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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

ClsabeUe Dafresne

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Suggested short title:

SHELF-LIFE AND SAFETY STUDIES ON RAINBOW TROUT FlLLETS

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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


5.8. 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


6.4. 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



Table 35: Estimated shelf-life (days) based on sensory analysis and pH analysis of



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


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


Figure 24: Changes in general appearance scores of ttout tillets packaged under


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


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


Figure 40: Changes in general appearance scores of inoculated trout fiUets packaged


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

•

•

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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

•

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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

•

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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,

•

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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).

•

•

Table 3: Suggested fasting period based on water temperature (Hansen and Landry,1982)

•

•

•

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

•

•

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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 -300Â» (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

•

•

•

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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37

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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38

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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39

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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40

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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43

%.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:

•

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


'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).


•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


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


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).


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.


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


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


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).


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


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


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.


•

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


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 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).


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..


•

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.


•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


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.


•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


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.


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


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.


•

•

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).


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

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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

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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

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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.

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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.

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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.

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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

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INFORMAnONTO USERS tram - McGill Universitydigitool.library.mcgill.ca/thesisfile30371.pdf · fumées à froid et 75% des truites fumées à chaud étaient toxiques ... FinaUy, 1owe

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