17: Changes in headspace 02 and CÛ2 ofcontrol and inoculated trout filletspackaged under different gas atmospheres and stored at 12°C
PackagiDl Inocul.tion Headspace lU composition ("_v/v)areatment
Initial FiDai(Day 0) (D.Y7)
Oa cOa Oa cOa
Air 20.7 < 1%1 2.0 31.7
CÛ2:N2 (85:15) < 1%1 85.8 < 1%1 77.4
Air + 20.7 < 1%1 < 1%1 42.7
C02:N2 (85:15) + < 1%1 85.8 < 1%1 76.1• i Below the detection limit ofthe apparatus
•
•
125
Deteriorative changes included discoloratioD, drip loss and changes in fisb texture. For
air and vacuum package<! control trout fillets, shelf-life was terrninated by the end of the
storage period (56 days) while it was still acceptable by day 56 for gas packaged control
trout fillets. Air and gas (C02:N2 (84.9:15.1» packaged inoculated trout fillets had a
shelf-life of -21 days while vacuum packaged inoculated trout fillets still bad acceptable
scores by the end ofstorage (> 56 days).
The color scores ofcontrol and inoculated trout fillets stored al 12°C are shown in
Table 19. For aU control trout fillets, color scores were unacceptable by the end of
stomge. Based on coJor, air packaged inoculated trout fillets had a shelf-Iife -S days
while vacuum and gas packaged inoculated trout filIets al 12°C had a shelf-life of-2 days
respectively.
Sensory texture scores of control and inoculated trout fillets stored al 12°C are
shown in Table 19.. Air, vacuum and gas packaged control trout fillets were aU
unacceptable based on texture scores by the end of storage. Air, vacuum and gas
packaged inoculated trout filIets had a sbelf-life of-6, -7 and -S days resPeCtively based
on texture and packaging treatment were not significandy ditTerent (p<O.OS) ftom one
anotber.
5.3.2.2. Changes in odor scores
•
Changes in sensory odor characteristics of control and inocu1ated trout fillets
packaged onder different atmospheres and stored at 4 and 12°C are shown in Tables 18
and 19. Shelf-life of aU control trout fillets was terminated by the end of storage,
irrespective of storage temPel'8ture. Air packaged, inoclIlated trout fillets stored al 4°C
bad a shelf-life, based on OOor, of -7 days. Odor scores were Dot significantly ditTerent
(P>O.OS) for vacuum and gas (C~:N2 (84.9:1S.1» packaged inoc1llated trout fillets
stored al 4°C. Their shelf-Iife was terminated after -16 days. At 12°C, all inoclliated
trout flUets bad a shelf-life of< 2 days, based on odor scores.
• • •Table 18: Estimated shelf-life (days) based on sensory analysis ofcontrol and inoculated trout flUets packaged under ditTerent gas
atmospheres and stored at 4°C
Plcuglnl trelment
Air
Vacuum
C02:N2 (85:15)
Air
Vacuum
C02:N2 (85:15)
Inoculation
+
+
+
Genenllppelrlncel
<562
<562
> 562
....21
>56
....21
Odorl
<562
< 562
< 562
-7
-16
....16
1 Time (days) to reach a score of3.5 on the hedonic scale of 1 to 7 (7=Extremely desirable, l=Extremely undesirable)
2 Sensory analyses for control trout flUets were perfonned al the bcginning (day 0) and at the end ofthe storage period (day 56)
• • •
Table 19: Estimated shelf-life (days) based on sensory analysis ofcontrol and inoculated trout flUets packaged under difTerent gasatmospheres and stored at 12°C
Pleuglnl freatment
Air
Inoculation Color' - Telture' .. Odor'
<7r-n
<72 <72
Vacuum
CO2:N2 (85:15)
Air
Vacuum
CO;z:N;z (85:15)
+
+
+
<72
<72
-5
-2
-2
<72
<72
....{)
-7
-5
<72
<12
-2
-1
-2
1 Time (days) to reach a score of3.5 on the hedonic scale of 1to 7 (7=Extremcly dcsirable, l=Extremcly undcsirablc);z Sensory analyses for control trout flUets were performed at the beginning (day 0) and at the end ofthe storage period (day 7)
•
•
•
128
5.3.3. Changes in pH values
Changes in pH values for control and inoculated trout fillets packaged under
various conditions and store<! at 4 and 12°C are shown in Figures S5 to 58. ft is evident
from these figures that pH changed very little from its initial value of 6.5-6.6 througbout
storage. In fact, changes in pH values of trout fiUets stored at 4 and 12°C were not
significantly different tbrougbout storage. The pH of gas packaged trout tillets decreased
slightly more than air and vacuum packaged trout filIets throughout storage. This might
he due to the dissolution of CÛ2 in the aqueous phase of the trout tissue and hence, a
reduCtiOD ofpH. However, final pH values remained close to the initial pH values.
5.3.4. Toxin Assay
The results of the toxin assay in trout tillets packaged onder various atmospheres
and stored at 4 and 12°C are summarized in Tables 20 and 21. Toxin was not detected in
any control samples store<! at 4°C (data not shown). Furthermore, toxin was Dot detected
in any of the inoculated trout fillets throughout storage when stored al 4°C even after 56
days. These results are in agreement with Garcia et al. (1987) who studied the risk of
growth and toxin production by C. botulinum types B, E and F in salmon flUets stored
onder modified atmospheres al low and abuse temperatures. They reported that toxin was
not detected in salmon fillets stored al 4°C for up to 60 days. Stier et al. (1981) also
reported that salmon fillets challenged with 104 sporeslg ofC. botulinum type E failed to
produce toxin at 4.4°C under modified atmosphere (C02, 6()O!'o : 02, 25% : N2, 15%) for
57 days. Baker et a/. (1990) also reported tbat botulinal toxin was inhibited in inoculated
salmon after 60 days at 4°C, even thougb C. botulinum type E is capable of growing al
temperatures as low as 3.3°C. These results confirm our observations that the shelf-life of
modified atmosphere packaged fresh trout fiUets can be extended without the risk of C.
hotulinum toxigenesis if temperature is maintained at or below 4°C. However, with
•
•
7...,.....-------------------.,6.8 -1----------------------1
6.2
6o
Figure 55: Changes in pH values of control trout fillets packaged under different gusatmospheres and stored at 4°C
7-r---------------------...,6.8 -I---------------------i
16.6 .f..-.----1i 6.4
6.2
6o 7 14
~
28
EJAiI
.VeaunaGes
• Figure 56: Changes in pH values of inocuJated trout fillets packaged under differentgas atmospheres and stored at 4°C
•7-r--------------------.......,
8.8 +---------------------1
8.2
80- 8
• Figure 57: Changes in pH values of control trout fillets packaged under different gasatmospberes and stored al 12°C
7
6.8
1 6.6
1~ 6.4
6.2
60 1 2 3 ..
o.ra5 6 7
•Figure 58: Changes in pH values of inoculated trout fillets packaged onder differentgas atmospheres and stored at 12°C
• • •
Table 20: rime to toxigenesis in trout flUets packaged under ditTerent gas atmospheres and stored at 4°C
OaYI to tOlln developDlenti-irackaglD! (nocalam level Storagetrea.ent temperature
-(IPOres/I) (C) 0 7 14 28 56
102Air 4 0/2 0/2 0/2 0/2 0/2
Vacuum 102 4 0/2 0/2 0/2 0/2 0/2
Gas 102 4 0/2 0/2 0/2 0/2 0/2
ln duplicate2 Trypsinized extract
• • •
Table 21: Time to toxigenesis in trout flUets packaged under different gas atmospheres and stored at 12°C
Days to tOIla developmenti.2raekagln§ Inoculum level Storagetreatlllent tempenture
-(sporeslg) (C) 0 1 1 3 4 5 6 7
Air 102 12 0/2 0/2 0/2 0/2 ~ 2/2 2/2 2/2
Vacuum Ur 12 0/2 0/2 0/2 0/2 2/2 2/2 2/2 2/2
Gas 102 12 0/2 0/2 0/2 0/2 0/2 • 2/2 2/2 2/2
2In duplicatcTrypsinized extract
•
•
•
133
existing refrigeration equipment and practices, a maximum temperature of 4°C
throughout distribution and sale cannot always he guaranteed.
Toxin was not detected in any control trout filIets stored at 12°C (data not shown).
However, toxin was detected in ail the inoculated samples stored al 12°C. Air and
vacuum pac:kaged inoculated trout flUets were toxic by day 4 while, gas packaged
inoculated trout fillets were toxie by day 5. For ail packaging treatments, spoilage
preceded toxigenesis (Table 21). This study is in agreement with Garcia et al. (1987)
who reported tbat toxigenesis occurred earlier in fish stored under vacuum than under
CO2-enriched atmospheres. Furthennore, it is in agreement with the observations of
Caon et al. (1983 and 1984) who showed that spoilage ofwhole trout, salmon fillets and
cod filIets packaged under gas atmospheres (40-6QOA. CCh) and vacuum and stored at
10GC preceded toxigenesis in challenge studies with non-proteolytic C. botu!inum type B
and E strains (l~ spores/g).
•
•
•
134
S.4. Conclusion
This study bas confirmed the importance of strict temperature control to ensure
the safety of MAP fish if contaminated witb C. botu/inum type E spores. In this study,
MAP fish wouId be regarded as safe if stored at 4°C or less. However, ail fish stored at
12°C were toxic by days 4-5 and spoilage preceded toxigenesis. Nevertheless, Lindsay
(1983) observed the opposite trend i.e., toxigenesis preceded spoilage. Tberefore,
spoilage cannot always be regarded as a reliable indicator of safety ofMAP fish at mild
temperature abuse storage conditioDS.
•
•
•
135
PARTH: CHALLENGE STUDIES ON MAP OF FUsa TROUT FlLLETSSTORED AT lZoC IN AN ENVIRONMENT OF DIFFERENT OXYGENCONCENTRATIONS
S.5. Introduction
In the Jast study, it was demonstrated tbat C. botulinum toxin was produced in air,
vacuum and gas (CÛ2:N2 (85.8:14.2» packaged ftesh trout fillets under mild temperature
abuse (12°C) conditions. To prevent the growth ofpsychrotrophic pathogens, sucb as C.
botulinum type E, strict temperature control is critical. However, surveys have shown
that the temperature of display cases in supermarkets was > 12°C with sorne as high as
17.5°C, while temperatures of borne remgerators ranged ftom 1.7 to 20.2°C (Wyatt and
Guy, 1981). It is evident that temperature aJone cao not be regarded as an adequate
barrier to control the growth of C. botulinum type E in MAP/chilled tish and an
additional banier sbould be included in such Products to ensure their safety. One sucb
barrier may be the addition of oxygen, either direcdy in the gas mixture or indirecdy
througb packaging tish in tilms of high oxygen transmission rates (OTRs). The
objectives of this study were to determine the effects of various Jevels of headspace
oxygen in the gas atmosphere on the growth and toxin production by C. botulinum type E
in fresh trout fillets stored al mild temperature abuse conditions (12°C).
•
••
•
136
5.'. Materials and Methods
5.'.1. Sample preparation and inoculation
Rainbow trout tillets were prepared and inocuJated with 1er sporeslg of C.
botulinum type E as described previously in sections 5.2.1. and 5.2.2.
5.'.2. Packaging and storage conditions
Trout fillets (control and inoculated) were packaged in dupücate in a high gas
banier bags (OTR = 12 cc/m2/day/atm @ 24°C, OOA. RH) (Cryovac Sealed Air
Corporation, Mississauga, Ontario) under various levels of Û2 (O-IOOOA.), balance CÛ2
(Table 22) as described in section 5.2.3. All packaged trout fillets were stored at 12°C for
7 days and analyzed daily.
5.6.3. Analyses
Headspace gas anaIysis, sensory analysis, pH measurements and toxin assay were
canied out as descnbed in sections 5.2.4.1., 3.2.3.3., 3.2.3.6. and 5.2.4.2. respectively.
5.6.4. Statistical aoalysis
A 2 X 6 X 4 factorial design was used throughout this study. Bach fi5h sample (in
duplieate) were subjected 10 6 packaging treatments and stored for 4 test days. A
Duncan'5 multiple range test was used to determine differences between meaus as
described in section 5.2.5.
•
Table 22: Packaging treatments ofcontrol and inoculated trout fillets stored al 12°C
Packagingtreatment
1 3.0
Gaseous cODiposition (~.vlv)
97.0
2 23.2 74.7 2.1
• < 1%13 50.1 49.8
4 73.5 26.5 < 1%1
5 100 < 1%1 < 1%1
6 (Air) 20.7 < 1%1 79.3
Below the detection limit ofthe apparatus
•
•
•
138
5.7 Resula and Discussion
5.7.1. Changes in headspace gas composition
Ali control and inoculated gas packaged trout showed similar trends in changes in
headspace gas composition i.e., a rapid decrease in headspace 02 and a CODStant increase
in headspace CÛ2 (Tables 23-24). At the end of storage, the final headspace Û2 levels
ranged from <1% to 11.3% while headspace CÛ2 levels ranged ftom 35.6 to 98.00A. for
bath control and inoculated trout fillets stored at 12°C (Tables 23-24). The results
confirm previous headspace gas changes in C. botu/inum challenge studies with value
added seafood products (Lyver et al., 1998) and are due to growth of aerobic and
facultative bacteria.
5.7.%. Sensory evaluation
Changes in the sensory scores for color, texture and OOor of trout fillets packaged
with various levels of 02 and stored at 12°C are shown in Tables 25-26. Trout fillets
were regarded as unacceptable when a score of 3.5 on a subjective scale of 7 was reached
(Greer, 1993). Color and texture scores of all packaged inoculated trout fillets were
unacceptable by - day 4 while OOor scores were unacceptable after ooly -2-3 days.
5.7.2.1. Changes in color acceptability scores
•
Changes in sensory color scores of control and inocuIated trout flUets stored al
12°C are shown in Tables 25-26. Inoculated trout fillets packaged under various levels of
headspace Û2 cao be divided ioto 2 groups. In group 1 i.e., fish packaged under lower
levels ofCÛ2 and higher levels ofCh (packaging treatments 4,5 and 6 (air» shelf-life was
•Table %3: Changes in headspace Ch and C(h ofcontrol trout tillets packaged onder
different gas atmospheres and stored at 12°C
Packaging Headspaee 1•• compositioD (%vlv)treatmeDt
Below the detection limit ofthe apparatus
1 3.0 97.0 < 1%1 94.72 23.2 74.7 < 1%1 98.03 50.1 49.8 3.4 92.44 73.5 26.5 5.8 86.95 100 < 1%1 9.0 67.6
6 (Air) 20.7 < 1%1 < 1%1 35.6
_________......I_D...,iti...,·a....I..,a,(D__...ylllllllioll0) Fi_._D_.__1(Day ,, _
<b cOa Oa cOa
•Table %4: Changes in headspace 02 and C02 of inoculated trout tillets packaged
under difJerent gas atmospheres and stored at 12°C
PackagiDg Headspace 1•• compositioD (%v/v)treatmeDt
1 3.0 97.0 < 1%1 95.62 23.2 74.7 < 1%1 95.63 50.1 49.8 11.3 74.24 73.5 26.5 2.7 83.05 100 < 1%1 7.9 68.0
6 (Air) 20.7 < 1%1 < 1%1 43.4
__________I,,;,;,Di;.;,ti;,;,;8-.1(Day Ol --..;Fl.........D..a...1 (Day 6) _
Oa COa 02 COz
•Below the detection limit ofthe apparatus
•140
terminated after - 2-3 days. In group 2 i.e., fish packaged under lower levels of 02 and
higher levels OfCÛ2 (packaging treatments 1,2 and 3) shelf-life was tennioated after -5
6 days. The lower shelf-life observed for trout fillets packaged under higher Ch levels
was probably due to an accelerated oxidation of carotenoid pigments and hence,
discoloration ofthe fish tissue.
Changes in the texture scores ofcontrol and inoculated trout fillets stored al 12°C
are shawn in Tables 25-26. For all control trout fillets, texture shelf-life was <7 days,
with the exception of trout fillets packaged under CCh:(h (26.5:73.5) which was still
acceptable after 7 days. The estimated texture shelf-life of all inoculated trout fillets
ranged from -3 to 4 days for aU packaging treatments.
•
5.7.2.2.
5.7.2.3.
Changes in texture scores
Changes in odor scores
•
Changes in odor scores of control and inoculated trout fillets stored at 12°C are
shown in Tables 25-26. Odor scores decreased dramatically during the fust few days at
12°C. AIl the inoculated trout fillets had an odor shelf-life of-2-3 days for all packaging
conditions with the exception of inoculated trout fillets sas packaged in C(h:02
(49.8:50.1) which had an odor shelf-life of-- 4 days al 12°C (Table 26).
5.7.3. Changes in pH values
Changes in pH value ofcontrol and inoculated trout fillets packaged under various
levels of headspace Û2 and stored al 12°C are shown in Figures 59 and 60. No major
changes in pH values were observed for control trout fillets with pH values remaining
fairly constant (pH 6-7) througbout storage. Two pH patterns were observed for
inoculateel trout fillets. In group 1, pH values for inoculated trout fiUets packaged in
•
•
•
Table 25: Estimated shelf-life (days) based on sensory analysis ofcontrol trout flUetspackaged under ditTerent gas atmospheres and stored at 12°C
PackagiRg Colorl-Z Texture'-! Odorl-ztreatmeDt
1 >7 <7 <7
2 <7 <7 <7
3 <7 <7 <7
4 <7 >7 <7
S <7 <7 <7
6 (Air) >7 <7 <7
Time (days) to reach a score of 3.5 on the hedonic scale of 1 to 7 (7=Extremelydesirable, 1=Extremely undesirable)2 Sensory analyses for control trout filIets were perfonned al the beginning (day 0) and stthe end of the storage period (day 7)
Table 26: Estimated shelf-tife (days) based on sensory analysis ofinoculated troutflUets packaged onder different gas atmospheres and stored al 12°C
Paekaging Colorl Texturel Odorl
treatlnent1 -5 >7 -2
2 -5 -4 -3
3 -6 -4 -4
4 -3 -4 -3
S -2 -3 -26 (Air) -3 -4 -2
Time (days) 10 reach a score of 3.5 on the hedonic scale of 1 10 7 (7=Extremelydesirable, 1=Extremely undesirable)
•1-r-----------------------,
.~1
.G8a2DG8a3DG8a4DG8a5aAr
1o
6.8 +-----------------------t r------.
• Figure 59: Changes in pH values ofcontrol trout fillets packaged onder various levelsofheadspace Û2 and stored at 12°C
1-r-----..-;..---------------,6.8 +-----------------------t ...--........
Figure 60: Changes in pH values of inoculated trout fillets packagcd under variouslevels ofbeadspace Û2 and stored at 12°C
• G8a1.G8a2a~3
D~4
a~5
DAir
653o6
6.2
1 6.6 +---------.----Ji. 6.4
•
•
•
•
143
50, 75, lOOOA. Ch (balance CCh) and in air remained sligbdy higher than the iDitial pH
value of6.46 throughout storage. In group 2, pH values of fillets packaged in 0 and 25%
02 (balance CCh) remained sligbdy lower than the initial pH value throughout storage.
However, ail inoculated trout fillets had pH values ranging ftom pH 6-7 throughout
storage i.e., not significandy different for the various packaging treatments.
5.7.4. Toxin Assay
The results ofthe toxin assayare sumrnarized in Table 27. No C. botulinum toxin
was detccted in any control samples stored at 12°C (data not shown). However, toxin was
detected in ail the inoculated samples stored at 12°C by clay S. The earliest lime to toxin
detection (day 4) was observed ftom trout fillets packaged in ()oA. Ch, 75% Ch (balance
C02) and in air. Other packaged trout fillets were toxic after 5 days. For all packaging
conditions, spoilage preceded toxigenesis as Most products had unacœptable odor scores
after -2 days (Table 26). These results showed that althougb trout was packaged in
various levels of Û2, headspace Û2 can not he regarded as an additional barrler to ensure
the safety of MAP trout. These results confirm the previous studies by Lambert (1991)
who showed toxin production in pork packaged under various levels of headspace
oxygene In addition, the probability of spore germination and toxin production was
higher in pork packaged under (h-enriched atmospheres compared to anaerobic
packaging conditions. ft can be concluded that from these studies, the addition of
headspace Û2 to the gas mixture docs not inhtbit the growth or toxin production of C.
botulinum type E in MAP fish. A more important factor is probably the redox potential
within the product. This potential probably decreases due to the growth of spoilagc
bacteria and the depletion of headspace <h. Therefore, the redox potential witbin the
product can he conducive to the growtb of C. botu/inum type E even though there may be
residual headspace Ch in the package. Previous studies have shown tbat the actual
composition of the gas mixture is less important than storage temperature when the risk
ofbotulism ftom MAP fish products are being investigated (Reddy et a/., 1992). Several
researchers showed that the inclusion ofOz in the gas mixture did not provide more safety
•
•
•
144
than the elevated CÛ2 atmospherc (Stier et a/., 1981; Post et a/., 1985; Garcia et a/.,
1987). Therefore, oxygen in MA gas mixture may otrer a false sense of security
(Lindsay, 1982).
• • •
Table 27: Time to toxigenesis in trout flUets packaged under different gas abnospheres and stored al 12°C
Paekaglng treatment1 Day. to tODR developmenti-!IRoculum level
Ga. %C02 -402 (.poresll> 0 3 4 S 6 7
1 97.0 3.0 JOr- 0/2 0/2 112 2/2 2/2
2 74.7 23.2 102 0/2 0/2 0/2 2/2 2/2
3 49.8 SO.l 102 0/2 0/2 0/2 112 1/2 212
4 26.S 73.5 I~ 0/2 0/2 2/2 2/2 2/2
5 0 100 l<r 0/2 0/2 0/2 212 2/2
Air 0 20.1 102 0/2 0/2 2/2 2/2 2/2
ln duplicate2 Trypsinized extract
•
•
•
146
S.8. CODelulloD
It cao be concluded from this study that packaging of trout under different <h
concentrations did not really influence time of toxigenesis at mild temperature abuse
(12°C) conditions. The inclusion ofCh in the package headspace did not inhibit or delay
the development of C. botulinum type E 10xin compared to previous studies in high gas
barrier films. In fact, aIl inoculated trout fiIIets were toxie by day S Le., similar 10 our
previous studies. Furthermore, the inclusion ofÛ2 in the package headspace did not have
a significant etTect on spoilage cbaracteristics. This is in agreement with previous studies
done on park (Lambert et a/., 1991). These results also agree with the previous
observations of Lindsay (1982) who reported that the addition of headspace oxygen does
not ensure the safety ofMAP fish at mild temperature abuse COnditiODS•
•
•
•
147
PARTe: CHALLENGE STUDIES ON MAP FREsa TROVT FO.LETSSTORED AT 12°C IN FILMS OF DIFFERENT OXYGEN TRANSMISSIONRATE
S.9. Introduction
ln the previous study, it was shown that C. botu/inum toxin was detected in ail
fresh trout fillets packaged onder ditTerent oxygen enriched atmospheres al mild
temperature abuse conditions (12°C). Oxygen was added direcdy to the package
headspace and the products were packaged in high barrier films i.e., films ~th a low
OTR. In tbat study, Û2 was depleted to < 4% while CCh increased to -800A. in ail
packaged tish. An indirect method of adding Û2 to the package headspace and
maintaining higher levels throughout storage may be achieved by packaging in films of
higher OTR i.e., films of lower barrier. Therefore, the objectives of this study were to
determine the effect of films of various oxygen transmission rates (OTR) on the growth
and toxin production by C. botu/inum type E in fresh trout fillets stored al mild
temperature abuse conditions (12°C).
•148
5.10. MAterials ABd Methodl
5.10.1. Sample preparation and inoculation
Rainbow trout fillets were prepared and inoculated with 1cf sporeslg of c.botu/inum type E as descnDed previously in section 5.2.1. and 5.2.2..
5.10.2. Packaging and storage conditions
•
Trout fiUets were packaged in films of various oxygen transmission rates (Table
28). AlI films were obtained from Cryovac Sealed Air Corporation (Mississauga,
Ontario). Control and inoculated trout fillets were packaged in duplicate under the
following treatments: air, vacuum and gas (84.3% C02/15.70A. N2) as descn"bed in section
5.2.3. Fillets were stored al mild temperature abuse conditions (12°C) for 7 days and
monitored after 3,4,5,6 and 7 days.
5.10.2.1. Film permeability: Oxygen transmission rate (OTR)
•
For the measurement of the oxygen transmission rate (OTR), a standard method
(ASTMD 3985-81) was used, with air as the permeant gas. The OTR of the film was
measured by cutting a film ofuniform size and placing it direcdy in the test ceU. A 25J.111l
thick piece ofMylar (polyester) was used as a standard. AlI films were conditioned al the
test temperature and at OOA. relative humidity for 3 hours prior to testing. For the OTR
test, an Oxtran 2120 Master (Macon, Minnesota, USA) was used with an Oxtran 2120
software package to monitor aU the phases of testing, including entering test conditions
(parameters), monitoring tests, and printing reports. Once the parameters were set, the
computer eontroUed the components, gathered and logged data, printed aU the data in the
form of tables and bar charts. Both eeUs of the Oxtran 2120 are divided into two
chambers separated by the test film. Air was passed through the upper chamber and
humidified carrier gas (98% Nitrogen and 2% Hydrogen) passed tbrough the boUOm
•149
ehamber to sweep the permeant gas to the sensor. A scanning automatic valve sent the
sample gas, oxygen, 10 a specifie coulometrie sensor detector. The detector gave a
current output directly proportional to the rate ofoxygen anival at the sensor. Therefore,
the oxygen flux across the film was dynamically measured, the OTR expressed as
cc/m2/day at 001'0 RH.
5.11.3. Analyses
Headspace gas analysis, sensory analysis, pH measurements and toxin assay were
carried out as descnbed in section 5.2.4.1., 3.2.3.3., 3.2.3.6. and 5.2.4.2. respectively.
5.10.4. Statistical analysis
•
•
A factorial design of 2 X 3 X 3 X 5 was used througbout this study. Each fisb
sample (in duplicate) was subjected to 3 films of different oxygen transmission rate and
packaged onder 3 treatments during 5 test days. A Duncan's multiple range test was used
for eomparison of the meaDS as descn"bed in section 5.2.5.
•
Table 28: Oxygen transmission rate (OTR) of tilms used to package control andinoculated trout flUets stored at 12°C
•
•
PackagiDg films
A
8
C
OxygeD Transmission Rate
(eclm2/day/atm @ 24·C, O%RH)
4,370
4,920
10,040
•151
S.ll. Results and DiseuaiOD
S.11.1. Changes in headspace gas composition
•
Changes in headspace gas composition of control and inoculated trout fillets
packaged in air or in a CÛ2:N2 (84.3:15.7) gas mixture in films of different OTRs and
stored at 12°C are shown in Table 29. In all control air packaged trout fillets, simüar
trends were observed, i.e., a decrease in headspace Û2 to between 1.9-6.2% after 7 days
and an increase in headspace C02 to between 4.2...7.8% depending on the on of the
film. The opposite trend was observed for ail control gas packaged trout fillets i.e.,
headspace Û2 increased to -7% throughout storage while headspace CÛ2 decreased
significandy ftom 84.3% to between 2.6-6% after 7 days depending on the OTRs of the
packaging films.
Similar b'ends were observed for all inoculated trout fillets. In air packaged
inoculated trout fillets Ch levels ranged from 1.3...7% while CÛ2 levels ranged from 7.1 ...
11.00,4 in films A,B and C respectivelyafter 7 days. For inoculated gas packaged trout
fillets, final headspace Ü2 ranged from 2.7-4.4% while CÛ2 levels decreased steadily
throughout storage to between 5.6-12.2% i.e., a decrease in -7Q...8001'a. This may be
attributed to the higher on of this film alloWÏDg more gaseous exchange with the
outside environment. A1so, a film C02 transmission rate is generally -3-4 times its OTR
rate.
5.11.2. Sensory evaluation
•Changes in the sensory scores for oolor, texture and odor of trout fillets packaged
onder various atmospheres in films of different OTRs and stored al 12°C are shown in
Table 30. Trout filIets were regarded as unacceptable when a score of 3.5 on a subjective
scale of7 was reached (Greer, 1993).
• • •Table 29: Changes in headspace O2 and CO2 levels ofcontrol and inoculated trout flUets packaged onder different gas
atmospheres in films ofdifferent OTa and stored at 12°C
OTR Film Packaging Inoculation Head.pace ga. composition ("'.v/v)treatment
(wfl},.y,••• Initiai (Day 0) Final (Day 7)EGI%RH)
OZ COz Oz COzAir . 20.7 < 1%' 4.0 4.3
4,370 ACO2:N2 (84.3:15.7) - < 1%1 84.3 7.1 2.6
Air . 20.7 < 1%1 1.9 7.84,920 B
COZ:N2 (84.3:15.7) - < 1%1 84.3 7.2 6.0
Air - 20.7 <1%1 6.2 4.210,040 C
CO2:Nz(84.3:1S. 7) - < 1%1 84.3 7.4 3.4
Air + 20.7 < 1%1 7.9 8.44,370 A
COz:Nz(84.3:15.7) + < 1%1 84.3 2.7 6.6
Air + 20.7 <1%' 1.3 11.04,920 B
COz:Nz (84.3: 1S.7) + <1%' 84.3 3.0 12.2
Air + 20.7 < 1%1 4.5 7.110,040 C
CO2:N2 (84.3:15.7) + < 1%' 84.3 4.4 5.6
1 Below the detection limit ofthe apparatus
Changes in sensory color scores ofcontrol and inoculated trout fillets packaged in
air, vacuum or gas (C(h:N2 (84.3:15.7) in tiIms ofdifferent OTRs and stored at 12°C are
shown in Table 30 respectively. For ail control packaging treatments, the shelf-Iife, based
on color, was tenninated by the end of storage, irrespective of film OTR. A similar
decrease in color scores was observed for inoculated trout filIets. Air packaged
inoculated trout flUets had an estimated color shelf-üfe of-4, -3 and -2 days in films A,
B and C respectively. Vacuum packaged inoculated trout flUets had an estimated color
shelf-Iife of >7, -5 and -3 days in film ~ 8 and C while gas packaged inoculated trout
fillets had a color shelf-üfe of -3, -4 and -3 days in films A, B and C respectively.
Similar trends were observed for trout flUets packaged with higher Û2 levels. Il would
appear that packaging in films of higher OTR increased the rate of pigment oxidation in
trout filIets.
•
•
5.11.2.1.
5.11.2.2.
Changes in color acceptability scores
Changes in texture scores
153
Changes in texture scores are shown in Table 30. The film OTR influenced the
texture of fish i.e., the > the OTR of the film, the lower the texture scores. The observed
shelf-life, based on texture, for an inoculated trout filIets ranged from -2-4 days. Trout
flUets packaged in film C had the shortest texture shelf-life and were rejected after -2-3
days based on texture scores.
5.11.2.3. Changes in odor scores
•AIl inoculated trout fiIIets were rejected, on the basis of odor scores, after -2-3
days at 12°C, irrespective ofpackaging treatment and packaging films (Table 30). Tbese
odor scores were similar to those of trout filIets packaged in higb Ü2 levels. Again,
higher OTRs appeared to enhance the growth ofspoilage~ resulting in strong off
odors.
• • •Table 30: Estimated shelf-life (days) based on sensory analysis and pH analysis ofcontrol and inoculated trout flUets packaged
under ditTerent gas abnospheres in fllms ofditTerent OrR and stored at 12°C
on Film rackaciDS tre.ment Inoculation SenlOry .bel'-li'e l!!!l!)1 pH values(ce/rIt'da,'.ha @ Color Tellure Odor Final (Day 7)
U-c.I%RH)
Air < 72- < 72 < 72 6.95
4,370 A Vacuum - < 72 < 72 < 71 7.00CO2:N2 (84.3:15.7) < 72 < ,2 < 72 6.92
4,920
10,040
4,370
4,920
B
C
A
B
AirVacuum
C(h:N2 (84.3:15.7)
AirVacuum
CO2:N2 (84.3: 1S.7)
AirVacuum
C02:N2 (84.3:15.7)
AirVacuum
CO2:N2 (84.3:15.7)
+
+
<72
<72
<72
<72
<72
<i
-4>7-3
....3-s-4
<72
<72
< 72
< 72
<72
< 72
-4....()
-4
-3-4-4
<72
<72
<72
< 72
<72
<72
-2....3....3
-2-3-2
6.917.036.88
7.086.837.22
6.686.566.76
6.786.526.75
~ ~ ~ ~ ~M
10,040 C Vacuum + ....3 3 -2 6.72C(h:N2 (84.3:15.7) -3 3 ....2 6.82
1 Time (ciays) to re8Ch a score of3.S on the hcdonic scale of 1to 7 (7=Extremely desirablc, I=Extremely undesirable)2 Sensory analyses for control trout flUets were performed at the beginning (day 0) and at the end ofthe storage period (day 7)
• 5.11.3. Changes in pH values
ISS
Changes in the pH values of control and inoculated trout fillets packaged under
various 88S atmospheres in films ofdifTerent OTRs and stored al 12°C are shown in Table
30. The initial pH value for trout tiUets was of 6.44. These results are consistent with
previous studies which showed that pH remained fairly constant (pH 6.4-6.9) throughout
stomge in both control and inoculated trout fillets.
5.11.4. Toxinassay
•
•
Time to toxin production in ail inoculated trout packaged onder difTerent
atmospheres in films of different OTR are summarized in Table 31. Toxin was not
detected in any control samples stored at 12°C (data not shown). However, toxin was
detected in aIl inoculated samples store<! at 12°C by day 5 which is consistent with
previous studies. The eartiest lime until toxin detection (4 days) was observed for air
packaged trout fiUets in film A and for gas packaged (C(h:N2 (84.3:15.7» trout fiIlets in
film B and C. Toxin was detected in ail the other samples by clay 5. For ail packaging
conditions, spoilage again preceded toxigenesis (Tables 30). These results are in
agreement with our previous studies with trout fiUets packaged with various levels of
headspace 02. Our studies also agree with previous observation by Post el al. (1985) who
showed that the packaging film permeability did Dot inhibit toxin production by C.
botulinum type E. In their studies with cod, whiting and tlounder fillets, toxin production
occurred within 10-12 days al 8°C and witbin 8-11 days al 12°C. In ail cases, spoilage
preceded toxigenesis. However, the authors did not report the OTR of the films used in
their study or the headspace gas composition of the packaged product al the time of
toxigenesis. Garren el al. (1995) reported that toxin was produced by day 6 in rainbow
trout inoculated with C. botulinum vacuum packaged in a high gas banier film and stored
at lOOC. This study a1so confirms the observations ofBaker and Genigeorgis (1990) who
reported that storage of fish under abusive time/tempcraturc conditions may lead to
toxigenesis, regardJess of packaging conditions. Therefore, temperature had a greater
• • •
Table 31: Time to toxigenesis in trout flllets challenged with C. botulinum type E(102 cellslg) packaged onder ditTerent gasatmospheres in films ofdifferent OTR and slored al 12°C
OTR Film Paekagln~
treatmentInoealaUon Day. to tODn developmentl
-2
(eeJm2/day/atm 0 3 4 S 6 7@U·C,O%__RH> _
Air + 0/2 0/2 112 2/2 2/2 2/24,370 A Vacuum + 0/2 0/2 0/2 112 0/2 2/2
C02:N2 (84.3: 15.7) + 0/2 0/2 0/2 2/2 2/2 2/2
4,920 B
10,040 C
Air + 0/2 0/2 0/2 1/2 0/2 2/2Vacuum + 0/2 0/2 0/2 1/2 1/2 2/2
C02:N2 (84.3: 15.7) + 0/2 0/2 2/2 2/2 2/2 2/2
Air + 0/2 0/2 0/2 1/2 1/2 2/2Vacuum + 0/2 0/2 0/1 2/2 1/2 2/2
C02:N2 (84.3: IS.7) + 0/2 0/2 112 2/2 2/2 2/2
1 In duplicate2 Trypsinized extract
•
•
•
157
impact on the time to toxigenesis and safety ofMAP fresh rainbow trout tillets tban either
headspace Û2 or film OTR, empbasizing the need for strict temperature control al all
stages of the distribution chain. The addition ofÜ2 directly to the package headspace or
indirecdy through packaging in films of OTRs > 4000 cc1m2/day/atm @ 24°C, OOA. RH
had little eifect on tinte to toxigenesis or on enhancing spoilage.
•
•
•
IS8
5.12. CORel_Ion
The growth of: aDd toxin production by C. botu!inum, in modified atmosphere
packaged trout flUets in films of difTerent OTR, ranging from 4, 370 to 10, 040
cclm2/day/atm @ 24°C, OOJORH and stored at 12°C occurred irrespective of the packaging
films used. It can he concluded that the addition of headspace 02, either directly 10 a
package or indirectly through the use. of films of higb OTR had little influence on lime to
toxigenesis. Clearly, the effect of storage temperature bas a greater influence on spoÜ8ge
and lime to toxigenesis than does headspace gas composition or OTR of the packaging
films. Therefore, additional baniers, other than headspace ~, should be recommended 10
enhance the safety ofMAP fish particularly at mild temperature abuse conditions.
•
•
•
159
CBAPTER6
CHALLENGE STUDIES ON COLD AND HOT SMOKED TROUY FlLLETS
6.1. IntroductioD
Trout is a highly perishable food produet with a limited shelf-life. One method of
extending the shelf-life of ftesh trout is through smoking. Fish may be ~~cold-smoked",
i.e., processed at low temperatures so the produet does not show signs ofheat coagulation
of the protein, or ~~ot-smoked", i.e., processed at high temperatures for a sufficient time
to obtain heat coagulation of fish protein. The process of smoking fish imparts a degree
of microbiological stability to the product, but this stability is a fimetion of several
factors, including the salt level reached after brining, the amount 0' heat applied, the
inhIbitory action of sorne smoke components as weil as the dehydrating effcct of the
smoking process (Dodds et al., 1992). Nowadays, smoked fish is processed with Jess
smoke and salt and more moisture, thus providing an ideal substrate for the growth of C.
botulinum compared to the traditional heavily smoked, salted and dried produets (Cano
and Taylor, 1979). Furthermore, since they are minimaIJy processed, smoked products
are regarded as hazardous and the presence of Clostridium botu/inum type E, and its
toxin, bas been demonstrated in smoked fish (Hayes et al., 1969).
From a public health point of view, ail smoked fish should be assumed ta be
camer ofC. botulinum (Huss et al., 1974). In the United Kingdom 5 out ofa total of646
vacuum packed smoked fish products were found to contain C. botulinum type E (Hobbs
et al., 1965; Caon et al., 1966). In Denmark, an incidence rate of 1.68% of C. botulinum
type E W8S reported in smoked salmon purchased from retail outlets (Nielsen and
Pedersen, 1967). Cano et al. (1980) reported toxin production by C. botulinum type E in
vacuum packed cold smoked salmon after 13 days al 10oe •
•
•
•
160
The prevention of botulism from smoked fish depends on (i) the inhibition of
gennination and outgrowth of the spores tbat may he present in the smoked product and
(ü) proper control at temperature <4°C (Christiansen et al., 1968). To ensure the safety of
MAP smoked tish products, recommendatioDS bave been made by Health Canada to
package these products in a film with an OTR > 2 000 cc/m2/day/atm @ 24°C, OOA.RH and
store at S 4°C for less than 14 days. However, this recommendation is not empirical1y
based.
The objectives of this study were to monitor the physical, chemical,
microbiological and sensorial changes in control and inoculated studies with C.
botulinum type E in vacuum packaged cold and hot smoked rainbow trout tillets in films
ofditTerent OTRs ranging ftom 12 to 10,040 cc/m2/day/atm @ 24°C, OOA.RH) and stored
at 4, 8 and 12°C.
•
•
•
161
6.2. Materi. and Metllodl
6.2.1. Sample preparation
Cold and hot smoked rainbow trout fiUets were obtaincd commercially ftom a
local fish processor (Levitts" Montreal, Quebec). Fillets were immediately stored on ice
and transported to our Iaboratory in expanded polystyrene (Styrofoam~ boxes. The
fillets were than cut into uniform pieces each weigbing -100g.
6.2.2. Bacterial strains and inoculation
AlI trout fiUets were inoculated with l~ sporeslg of C. botulinum type E as
descnbed previously in section 5.2.2. Control trout fillets were a1so inoculated with a
similar volume ofsterile gelatin phosphate bufJer al pH 6.6.
6.2.3. Packaging and storage conditions
Trout fillets were vacuum packaged in films of difJerent oxygen transmission rate
as shown in Table 32. Ali films were obtained ftom Cryovac Sealed Air Corporation
(Mississauga, Ontario) with the exception of film B which was obtained ftom Levitts
(Montreal, Quebec) and is used commercially to package smoked trout. AlI control and
inoculated trout filIets were vacuum packaged in duplicate, as described in section 5.2.3.
and stored al 4, 8 and 12°C for 28 days and monitored at weekly intervals.
6.2.4. Analyses
Sensory anaIysis, pH measurement and toxin assay were carried out as described
in section 3.2.3.3.,3.2.3.6. and 5.2.4.2. respectively.
• 6.2.4.1. Water setivity detennination
162
The 8w was detennined using a previously calibrated Aqua Lab water 8Ctivity
Meler (Model CX3, Decagon Devices InC., Pullman, WA, USA).
6.2.4.2. Salt concentration
•
•
Salt concentration was determined by the AOAC method by Bio-Lalonde
Laboratories, Pointe-Claire, Quebec. AlI salt concentrations were expressed as %w/w of
aqueous phase.
6.2.5. Statistical anaIysis
Two 2 X 4 X 4 fsetorial designs, for cold and bot smoked trout filIets, were used
throughout this study. Each fish sample (in duplicate) was packaged in 4 films of
different oxygen transmission rate and analyzed during 4 test days. A Duncan's multiple
range test was used for comparison ofthe means as described in section 5.2.5..
•
Table 32: Oxygen transmission rate of films used for vacuum packaged control andinoculated cold and hot smoked trout fillets stored at 4, 8 and 12°C
PackagiDg rdms O~geDTransmission Rate(cclm Iday/atm @ 24·C,O%RH)
A 12
• B 2,950
C 4,920
D 10,040
•
•164
6.3. Resulta and DiseuaiOD
6.3.1. Sensory evaluation
Changes in the sensory scores for color, texture and odor of vacuum packaged
cold and hot smoked trout fillets in films of different OTRs and stored al 4, 8 and 12°C
are shown in Tables 33...35. Trout fillets were regarded as unacceptable when a score of
3.5 on a subjective scale of7 was reached (Greer, 1993).
6.3.1.1. Changes in rolor acceptability scores
•
•
Changes in sensory color scores of control and inoculated smoked trout tillets
vacuum packaged in films of different OTR and stored al 4, 8 and 12°C are shown in
Tables 33-35 respectively. The sensory color shelf...life was terminaled for Most control
vacuum packaged cold and hot smoked trout fillets stored al 4, 8 and 12°C by the end of
storage with the exception ofvacuum packaged cold smoked trout fillets in film B which
had acceptable color scores after 28 days. The estimated color shelf-life was longer when
smoked trout fillets were stored al 4°C than at 8 or 12°C.
At 4°C, color shelf-Iife was estimated to be ~ 28 days for most vacuum packaged
inoculated cold and hot smoked trout fillets with the exception of inoculated cold smoked
trout filIets vacuum packaged in film A or C and inoculated hot smoked trout fillets
vacuum packaged in film D. Their color shelf-life was estimated to he -20, -20 and -18
days respectively (Table 33).
At 8°C, the longest color shelf-life (-27 days) was observed for inoculated hot
smoked vacuum packaged trout fillets in film A while the shortest color shelf-life (-12
days) was noted for inoculated cold smoked fillets packaged in tbis film (Table 34).
These results appear contradictory since film A resulted in better color for hot smoked
trout and a paler color for cold smoked trout. This variability in color
• • •Table 33: Estimated shelf-life (daya) based on sensory anaIyais and pH analysis ofcontrol and inoculated smoked trout flUets
vacuum packaged in films ofdifferent oxygen transmission rate and stored at 4°C
FUm' Smoldng type Inoculadon SeDlOry .hel'-U'e (day.)2 pH value
Cotor Testure Odor Final (Day 2aLA Cold .. < 283 >283 < 283 6.2B Cold - >283 >283 < 283 6.1C Cold - < 283 < 283 < 283 6.0D Cold - < 283 < 283 < 283 6.2
A Cold + ....20 ....24 >28 6.1B Cold + >28 >28 >28 6.1C Cold + -20 ....24 -22 6.20 Cold + >28 ....16 ....18 6.3
A Hot - <283 >283 >283 6.4
B Hot - <283 >283 <283 6.3C Hot - <283 >283 <283 6.4D Hot - <283 >283 <283 6.4
A Hot + >28 >28 >28 6.6B Hot + ....28 >28 >28 6.SC Hot + >28 >28 >28 6.3D Hot + ....18 >28 >28 6.S
i Film A (OTR=12 cc/ml/day/atm @24°C, OOt'oRH); film B (OTR=2, 950 cclm1iday/atm @24°C, OOt'oRH); film C (OTR=4, 920cc/m2/day/atm@24°C,OOA,RH)and film D (OTR=10, 040 cc/m2/day/atm@24°C,OOA,RH)2 Time (days) to reach a score of3.5 on the hedonic scale of 1 to 7 (7=Extrcmely desirable, l=Extrcmely undesirable)3 Seosory analyses for control trout flUets were perfonned at the beginning (day 0) and at the end ofthe storage period (day 28)
• • •Table 34: Estimated shelf-life (days) based on sensory analysis and pH anaIysis ofcontrol and inoculated smoked trout flUets
vacuum packaged in films ofditTerent oxygen transmission rate and stored at 8°C
FIIDlI Smoking type Inoculation SenlOry theil-ille (dayt)z pH value
Coler Tellure Oder Final (Day 28)_A CoId - < 283 < 283 < 283 6.0B Cold - < 283 < 283 < 283 S.9C Cold - < 283 < 283 < 283 6.1D Cold - < 283 < 283 <283 6.2
A Cold + -12 -14 -14 6.3B Cold + -19 -28 -13 6.2C Cold + -13 -21 -16 6.4D Cold + -18 -14 -6 6.S
A Hot - <283 >283 >283 6.4B Hot - <283 >283 >283 6.3C Hot - <283 >283 <283 6.0D Hot - <283 <283 <283 6.2
A Hot + -27 >28 >28 6.SB Hot + -21 >28 >28 6.4C Hot + -18 >28 -23 6.4D Hot + -16 >28 -28 6.4
1 Film A (OTR=12 cclm2/day/atm @24°C, OOiORH); film B (OTR=2, 950 cclm2/day/atm @24°C, OOiORH); film C (OTR=4, 920cc/m2/day/atm @24°C, OOIORH) and film D(01R=10, 040 cc/m2/day/atm @24°C, OOAtRH)2 rime (days) to reach a score of15 on the hedonic scale of 1to 7(7=Extremely desirable, I=Extremely undesirable)3 Sensory analyses for control trout flUets were perfonned at the beginning (day 0) and at the end of the storage period (day 28)
• • •Table 35: Estimated shelf-life (days) based on sensory analysis and pH analysis ofcontrol and inoculated smoked trout fiUets
vacuum packaged in films ofditTerent oxygen transmission rate and stored at 12°C
FIlm' Smoking type Inoculation SenlOry tbelf-Ufe (daYI)z pH value
Color Telture Odor Final (Day 2aLA Cold - < 283 < 283 < 283 6.1B Cold • < 283 < 283 < 283 5.9C Cold • < 283 < 283 < 283 6.1D Cold - < 283 < 283 < 283 6.3
A Cold + -II -12 -II 6.4B Cold + -17 -18 -12 6.SC Cold + -12 -12 -Il 6.5D Cold + -10 -14 -6 6.5
A Hot . <283 <283 >283 5.8B Hot - <283 >283 <283 6.1C Hot - <283 >283 <283 6.0D Hot . <283 <283 <283 5.9
A Hot + -19 >28 -24 6.3B Hot + -15 >28 -21 6.2C Hot + -16 >28 -16 6.3D Hot + -10 -28 -14 6.2
, Film A (01R=12 cc/m'l/day/atm @24°C, OOA»RH); film B (OTR=2, 950 cc/m2/day/atrn @24°C, OOARH); film C (01R=4, 920cclm2/day/atm@24°C, OOARH) and film D(OTR=10, 040 cc/m2/day/atm@24°C,OOI'oRH)2 Time (days) 10 reach a score of3.5 on the hedonic scale of 1to 7 (7=Extremely desirable, I=Extremely undesirable)3 Sensory analyses for control trout flllets were perfonned at the beginning (day 0) and al the end ofthe stomge period (day 28)
e·
•
168
retentionldiscoloration for trout may not be solely due to packaging film but aIso to the
greater stability of pigments in hot smoked products. The estimated color shelf-Iife of
inoculated cold smoked vacuum packaged trout fiUets in film B, C and D was of-19, -13
and -18 days respectively compared to -21, -18 and -16 days for inoculated hot smoked
vacuum trout fillets packaged in similar tiIms (Table 34).
At 12°C, the longest color shelf-life (-19 days) was observed from inoculated hot
smoked vacuum packaged trout tillets in film A while the shortest shelf-Iife (-10 days)
was recorded for inoculated hot smoked vacuum. packaged trout fillets in film D (Table
35). The higher OTR of film D may result in a more rapid oxidation of the carotenoid
pigments and hence, discoloration of the product Inoculated cold smoked trout tillets
vacuum packaged in film B had an estimated color shelf-life of-17 days i.e., significandy
different (P<O.OS) from inoculated cold smoked vacuum packaged trout tillets in film A,
C or D which had an estimated color shelf-life of -11, -12 and -10 clays respectively.
Vacuum packaged inoculated hot smoked trout fillets had an estimated coJor shelf-life of
-15 and -16 days respectively in films B and C (Table 35).
6.3.1.2. Changes in texture scores
•
ln general, the texture of hot smoked trout fillets was more acceptable than cold
smoked trout throughout storage (Tables 33-35). This may be attnbuted to the higher salt
concentration (2.1% versus 1.7%) and lower 8w (0.978 versus 0.985) of hot smoked trout
fiUets. This may have slowed down microbial spoilage and hence, proteolysis of the fish
muscle. However, only two lots ofcontrol cold smoked trout fillets packaged in films of
high OTRs Le., C and D had unacceptable color scores «3.5) before 28 days. AIl texture
scores decreased as storage tinte increased, however, even after 28 clays at 12°C, the
texture ofcontrol hot smoked vacuum. packaged trout fillets was still acceptable.
At 4°C, the texture shelf-life of vacuum packaged inoculated cold smoked Irout
fillets was of-24, -24 and -16 days respcctively in films ~ C and D. Vacuum. packaged
•169
inoculated cold smoked trout flUets in film B had a shelf-Iife of >28 days as did ail
vacuum packaged inoculated hot smoked trout filIets (Table 33). A similar pattern was
observed for aU inoculated smoked vacuum packaged trout fillets stored al 8 and 12°C.
The estimated texture shelf-life of inoculated cold smoked vacuum packaged trout fillets
was -14, -21 and -14 days respectively in film A, C and D at SoC while al 12°C, the
estimated texture shelf-life was -12, -18, -12 and -14 days for inoculated cold smoked
vacuum packaged trout tillets in film A, B, C and D respe<:tively (Tables 34-35). Even at
mild temperature abuse conditions (12°C), the texture of inoculated hot smoked trout
fillets remained acceptable after 28 days, irrespective ofOTR ofthe packaging films.
6.3.1.3. Changes in odor scores
•
•
Changes in sensory odor scores are shown in Tables 33-35 respectively. Again,
sensory scores decrease as storage temperature increased for all cold and hot smoked trout
fillets.
At 4°C, the odor shelf-life for Most inoculated cold and hot smoked vacuum
packaged trout fillets was >28 days, with the exception of inoculated cold smoked
vacuum packaged trout tillets in film C and D which had an odor shelf-life of -22 and
-18 days respectively (Table 33). At 8°C, the odor shelf-life of inoculated cold smoked
vacuum packaged trout fillets was consistendy less tban inoculated hot smoked trout
fillets. Cold smoked trout fillets had a salt content of 1.7% (w/w) and an 8w of 0.985
while hot smoked trout fillets had a higher salt content 2.1% (w/w) but a lower 8w of
0.978. These differences MaY have enhanced spoüage in the cold smoked product
resulting in more off-odors and shorter shelf-life. The estimated odor shelf-life of
inoculated cold smoked vacuum packaged trout flUets was -14, -13, --16 and -6 days in
films A, B, C and D respectively. This compared 10 an odor shelf-Iife of inocoJateel hot
smoked vacuum packaged trout fillets of -23 to > 28 days (Table 34). At 12°C, the odor
shelf-üfe of inoculated cold smoked vacuum packaged trout fiUets ranged ftom -11-12
days in films A, B and C respectively. These treatments had significandy different
•
•
•
170
(p<O.05) odor scores than inoculated cold smoked vacuum packaged trout fillets in film
o which had an estimated odor shelf..life of -6 days. The estimated odor sh"lf-Iife for
inoculated hot smoked vacuum packaged trout filIets in films~ B, C and 0 ranged ftom
-14-24 days respectively (Table 35).
6.3.2. Changes in pH values
Changes in pH values of control and inoculated smoked trout fillets vacuum
packaged in films ofdifferent OTRs and stored at 4, 8 and 12°C are shown in Tables 33
35. The initial pH values for cold smoked trout flUets was of 6..2 and hot smoked trout
flllets was of6.S. The pH changes in both control cold and hot smoked vacuum packaged
trout fillets were not significandy ditTerent (P>O.05) and remained fairly constant
throughout storage. Simîlar trends were observed for inoculated cold and hot smoked
trout fillets. Inoculated cold smoked vacuum packaged trout fillets in film D had
consistently higher pH values compared ta vacuum packaged inoculated cold smoked
trout flUets in films A, B or C. A similar pattern was observed for inoculated hot smoked
trout flUets vacuum packaged in film A which had consistendy higher pH values than hot
smoked trout flUets vacuum packaged in film B, C or D. While the pH of both cold and
hot smoked trout flUets remained fairly constant at 4°C, it decreased slightly in bath cold
and hot smoked trout flUets at higher temperatures (8 and 12°C). This may be attributed
to greater microbial spoilage at these temPeratures. However, film OTR did not appear to
influence pH changes and no significant differences were observed for pH changes in any
packaging treatment.
6.3.3. Toxin assay
The results of toxin assay for both cold and hot smoked trout flUets inoclllatecf
with 1er sPOreslg ofC. botulinum type E are summarized in Tables 36-37. Toxin W8S not
deteeted in any control samples stored st 4, 8 or 12°C (data not shown). None of the
inoculated cold and hot smoked trout fillets at 4°C produced toxin throughout the 28 days
• • •
Table 36: Time to toxigenesis in cold smoked trout flUets vacuum packaged in films ofdifferent OTR and stored al 4, 8 and 12°C
Filml Inoculum Storage Day. to tOllgeneslsl :]
level temmperatDre_____(Iporeslc) cg 0 7 14 11 18
A 102 4 0/2 0/2 0/2 0/2 0/2B 1fil 4 0/2 0/2 0/2 0/2 0/2C 102 4 0/2 0/2 0/2 0/2 0/2D lOZ 4 0/2 0/2 0/2 0/2 0/2
ABCD
urur1filur
8888
0/20/20/20/2
0/20/20/20/2
0/20/20/20/2
0/20/20120/2
0/20/2112112
A ur 12 0/2 0/2 1/2 2/2 1/28 102 12 0/2 0/2 1/2 2/2 2/2C ur 12 0/2 0/2 112 2/2 2/2D ur 12 0/2 0/2 1/2 2/2 212
1 Film A(OTR=12 ecIm2/day/atm @24°C, OOA.RH); Film B(OTR=2 950 cc/m2/day/atm @ 24°C, OOA.RH); Film C (OTR=4 920cclm2/day/atm @ 24°C, OO.4RH); Film 0 (OTR=IO 040 cc/m2/day/atm @24°C, OOA.RH)2 ln duplicatel Trypsinized extract
• • •
Table 37: Time to toxigenesis in hot smoked trout fiUets vacuum packaged in films ofdifTerent OTR and stored at 4, 8 and 12°C
PU_' Inoeulum Storage Day. to tOllgenal.2OO3
level temperature_____(Ipom/c) cg 0 7 14 11 18
A 1()2 4 0/2 0/2 0/2 0/2 0/2B 1()l 4 0/2 0/2 0/2 0/2 0/2C 1<r 4 0/2 0/2 0/2 0/2 0/2D 1cr 4 0/2 0/2 0/2 0/2 0/2
ABCD
ururur102
8888
0/20/20/20/2
0/20/20/20/2
0/21/20/20/2
0/20/21120/2
1/2 .1120/20/2
A ur 12 0/2 0/2 212 112 2/2B 102 12 0/2 0/2 2/2 2/2 112C ur 12 0/2 012 112 112 1/2D ur 12 0/2 0/2 0/2 2/2 1/2
1 Film A (OTR=12 cc/mz/day/atm @24°C, OOI'oRH); Film 8 (OTR~ 950 cc/m'l/day/atm @24°C, OOI'oRH); Film C (OTR=4 920cclm2/day/atm @ 24°C, OOI'oRH); Film D (OTR=10 040 cc/m2/day/atm @24°C, ()oI'oRH)2 ln duplicatc3 Trypsinized extract
•
•
•
173
ofstorage, again showing the emphasis of strict temperature control (Tables 36-37). The
lack of toxin production al 4°C &greCS with previous challenge studies on fresh trout
fillets (section 5.3.4.).
At 8°C, cold smoked trout fillets vacuum packaged in films C and D were toxie by
day 28 (Table 36). In these 2 cases, spoilage preceded toxigenesis (Table 34). Toxin was
not deteeted in any other eold smoked trout fillets stored al 8°C. However, al 12°C, ail
cold smoked trout filIets were toxie by day 14. They were ail rejected based on color and
odor scores before day 14, with the exception of inoculated cold smoked trout fillets
vacuum packaged in film B which had an estimated eolor shelf..üfe of -17 days (Table
35). Based on color, this latter packaging treatment may constitute a potential hazard as
smoked fish would appear acceptable while being toxie.
At 8°C, the earliest lime until toxin detection was clay 14 for hot smoked trout
fillets vacuum packaged in film B (Table 37). Toxin W8S detected by day 21 in hot
smoked trout fillets vacuum packaged in film C (Table 37). In the latter 2 packagiDg
treatments, toxin production preceded spoilage (Table 34). Therefore, products may be
perceived acceptable by the consumer while being toxic. Hot smoked trout fillets
vacuum packaged in film A were toxie by day 28 (Table 37) however, spoilage preceded
toxigenesis (Table 34). At 12°C, inoculated hot smoked vacuum packaged trout flUets
were toxic by day 14 in film A, B and C. The latter three packaging treatments eould
pose a POtential health risk since toxin production preceded sensory rejection (Table 35).
For inoculated hot smoked trout flUets vacuum packaged in film D, toxigenesis (after 21
days) was preceded by spoilage; color, texture and odor shelf-üfe being -10, -28 and -14
days respcctively (Table 35).
The lime to toxin detection in both inoculated cold and hot smoked vacuum
packaged trout fillets was similar. In fact, even tbough the salt content was higher and 8w
was lower in hot smoked trout fiUets (2.1% (w/w) and aw=O.978) compared to eold
•
•
•
174
smoked trout fillets (1.7% (w/w) and aw=O.98S), the difference was not significant to
have any etTect on tinte to toxigenesis.
Eldund (1992) reported toxin production in vacuum packaged white tish sticks
packaged in both 02-permeable and Û2-imPe11lleable films. However, toxin production
was always less in films packaged with Ch-permeable films which could he attributed to
the combined eiTeet of salt concentration in the aqueous phase of the produet (1.8-3.5%)
and the OTR of the packaging film rather than the etTect of film permeability alone.
Lambert (1991) also reported that toxin production occurred in pork challenged with C.
botulinum type B spores after 19 days al 1SoC, irrespective of the OTR of the packaging
films.
•
•
•
175
6.4. CORdasioB
This study indicates that if smoked fish are contarninated by C. botulinum type E
they may pose a public health risk since these are ready-to-eat foods. In order to ensure
the safety ofMAP smoked trout fillets, Health Canada recommends that such produets he
packaged in a film with an OTR of> 2000 cc/m2/day/atm @ 24°C, OO/aRH and stored at S
4°C for less tban 14 days. At 4°C, temperature is more important than film OTR with
respect to toxin production by C. botulinum type E. A shelf-life of 14 days is possible for
both cold and hot smoked trout fillets al 4°C, ÏlrespectÏve of film On. However, al mild
temperature abuse conditions (8-12°C) packaging film OTR cannot be regarded as a
safety barrier for cold or hot smoked trout fillets. Strict temperature control is the only
way to ensure safety of vacuum packaged cold and hot smoked trout filIets. To achieve
this, monitoring documentation and the use of time-temperature indieators should be
mandatory to ensure strict storage temperature control (S4°C) from processing to
consumption (Korkeala et al., 1998). A higher NaCI concentration in the aqueous phase
of the Product may prevent the outgrowth of C. botulinum type E spores al abusive
temperatures. Heinitz and Johnson (1998) recommended a water phase salt level of ~
3.5% to ensure the safety ofMAP smoked fish. These recommendations are in agreement
with previous studies by Simpson et al. (1995) who showed that > 3% salt was sufficient
to ensure the safety ofsous-vide products stored at mild temperature abuse conditions.
•
•
•
176
GENERAL CONCLUSION
The seafood processing industry is an extremely important component of the
Canadian food industry, generatïng approximately 1 billion doUars in revenue annually.
However, with increasing energy costs associated with freezinglftozen storage and
distribution, and increasing consumer demands for fresh fish products, the seafood
industry is constantly searching for new preservation technologies. One such approach is
modified abnosphere packaging (MAP).
This study bas shown that an appreciable shelf-life extension is possible by
packaging trout fillets under modifled atmospheres. As color being one of the most
important parameter consumers use to judge trout fillets, an understanding of the
variables that affect trout color was necessary to achieve the desired extension in shelf
life. It was found that the color of fresh rainbow trout fillets was highly dependable on
bath packaging atmospheres as weU as storage temperature. In Cact, vacuum and gas
packaging (85% C(h:lS%N2) resulted in the longest color shelf-life at 4°C.
Shelf-life studies have showed that MAP could he used tQ extend the
microbiological shelf-life of fresh trout flUets for approximately 6 days al 4°C. However,
at mild temperature abuse conditions (12°C), the shelf-life oftrout fillets was terminated
after -2 days in ail packaged trout fillets.
While MAP cao he used to extend the shelf-life and keeping quality of trout
fiUets, there are concems express by the scientific community about the microbiological
safety of this technology, particularly with respect to the groWth of Listeria
monocytogenes and Clostridium botulinum type E. Since adequate remgeration of foods
throughout the distribution chain cannot he guaranteed, MAP products MaY he subjected
to temPel'8ture abuse storage conditions and become a health treat to the consumers.
•
•
•
177
CbalIenge studies with L. monocytogenes showed tbat wbile gas packaging (S5%
COz: lS%N2) had an inhibitory effect on the growth ofL. monocytogenes, it grew weU in
both air and vacuum packaged trout fillets stored al 4°C and in all gas atmospheres al
12°C.
Challenge studies witb C/ostridium botu/inum type E showed that no toxin was
produced at 4°C in trout fillets packaged in air, vacuum and in a gaseous mixture (S5%
COz: lS%N2). However, C. botu/inum type E grew and produced toxin in all inocu1ated
fresh trout fillets packaged under similar conditions at 12°C. A consensus of opinion
have heen demonstrated by the scientific community that packaging foods with
atmospheres containing Oz would inhibit the growth of this patbogen (Schvester, 1989).
However, this study bas shown that C. botulinum can grow and produce toxin in
inoculated fresh trout fillets packaged under various levels of oxygen (0-1()()oA., balance
CO2) or films of different oxygen transmission rate (4, 370 cc/m2/day/atm @ 24°C, OOAt
RH < OTR < 10, 040 cclm2/day/atm @ 24°C) and stored under conditions of mild
temperature abuse (12°C). Therefore, the addition of Û2 in the packaging atmosphere
cannot he considered an additional safety banier against C. botu/inum type E in ftesh
trout fillets. The Cact that fresh fish is contarninated with aerobic sPOilage organisms
which consume 02 and produce CÛ2 contribute to the ~on of a more favorable
environment for the growth ot: and toxin production by, this pathogen. In fact, this study
bas shown that C. botulinum will produce toxin onder temperature abuse condition,
irrespective of the initial gas atmosphere, enhancing the importance of strict temperature
control.
There is also concem about the public health safety of rnjnimaIJy processed food
sucb as MAP smoked fish. Challenge studies witb C. botu/inum type E were carried out
on vacuum packaged cold and hot smoked trout fillets in films of different OTR and
stored at 4, 8 and 12°C. While no samples were toxie at 4°C, 25% of the samples were
toxic al SoC and ail produets were toxic alter 21 days al 12°C. In some cases, spoilage
preceded toxigenesis, while in other cases, toxigenesis preœded spoilage.
•
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•
178
While the growth of: and toxin production by, non-proteolytic strains of C.
botulinum in MAP trout filIets cao be prevented by storage al proper reftigeration
temperatures, temperature alone cannot he reüed upon to ensure the microbiological
safety of MAP fresh and smoked trout tiUets. There is a risk of toxin production if C.
botulinum spores are present and the product is temperature abused. Therefore, furtber
studies need to he done to determine" the additional barriers necessary to inhibit this
pathogen should contamination ofthe produet occur.
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•
179
REFERENCES
Amlacher, E. (1961). Rigor mortis in tish. In: Fish as food, (ed. G. Borgstrom).Academic Press, New York and London, pp. 385-409.
Andersen, H.J., Bertelsen, G., Cbristophersen, A.G., Ohlen, A. and Slabsted, L. (1990).Development of rancidity in salmonid steaks during tetail display; a comparison ofpractical storage life of wild salmon and farmed rainbow trout. Zeitschrift furLebensminel-Untersuchung und-Forschung. 191: 119-122.
Ang, J. and Hsard, N.F. (1985). Chemical composition and postmortem changes in softtextured muscle ftom intensely feeding Atlantic cod, Gadus morhuo. Journal ofFoodBiochemistry. 9: 49-64.
Avery, S.M., Hudson, I.A. and Penney, N. (1994). Inhtbition of Listeria monocytogeneson normal ultimate pH beef (pH 5.3-5.5) al abusive storage temperatures by saturatedcarbon dioxide controlled atmosphere packaging. Journal ofFood Protection. 57: 331333,336.
Bahk, J. and M~ E.H. (1990). Listeriosis and Listeria monocytogeneses. InFoodborne diseases, (ed. 0.0. Cliver.). Academic Press Inc., San Diego, CA, pp. 249250.
Baker, D.A. and Genigeorgis, C. (1990). Predicting the safe storage of fresh fish undermodified atmospheres with respect to Clostridium hotulinum toxigenesis by modelinglength of the lag phase ofgrowth. Journal o/FoodProtection. 53:131-140.
Baker, D.A., Genigeorgis, C., Glover, J. and Razavilar, V. (1990). Growth andtoxigenesis of C. botulinum type E in fishes packaged under modified atmospheres.International Journal ofFood Microbiology. 10:269-290.
Barnett, H.J., Conrad, J.W. and Nelson, R.W. (1987). Use of larnjnated high and lowdensity polyethylene fleXIble packaging to store trout (5almo gairdnerf) in a modifiedatmosphere. Journal ofFood Protection. SG:645-651.
Barnett, HJ., Stone, F.E., Roberts, G.C., Hunter, PJ., Nelson, R.W. and Kwok, J.,(1982). A study in the use of high concentration of CCh in a modified atmosphere 10preserve salmon. Marine Fisheries Review. 44: 7·11.
Bauemfeind, J.C., Brobacher, G.B., Klaui, H.M. and Marusich, W.L. (1977). Use ofcarotenoids. In: Carotmoitis. (ed. O. Isler). Basel: Birkbauser Verlag, pp. 743·770.
Billard, R. and Nadot, F. (1989). Changement de nom de la truite arc-en-ciel Salmogairdneri (lUchardson) en Onchorhynchus mylciss (Walbaum). La pisciculture francaise.97: 911-917.
•
•
•
180
Bjomdahl, T. (1990). The economics of salmon. In: Aquaculture. Blackwell Scientific,Oxford, U.R. p.l.
Black, D. and Love, R.M. (1986). The sequential mobilization and restoration of energyreserves in tissues of Atlantic cod during starvation and refeeding. Journal ofComparative Physiology. Biochemical, Systemic, and Environmental Physiology. 156:469479.
Blocher, J.C. and Busta, F.F. (1983). Bacterial spore resistance to acid. FoodTechnology. 37:87-89.
Blokhus, H. (1986). Aspects related ta the quality of farmed Norwegian salmon (Salmosalar). In: Seafood Quality Determination, (eds. D.E. Kramer and J. Liston). ElsevierScience Publishers B.V., Amsterdam, pp.615-628.
Bramstedt, F. and Auerbach, M (1961). The spoilage of fresh-water tish. In: Fish asfood, (ed. G. Borgstrom). Academie Press, New York and London, pp. 613-637.
Brady, A.L. (1989). Modified atmosphere packaging of seafoods. In:ControllediModified atmosphere paclcaging of foods, (ed. A.L. Brody). Food andNutrition Press, Inc., Trumbull, CT., pp.59-66.
Brown, M.H. and Emberger, O. (1980). Oxidation-reduetion potential. In: Microbialec%gy offoods. Vol. l, Academie Press, New York.
Brown, W.D., Albright, M., Heyer, D.A., Spruce, B. and Price, R.J. (1980). Modifiedatmosphere storage ofrock fish and silver salmon. Journal ofFood Science. 45:93-96.
Cano, D.C. and Taylor, L.Y. (1979). The control of the botulism hazard in hot-smokedtrout and mackerel. Journal ofFoodScience. 14:123-129.
Cano, D.C. and Taylor, L.Y. (1982). An outbreak of botulism in rainbow trout, Salmogairdneri Richardson, farmed in Britain. JournalofFish Diseases. 5:393-399.
Cano, D.C., Houston, N.C., Taylor, L.Y., Smith, G.L., Thomson, A.B. and Craig, A.(1984). Studies of salmonids packed and stored under a modified atmosphere. TorryResearch Station. Aberdeen, 53pp.
Cano, D.C., Smith, G.L. and Houston, N.C. (1983). Further studies on marine fish storedunder modified atmosphere packaging. Tony Research Station, Aberdeen, 61pp.
Cano, D.C., Taylor, L.Y. and CoUett, J.M. (1980). Botulism and tishery products:evaluation ofthe role ofvacuum packaging. Proceeding]n World Congress. FoodbomeInt: Intox., Robert von Ostertag Institute, Berlin, pp.826-833.
•
•
•
181
CaDn, D.C., Wilson, B.B., Shewan, JM. and Robbs, G. (1966). Clostridium hotulimnntype E in fish products in the U.K. Nature (London). 111:205-206.
Chen, H., Meyers, S.P., Hardy, R.W. and Biede, S.L. (1984). Color stability ofastaxanthin pigmented rainbow trout under various packaging conditions. Journal ofFood Science. 49:1337-1340.
Choubert, G. Blanc, J.M. and Courvalin, C. (1992). Muscle carotenoid content andcolour of farmed rainbow trout fed astaxanthin or canthaxanthin as affected by cookingand smoke-curing procedures. International Journal ofFood Science and Technology.17: 277-284.
Christiansen, L.N., Deffiler, J., Foster, E.M. and Sugiyama, H. (1968). Survîval andoutgrowth of Clostridium botulinum type E spores in smoked fish. ÂppliedMicrobiology. 1:133-137.
Coyne, F.P. (1933a). The effect of carbon dioxide on bacterial growth with specialreference ta the preservation of tish Part n. Journal Society of Chemical Industry(London). 51: 19-24.
Coyne, F.P. (1933b). The effect of carbon dioxide on bacterial growth with specialreference to the preservation of fish Part 1. Journal Society of Chemical Industry(London). 51: 119-121.
Daewood, A.A., Roy, R.N. and Williams, C.S. (1986). Effect of delayed icing on thestorage life ofrainbow trout. Food Techn%gy. 11: 159-166.
Davis, H.K. (1995). Modified atmosphere packaging (MAP) of fish and seafoodproducts. In: Proceedings modified a1mosphere packaging (MAP) and relatedtechnologies. Campden and Chorleywood Food Research Association, Gloucestersbire,VI(. pp.l-!3.
Davison, W. and Goldspink, G. (1977). The etTect of prolonged exercise on the lateralmusculature ofthe brown trout (Sa/mon rruUa). Journal ofExperimental Biology. 71: 112.
Dodds, K.L., Brodsky, M.H. and Warburton, D.W. (1992). A retail survey of smokedready-to-eat fish to determine their microbiological quality. Journal ofFood Protection.55:208-210.
Eklund, M.W. (1982) Effect ofCÛ2 atmospheres and vacuum packaging on Clostridiumbotulinum and spoilage organisms of fishery products. In: Proceedings FinI NationalConference Seafood Pacmging Shipping, (ed. R.E. Martin). National Fisheries Institute,Washington, D.C., pp. 298.
•
•
•
182
Eklund, M.W. (1982). SignificaŒe of C/ostridium hotu/;lIIIm in tishery productspreserved short ofsterilization. Food Techn%gy. 36:107-112, 115.
Eklund, M.W. (1992). Control in fishery produets. In: C/ostridium botulinum-ecologyand control infoods. (eds. A.H.W. Hauschild and LL. Dodds). Marcel Dekker, NewYork, pp.209-232.
Eklund, M.W., Poysky, F.T., Paranjpye, R.N., Lashbrook, L.C., Peterson, M.E. andPelroy, GÂ. (1995). Incidence and sources of Listeria monocytogenes in cold-smokedtishery products and processing plants. Journal ofFoodProtection. 58:502-508.
Eklund, M.W., Poysky, F.T., Peterson, M.E., Peck, L.W. and Bnmson, W.D. (1984).Type E botulism in salmonids and conditions contributing to outbreaks. Aquaculture.41:293-309.
Enfors, S.O. and Molin, G. (1978). The influence of bigh concentrations of carbondioxide on the genninarion ofbacterial spores. Journal ofÂpplied Bacteriology. 4: 279285.
Fantasia, LD. and Duran, A.P. (1969). Incidence of Clostridium hotulinum type E incommercially and laboratory dressed whitetish chubs. Food Technology. 23:793-794.
Farber, J.M. (1991). Microbiological aspects of moditied atmosphere packagingtechnology -A review. Journal ofFood Protection. 54(1): 58-70.
Farber, J.M. and Harwig, l. (1996). The Canadïan position on Listeria monocytogenes inready-to.eat foods. Food Control. 7:253-258.
Farber, J.M. and Peterkin, P.I. (1991). Listeria monocytogenes, a food-borne pathogen.Microbiological Review. 55:476-5II.
Fauconneau, M., Corraze, G., Lebail, P.Y. and Vernier, l.M. (1990). Les lipides de dépôtchez les poissons d'élevage: contrôle cellulaire, métabolique et hormonal. lNRA.production animale. 3: 369-381.
Fernandes, C.F., Flick, GJ. and Thomas, T.B. (1998). Growth of inoculatedpsychrotrophic patbogens on refrigerated fiUets of aquacultured rainbow trout andchannel catfish. Journal ofFoodProtection. 61:313..317.
Fine, G. (1992). Non-protein nitrogen compounds in fish and shellfish. In: SeafoodBiochemistry. Composition and Quality, (eds. GJ. Flick and R.E. Martin). TecbnomicPublishing Co., Lancaster, PA, pp.393-399.
Gabriel, R. (1990). Une étude de marché de la truite en Europe. La pisciculturefrancaise. 102:4--100.
•
•
183
Garcia, G.W. and Genigeorgis, C. (1987). Quantitative evaluation of C/ostridiumbotu/inum nonproteolytic types B, E and F growth risk in fresh salmon tissuehomogenates stored under modified atmospheres. Journal ofFood Protection. 58:390397.
Garcia, G.W., Genigeorgis, C., and Lindroth, s. (1987). Risk of growth and toxinproduction by Clostridium botulinum nonproteolytic types B, E, and F in salmon filletsstored under modified atmospheres at low and abused temperatures. Journal of FoodProtection. 50:330-336.
Garrett, D.M., Harrison, M.A.. and Huang, Y. (1995). Growth and production oftoxin ofClostridium botu!inum type E in rainbow trout under various storage conditions. JOU17Ul/ofFood Protection. 58:863-866..
Gendron, A., Boyer, J., Rioux, J, Coulombe, N. and Blais, J. (1993).. Méthodesd'évaluation de la fraîcheur de la truite arc-en-ciel (Oncorhynchus mylciss) sur glace.Cahier d'information / Ministère de l'agriculture, des pêcheries et de l'alimentation.Direction de la recherche scientifique et technique. 131:1-60.
Glass, K.A. and Doyle, M.P. (1989). Fate of Listeria monocytogenes in processed Meatproducts during refrigerated storage. Applied Environmenta/ Microbiology. 55:15651569.
Gobantes, 1., Choubert, G. and Gomez, R. (1998). Quality ofpigmented (astaxanthin andcanthaxanthin) rainbow trout (Oncorhynchus mylciss) fillets stored under vacuumpackaging during chilled storage. Journal 01A.gricultural and Food Chemistry. 46:43584362.
Gordon, M.H. (1990).. The mechanism of antioxidant action in vitro. In: Foodantioxidants, (ed.. B.J.F.. Hudson). Elsevier Science Publishers, England, pp. 1-15.
Greer, G.G. (1993).Research Branch,Communication.
Estimation of chilled meat storage life.Lacombe Research Station, Lacombe,
Agriculture CanadaAlberta, Personal
•
Haard, N.F. (1992a). Control of chemical composition and food quality attnbutes ofcultured fish. Food Research International. 25: 289-307.
Haard, N.F. (1992b). Biochemistry of color and color change in seafoods. In: SeafoodBiochemistry: Composition and Qua/ity, (cds. R.. Martin, R. Ory and G. Flick).Technomic Publishing Co., Lancaster, PA, pp.305-361.
Haard, N.F.. (1992c). Technological aspects of extending prime quality of seafood: Areview. Journal ofAquatic FoodProduction and TechllOlogy. 1: 9-27.
•
•
•
184
Haan!, N.F. and Arcilla, R. (1985). Precursors ofMaillard browning in dehydrated squid,Ria illecehrosus. Canadian Institute ofFood Science and Technology Journal. 18: 326331.
Hansen, L. and Landry, P.L. (1982). Qualités requises pour différentes mises sur lemarché de saImonidé au QueDec. Gouvernement du Québec. Ministère de 1~griculture.des Pêcheries et de 1~/imentation. pp. 1-37.
Hauschild, A.H.W. and Gauvreau, L. (1985). Food-borne botulism in Qtnada, 19711984. Journal ofthe Canadian Medical Association. 133:1141-1146.
Hauschild, A.H.W., Aris, B.J. and Hilsbeimer, R. (1975). Clostridium botulinum inmarinated products. Canadian Institute ofFood Science and Technology Journal. 8:8487.
Hayes, S., Craig, J.M. and Pilcher, K.S. (1969). The detection of Clostridium botulinumtype E in smoked tish products in the Pacific Northwest. Canadian Journal ofMicrobiology. 16:207-209.
Heinitz, M.L. and Johnson, J.M. (1998). The incidence of Listeria spp., Salmonella spp.,and Clostridium botu/inum in smoked fish and sbeUfish. Journal ofFood Protection.61:318-323.
Henmi, H, Iwata, T. and Hata, M. (1987). Studies on the carotenoids in the muscle ofsalmons 1- Intracellular distribution of carotenoids in the muscle. Toholcu Journal ofAgricultura/ Research. 37: 101-111.
Hobbs, G. (1976). Clostridium botu/inum and its importance in fisbery products.Advances in Food Research. 12:135-185.
Hobbs, G., Roberts, T.A. and Walker, P.D. (1965). Some observations on OS variants ofClostridium botulinum type E. Journal ofAppliedBacteriology. 28:147-152.
Homer, B. (1992). Fish smoking: ancient and modern. Food Science and TechnologyToday. 6: 166 -171.
Hsieh, R.J., German, JB. and Kinsella, J.E. (1988). Lypoxygenase in fish tissue: Someproperties of the 12-lipoxygenase ftom trout gille Journal of Agriculture and FoodChemistry. 36:680-685.
Huss, R.H. (1976). Konsumfisk- biologi, teknologi, kvalitet og holdbarbed. Dans/c VetoTidss/cr. 59: 165-175.
Huss, R. (1980). Distribution of Clostridium botulinum. Applied and EnvironmentalMicrobiology. 39:764-769.
185
• Huss, H.H. (1995). Post mortem changes in tish. In: Qua/ity and qua/ity changes infreshfish, (eds. H.H. Huss Technologicallaboratory Ministry ofAgriculture and FisheriesDenmark). FAO Fisheries technical paper, pp. 35-67.
Huss, H.H. and Borresen, T. (1995a). Chemical composition. In: Qua/ity and qua/itychanges infreshfish, (eds. H.H. Huss Technologicallaboratory MiDistry of Agricultureand Fisheries Denmark). FAO Fisheries technical paper, pp. 20-34.
Huss, H.H. and Borresen, T. (l99Sb). Biological aspects. (section 3.2 Muscle anatomyand function) In: Qua/ity and qua/ity changes in fresh fish, (eds. H.H. HussTechnological laboratory Ministry of Agriculture and Fisheries Denmark). FAOFisheries technical paper, pp. 11-17.
Huss, H.H. and Eskildsen, U. (1974). Botulism in farmed trout caused by C/ostridiumbotu/inum type E. Yeterinarni medicina. 26:733-738.
•
•
Huss, H.H. and Gram, L. (1995a). Post mortem changes in fish. (section 5.3Bacteriological changes) In: Qua/ity and qua/ity changes in fresh flSh, (cds. H.H. HussTechnological laboratory Ministry of Agriculture and Fisheries Denmark). FAOFisheries technical paper, pp. 51-64.
Huss, H.H. and Gram, L. (l995b). Post mortem changes in tish. (section 6.5 The effectof fish species, fishing ground and season) ln: Qua/ity and qua/ity changes in fresh fish,(eds. H.H. Huss Technological laboratory Ministry of Agriculture and FisheriesDenmark). FAO Fisheries technical paper, pp. 86-92.
Huss, H.H. and Jorgensen, (1995). Post mortem changes in tish. (section 5.4 Lipidoxidation and hydrolysis) In: Quality and qua/ity changes infreshfish, (cds. H.H. HussTechnological laboratory Ministry of Agriculture and Fisheries Denmark). FAOFisheries technical paper, pp.64-67 .
Huss, H.H. and Nielsen, J. (1995a). Assessment of fish quality. In: Quality and qua/itychanges in fresh fish, (cds. H.H. Huss Technological laboratory Ministry of Agricultureand Fisheries Denmark). FAO Fisheries technical paper, pp. 130-153.
Huss, H.H. and Nielsen, J. (I99Sb). Post mortem changes in tish. (section 5.1 Sensorychanges) In: Qua/ity and qua/ity changes in fresh fish, (cds. H.H. Huss Technologicallaboratory Ministry of Agriculture and Fisheries Denmark). FAO Fisheries technicalpaper, pp. 35-39.
Huss, H.H., Pedersen, A. and Cano, D.C. (1974). The incidence of C/ostridiumbotu/inum in Danish trout farms n. Measures to reduce contamination of the fish.Journa/o/Food Techn%gy. 9:451-458.
•
•
•
186
Inoue, T., SimpsOn, K.L., Yoshito, T. and Sameshima, M. (1988). Condensedastaxanthin ofpigmented oil ftom crayfish carapace and its feeding experimenL NipponSu/san Galc/œishi. 54: 103-106.
International Commission on Microbiological Specifications for Foods (1978). Samplingfor microbial anaIysis: principles and specific applications. In: Microorganisms in Foods,Vol. 2. (ed. International Commission on Microbiological Specifications for Foods).University ofToronto Press. Canada p.92.
International Commission on Microbiological Specifications for Foods (1996). InMicroorganisms in Foods. 5. Microbiological Specifications of Food Pathogens, BlackieAcademie & Professional, London.
Jacober, L.F. and Rand, Jr. AG. (1982). Biocbemical evaluation of seafood. In:Chemistry and biochemistry ofmarine food products. (cds. LE. Martin, GJ. Fliek, C.E.Hebard and D.R. Ward). Avi Publishing Company, Westport, Connecticut pp.347-36S.
Jacquot, R. (1961). Organic constituents offish and other aquatic animal foods. In: Fishasfood, (ed. G. Borgstrom). Academie Press, New York and London, pp. 145-209.
Jemmi, T. and Keusch, A. (1994). Occurrence of Listeria monocytogenes in fteshwaterfisb farms and fisb-smoking plants. Food Microbi%gy. 11:309-316•
Johnson, E.A. and An, G. (1991). Astaxanthin from microbial sources. Critical Reviewsin Biotechnology. II: 297-326.
Josephson, 0.8. and Lindsay, R.C. (1986). Enzymic generation of volatile aromacompounds from fresh fish. In: Biogenesis of aromas, (cds. T.H. Parliament and R.Croteau). ACS Symposium Series No. 317, Washington OC, pp.201-219.
Josephson, D.8., Lindsay, R.C. and Stuiber, D.A. (1984). Biogenesis of Iipid derivedvolatile aroma compounds in emerald shiner (Notropis atherinoides). Journal ofAgriculture and Food Chemistry. 32:1347-1352.
Kiessling, A., Asgard, T., Storebakken, T., Johansson, L. and Kiessling, K.H. (1991).Changes in the stnlcture and fimction of the epaxial muscle of rainbow trout(Oncorhynchus my/ciss) in relation to ration and age. m Cbemical composition.Aquaculture. 93: 373-387.
K1aui, H. and Bauernfeind, J.e. (1981). Carotenoids. In: Carotenoids os colorants andvitamin Aprecursors, (cd. J.C. Bauernfeind). Academie Press, New York, pp. 47-317.
Konosu, S. (1979). The tastc offisb and sheUfish. In: Food Taste Chemistry, (ecI. J.C.Boudreau). ACS Symposium Series, No. 115, Washington OC, pp.185-203.
•
•
•
187
Korkea1a, H., Stengel, G.,H~ E., Vogelsang, B., Boh1, A., Wiblman, H., pakkala, P.and Hielm, S. (1998). Type E botulism associated with vacuum-packaged hot-smokedwhitetish. International Journal o/Food Microbiology. 43:1-5.
Lambert, A. (1991). Effects of modified atmosphere packaging and low-doseirradiationon the shelf-Iife and microbiological safety of ftesh port. Ph.D. Thesissubmitted to McGiIl University, Department ofFood Science·and Agricultural Chemistry,Mon~,~,~nada
Lambert, A.D., Smith, J.P. and Dodds, K.L. (1991). Combined effects of modifiedatmosphere packaging and low-dose irradiation on toxin production by Clostridiumbotu/inum in ftesh pork. Journal o/FoodProtection. 54:94-101.
Laycock, R.A. and Loring, O.H. (1972). Distribution of Clostridium botulinum type E inthe Gulf of St. Lawrence in relation to the physical environment. CQ1IQdian Journal ofMicrobi%gy. 18:763-773.
Liaaen-Jensen, S. (1991). Marine carotenoids: rec::ent progress. Pure and AppliedChemistry. 63: 1-12.
Lindsay, R.C. (1981). Recent developments in packaging. In: Integration PromisesProhlems Northwest Seafood Industry, (cd. R.R. Winget). Small Tnbes Org. of W.Washington, Sumner, WA, p. 118.
Lindsay, R.C. (1982). Sorbate and botulism safety aspects for modified atmospherepackaging. In: Preceedings in First National Conference Sea/ood Packaging Shipping.(cd. R.E. Martin). National Fisheries Institute, Washington D.C. p. 332.
Lindsay, R.C. (1983). Safety and technology ofmodified atmosphere packaging offreshfish. Abstract from the 43rd Annual Meeting ofthe Institute ofFood Technology. No. 152.
Love, R.M. (1980). Biological factors affccting processing and utilization. In: Advancesin Fish Science and Techn%gy, (cd. J.J. Connell). Fishing News Books, Farnham, UK,pp.135-136.
Love, R.M. (1988). The Food Fishes. The;r Intrinsic Variation and PracticalImplications. Farrand Press, LondonIVan Nostrand Reinhold, New York.
Love, R.M., Munro, L.J. and Robertson, 1. (1977). Adaptation of the dark muscle ofcodto swimming activity. Journal ofFish Bi%gy. Il:431-436.
Lovern, J.A. (1950). Some causes ofvariations in the composition of fish oils. Journal.Society 01Leather Trades Chemists. 34: 7-23.
•
•
•
188
Lund, BM. and Wyatt, G.M. (1984). The effect of redox potential, and its interactionwith sodium chloride concentration, on the probability of growth of Clostridiumbotulinum type E &am spore inocula. Food Microbiology. 1:49-65.
Lyot, R.K., Kautter, D.A. and Solomon, H.M. (1982). Differences and similarities amongproteolytic and nonproteolytic strains of Closlridium botulinum types A, B, E and F: areview. Journal ofFood Protection. 45:466-473.
Lyver, A., Smith, J.P., Nattress, F.M., Austin, J.W. and Blanchfield, B. (1998).Challenge studies with Clostridium hotulinum type E in a value-added surimi produetstored under a modified atmosphere. Journal ofFood Safety. 11:1-23.
Malak, N.A., Zwingelstein, G., Jouanneteau, J. and Koenig, J. (1975). Influences decertains facteurs nutritionnels sur la pigmentaiton de la truite arc-en-ciel par lacanthaxanthine. A.nnales de la nutrition alimentaire. 29: 459-475.
March, B.E. and MacMillan, C. (1996). Muscle pigmentation and plasma concentrationsof astaxanthin in rainbow trout, chinook salmon, and Atlantic salmon in response todifferent dietary levels ofastaxanthin. The Progressive Fish-Culturïst. 58:178-186.
Marshall, D.L., Wiese-Lehigb, P.L., Wells, J.H. and Farr, A.J. (1991). Comparativegrowth of Listeria monocytogenes and Pseudomonas fluorscens on precooked chickennuggets stored onder modified atmospheres. Journal ofFood Protection. 54:841-843,851.
Matsuno, T. and Hirao, S. (1989). Marine carotenoids. In: Marine biogenic lipids, fats,and oi/s, (Volt), (cd. R.G. Ackman). CRC Press, Boca Raton, FI, USA, pp 251-388.
McAdams, T.I. (1996). The determination of microbial quality and presence ofPathogens and chemica1 contaminations in aquacultured rainbow trout (Onchorynchusmykiss) fillets and whole fish from different aquacultured Productions systems. M.S.Thesis submitted to Virginia Polytechnic Institute and State University, Blacksburg, VA.
McCarthy, S.A. (1996). EtTect of sanitizers on Listeria monocytogenes attaehed to latexgloves. Journal ofFood Safety. 16: 231-237.
McCarthy, S.A. (1997). Incidence and survival ofListeria monocytogenes in ready-to-eatseafood produets. Journal ofFoodProtection. 60: 372-376.
McLauchlin, J. (1993). Listeriosis and Listeria monocytogenes. Environmental PolicyandPractice. 3:201-214.
Miki, w. (1993). Recent progress in the research ofbiological activities. In: Carotenoidsin Marine Organisms, (ed. Koseisya Koseikaku). Tokyo, Cbapter 7.
•
•
•
189
Miller, L.G., Clark, P.S. and Kunlde, G.A. (1972). Possible origin of C/ostridiumbotu/inum contamination of Eskimo foods in northwestem Alaska. App/iedMicrobi%gy. 23:427-428.
Minolta. (1994). Precise color measurement. Minolta Corp., Ramsey, NJ, U.SA.
Moe, N.H. (lm). Key factors in marketing fanned salmon. Proceedings of theNutrition Society ofNew Zea/and 15: 16-22.
Mohr, V. (1986). Control ofnutritional and sensory quality ofcultured tisa ln: SeafoodQua/ity Determination, (ed. E.D. Kramer and J. Liston). Elsevier Science Publishers,Amsterdam, pp.487-496.
Murray, J. and Burt, J.R. (1969). The composition of tish. Torry Advis. Note 38, TonyResearch Station, Aberdeen, Scotland.
Newton, K.G. and Rigg, W.I. (1979). The effect of film permeability on the storage lifeand microbiology of vacuum-packed Meat. Journal ofApp/ied Bacteriology. 47:433441.
Newton, L., Hall, S.M., Pelerin M. et al. (1992). Listeriosis surveillance:l991.Communicable Disease Report. 2: 142-144.
Nielsen, S.f. and Pedersen, H.O. (1967). Studies on the occurrence and gennination ofClostridium hotulinum in smoked salmon. In: Botulism J966. (eds. M. Ingram and T.A.Roberts). Chapman and Hall, London. pp.66-72.
No, H.K. and Storebakken, T. (1992). Pigmentatio~ of rainbow trout with astaxanthinand canthaxanthin in freshwater and saltwater. Aquaculture. 111: 123-134.
Obata, Y. (1952-1953). The smell and odor of marine products. Journal ofthe JapaneseSociety ofFood andNutrition. 5: 115-120.
Ogrydziak, D.M. and Brown, W.D. (1982). Temperature effects in modified-atmospherestorage ofseafoods. Food Technology. 36:86-96.
Pelroy, G., Peterson, M., Paranjpye, R., Almond, J. and Eldund, M. (1994). Inhtbition ofLisleria monocytogenes in cold-process (smoked) salmon by sodium nitrite andpackaging Methode Journal ofFood Protection. 57:114-119.
Post, L.S., Lee, D.A., Solberg, M., Furang, D., Specchio, J. and Graham, C. (1985).Development ofbotulinal toxin and sensory deterioration during storage of vacuum andmodified atmosphere packaged fish fillets. Jouma/ ofFood Science. 50:990-996.
•
•
•
190
Przybyrski, LA., Finerty, M.W., Grodner, R.M. and GenIes, D.L. (1989). Extension ofshelf-life of iced fresh channel catfish fillets using modified atmospheric packaging andlow dose irradiation. Journal ofFood Science. 54:269-273.
RandeU, K., Ahvenainen, R. and Hattula T. (1995). Effect of the gaslproduct ratio andCOz concentration on the shelf-life of MA packed fish. Packaging Technology andScience. 8:205-218.
Reddy, N.R., Armstrong, D.J., Rhodehamel, E.L. and Kauter, DA. (1992). Shelf-Iifeextension and safety concems about fresh fishery products packaged under· modifiedatmospheres; a review. Journal ofFood Safety. 12: 87-118.
Reddy, N.R., Paradis, A., Roman, M.G., Solomon, H.M. and Rhodehamel, E.J. (1996).Toxin development by Clostridium botulinum in modified atmosphere packaged fteshTilapia fillets during storage. Journal ofFood Science. 61:632-635.
Rhodehamel, E.J., Solomon, H.M., Lilly, Jr. T., Kautter, D.A. and Peeler, J.T. (1991).Incidence and heat resistance of Clostridium botulinum type E spores in Menhadensurimi. Joumal o/FoodScience. 56:1562-1563, 1592.
Riedel, D. (1961). World fisheries. In: Fish as food, (ed. G. Borgstrom). AcademicPress, New York and London, pp. 41-75.
Riemann, H. (1969). Botulism-Types A, B and F. In: Food-Borne Infections andIntoxications, (ed. H. Riemann). Academic Press, New York, p.291.
Rocourt, J. (1996). Risk factors for listeriosis. Food Control. 7: 195-202.
Rumsey, G.L. (1988). Fish farming as a fonn of animal agriculture: recent advances infish nutrition. In: Proceedings A.lltech's 4''' Ânnual Symposium ofBiotechnology in theFeed lndustry, (ed. T.P. Lyons). AlItech Technical Publications, NicholasviUe, KY, p.235.
Ryser, E.T. and Marth, E.H. (1991). Incidence and behavior of Listeria monocytogenesin fish and seafood. In: Listeria. listeriosis andfood safety, (ed. Marcel Dekker). NewYork, p. 246.
Sainclivier, M. (1983). L'industrie alimentaire halieutique. vol l. Le poisson matièrepremière. Bulletin scientifique et technique de l'Ecole 1IDtionaie supérieure agronomiqueet du Centre de rechreche de Rennes: 263p.
SAS (1988). SAS user's guide. SAS Institute, me. Carry, NY., U.S.A.
Schmidt, C.F., Lechowich, R.V. and Folinazzo, J.F. (1961). Growtb and toxin productionby type E Clostridium botulinum below.woC. Journal ofFood Science. 26:626-630.
•
•
•
191
Sebvester, P. (1989). Safety and seafood. Canadian Paclroging. 42:32-33.
Sea Fisb Industry Authority (1985). Guidelines for the handling of fish packed ineontroUed atmosphere. Sea Fisheries House, Edinburgb, Scotland.
Sears, D.F. and Eisenberg, D.F. (1961). A model representüig a physiological role ofcarbon dioxide at the eeU membrane. Journal ofGeneral Physi%gy. 44: 869-877.
Setser, C.S. (1984). Color: Retlections and transmission. Joumal ofFood Quality. 6:183-197.
Sheridan, J.J., Doherty, A., Allen, P., MeDowell, DA., Blair, I.S. and Hanington, D.(1995). Investigations on the growth ofListeria monocytogenes on lam.b packaged undermodified atmospheres. Food Microbiology. 12: 259-266.
Sigurgisladottir, S., Parrish, C.C., Lall, S.P. and Ackman, R.G. (1994). Effects offeediDgnatural tocopherols and astaxanthin on Atlantic salmon (Salmo salar) fiIlet quality. FoodResearch International. 27: 23-32.
Simpson, K.L., Katayama, T. and Chichester, c.a. (1981). Carotenoids in fish feeds. In:Carotenoids as colorants and vitamin A precursors, (ed. J.C. Bauemfeind). AeademiePress, New York and London, pp.463-538.
Simpson, M.V., Smith, J.P., Dodds, K., Ramaswamy, H.S., Blanchfield, B. and Simpson,B.L (1995). Challenge studies with Clostridium botulinum in a sous-vide spaghetti andmeat-sauce produet. Jou17lDiofFood Protection. 58:229-234.
Simunovic, J., Oblinger, J.L. and Adams, J.P. (1985). Potentials for growth ofnonproteolytie types of C/ostridium botulinum in pasteurized restructured meat products.Journal ofFood Protection. 48:265-276.
Sinnhuber, R.a. and Yu T.C. (1958). 2-thiobarbituric acid method for the measurementof rancidity in tishery products II-The quantitative detennination of malonaldehyde.Food Technology. 1:9-12.
Skrede, G., Storebakkcn, T. and Naes, T. (1989). Color evaluation in raw, baked andsmoked tlesh of rainbow trout (Onchorhynchus myldss) fed astaxanthin or cantbaXantbin.Journal o/FoodScience. 55:1574-1578.
Smith, J.P., Ramaswamy, H.S. and Simpson, B.K. (19908). Developments in foodpackaging technology. Part 1: Proccss and cooking consideration. Trends in Food Scienceand Technology. 1:107-111.
•
•
•
192
Smith, J.P., Ramaswamy, H.S. and Simpson, BK. (199Ob). Developments in foodpackaging technology. Part fi: Storage aspects, Trends in Food Science and Technology.1:112-119.
Smith, L. and Sugiyama, H. (1988). Botulism: the organism. ils toxins. the disease, (ecI.A. Balows). Second edition, Charles C. Thomas Publisher, Springfield, IL.
Sperber, W.H. (1982). Requirements of Clostridium botulinum for growth and toxinproduction. Food Techn%gy. 36:89-94.
Stammen, K., Gerdes, o. and Caporaso, F. (1990). Modified atmosphere packaging ofseafood. Critica/ Review in Food Science andNutrition. 29:310-316.
Stansby, M.E. and Hall, A.S. (1967). Chemical composition of commercially importantfish of the USA. Fishery lndustrial Reseorch. 3: 29-34.
Stier, R.F., Bell, L., Ito, K..A., Shafer, B.D., Brown, L.A., Seeger, M.L., Allen, B.H.,Procuna, M.N. and Lerke, PA. (1981). Effect of modified atmosphere storage on C.botulinum toxigenesis and the spoilage of salmon fillets. Journal of Food Science.46: 1639-1642.
Sugiyama, H. (1990). Botulism. In: Foodbome diseases, (e<!. 0.0. Cliver). AcademicPress, Inc., San Diego, CA, pp. 107-125.
Suzukï, T. and Suyama, M. (1980).· Changes in free amino acids and phosphopeptides ofrainbow trout during development. Nippon Suisan Galckaishi. 46: 591-597.
Tarladgis, B.G., Watts, B.M. and Younathan, M.T. (1960). A distillation method for thequantitative deterrnination of malonaldehyde in rancid foods. The Journal of theAmerican Oil Chemisls •Society. 37: 44-48.
Teskeredzic, Z. and Pfeifer, K. (1987). Determining the degree of freshness of rainbowtrout (Sa/mo gairdnen) cultures on brackish water. Journal ofFood Science. 52: 11011102.
Thurman, H.V. and Webber, H.H. (1984). Marine Bio%gy. Charles E. MeriillPublishing C.A. Bell and HoweU Co. Colombus Ohio.
Torrissen, O.J. and Tonissen, K.R. (1985). Protease activites and carotenoid levelsduring the sexual maturation of Atlantic salmon (Salmon salar). Aquaculture. 50: 113122.
Torrissen, OJ., Hardy, R.W. and Shearer, K.D. (1989). Pigmentation of salmonidscarotenoids deposition and metabolism in salmonids. Critical Reviews i" AquaticSciences. 1: 209-225.
193
• TUlUjman, S.A., Wamer, W.G., Wei, R.R. and Albert, R.H. (1997). Rapid liquidchromatographie method to distinguish wild salmon ftom aquacultured salmon feclsynthetic astaxanthin. Journa/ ofADAC International. 81: 622-632.
Valley, G. and- Rettger, L.F. (1927). The influence of carbon dioxide on bacteria.JournalofBacteriology. 14:101-137.
Via-Mer (1998). Personal Communication. St-Hyacinthe, Quebec.
Ward, D.R. and Baj, NJ. (1988). Factors affecting microbiological quality of seafoods.Food Technology. 42:85-89.
Withler, LE. (1986). Genetic variation in carotenoid pigment deposition in the redtleshed chinook salmon (Oncorhynchus tshawytscha) of Quesnel River, BritishColumbia Canadian Journal ofGenetic and Cyr%gy. 28: 587-594.
Wolfe, S.K. (1980). Use of CO and C(h enriched atmospheres of meats, fisb andproduce. Food Technology. 34:55-58.
•
•
Wyatt, C.J. and Guy, V.H. (1981). Incidence and growth of Bacil/us cereus in retailpumpkin pies. Journal ofFood Protection. 44:422-424, 429.
Yasuda, M., Nishino, H., Tanaka, M., Chiba, T., Nakano, H., Yodoyama, M. and Ogawa,S. (1992). Preservation of freshness of rainbow trout fillets packaged with carbondioxide-nitrogen gas mixture (Studies on gas-excbange packaging of fresh fis~ Part 2).Packagïng Technology andScience. 5: 109-113.
Young, L.L., Revuere, R.D. and Cole, A.B. (1988). ~~resh red Meats: A place to applymodified atmospheres". Food Techn%gy. 42: 65~9.
Youngson, A.F., Mitchell, A.I., Noack, P.T. and Laird, L.M. (1997). Carotenoid pigmentprofiles distinguish anadromous and nonanadromous brown trout (Salmotrutta).Canadian Journal ofFisheries andAquatic Sciences. 54: 1064-1066.
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