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The influence of ration level and swimming speed on sensory attributes, gas chromatographic properties,… Siemens, Beverly Ruth 1997

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T H E I N F L U E N C E OF R A T I O N L E V E L A N D S W I M M I N G SPEED O N  SENSORY  A T T R I B U T E S , GAS C H R O M A T O G R A P H I C PROPERTIES, I N S T R O N T E X T U R E PROFILE A N A L Y S I S A N D P H OF C O O K E D M U S C L E F R O M F A R M E D C H I N O O K S A L M O N (Oncorhynchus tshawytscha) C U L T U R E D I N S E A W A T E R by Beverly R u t h Siemens B.Sc. The University o f Manitoba, 1990 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE  in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f F o o d Science)  W e accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A December 1996 © Beverly Siemens, 1996  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia,  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and  study.  scholarly  or  her  for  purposes  gain  Department .of  .Date  DE-6 (2/88)  £3  that  be  It  shall not  that  the  by  understood be  allowed  an  advanced  Library shall  permission  granted  is  for  for  the that  without  make  it  extensive  head  of  my  copying  or  my  written ...  PcT)c\  The University of British Vancouver, Canada  :  requirements  agree  may  representatives.  financial  the  I agree  I further  permission.  :  of  $)Cl<?riC<C Columbia  Se^esnhor  fflf.  Abstract Representative samples o f post-juvenile C h i n o o k salmon were obtained from the Department o f Fisheries and Oceans Canada - West Vancouver Laboratory. The fish were part o f a study directed t o assessing the influence o f t w o ration levels ( 7 5 % and 1 0 0 % o f m a x i m u m ration) and three swimming speeds (0.5, 1.0, and 1.5 body lengths/second) o n g r o w t h , body composition and thyroid function o f chinook salmon i n seawater.  A l l analyses in the present study were conducted using cooked muscle.  Sensory analysis, conducted in 19 sessions (10 days), was performed by 6 trained panellists.  The  treated and reference samples, composed o f randomly mixed slices o f muscle from farmed chinook salmon obtained at a local fishmonger, were graded for 28 sensory attributes; 9 aroma, 10 flavour, 8 texture as w e l l as "overall acceptability".  A f t e r completing preliminary analyses o f the sensory data,  data from panellist 6 and the first panel session were eliminated due to excessive inconsistencies in the results.  A N O V A revealed that 8 attributes were significantly affected by ration level.  After  standardising the significantly affected attributes' data, using a z-transformation to remove the panellist effect, one aroma t e r m was no longer significant. Principal Component (PC) Similarity graphs using the standardised data clearly illustrated the effect o f ration level o n these sensory attributes. The effect o f using a replacement panellist f o r panellist 5 o n t w o occasions became apparent from a P C 1 vs. P C 2 graph o f that panellist's data. Purge and trap extracts were used for gas liquid chromatographic analysis o f volatile compounds from cooked salmon.  A n A N O V A o f consistently appearing peaks  revealed that 27 o f these were significantly affected by either SS or R L .  Principal Component  Similarity graphs o f data from these peaks showed a clear separation o n the basis o f R L but not SS. The Instron Texture Profile Analysis statistics differed sharply from the other results since they indicated that SS and not R L significantly affected the texture o f the cooked salmon. The p H values  ii  for cooked fish were significantly affected by R L .  The results o f this study, w i t h the exception o f  those from Instron T PA , agreed w i t h those o f Kiessling et al. (1994a,b) w h o generally found that R L and not SS significantly affected the g r o w t h and whole-body and muscle proximate composition o f chinook salmon in seawater.  iii  Table of Contents Abstract  ii  Tables  viii  Figures  x  Acknowledgements  xi  1. Introduction  1  2. Literature Review  2  2.1 Exercise L e v e l  2  2.1.1 Types o f exercise experiments  2  2.1.2 Effect o f exercise training o n fish  3  2.1.3 Effect o f water current o n fish behaviour  3  2.1.4 Fuel use f o r fish locomotion  5  2.1.5 Effects o f exercise training o n  fish  6  2.1.5.1 Increased stamina and m a x i m u m swimming speed 2.1.5.2 Hypertrophy o f fish muscle  6 fibre  7  2.2 Physiological factors i n fish affected b y r a t i o n level  9  2.2.1 T h e effect o f ration level o n fish size 2.2.2 The effect o f ration level o n the fat content o f  9 fish  10  2.2.3 The effect o f ration level o n the protein content o f f i s h  10  2.2.4 The effect o f ration level o n fish muscle fibre size  10  2.2.5 The effect o f ration level o n the muscle glycogen content o f  fish  2.2.6 The effect o f ration level o n fish g r o w t h hormone levels 2.3 T h e c o m b i n e d effect o f r a t i o n level a n d s w i m m i n g speed o n 2.4 Sensory testing  11 11  fish  12 12  2.4.1 Quantitative Descriptive Analysis ( Q D A )  12  2.4.2 Unstructured line scale  14  2.4.3 The difficulty in the analysis o f sensory data  14  2.5 T h e m e a s u r e m e n t o f f o o d t e x t u r e  14  2.5.1 Instron Universal Testing Machine  17 iv  2.5.2 Instrumental Texture Profile Analysis ( T P A )  18  2.5.2.1 T P A o n cooked salmon  18  2.6 T h e factors affecting t h e t e x t u r e o f c o o k e d 2.6.1 The effect o f p H o n the texture o f cooked  fish  19  fish  19  2.6.2 T h e effect o f muscle fibre size o n the texture o f c o o k e d  fish  2.6.3 The effect o f the level o f connective tissue o n the texture o f cooked 2.6.4 The effect o f the fat content o n the texture o f cooked  20 fish  fish  2.7 Gas C h r o m a t o g r a p h i c flavour volatile analysis 2.7.1 Purge and trap analysis  20 21 21 21  2.7.1.1 The effect o f fat content o n purge and trap extractions  22  2.7.1.2 The choice o f Tenax G C , a porous polymer, for use i n purge and trap extraction 23 2.7.1.3 The elution o f adsorbed volatiles from porous polymers w i t h ethyl ether  23  2.7.2 The relationship between Gas Chromatography ( G C ) data and quantity and intensity judgements from trained sensory panellists  24  2.8 Statistical analysis  25  2.8.1 The b o x plot  25  2.8.2 Principal component similarity (PCS)  25  3. M a t e r i a l s a n d M e t h o d s  27  3.1 E x p e r i m e n t a l c o n d i t i o n s used i n t h e r e a r i n g o f s a l m o n used i n t h i s s t u d y  27  3.2 Sensory A n a l y s i s  29  3.2.1 The selection o f sensory panellists f o r Q D A analysis  29  3.2.2 Sensory panel training  30  3.2.3 The selection o f sensory attribute terms  30  3.2.4 Ballot familiarisation by sensory panellists  31  3.2.5 The use o f composite samples  32  3.2.6 Sensory panel set-up  32  3.2.7 Sensory panel session scheduling  33  3.2.8 The preparation o f samples f o r sensory panels  33  3.2.9 The reference samples used during sensory panels  35  3.2.10 T h e sampling procedure employed by panellists  35  3.2.11 Generation o f numerical scores from the sensory ballot judgements  36  3.3 I n s t r u m e n t a l analysis o f cooked s a l m o n samples 3.3.1 Preparation o f salmon samples for instrumental analysis  36 36  3.3.2 G C headspace analysis o f cooked salmon samples  40  3.3.3 Instron T P A analysis o f cooked salmon samples  43  3.3.4 p H measurement o f cooked salmon samples  45  v  2.5.2 Instrumental Texture Profile Analysis ( T P A )  19  2.5.2.1 T P A o n cooked salmon  19  2.6 T h e factors a f f e c t i n g t h e t e x t u r e o f c o o k e d fish 2.6.1 The effect o f p H o n the texture o f cooked  20 fish  2.6.2 The effect o f muscle fibre size o n the texture o f cooked  20 fish  2.6.3 The effect o f the level o f connective tissue o n the texture o f cooked 2.6.4 The effect o f the fat content o n the texture o f cooked  21 fish  fish  2.7 Gas C h r o m a t o g r a p h i c flavour volatile analysis 2.7.1 Purge and trap analysis  21 22 22 22  2.7.1.1 The effect o f fat content o n purge and trap extractions  23  2.7.1.2 The choice o f Tenax G C , a porous polymer, for use i n purge and trap extraction 2 4 2.7.1.3 The elution o f adsorbed volatiles from porous polymers w i t h ethyl ether  25  2.7.2 The relationship between Gas Chromatography ( G C ) data and quantity and intensity judgements from trained sensory panellists 25 2.8 Statistical analysis  26  2.8.1 The box plot  26  2.8.2 Principal component similarity (PCS)  26  3. Materials and Methods  28  3.1 E x p e r i m e n t a l c o n d i t i o n s used i n t h e r e a r i n g o f s a l m o n used i n t h i s s t u d y  28  3.2 Sensory A n a l y s i s  31  3.2.1 The selection o f sensory panellists for Q D A analysis  31  3.2.2 Sensory panel training  32  3.2.3 The selection o f sensory attribute terms  32  3.2.4 Ballot familiarisation by sensory panellists  33  3.2.5 T h e use o f composite samples  34  3.2.6 Sensory panel set-up  34  3.2.7 Sensory panel session scheduling  35  3.2.8 The preparation o f samples f o r sensory panels  35  3.2.9 The reference samples used during sensory panels  37  3.2.10 The sampling procedure employed by panellists  37  3.2.11 Generation o f numerical scores from the sensory ballot judgements  38  3.3 I n s t r u m e n t a l analysis o f cooked s a l m o n samples  38  3.3.1 Preparation o f salmon samples f o r instrumental analysis  38  3.3.2 G C headspace analysis o f cooked salmon samples  43  3.3.3 Instron T P A analysis o f cooked salmon samples  47  3.3.4 p H measurement o f cooked salmon samples  49  vi  3.4 D a t a A n a l y s i s  51  3.4.1 Analysis o f sensory data  51  3.4.1.1 Exploratory analysis  51  3.4.1.2 Principal component analysis ( P C A ) 3.4.1.3 A N O V A  51 ..52  3.4.1.4 Z-transformation o f significant sensory attribute scores  52  3.4.2 Calculation o f Instron T P A parameters  53  3.4.2.1 Calibration o f results  53  3.4.3 Calculation o f T P A 'Tirmness"  53  3.4.4 Calculation o f peak area  54  3.4.5 A N O V A o f Instron T P A and p H data  54  3.4.6 Principal Component Similarity (PCS) analysis o f Sensory, and G C headspace volatile data  54  4. Results and Discussion  57  4.1 Sensory analysis o f cooked s a l m o n samples  57  4.1.1 Sensory panel reference samples  57  4.1.1.1 Purpose o f reference sample  57  4.1.1.2 Reference sample observations  59  4.1.2 Treated samples  59  4.1.2.1 Exploratory analysis  59  4.1.2.1.1 Boxplots  60  4.1.2.1.2 Deletion o f unacceptable data  60  4.1.2.2 The use o f replacement panellists  63  4.1.2.3 Summary statistics o f treated samples  78  4.1.2.4 Three factor A N O V A o f sensory attribute data  79  4.1.2.5 Z-transformation o f sensory attributes significantly affected by the treatment  84  4.1.2.6 P C A and P C S o f sensory data  84  4.1.2.7 Effect o f SS o n the sensory attributes  92  4.1.2.8 Effect o f R L o n the sensory attributes  92  4.1.3 Instrumental analysis  93  4.1.4 G C headspace analysis  93  4.1.5 Instron T P A analysis  102  4.1.6 p H analysis  110  4.1.6.1 Comparison o f p H and sensory analysis results  5. Conclusions  113  115  5.1 Sensory Analyses  115  5.2 G C headspace analyses  116 vii  5.3 I n s t r o n T P A  116  5.4 p H  117  5.5 O v e r a l l Conclusions  117  References  119  Appendix A: Samples of sensory exploratory analysis boxplots  viii  126  Tables T a b l e 1 Comparative behaviour o f instruments and human subjects (Pangborn, 1987) T a b l e 2 Experimental design used by Kiessling et al (1994 a, b) t o assess the influence o f  16 sustained  exercise and t w o ration levels o n g r o w t h o f chinook salmon in seawater. A 2 X 3 factorial design was used w i t h t w o ration levels and three swimming speeds and their treatment numbers have been used as identifiers i n statistical analyses.  30  Table 3  Conditions used i n the extraction o f cooked salmon using a purge and trap procedure  Table 4  G C conditions used i n the analysis o f purge and trap extracts from cooked chinook salmon  46  samples  48  Table 5  Conditions used for Instron measurements o f minced cooked chinook salmon samples —  50  Table 6  Calculation o f Instron T P A parameters  55  Table 7  Range, mean, and standard deviation (St. Dev.) o f sensory attributes o f the cooked, farmed chinook salmon reference samples (5 panellists, 9 panel days)  Table 8  58  A N O V A results o f judge and panel day effect o n reference samples for 28 sensory attributes o f cultured chinook salmon (5 panellists, 9 panel days)  Table 9  62  Range, mean, and standard deviation (St. D e v . ) o f cooked, cultured chinook salmon sensory attributes (all treatments combined; 5 panellists, 9 panel days)  65  T a b l e 10 M e a n sensory scores and standard deviation o f cultured chinook salmon aroma attributes for each ration level X swimming speed treatment ( 5 panellists; 9 panel days)  66  T a b l e 11 Range o f sensory scores o f cultured chinook salmon aroma attributes f o r each ration level X swimming speed treatment (5 panellists; 9 panel days) T a b l e 12  67  M e a n sensory scores and standard deviation o f cultured chinook salmon flavour attributes  for each ration level X swimming speed treatment (5 panellists; 9 panel days)  68  T a b l e 13 Range o f sensory scores o f cultured chinook salmon flavour attributes f o r each ration level X swimming speed treatment (5 panellists; 9 panel days) T a b l e 14  69  M e a n sensory scores and standard deviations o f cultured chinook salmon texture  attributes for each ration level X swimming speed treatment (5 panellists; 9 panel days)  70  T a b l e 15 Range o f sensory scores o f cultured chinook salmon texture attributes f o r each ration level X swimming speed treatment (5 panellists; 9 panel days) T a b l e 16  sensory aroma attributes o f cultured chinook salmon (5 panellists, 9 panel days) T a b l e 17  71  Summarised A N O V A results o f ration level, swimming speed and panellist effect o n —  sensory flavour attributes o f cultured chinook salmon (5 panellists, 9 panel days) T a b l e 18  85  Factor score coefficients o f the first 6 principal components o f the z-transformed sensory  attribute scores o f the R L X SS treated chinook salmon samples Table 21  83  Summarised A N O V A results o f ration level and swimming speed effect o n standardised  data from significant sensory attributes o f cultured chinook salmon T a b l e 20  82  Summarised A N O V A results o f ration level, swimming speed and panellist effect o n  sensory texture attributes o f cultured chinook salmon (5 panellists, 9 panel days) T a b l e 19  81  Summarised A N O V A results o f ration level, swimming speed and panellist effect o n  86  Peak labels, retention times and level o f significance for ration level and swimming speed  o f G C peaks that consistently appeared i n purge and trap G C headspace analysis o f cultured, cooked chinook salmon  94  ix  T a b l e 22  Factor score coefficients o f the first 7 principal components from G C headspace peaks  significantly affected by either ration level or swimming speed T a b l e 23  Summarised A N O V A results o f ration level and swimming speed effect o n Instron T P A  parametersof cooked cultured chinook salmon T a b l e 24  97 109  A N O V A table o f ration level and swimming speed effect o n the p H o f cultured cooked  chinook salmon  112  x  Figures Figure 1  Sensory ballot that was used t o evaluate samples o f cooked, farmed chinook salmon  Figure 2  PC 1 versus PC 2 using sensory aroma attribute scores from panellist 5, data points  40  labelled w i t h treatment numbers Figure 3  72  P C 1 versus PC 2 o f panellist 5 sensory flavour attribute scores, data points labelled w i t h  treatment numbers Figure 4  73  PC 1 versus P C 2 o f panellist 5 sensory texture attribute scores, data points labelled w i t h  treatment numbers  74  F i g u r e 5 P C 1 versus P C 2 o f panellist 5 sensory aroma attribute scores, data points labelled w i t h panel day number  75  F i g u r e 6 PC 1 versus P C 2 o f panellist 5 sensory flavour aroma attribute scores, data points labelled w i t h panel day numbers 76 Figure 7  P C 1 versus P C 2 o f panellist 5 sensory texture attribute scores, data points labelled w i t h  panel day number  77  Figure 8  PCS graph o f significant sensory attributes, data points labelled w i t h treatment numbers— 88  Figure 9  PCS graph o f significant sensory attributes, data points labelled w i t h ration level numbers  (1 = 7 5 % ; 2 = 1 0 0 % ration level)  89  F i g u r e 10 PCS graph o f significant sensory attributes, data points labelled w i t h swimming speed numbers ( 1 = 0 . 5 , 2 = 1 . 0 , 3=1.5 bl/s)  90  F i g u r e 11 PCS graph o f significant sensory attributes, data points labelled w i t h panel day number — 91 F i g u r e 12 Sample gas chromatogram from a purge and trap extract o f cultured chinook salmon —  96  F i g u r e 13 PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h treatment numbers  99  F i g u r e 14 PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h ration level number (1 = 7 5 % , 2 = 1 0 0 % ration level)  100  F i g u r e 15 PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h swimming speed number (1 = 0 . 5 , 2 = 1.0,3 = 1.5 bl/s)  101  F i g u r e 16 B o x p l o t o f the Instron T P A parameter Hardness 1 for cooked, cultured chinook salmon samples, results by treatment number  103  F i g u r e 17 B o x p l o t o f the Instron T P A parameter Hardness 2 f o r cooked, cultured chinook salmon samples, results by treatment number  104  F i g u r e 18 B o x p l o t o f the Instron T P A parameter Firmness 1 for cooked, cultured chinook salmon samples, results by treatment number  105  F i g u r e 19 B o x p l o t o f the Instron T P A parameter Firmness 2 f o r cooked, cultured chinook salmon samples, results by treatment number  106  F i g u r e 20 B o x p l o t o f the Instron T P A parameter Cohesiveness for cooked, cultured chinook salmon samples, results by treatment number  107  F i g u r e 2 1 B o x p l o t o f the Instron T P A parameter Gumminess for cooked, cultured chinook salmon samples, results by treatment number  108  F i g u r e 22 B o x p l o t by treatment number o f the p H o f t h e cooked chinook salmon samples xi  111  Acknowledgements I wish to thank the Department of Fisheries and Oceans for providing the fish for these experiments, my advisory committee for their patience and advice, and my parents and friends for their love and support.  xii  1.  Introduction Changes i n fish that result from constant high levels o f swimming have long been observed.  Recently, several researchers w o r k i n g o n this topic, talking informally, agreed that fish kept i n  flowing  water l o o k subtly healthier, for instance their skin appeared shinier, flesh seemed firmer, also their eyes seemed t o be brighter ( L o v e , 1988). This is not a recent revelation. I n 1650, Venner observed this phenomenon and w r o t e "...of sea-fish the best swimmeth in a pure sea, and is tossed and hoist w i t h the w i n d and surges; f o r by reason o f continual agitation it becometh o f purer and less slimey sic substances" (Love, 1988).  Certainly, a clear-cut demonstration o f a favourable effect o f exercise  (swimming speed) o n one or more quality attributes o f the flesh o f farmed salmon w o u l d be o f interest t o the salmon farming industry. This is because the market values o f farmed salmon can approach their cost o f production and consequently any approach that raises market value o r reduces production costs is o f importance for economic viability o f the industry. This thesis examines the effect o n the eating quality o f the cooked muscle o f cultured chinook salmon o f rearing these fish w i t h different combinations o f swimming speed and ration levels.  Sensory  analysis was used to quantitate the changes i n aroma, flavour and texture attributes. Various forms o f instrumental analysis were employed t o obtain objective measurements o f the treatment effects o n the fish.  Textural changes were quantified using the Instron Texture Profile Analysis and p H .  The  treatment effect o n the flavour volatiles was quantified by gas liquid chromatographic analysis o f purge and trap extracts.  1  2. Literature Review 2.1 2.1.1  Exercise L e v e l T y p e s o f exercise e x p e r i m e n t s Three types o f exercise tests have been performed o n fish. These include sprint, sustained  svWmming and training. Sprint swimming, otherwise k n o w n as "Burst" swimming tests, are conducted by forcing the fish t o s w i m against very h i g h water velocities for a short time; usually measured i n seconds. Sustained swimming tests, w h i c h have been the subject o f the greatest amount o f research, involve forcing the fish t o s w i m for several hours (Davison and Goldspink, 1978). The fish obtained from Fisheries and Oceans Canada, West Vancouver laboratory for m y study had undergone sustained exercise training.  I n experiments o f this type, the fish first go through a  period o f training where they s w i m against increasing currents until reaching the desired water velocity. T h e n fish maintain this swimming velocity for the duration o f the experiment. The experimenter notes any adaptive change(s) that occur (Davison and Goldspink, 1978). M o s t o f the research involving this type o f exercise training i n fish has involved salmonids i.e. salmon, t r o u t and charr. There are several reasons f o r this. First salmonids are reared commercially, they are easy t o obtain and generally respond w e l l to captivity. Second, their behaviour is predictable unlike many other species (Davison, 1989). T h i r d , salmonids respond readily to training under artificial conditions (Davison and Goldspink, 1977; Greer Walker and Emerson, 1978). Finally, since salmonids are a commercially important group o f fish, research funding is more readily available than f o r other types o f fish (Davison, 1989).  2  2.1.2  Effect o f exercise t r a i n i n g o n fish Fish do not respond to exercise training i n the same w a y as mammals. Changes i n fish do occur  as a result o f training, but they are comparatively modest.  Training has been shown t o affect fish  g r o w t h rates as w e l l as their ability to s w i m (reviewed by Davison, 1989). Unfortunately, as noted by b o t h Davison (1989) and B r o u g h t o n and Goldspink (1978), due t o their limited number, comparisons between studies are difficult.  There are several factors that  contribute t o this problem. Some o f the difficulty is due to differences i n the studies w i t h regards t o the species, size, sex and life history o f the fish ( B r o u g h t o n and Goldspink, 1978). There is also a diverse array o f apparatus that has been employed. Other areas o f disparity include the dissimilar durations and intensities o f exercise between studies and i n many studies there have been differences i n the types o f tests that have been used t o detect changes. A s a result, there is a great deal o f variability i n the results, and conflicting data are often presented (Davison, 1989).  2.1.3  Effect o f w a t e r c u r r e n t o n fish b e h a v i o u r I n many previous studies (Davison and Goldspink, 1977; Davison and Goldspink, 1978; Greer  Walker and Emerson, 1978; East and Magnan, 1987; Houlihan and Laurent, 1987; Christiansen et al., 1989) the control fish have been held in calm water in an attempt to reduce the amount o f energy required f o r l o c o m o t i o a This p r o t o c o l , however, has led t o behavioural problems, such as increased aggressive responses as manifested by biting and  fin-nipping.  Christiansen and Jobling (1990) found  that fish w i t h o u t bite marks g r e w significantly better than those w i t h evidence o f bite marks. Elevated plasma Cortisol levels w h i c h are k n o w n t o suppress g r o w t h (Pickering, 1990) and feed efficiency (Vijayan and Leatherland 1989), have also been found. I n contrast, salmonids reared i n water w i t h a  3  current, school, s w i m less randomly, and they also exhibit less aggressive behaviour (Christiansen et al. 1989; Christiansen and Jobling 1990) and have lower levels o f plasma  Cortisol adrenaline, and  noradrenaline ( W o o d w a r d and Smith, 1985). This leads to questions whether the improved g r o w t h , noted i n the studies w i t h the still water controls, was a result o f the exercise per se o r was due t o the increased energy demands and stress that accompany aggression i n the control  fish (Kiessling et al.,  1994b). Davison and Goldspink  (1977, 1978) conducted t w o experiments that address this issue.  These researchers studied the effect o f different levels o f exercise training o n the g r o w t h o f b r o w n trout, a member o f the salmonid family, and goldfish, a fish normally found in still water. these experiments the control fish were held in still water.  I n both o f  The results o f these t w o experiments were  dramatically different. I n the trout experiment, fish at the lowest swimming speed g r e w m u c h more rapidly than the control fish. Moreover, they had large stores o f glycogen and lipids and other physiological changes. The fish at the medium swimming speed had decreased f o o d utilisation due to increased energy demands. M a n y fish at the highest swimming speed were unable to survive. B y contrast, in the goldfish study the fish subjected t o exercise often g r e w less than the controls and they also consumed substantially more f o o d . Surprisingly, most o f the goldfish survived at the highest swimming speed. These preceding results higher exercise  led the researchers t o speculate that once the fish acclimates t o a  rate, elevated levels o f anabolic hormones such as thyroxine are produced.  They  theorised that the goldfish were unable t o acclimate to the flowing water w h i c h resulted i n the fish having elevated levels o f stress-related hormones such as adrenaline, noradrenaline, and  4  Cortisol.  However, they never considered the possibility that the trout control may have also been under stress. Hence, they drew some incorrect conclusions from their w o r k .  2.1.4  F u e l use f o r fish l o c o m o t i o n The fish's use o f fuels f o r locomotion can be quite complicated; protein, lipids as w e l l as  glycogen may be used as sources o f energy (reviewed by Davison, 1989). I n fish, red muscle has an aerobic f o r m o f metabolism and is used for normal l o c o m o t i o n while the white muscle is used f o r burst (sprint) swimming, or w h e n the fish is involved i n strenuous exercise. W h i t e muscle uses glycolysis o r the anaerobic degradation o f glucose to yield lactic acid and energy. The overall rise i n lactate is accompanied by a fall in glycogen concentration f o l l o w i n g exercise (reviewed by B r o u g h t o n and Goldspink, 1978). The findings o f Johnston and M o o n (1980a, b) suggest that training produces a shift towards fat utilisation rather than use o f glucose as an energy source. Davison and Goldspink (1977) had similar findings.  They observed that b r o w n t r o u t w h e n exercised at a slower swimming speed  (1.5 body  lengths / second (bl/s)) had elevated levels o f b o t h glycogen and lipids. The trout at the intermediate speed (3 bl/s) still had elevated glycogen levels, but the lipid levels had fallen. This suggests that lipids were the major source o f fuel f o r the fish at that swimming speed. White and L i (1985) also found that lipids were the primary source o f fuel during training. They f o u n d that chinook salmon at all speed by ration level combinations, except the slowest swimming speed by highest ration l e v e l experienced a net decrease i n body fat. The fish exercised at 2, 3 and 4 bl/s, at b o t h the 2.5 and 6 % ration levels (% dry body weight/day), exhibited greater decreases in their percentages  o f fat than noted for the unfed fish ( 0 % dry body weight/day).  5  Alternatively,  Kiessling et al. (1994b) found that the fat contents i n the fillet and whole bodies o f chinook salmon were not significantly affected by changing the swimming speed over the range o f 0.5-1.5 bl/s. Several studies indicate that protein may also serve as a source o f fuel f o r fish i n training experiments.  I n the Davison and Goldspink (1977) study o n trout, the protein content o f the fish  decreased as swimming speed increased. This suggested that protein might have been utilised as a fuel source. East and M a g n a n (1987) obtained similar findings w i t h b r o o k charr. I n another study, White and L i (1985) observed that chinook salmon forced t o s w i m f o r 10 days without f o o d , had decreased protein levels while their lipid levels were unaffected.  2.1.5 Effects of exercise training on fish 2.1.5.1 Increased stamina and maximum swimming speed Fish, like athletes, must be subjected t o a period o f conditioning before the full expression o f their capacity f o r swimming is realised (Farlinger and Beamish, 1978). I t has been s h o w n that training generally increases stamina and aerobic capacity (Hochachka, 1 9 6 1 ; Farlinger and Beamish, 1978; B r o u g h t o n et al. 1980) as w e l l as m a x i m u m swimming speed (Davison and Goldspink, 1977). H a m m o n d and H i c k m a n (1966) showed that conditioned fish can n o t only tolerate higher levels o f blood lactate, but they can also remove lactate  from  the b l o o d more quickly than  unconditioned fish. B o t h total accumulations o f muscle lactate during exercise and its subsequent rate o f removal during recovery were found t o vary directly w i t h the degree o f physical conditioning. Hochachka (1961) demonstrated that trained fish could acquire an oxygen debt that was three times higher than that o f untrained fish before becoming fatigued.  H e postulated that the increased  ability o f trained fish t o resist fatigue was due t o increased buffering capacity. 6  This hypotheses was  based o n the observation that the trained fish had higher haemoglobin levels.  W i t h respect to this,  haemoglobin has t w o functions, first as a carrier for oxygen, and second as a buffer.  Hochachka  theorised that i n trout, the primary function o f the haemoglobin may be t o act as a buffer, and the respiratory function might be secondary in nature during resting metabolism However, during more extreme conditions, e.g., higher exercise levels, the respiratory function may become more important. Under these conditions the increased oxygen carrying capacity w o u l d be invaluable. L o v e (1988) also postulated that other buffers may be present i n larger quantities in trained  fish.  I n addition, L o v e  (1988) speculated that i f buffers, such as anserine, were present in greater than normal quantities, there could also be an effect o n the flavour o f the fish.  2.1.5.2 Hypertrophy of fish muscle fibre W h e n a fish swims for long periods o f time, there are often significant changes i n the number and diameter o f red and white muscle fibres, w h i c h together result in increased muscle size (reviewed by Davison, 1989). Unlike mammals where the fibre number in the body remains constant, the fibre number in many species o f fish increases during their lifetime, paralleling the increases i n body size (Weatherly and Gill, 1981, 1984).  Davison and Goldspink (1977) noted that the initial size o f the  muscle fibre i n the fish w i l l determine whether the fibre splits or enlarges.  For instance, w h e n they  exercised fish w i t h small fibres, there was an increase i n fibre size. Conversely, w h e n fish w i t h large muscle fibres were exercised, there were concomitant increases i n muscle mass that were due t o an increase i n fibre number.  Similarly, Patterson and Goldspink (1976), w o r k i n g w i t h saithe, observed  that the muscle fibres split longitudinally once they reached a diameter o f 1.2 micrometers.  7  Greer Walker and Pull (1973) observed that the extent o f fibre hypertrophy in coalfish varied w i t h swimming speed. A l s o , they noted that different muscle types became active as the swimming speed was changed. I n this regard, Johnston et al. (1977), w o r k i n g w i t h carp, noted that red muscle fibres were used predominately at lower swimming speeds. However, as the speed was increased, the red fibres became progressively less important, whereas the pink, and then the white muscle fibres became increasingly more active. Collectively, the studies o n fish suggest that  white muscle is used  primarily w h e n the speed is near or above the threshold for sustained speed. A t lower speeds b o t h the red and white muscles are used (Greer Walker and Pull, 1973). Greer Walker and Emerson (1978), i n an experiment involving rainbow trout found that oxidative metabolism occurred predominantly in the red muscle at swimming speeds up t o 1.4 bl/s.  B e y o n d this, the white fibres became increasingly  active. I n another experiment, Johnston and M o o n (1980a) subjected b r o o k trout t o a water current o f 1 bl/s and noted that electrical activity occurred only in the red muscle. A t water speeds above 1.8 bl/s, however, electrical activity was exclusively observed i n the white muscle . Changes i n muscle fibre number and hypertrophy provide g o o d indications o f the different muscle types that are active at a given swimming speed (Greer Walker and Pull, 1973). Generally, red muscle fibres hypertroph to a greater extent than white fibres at any given swimming speed (Greer Walker and PulL 1973; Davison and Goldspink, 1977).  Kiessling et al. (1994b) found that the red  muscle o f chinook salmon trained at 1.5 bl/s showed significant fibre hypertrophy ( 2 5 % ) i n the rostral region o f the fish. Also, they observed i n another experiment that fish that s w a m at their critical swimming velocity every other day f o r 120 days had doubled the total red muscle area i n their caudal region. Kiessling et al. (1994b) speculated that the dissimilarity i n red muscle area between rostral and caudal regions was probably due t o the differences i n swimming patterns between the t w o experiments.  8  I n situations where swimming speed is excessive, muscle fibre hypertrophy may only be evident t o a small extent o n not at all.  For example Greer Walker and Emerson (1978) observed that red  muscle fibre hypertrophy decreased w h e n t r o u t were forced t o s w i m between 2 and 3 bl/s.  They  speculated that the reason for this response was due to the fibres contracting above their optimal frequency. Other experiments have not shown any significant increase in muscle mass i n relation t o swimming speed. Davie et al. (1986), for example, did not find any alteration i n the ratio o f total muscle t o t o t a l body weight o f trout that had been forced t o s w i m continuously at a l o w svsdmming speed for 200 days. They did, however, observe an increase in the p r o p o r t i o n o f red fibre muscle.  2.2 Physiological factors in fish affected by ration level 2.2.1 The effect of ration level on fish size Level o f dietary intake can have a profound effect o n numerous aspects o f fish physiology. N o t unexpectedly, an increase o r decrease i n ration level can significantly affect fish size (weight and length). Kiessling et al. (1991a) found significant differences i n the weights o f 1 - 2 year o l d rainbow trout maintained o n ration levels o f 50, 75 and 1 0 0 % ( 1 0 0 % ration level defined as the ration level required for o p t i m u m g r o w t h ) . Numerous other studies o n various fish species have also shown that g r o w t h is positively correlated w i t h increased ration level until feed intake becomes excessive (Kiessling et al., 1989a; Storebakken et al., 1 9 9 1 ; L i and L o v e l l 1992). Kiessling et al. (1994b) also f o u n d that chinook salmon weight increased significantly as ration level was increased from 7 5 % t o 1 0 0 % o f maximum.  9  2.2.2 The effect of ration level on the fat content of fish Fish maintained o n higher ration levels often have increased body fat content. Kiessling et al. (1994b) f o u n d that the fat content o f chinook salmon o n m a x i m u m ration was significantly higher than noted for those given 7 5 % o f m a x i m u m ration. Storebakken et al. (1991) and Kiessling et al. (1991a) also showed a trend towards increased fat accretion in trout as ration level was increased. I n contrast, Kiessling et al.(1989a) did not find any significant effect o f fixed rations ( 2 5 % , 5 0 % and 100%) o n total fat levels i n either the white o r the red muscle tissue o f trout.  They d i d find, however, that the fat  content i n the dorsal muscle fat depot rose in direct relation to ration level.  2.2.3 The effect of ration level on the protein content of fish Changes in ration level generally alter the absolute but not the relative (%) amounts o f protein in fish (Kiessling et al. 1991b; Storebakken et al., 1991). For instance, Kiessling et al. (1991b), noted that the increase i n protein content o f rainbow trout epaxial muscle was independent o f ration level once the fish had reached 50g. They also found that a profound drop in muscle protein content only t o o k place i n mature fish, and that this was only i n response t o decreased feed availability i n combination w i t h prolonged physical activity.  2.2.4  The effect of ration level onfishmusclefibresize Fibre size has been found t o be highly correlated w i t h fish size (Kiessling et al. 1989b; 1991a)  which, i n turn, is significantly affected b y ration level.  10  I n the study performed b y Kiessling et al.  (1989b), significant changes were seen in both white and red muscle fibre areas. The white muscle fibre area was positively correlated w i t h slaughter weight. Kiessling et al. (1989b) concluded from this observation that the pattern o f g r o w t h was based mainly o n enlargement o f single fibres rather than o n increases i n fibre number. There was also a shift t o w a r d dominance o f fibres w i t h larger areas as fish weight increased.  They went o n t o speculate that fibres also g r e w i n length as ration level was  increased.  2.2.5 The effect of ration level on the muscle glycogen content offish Glycogen content appears to be affected by ration level, but this relationship is less clear. Kiessling et al. (1989b), for example found that the glycogen levels i n b o t h red and white muscle o f rainbow trout increased as ration level was varied between 50 and 100%.  I n the same experiment,  however, the glycogen content o f the white muscle from trout o n the 2 5 % ration level was observed to be significantly higher than noted for trout o n the 1 0 0 % ration.  I n an experiment performed by  Storebakken et al. (1991), b l o o d glucose levels were f o u n d t o increase between the 0 - 1 % ration levels but then dropped at the 2 % ration level.  Finally, the data o f Kiessling's et al. (1991b) did not reveal  any effect o f ration level o n the glycogen content o f trout.  2.2.6 The effect of ration level on fish growth hormone levels G r o w t h hormone levels tend t o decrease as ration levels increase.  Storebakken et al. (1991)  found a 3.5-4.0 f o l d reduction i n the circulating concentrations o f g r o w t h hormone i n trout maintained o n a ration o f 2 % o f body weight relative to those deprived o f feed. Elevations i n g r o w t h hormone levels i n fish maintained o n reduced levels o f dietary intake have been linked t o depressed fat content. 11  Indeed, it is generally accepted that there is an inverse relationship between growth hormone level and fat deposition in fish (Storebakken et al. 1991).  2.3  T h e c o m b i n e d effect o f r a t i o n level a n d s w i m m i n g speed o n fish  Several studies have evaluated the combined effect of ration level and swimming speed on fish growth and other aspects of performance. One of the most recent of these was conducted by Kiessling et al. (1994b), andfishfromthis study were used for this thesis. White and Li (1985) and Leon (1986), also conducted similar projects on this theme using juvenile chinook salmon and brook trout, respectively. White and Li (1985) found that nearly all of the variation associated with growth could be accounted for by differences in level of dietary intake. In addition to the energy required for standard metabolism, which is constant at all swimming speeds, the amount of energy required for activity (swimming) increased steadily as swimming speed was raised. As a result, the fish had to ingest more feed (energy) at the higher swimming speeds to maintain their body weight. Kiessling et al. (1994b) arrived at the same conclusion.  2.4  Sensory testing  2.4.1  Quantitative Descriptive Analysis ( Q D A )  Traditionally, each food manufacturing company employed one or a group of experts to perform sensory analysis. These expert(s) judgements were relied upon in all facets of production, from the choice of ingredients, to the choice of which products were ready for release into the  12  marketplace.  I n recent years, our society has become increasingly multicultural.  markets have become m u c h more complex and competitive.  Consequently, the  The judgements o f the expert are still  useful, but there are limits t o one person's ability (Stone et al. 1974). A r o u n d 1949, the A r t h u r D. Little Co. proposed the Flavour Profile M e t h o d ( F P M ) as a means o f dealing w i t h the complex w o r l d o f f o o d flavours ( A n o n . 1963). I n this method, a small group o f judges evaluates the product together i n a conference style meeting.  Before testing, the judges  undergo some descriptive training. T o accomplish this, a broad selection o f references is presented t o the judges t o prepare t h e m for subsequent evaluation o f the intensity o f flavour and aroma attributes i n one or more test samples. F P M allows, in many cases, the successful replacement o f the individual expert by the expertise o f the group (Stone et al., 1974). Quantitative Descriptive Analysis ( Q D A ) , developed by T r a g o n Corp., has brought additional improvements t o sensory evaluation (Stone et al., 1974).  I n Q D A , trained individuals identify and  quantify the sensory properties o f a product or an ingredient (Stone et al., 1974).  This method uses an  interval scale w i t h anchor points located one half inch from each end. The panellist places a vertical mark at the point that she/he feels best represents the magnitude o f the intensity o f the attribute (Stone et al., 1974). Use o f Q D A makes it possible to statistically analyse data. For example, a researcher can p e r f o r m a one o r t w o w a y analysis o f variance to analyse individual and group performance.  He/She  could also use principal component analysis ( P C A ) to determine w h i c h are the primary sensory variables and then redundant terms could be identified and removed (Rutledge and Hudson, 1990; Stone e t a l . , 1974).  13  2.4.2  U n s t r u c t u r e d line scale Using an interval scale can lead to problems due t o the panellist's difficulty i n attributing the  same psychological w i d t h t o the various intervals o n the scale. This problem can be alleviated t o a large extent by using unstructured graphic scales that consist o f a 10 c m horizontal line anchored at the ends. The panellist response is indicated by a vertical mark o n the line (Giovanni and Pangborn, 1983). Unfortunately, as Gacula (1987) has pointed out, panellists have a tendency t o underestimate the score at the lower end and overestimate the score at the higher end o f the unstructured scale.  2.4.3  T h e d i f f i c u l t y i n t h e analysis o f sensory d a t a There are many differences between sensory and instrumental analyses (Table 1) that can create  problems i n data analysis.  Gacula (1987) found that sensory data were among the most difficult  scientific data to statistically analyse and interpret since there are often untested assumptions about the data and the analysis procedure. Some o f the difficulties are as follows. First, panellists tend t o use the scale differently. The data are relative, easily skewed, and very difficult t o replicate (Gacula, 1987). Third, panellists are prone to fatigue, time-order effects, and subject t o drifts (Pangborn, 1987).  Even  w h e n panellists have been screened and w e l l trained, there is still the chance that a panellist by treatment interaction w i l l stem from differences i n motivation, sensitivity o r psychophysical response behaviour (Lundahl and M c D a n i e l , 1990).  2.5  The measurement o f food texture Szczesniak (1963) defined texture as "the sensory manifestation o f the structure o f the f o o d  and the manner i n w h i c h this structure reacts t o the applied forces and specific senses involved being  14  Table 1 Comparative behavior  (Pangborn, 1987)  Instrumental  Sensory  Separator  Integrator  Univariate  Multivariate  Absolute  Relative  Fast  Slow  Calibratable  Difficult to calibrate  Precise  Subject to drift  Doesn't Fatigue  Fatigues, Adapts  No time-order effects  Time-order effects  Equal-interval Units  Unequal-interval units  Expensive to purchase and maintain  Expensive to hire judges  Cannot measure hedonics  Biased by hedonics  Cannot mimic sensory  Artificial to mimic instrumental  15  vision, Anaesthetics and hearing." I n 1990 she simplified this definition t o " h o w the f o o d feels i n the m o u t h o n manipulation and mastication, and h o w it handles during transport, preparation, and o n the plate" (Szczesniak, 1990). Texture for many years has been considered by some t o be an overlooked f o o d attribute. There are several reasons for this. First, there has been a lack o f government funding for research into f o o d texture. A second problem has been that off-texture is not a signal that a f o o d is unsafe, unlike attributes such as smell, colour and taste.  Finally, changes in texture are often more difficult t o  accomplish and often affect other quality parameters such as taste; these changes can not just be "added from a bottle" whereas those related to aroma and flavour can (Szczesniak, 1990). Attempts have been made to measure texture quantitatively since the 1860's.  According t o  Szczesniak (1990), the first texture measuring device was developed i n Germany by L i p o w i t z in 1861 w h i c h was an instrument, designed to quantify the consistency o f jelly. Since then, other instruments, designed t o measure textural qualities o f various types o f f o o d , have been developed and these have evolved into the instruments used today. Some examples o f texture measuring devices currently in use include, the Instron Universal Testing Machine, the General Foods Texturometer, and the Brabender Farinograph. According t o Szczesniak (1963), textural measurements can be grouped into three types o f characteristics:  (1)  mechanical,  (2)  geometrical  and  (3)  other  characteristics.  Mechanical  characteristics result from pressure exerted o n the teeth, tongue and r o o f o f the m o u t h during eating. These characteristics include the hardness, cohesiveness, viscosity, elasticity and adhesiveness, etc. o f the f o o d . Geometrical characteristics are related to the size, shape, and arrangement o f the particles  16  w i t h i n a f o o d (Brandt et al., 1963).  The last group is 'other' characteristics w h i c h includes mainly  moisture and fat; qualities concerned w i t h lubricating properties o f the f o o d product. Brandt et al. (1963) developed the Texture Profile M e t h o d ( T P A ) , patterning it after the flavour profile method developed by Cairncross and Sjostrom (1950). They used the standard rating scales developed by Szczesniak (1963), and systematically examined various textural attributes, breaking them d o w n into initial (textural attributes perceived o n the first bite), masticatory (perceived during chewing), and residual characteristics (changes that occur during mastication).  In TPA,  additional scales can be added t o enable the judgement o f moisture and fat content (Brandt et al. 1963). T P A requires that the panellists be trained thoroughly w i t h respect t o the texture classification system and the use o f standard evaluation procedures for assessment o f the product.  Panellists must also  become reliable i n recognising and identifying the degrees o f each characteristic (Brandt et al., 1963).  2.5.1  Instron Universal Testing M a c h i n e The Instron is an instrument designed t o study stress-strain properties o f materials (Bourne,  1982). I n addition t o f o o d , it can also be used to study texture i n other materials such as fabric, metals, w o o d , rubber, plastics, etc. (Bourne, 1982).  W i t h an assortment o f accessories available (Bourne,  1982), this machine can p e r f o r m various types o f tests such as penetration, shear, bending, compression, and extension (Segars and Kapsalis, 1987). The Instron generates b o t h force-time and force-distance curves, allowing w o r k function to be calculated i n pounds, kilogram, o r N e w t o n ' s (Bourne, 1978). The curve(s) can become the basis f o r calculating various mechanical properties o f the material.  These values may be used t o correlate or  predict sensory response t o texture (Segars and Kapsalis, 1987).  17  2.5.2  Instrumental Texture Profile Analysis ( T P A ) T P A was a major breakthrough in the quest to produce a machine that could imitate  mastication. The General Foods Texturometer attempts to imitate mastication by twice compressing a bite sized piece o f f o o d to 2 5 % o f its original height; mimicking a person taking t w o bites. F r o m this, a force-time curve is produced w h i c h captures the entire force history o f this simulated masticatory action. Several textural parameters can be determined from the force-time curve. described seven parameters; five measured and t w o calculated. fracturability,  hardness, cohesiveness, adhesiveness, and  Bourne (1978)  The measured parameters include  springiness. The t w o calculated parameters  are gumminess and chewiness (Bourne, 1978). Firmness may also be calculated from the curve by measuring the m a x i m u m slope o n each compression cycle (Durance and Collins, 1991).  2.5.2.1  T P A o n cooked salmon I t is difficult to obtain meaningful, reproducible instrumental texture measurements o n cooked  fish.  M o s t o f the devices that are commonly used in the rheological testing o f foods, even those that  are used for red meat, are generally unsuitable f o r fish. D u r i n g eating, almost all o f the energy required to prepare the fish f o r swallowing is used for mastication.  A s a result, instrumental testing o f fish  samples needs to measure resistance o f the muscle fibres to mechanical disintegration (Dunajski, 1979). Durance and Collins (1991), and Reid and Durance (1992) examined textural changes o f canned late r u n salmon by using Bourne's (1978) T P A and an Instron Universal Testing Machine ( M o d e l 1122, Instron Corp. Canton M A ) . I n their experiments, a modified syringe was used t o f o r m cylinders o f flaked fish o f u n i f o r m size.  Using samples composed o f thoroughly flaked fish, they  managed t o gain a greater degree o f homogeneity between replicates. Borderias et al. (1983) tested  18  b o t h minced fish and intact fillets using various Instron attachments and found a higher coefficient o f variation for the fillets relative to the minced fish. They speculated that this occurred because w h e n the force o f compression was applied to the fish fillets, the myotome layers slid away from the force. Hence, it was more difficult to obtain reproducible results in separate detenninations.  2.6  T h e factors affecting t h e t e x t u r e o f cooked fish  2.6.1  T h e effect o f p H o n t h e t e x t u r e o f cooked fish L o v e (1988) and Dunajski (1979) both stated that the p H o f fish muscle is probably the most  important factor affecting the rheological properties o f a given muscle. L o v e (1988) postulated that muscle from exercised fish w o u l d have a lower post-mortem p H due t o an elevated glycogen content. H e went o n t o theorise this w o u l d lead to firmer muscle texture. Feinstein and B u c k (1984) f o u n d a linear relationship between p H and the texture o f flounder but only in the head section o f the fish. They also looked for a relationship between p H and texture in cusk without success. A s w i t h most animals, after the death o f a fish, glycogen is degraded t o lactic acid via the Embden-Meyerhof-Parnas glycolytic cycle. This causes the p H i n the muscle t o fall dramatically w i t h i n the first several hours post-mortem. I n most species o f fish, the final p H is usually around 6.5 - 6.2, but is also can be as l o w as 5.4. A s the p H approaches the isoelectric point o f the myofibrillar  proteins,  there is a change in the ionisation o f the polar groups o f the protein molecules. Originally these were negatively charged but after death they become neutral. This causes a decrease i n the repelling forces between proteins, resulting in a tightening in the protein structure. A s the myofibrillar proteins become more concentrated, the muscle becomes increasingly tougher and drier. Dunajski (unpublished), f o r  19  example, noted that there was a 2.5 fold increase i n the toughness o f the fish as the p H changed  from  6.7-5.7(Dunajski, 1979).  2.6.2  T h e effect o f muscle f i b r e size o n t h e t e x t u r e o f cooked fish Fibre size has also been found to affect the texture o f fish muscle. K a n o h et al. (1988), i n an  experiment using yellowfin tuna, found that the fish had a firmer texture w h e n the fibre diameter was less than that o f ordinary muscle. Hatae et al. (1990), d r e w the same conclusion after examining the role o f muscle fibre contribution to firmness i n the cooked flesh o f five species o f fish. Dunajski (1979) reported an increase i n the coarseness o f the muscles w h e n the diameter and length o f fibres increased.  2.6.3  T h e effect o f t h e level o f connective tissue on t h e t e x t u r e o f cooked fish Unlike red meat, connective tissue i n fish muscle is present i n l o w quantities and hence does  not play an important role in the texture o f fish. Collagen is thermally denatured during cooking and as a result generally has very little influence o n fish texture. The texture o f muscle after cooking is more a consequence o f the state o f the myofibrillar proteins (Dunajski, 1979). Hatae et al. (1990) did, however, report an effect o f muscle collagen content o n texture.  In  this regard, they observed that w h e n c o o k e d fish tissue was masticated the coagulated proteins tended to impede the sliding o f the muscle fibres over each other. F r o m this they concluded that, i n fish w i t h a lower muscle collagen content, the muscle fibres slide more easily over one another, softer texture.  20  resulting i n a  2.6.4  T h e effect o f t h e fat c o n t e n t o n t h e t e x t u r e o f cooked fish Fat content can also affect the texture o f fish samples. Samples o f fish muscle w i t h a higher fat  content are often perceived as being more tender (Dunajski, 1979). Dunajski (1979) explained that, among other post-mortem changes i n fish, the liquid neutral lipids are immobilised by the physical structure o f the muscle.  This tends t o dilute the structural elements and decrease the overall  mechanical strength o f the fish meat.  A higher fat content w i l l also impart an oily mouthfeel  (Szczesniak, 1963).  2.7  Gas C h r o m a t o g r a p h i c f l a v o u r volatile analysis  2.7.1  P u r g e a n d t r a p analysis The principle behind purge and trap extraction is quite simple. First, the sample is placed i n a  sealed container that is flushed w i t h an inert gas. This gas then passes t h r o u g h a trap containing a small amount o f adsorbent, such as tenax, w h i c h retains the volatiles. Following the extraction, the trap is removed and adsorbed compounds are eluted w i t h a small amount o f solvent, and a sample o f this is then analysed by gas liquid chromatography ( G C ) (Gilbert, 1990).  Heikes and Hopper (1986) outlined several advantages o f this method. First, it is n o n labourintensive and may be carried out unattended.  Furthermore, it does not require highly specialised  equipment (though n o w available); a suitable apparatus can be constructed quite simply w i t h materials already available i n most analytical laboratories. The extract is concentrated and relatively clean. The limits o f detection that can be achieved are m u c h l o w e r relative t o solvent extraction o r static headspace analysis; b o t h G C quantitation at l o w parts per billion and sub-parts per billion levels as w e l l  21  as G C / M S corifirmation are possible. Generally, this technique provides an inexpensive alternative to other methods (Olafsdottir et al., 1985). There are conflicting opinions as to who originally developed purge and trap extraction. According to Gilbert (1990), this method was developed by Heikes in 1985 for the analysis o f ethylene dibromide in grains.  However, it is noteworthy that Josephson and co-workers published an  experiment in 1983 that described the use o f purge and trap extraction to identify aroma compounds from fresh white fish. In recent years, this extraction method has been employed in several studies that have examined volatiles produced by several types o f seafood. For example, Josephson et al. (1983, 1985, 1991) used purge and trap extraction to study the volatiles produced by fresh seafood such as fresh Whitefish, Great Lakes salmon and Atlantic and Pacific oysters. Shamaila et al. (1995) also used this technique to evaluate the volatiles in Pacific ocean perch (Sebastes alutus).  2.7.1.1  The effect of fat content on purge and trap extractions Some studies have shown that the fat content o f a food product undergoing purge and trap  extraction affects the amount and types of compounds found, while others have not. Heikes (1985), in a study on the determination o f ethylene dibromide (EDB) in table-ready foods, where various foods ranging from boiled cabbage to chocolate cake icing were spiked with E D B , did not find that fat content had an effect on the efficiency o f the extraction. Persson and von Sydow (1973), on the other hand, in their study of the aroma o f canned beef did find that fat content affected the amount and types o f compounds found. It was also noted that when frit was added to some samples, some volatile compounds were more lipid soluble than others and consequently they were detected to a lesser  22  degree. Also, in those samples, some compounds such as straight chain aldehydes, furan and 2-methyl furan were detected i n higher concentrations; possibly because fat is a precursor f o r those compounds.  2.7.1.2  T h e choice o f T e n a x G C , a p o r o u s p o l y m e r , f o r use i n p u r g e a n d t r a p e x t r a c t i o n Tenax G C (2,6-diphenyl-p-phenylene oxide polymer) is one o f a group o f porous polymers that  are often used in research because o f their ability t o trap organic compounds. W h e n the sample o f gas is passed through the porous polymer, the organic compounds are retained and concentrated.  Once  these compounds are collected, they can either be thermally desorbed or eluted w i t h a solvent (Butler and B u r k e , 1976; Olafsdottir et al. 1985). Butler and B u r k e (1976), looking at the capacities and efficiencies o f several porous polymers, concluded that no single porous polymer was universally suitable. One needs t o examine the pros and cons o f each and then choose the polymer that is most suitable for the application.  Tenax G C has  emerged as a widely used porous polymer for f o o d , beverage, and environmental applications (Olafsdottir et al. 1985).  I t is particularly advantageous for samples consisting o f only high boiling  point components (Butler and B u r k e , 1976; Jennings and Fisloof 1977). This is due to this polymer's high temperature limit and relatively l o w retention volumes, w h i c h allow the trapped compounds to be desorbed more rapidly than from other adsorbents (Butler and B u r k e , 1976). I n addition, it also has the advantage o f having shorter recovery times (Jennings and F i l s o o f 1977).  2.7.1.3 T h e e l u t i o n o f a d s o r b e d volatiles f r o m p o r o u s p o l y m e r s w i t h e t h y l e t h e r Olafsdottir et al. (1985) examined the reproducibility and absolute recoveries o f volatiles  from  Tenax G C desorbed w i t h ethyl ether. They f o u n d that this method resulted in a variability in analysis o f 23  less than 2 0 % w h i c h is comparable t o other adsorbents and solvents. Thus, this procedure was f o u n d to be suitable for many objectives and applications.  2.7.2  T h e r e l a t i o n s h i p between Gas C h r o m a t o g r a p h y ( G C ) d a t a a n d q u a n t i t y a n d i n t e n s i t y j u d g e m e n t s f r o m t r a i n e d sensory panellists Persson et al. (1973 a, b) and v o n S y d o w et al. (1970) were among the first researchers t o  present a clear-cut relationship between quality and intensity judgements from a trained panel and the G C / M S output for the product. Comparisons o f these t w o sources o f data are, however, not w i t h o u t their pitfalls.  V a n Gemert et al. (1987) found that the relationship between sensory analysis and  chemical, physical and instrumental parameters was complex.  The characteristic aroma o f a f o o d is  often not the result o f one compound alone, but rather, results from an interaction between several compounds. Consequently, an increase o r decrease in only one odour compound might result i n b o t h increases and decreases in several o f the sensory odour qualities ( v o n S y d o w et al., 1970). There is a second difficulty in comparing these t w o types o f data. Sometimes compounds that are highly correlated w i t h flavour w i l l not be flavour substances, although, more commonly they w i l l be (Powers and Keith, 1968). A t times this can occur i f more than one compound co-elutes, or i f the compounds responsible for the aroma are being adsorbed by an active site near the exit port and wash o f f w h e n a major peak elutes (Williams and Tucknott, 1977).  24  2.8  Statistical analysis  2.8.1  The box plot The b o x plot, first introduced i n 1977, has proven t o be an effective means o f producing a  visual summary o f data. In this type o f plot, there is a box that is divided w i t h a horizontal line, and there are also t w o protruding "whiskers" that extend vertically from the t o p and b o t t o m o f the box. The box p o r t i o n is comprised o f the t w o middle quartiles o f data, that is, the data that falls between the 25  th  and 7 5  th  percentiles. This interquartile range is computed as  measure the amount o f variation in the data.  IQR=  Q0.75  - Qo 25, w h i c h serves t o  A horizontal line splits the b o x at the median ( 5 0  th  percentile). The lower whisker is defined as the smallest observation that is greater than o r equal to the lower quartile minus 1.5 X IQR.  Similarly, the upper "whisker" is defined as the largest observation  that is less than or equal t o the upper quartile plus 1.5 X . IQR, w h i c h may be the upper extreme o f the data. A n y values that fall outside this range are considered t o be outliers and are plotted as individual points ( M a , 1992).  2.8.2  Principal component similarity (PCS) PCS is a technique that was developed by combining principal component analysis ( P C A ) , a  data compression method w h i c h is based o n identifying the most important directions o f variability in a multivariate data space, w i t h pattern similarity. I n PCS, principal component (PC) scores are used for computing pattern similarity constants instead o f using the original data directly ( V o d o v o t z et al., 1993). Furtula et al. (1994b) considered PCS t o be an extended version o f P C A . PCS can utilise the information o n variation o f the P C A principal components f o r classification purposes ( V o d o v o t z et al., 1993).  25  W h e n V o d o v o t z et al. (1993) compared PCS w i t h P C A and other types o f multivariate analyses they found that they compared favourably. than P C A .  Also, they found that P C S had better resolution  PCS can also graphically illustrate a larger number o f computed P C outputs than P C A  (Furtula et al. 1994b). I n P C A it is customary to use only 2 PC scores for a 2-dimensional ( 2 - D ) P C plot o r three P C scores for a 3-dimensional ( 3 - D ) plot. H o w e v e r , portions o f the original data that may have been important for classification can be ignored. This is particularly true i n flavour analysis where it is not u n c o m m o n for seemingly minor compounds to play an important role i n creating characteristic flavour notes. W i t h PCS, results from numerous PC scores can be displayed graphically i n a 2 D figure, minimising this problem ( V o d o v o t z et al., 1993). PCS is most useful w h e n the size o f the data matrix is large (Furtula et al. 1994a).  The  advantages offered by P C S diminish as the number o f P C scores for computation decreases (Furtula et al. 1994b).  26  3.  Materials and Methods  3.1  E x p e r i m e n t a l c o n d i t i o n s used i n t h e r e a r i n g o f s a l m o n used i n t h i s s t u d y The fish f o r this research were obtained from the Department o f Fisheries and Oceans, West  Vancouver Laboratory, West Vancouver, B . C . A total o f 660 salmon were used in the Fisheries and Oceans experiment. The all-female seawater-adapted hatchery-raised one year o l d Qualicum chinook salmon had been selected f o r u n i f o r m size and they originated from Sea Spring Salmon F a r m L t d . (Chemainus B C , Canada).  Before commencement o f the study, the fish were divided equally into 12  groups o f 55 fish. E a c h group was placed into a separate outdoor fibreglass tank. Each o f the 4 m circular tanks was fitted w i t h a 1.5 m diameter inner fibreglass h o o p to create 3  a circular swimming channel that was 45 c m w i d e and 55 c m deep. One h a l f o f the tank was covered w i t h plastic netting while the other half was covered w i t h black nylon cloth. This allowed the fish t o choose between dark and light areas. T o create a current, the seawater was pumped into each tank through a vertically placed pipe that was equipped w i t h three horizontally oriented pipes. A l l the tanks were designed w i t h a f l o w through system and there was no recirculation o f water (Kiessling, et al. 1994b). A two-by-three factorial design w i t h t w o ration levels ( m a x i m u m ration = R L 1 0 0 , 7 5 % o f m a x i m u m ration = R L 7 5 ) and three swimming speeds (SS), (0.5, 1.0, and 1.5 body lengths bl/s) w a s used (Table 2). Duplicate groups o f f i s h were assigned to each o f the six treatments. The actual amounts o f feed that the fish ingested varied between swimming speeds. A s the SS level was increased, the fish o n the R L 1 0 0 protocol, w h i c h were fed to satiety, consumed more feed.. A t each SS, the R L 7 5 fish were given 7 5 % o f the ration that was given t o their R L 1 0 0 counterparts (Kiessling, et al. 1994b). 27  Table 2  Experimental design used by Kiessling et al (1994 a, b) to assess the influence of sustained exercise and two ration levels on growth of chinook salmon in seawater. A 2 X 3 factorial design was used with two ration levels and three swimming speeds and their treatment numbers have been used as identifiers in statistical analyses.  Treatment No. 1 2 3 4 5 6  Ration Level 75 75 75 100 100 100  3  Swimming Speed (bl/s) 0.5 1.0 1.5 0.5 1.0 1.5  Ration levels: RL100 (100% ration level) is a ration sufficient for satiation, RL75 is 75% of the RL100 at a given swimming speed a  28  Careful records o f daily feed waste were maintained i n each case and these allowed accurate estimations o f the actual rations consumed.  A l l fish, irrespective o f treatment, were fed 4 to 6 m m  B i o d r y 2500 pellets (Bioproducts Inc., Warrenton, Oregon, U S A ) . The mean levels (% o f d r y matter) o f protein, lipid and ash in the B i o d r y pellets were 52.0, 20.2 and 12.6, respectively. For the purposes o f this thesis, five representative fish were removed from each tank at the end o f the 212-day study. Subsequently, the fish were killed by a b l o w to the head. The salmon were then filleted, labelled, and v a c u u m packaged i n mylar film. Thereafter, the packaged fillets were placed into a - 35°C freezer pending analysis months later. Fillets from the left side o f the fish were used for sensory analysis, whereas those from the right side were reserved for instrumental analysis.  3.2  Sensory A n a l y s i s  3.2.1  T h e selection o f sensory panellists f o r Q D A analysis Seven panellists, three men and four w o m e n , were recruited from the staff and students o f the  U B C F o o d Science Department.  A l t h o u g h seven people were trained, only six people could be  accommodated in any one sensory test due t o the limited amount o f sample. The extra trained person was available in the event that a regular panellist was unable t o attend a session. Interest i n the experiment and the availability o f personnel were the main criteria used for panel selectioa  I t was also important that the selected panellists generally liked t o eat salmon.  The  individual's experience o n sensory panels was also considered to be an asset. I t was fortunate that most o f the panel members had some previous sensory panel experience.  29  3.2.2  Sensory p a n e l t r a i n i n g Panel training was carried out over a three week period, during w h i c h the panellists met five  times. Training consisted o f first gathering descriptive terms from the panellists in a round table format followed by eliminating the less distinguishable or redundant terms.  That procedure produced 27  terms t o describe the aroma, flavour, and texture, as w e l l as the overall acceptability o f the salmon. Agreement was then reached amongst the panellists regarding the scoring o f those attributes. B o t h w i l d and farmed Spring (chinook) salmon were used i n the training sessions. These fish were purchased at a local fish market and they were o f similar size to the experimental  fish.  The  purchased fish were thought to be the best sources o f the characteristic flavour, texture, and aroma extremes needed for training. The w i l d salmon were assumed to have been m u c h more active and less w e l l fed than their pen-reared counterparts. The market fish were filleted. Subsequently, the fillets were vacuum packaged i n a barrier film, and then frozen at -35 ° C until the day before they were needed f o r analysis. A t that time they were placed i n a -4 °C freezer to partially t h a w overnight.  Samples o f approximately the same size  (approximately 1 c m by 3 cm) were wrapped in foil, and baked at 190 °C for approximately 15 minutes, o r until they were cooked before being presented t o the panellists.  3.2.3  T h e selection o f sensory a t t r i b u t e t e r m s The panellists were asked to list as many aroma, taste and texture notes as they could detect i n  the cooked salmon samples. W h e n a panellist observed a flavour note, all the panellists w o u l d retaste the samples, looking for that sensory note. They then discussed their individual observations. This list  30  o f attributes that was compiled was subsequently reviewed by the panel f o r the purpose o f eliminating the terms that were either ambiguous, o r redundant. The terms were defined b y the panellists t o ensure that all the panellists were measuring the same sensatioa Whenever it was possible, reference samples f o r the attributes were provided t o aid i n clarifying the terms f o r the panellists. F o r example, boiled milk, boiled potato, and seaweed samples were provided to the sensory panel as a reference f o r the corresponding aroma terms.  3.2.4 Ballot familiarisation by sensory panellists Additional training sessions were necessary to allow the panellists to become familiar w i t h using the ballot. A f t e r selection o f the terms f o r the study, a sensory ballot was produced and copies were presented t o the panellists. They were then given fish samples and asked to rate t h e m using this ballot and then discuss their scores. This procedure enabled assessment o f whether each panel member was using the same intensity scale i n the prescribed manner. One source o f disagreement i n the panellists responses occurred w h e n one o r more o f the panellists d i d not have the same conceptualisation o f a particular descriptive t e r m  I n this situation,  every effort was made t o clarify the t e r m in question. This was sometimes accomplished by producing a reference, o r b y having the panellists discuss the sensation amongst themselves. I f it became apparent that a t e r m was ambiguous, o r that there w o u l d never be any real agreement among panellists, the t e r m was discarded.  31  3.2.5  T h e use o f composite samples The training sessions also provided a f o r u m for the panellists to express any ideas that they felt  could improve the panel's performance.  One suggestion was t o construct composite samples  from  each fish, by combining slices from the anterior, middle and posterior sections o f the fillet before cooking. D u r i n g the course o f the first f e w training sessions it had become apparent that the intensities and profiles o f the sensory attributes changed substantially between the different portions o f the fillet; the anterior p o r t i o n being m u c h more  flavourful  then the posterior portion.  This finding is in  agreement w i t h Johnsen and K e l l y (1990) w h o found that anterior and posterior portions o f fish could have quite disparate flavour profiles.  3.2.6  Sensory p a n e l set-up Steps were taken to eliminate as many sources o f error as possible that may have influenced the  panellists perceptions. For instance the panellists were asked t o refrain from various activities such as drinking coffee, wearing after-shave, or perfume (Rutledge and Hudson, 1990). Since the appearance o f the fish was not being tested, the sensory testing was conducted under red lights t o mask any variation in appearance and, thus reduce the risk o f panellist bias. T o avoid any carry over o f  flavours  from one sample t o another, the panellists were given distilled water and unsalted crackers to help cleanse their palate between samples. A s m u c h as possible, background sound was kept t o a m i n i m u m t o prevent this from disturbing the panellists.  32  3.2.7  Sensory p a n e l session s c h e d u l i n g Sensory tests were performed o n ten different days.  each o f these days.  A l l six treatments were represented o n  Originally it was planned t o have one session per day during w h i c h all the  treatments and a reference w o u l d be rated. A f t e r the first session, however, it was found that having the task o f assessing samples from six treatments and a reference f o r 28 attributes at one sitting resulted in some o f the panellists becoming fatigued and making errors.  Steps were then taken t o adapt the  procedure t o reduce panellist error. I n this regard, it was decided that three o f the treatments w o u l d be selected at random for the morning session, and the remaining three were set aside for the afternoon. A reference was evaluated by the panellists at both the morning and afternoon sessions. Sometimes, a panellist w o u l d be unable t o attend one o f the t w o sessions o n a given day. W h e n this situation arose, the panellist(s) w o u l d rate b o t h sets o f samples at the session they attended. T o accomplish this the panellist was given a short break f o l l o w i n g the scheduled sensory panel session and then he/she was presented w i t h the samples from the session that could not be attended. A l l the sensory panels were carried out between June and August, 1992. Seven sensory panel days were carried out i n a t w o and a h a l f week period. This was followed by a three week break. A f t e r the break, the panellists participated i n a training session t o ensure consistency in judgements between the t w o periods. The three remaining sessions were then held over a one week period.  3.2.8  T h e p r e p a r a t i o n o f samples f o r sensory panels O n the day before a panel session was t o take place, one fillet from each o f the six treatments  was selected at random and transferred along w i t h packages o f reference fish, from the -35°C freezer  33  t o one set at -4°C. This allowed the fillets t o partially t h a w overnight so that they could be sliced the next day o n a meat slicer without becoming t o o mushy. The fillets samples were removed from the -4°C freezer just prior to being sliced using a Hobart meat slicer to a thickness o f approximately 3 m m . D u r i n g the slicing o f each fillet, at least 6 slices were taken from each o f the three sections referred to above i.e., anterior, middle and posterior. Slices from each section were then randomly distributed into six piles o f slices o n pieces o f aluminium foiL and efforts were made t o ensure that the six samples were as similar as possible.  The six  aluminium foil sheets were labelled previously w i t h a three digit r a n d o m code and samples from the same fillet had the same number inscribed, using a permanent ink felt marker. The dull side o f the foil was always o n the outside. Care was taken to pile the slices from the various sections so that the skin faced the same d i r e c t i o a  The samples w o u l d , once cooked, have the appearance o f a solid piece o f  fish. Samples for b o t h the morning and afternoon sessions were prepared i n the morning before the first panel sitting.  The afternoon samples were stored i n a 4 ° C cooler prior t o being c o o k e d and  presented t o the panel. The f o i l wrapped samples were placed o n a f o i l pan and placed i n a 190°C oven for 15-20 minutes. T h e n the cooked samples were served to the panellists as promptly as possible. Frequently, it was difficult t o have all six panellists assembled w h e n the samples were ready, even w h e n they were notified just prior to the fish being served. W h e n it was k n o w n that a panellist was going t o be a f e w minutes late, the samples were left over the vent from the oven to keep them w a r m .  34  3.2.9  T h e reference samples used d u r i n g sensory panels The reference consisted o f a composite sample from the fillets o f numerous farmed Spring  (chinook) salmon that had been obtained commercially from a local seafood market. These fillets were sliced t o u n i f o r m thickness using a meat slicer. A f t e r this, the slices were divided into four groups: the anterior, anterior and back midsections, and posterior o f the fillet. The slices from the corresponding sections o f all the fillet were pooled together and mixed thoroughly. Following this they were v a c u u m packaged w i t h each bag containing at least 12 slices o f fish. The packages were numbered either 1, 2, 3 or 4 depending o n w h i c h section o f fillets had been enclosed. A l l o f these packages were placed in a -35°C freezer. One package from each section was removed the day before a sensory panel session and.subsequently these were placed i n a -4°C freezer over night. O n the morning o f the sensory panel day, composite samples were prepared from the fish slices. Slices from each section were evenly distributed t o twelve samples i.e., six reference samples f o r the morning and six f o r the afternoon session.  The reference samples were wrapped i n f o i l and  labeUedwithan"R".  3.2.10  T h e s a m p l i n g p r o c e d u r e e m p l o y e d b y panellists The sensory tests were performed in the sensory panel r o o m located in the U B C F o o d Science  Building. The panellists first rated the reference and then the treatment samples i n r a n d o m order for the aroma attributes. The reference sample, and then the treatment samples were j u d g e d o n the remaining attributes.  Testing all the samples first for aroma helped t o insure that all the samples w o u l d be close  35  t o the same temperature.  This sequence was important because as the fish cools the amount o f  volatiles given o f f decreases. The panellists were asked t o take a f o r k full o f fish, including portions from all the slices i n the composite sample. This f o r k f u l o f fish was then placed i n the m o u t h and chewed. The panellists then evaluated the sample for the various taste and texture attributes. I f necessary, the panellist could take a second o r third forkful. The panellists then either expectorated or swallowed the fish. Distilled water was provided to the panellists t o rinse their mouths, and unsalted crackers were provided t o help cleanse the palate between samples.  3.2.11  G e n e r a t i o n o f n u m e r i c a l scores f r o m t h e sensory b a l l o t j u d g e m e n t s The ballot (Fig. 1) used b y each o f the panellists consisted o f a 10 c m unstructured scale  anchored w i t h a t e r m at both ends for each o f the attributes being tested. The panellists were asked t o indicate their score by placing a vertical line through the scale at the appropriate spot.  A numerical  score could then be generated by measuring w i t h a metric ruler from the left side o f the scale to the point where the panellist's line crossed the line.  3.3  I n s t r u m e n t a l analysis o f cooked s a l m o n samples  3.3.1  P r e p a r a t i o n o f s a l m o n samples f o r i n s t r u m e n t a l analysis D u e t o time and equipment constraints, it was only possible to p e r f o r m instrument analysis o n a  m a x i m u m o f t w o samples per day. Late i n the afternoon o n the day prior t o analysis, the salmon were placed i n a freezer that was set at -4°C.  fillets  This allowed t h e m to t h a w slightly overnight w h i c h  36  Figure 1  Sensory ballot that was used t o evaluate samples o f cooked, farmed chinook salmon A r o m a Profiling Score Sheet  Name:  Date:  Sample Number: Instructions 1. P E R F O R M A R O M A P R O F I L I N G O N A L L S A M P L E S B E F O R E M O V I N G O N T O F L A V O U R A N D TEXTURE PROFILING 2. O p e n the f o i l wrapper and smell contents, flake the fish i f necessary t o release more o f the aroma 3. M a r k the horizontal lines w i t h a vertical Erie t o indicate the intensity o f each o f the odours listed. 4. Record any meaningful observations either o n the b o t t o m o r the back o f the page. 4. D O U B L E C H E C K T O M A K E S U R E T H A T T H E S A M P L E N U M B E R R E C O R D E D O N T H E SCORE SHEET IS CORRECT. Seaweedy  1  "  1 very seaweedy  none  Boiled m i l k  1  1 1  1  none  Boiled potato  strong  1 1  1 1  none  Lemony  - strong  1 1  1 1  none  Sour  very lemony  1 1  1 1  none  Fishy  very sour  1 1  1 1  none  Chickeny  very fishy  1 1  1 1  none  Oily  very chickeny  1  1 I  1  very oily  1 oily not Fresh  old  fresh  37  1  Flavour Profiling Score Sheet Instructions 1. Taste the sample (note: texture analysis can be performed simultaneously) 2. M a r k the horizontal lines w i t h a vertical line t o indicate the intensity o f each o f the flavour terms listed below. 3. W r i t e any additional comments o n the b o t t o m o r back o f the page.  Flavour  1  1 intense  weak  Fishy  1  1 1  1  none  Earthy  very fishy  1 1  1 1  none  Papery  very earthy  1 1  1 1  none  Bitter  very papery  1 1  1 I  not bitter  Sour  very bitter  1 1  1 1  not sour  Lemony  very sour  1 1  1 I  not lemony  Salty  very lemony  1 1  1 1  not salty  Spicy  very salty  1 1  1  1 very spicy  not spicy  Brothy not 1 brothy  very brothy  38  Texture Profiling Score Sheet Instructions 1. C h e w sample. 2. M a r k horizontal line w i t h a vertical line t o indicate the intensity o f each o f the texture terms listed. 3. W r i t e any additional comments o n the b o t t o m o r back o f the page. 4. P L E A S E R E C H E C K A N D M A K E S U R E S A M P L E N U M B E R I S C O R R E C T . Moistness dry  very moist  not powdery  very powdery  not flaky  very flaky  firm  soft  not chewy  very chewy  loose mass  compact mass  not sticky  very sticky  not mushy  very mushy  Powderiness  Flakiness  Firmness  1  Chewiness  Cohesiveness  Adhesiveness  Mushiness  Overall Acceptance extreme dislike  extreme like  39  facilitated easier handling. cubes.  O n the day o f the test, the fillets were skinned and chopped into 1 c m  3  T o ensure that there w o u l d be sufficient sample for a m i n i m u m o f three replicates o f each  treatment f o r G C , Instron, and p H analysis, all the fish cubes from the same treatment were pooled. These cubes were mixed thoroughly to ensure that the samples were uniform. T o avoid having any refreezing o f the samples, the instrumental analyses were performed o n the same day that the fillets were removed from the freezer. The fillets were divided by weight into the sample sizes that were required for the various experiments. These samples were wrapped i n aluminium foil, dull side out, and stored i n a 4 °C cooler until required. A t that time the foil packages were placed i n a preheated 190 °C oven and baked until the fish muscle was no longer translucent (15-20 minutes).  3.3.2  G C headspace analysis o f cooked s a l m o n samples G C Headspace analysis, after using a purge and trap extraction, was performed o n the cooked  samples. D u e t o limitations in the amount o f fish available from each treatment, it was only possible to p e r f o r m this analysis i n triplicate. A series o f experiments that were designed to select the most appropriate conditions f o r this purge and trap extraction were performed before the test samples were analysed. The five variables examined were: sample size, extraction temperature, extraction time, nitrogen flow rate, and the amount o f Tenax packed into the traps.  T o determine the right l e v e l samples were extracted at  various levels w i t h i n a realistic range e.g. extraction times o f 1, 2, 3, and 4 hours were used. The four variables not being examined i n a particular experiment were kept at a moderate level (which, coincidentally, turned out to be the levels chosen for this study).  40  Once the results from these  experiments were graphed, the best level was determined based o n the level o f volatiles extracted and the size o f the increase in recovery between levels. For choice o f temperature and the extraction time, the risk o f sample loss due to moisture build-up i n the Tenax G C also was taken into account. I t was concluded that the best conditions for conducting this extraction were: a sample size o f 200 g, an extraction temperature o f 70 ° C , an extraction time o f 3 hours, a f l o w rate o f 50 ml/min, and 120 m g Tenax G C . (conditions used are summarised i n Table 3). Three 200 g foil packages o f the chopped fillets were prepared from each treatment and these were placed in a 190 °C oven until cooked (approximately 20 minutes). Once cooked, the fillets were gently flaked w i t h a f o r k and then promptly deposited, along w i t h any liquid that was released during cooking, into prewarmed 1 L extraction vessels (Wheaton, Millville, N J ) . The six extraction vessels were maintained at 70 °C w i t h a waterbath (Haake FS). The water passed from the waterbath through plastic tubing into a copper pipe that connected all the extraction vessels i n parallel. The water exited from the vessels in a similar fashion and was recirculated back t o the waterbath.  This was designed to ensure that all the vessels w o u l d be maintained at the same  temperature. The internal standard used i n this experiment was tetradecane (purity: 9 9 % , Aldrich) that was +  dissolved i n diethyl ether (spectranalyzed grade, Fisher Scientifie)(l: 10 v/v). A f t e r removing the tenax trap assembly, 20 microlitres o f this standard was injected into the extraction vessel t h r o u g h the open side arm. The trap assembly was promptly replaced and the extraction vessel was closed tightly. T h e vessels were left t o equilibrate for 30 minutes. A f t e r this time had expired, the volatile compounds that were produced b y the cooked fillet were purged from the flask w i t h prepurified N2 gas ( U H P grade, Linde U n i o n Carbide) into the tenax G C trap.  41  Table 3  Conditions used i n the extraction o f cooked salmon using a purge and trap procedure  sample size  200 g  no. o f replicates  3  extraction vessel temperature  70 °C  equuibrium time  30min.  extraction time  3 hours  N  2  gas f l o w rate  50 ml/min.  adsorbent used  Tenax G C  amount o f adsorbent  120 m g  internal standard  tetradecane  solvent used t o elute traps  diethyl ether  42  Approximately 120 m g o f Tenax G C (60/80 mesh, Alltech), a porous polymer, was packed into a glass tube (11.5 c m , 6 m m O.D., 4 m m I.D.), that was held i n place between t w o plugs o f glass wool.  The Tenax G C had been conditioned prior t o the extractions t o remove contaminants.  involved holding the Tenax G C at 200°C w i t h N  2  This  flowing t h r o u g h the tube at 30 ml/min f o r a m i n i m u m  o f 4 hours (Jennings and Filsoof, 1977). The n a r r o w end o f the trap was wrapped several times w i t h teflon tape before attaching it t o the extraction vessel to help ensure an air-tight fit. A f t e r the 3 hour extraction was completed, the Tenax traps were removed and eluted w i t h 2 m l diethyl ether (spectranalyzed grade, Fisher Scientific).  The extracts were stored in 3.7 m l glass vials  (screw t o p lid w i t h septa) (Supelco), w h i c h were placed i n a 4 ° C cooler until required f o r G C analysis. A t that time, the extract was concentrated by evaporating the ether using a gentle stream o f nitrogen, until approximately 100-200 microlitres remained. One microlitre o f this extract was then injected into a Varian 3700 gas chromatograph (Varian and Associates, Inc. Palo A l t o , C A ) set up and operated according to the specifications given i n Table 4. Relative amounts o f each compound were then determined by taking a ratio o f each peak area t o that o f the internal standard. These data were then subjected to an A N O V A (Systat 5 . 0 1 , Systat Inc.), t o determine i f there were any treatment effects. Only peaks that consistently appeared in all o f the chromatograms were examined.  3.3.3  I n s t r o n T P A analysis o f cooked s a l m o n samples Bourne's (1978) T P A method, based o n the compression o f the sample w i t h the Instron  Universal Testing Machine ( M o d e l 1122, Instron Corp., Canton, M A ) , was used t o achieve an objective quantitative measurement o f the c o o k e d salmon texture. Cylinders o f flaked salmon were  43  Table 4  G C conditions used i n the analysis o f purge and trap extracts from cooked chinook salmon samples  GC  Varian3700  Integrator  3 3 9 0 A Hewlett-Packard  Detector  Flame Ionisation  Split injection Column  100:1 capillary SPB-1 nonpolar  C o l u m n manufacturer  Supelco Inc.  Internal diameter  25 m m  F i l m thickness C o l u m n length  0.25 micro meters 30 m  Initial temperature  50 °C  T i m e initial temperature held  5 min.  Rate o f heating  5 °C/min.  Final temperature  220 °C  Injector port temperature  250 °C  Detector temperature  250 °C  H e ( U H P grade) f l o w rate  30 ml/rnin  A i r (Zero Gas) f l o w rate  300ml/min  H  30 m l / m i n  2  ( U H P grade) f l o w rate  V o l u m e o f sample injected  lul  44  formed using a 60 m l syringe (2.6 c m internal diameter) w i t h the end cut o f f at the zero line.  Ten  grams o f the cooked, deboned, flaked fish were poured into the t o p o f the syringe and then gently compressed w i t h a flat bottomed plunger to f o r m a cylinder 2 c m high. These fish samples were compressed twice w i t h the Instron, between t w o parallel plates (approximately 14.8 c m diameter), t o a height o f 0.5 c m ; 2 5 % o f their original height. This testing procedure creates a texture profile curve w i t h t w o peaks from w h i c h numerous textural parameters may be measured. These include hardness, firmness,  cohesiveness, chewiness and gumminess.  Test conditions (Table 5) were selected after  preliminary trials. The Instron was interfaced w i t h a personal computer, using JCL6000 software, the force/deformation curves at a rate o f 2 times per second were recorded. Prior t o analysis, the Instron was calibrated by measuring the difference i n load weight output w i t h no weight and w i t h a k n o w n weight. Quadruplet samples o f each treatment were used i n this p o r t i o n o f the experiment.  3.3.4  p H m e a s u r e m e n t o f cooked s a l m o n samples The p H o f the salmon was measured using the procedure outlined by Feinstein and B u c k  (1984). Samples were prepared by adding 3 g o f f i s h muscle t o 30 m l o f deionized, distilled water and homogenisedfor approximately 10 seconds using a Kinematica G m b H homogenizer (speed setting 6). p H was then measured using a Corning p H meter 220 (standardised t o p H 7). These analyses were performed i n quadruple.  45  Table 5  Conditions used for Instron measurements o f minced cooked chinook salmon samples  Analysis  TPA  N o . o f replicates  4  L o a d cell  1001b.  Crosshead speed  100 m m / m i n  N o . o f cycles o f crosshead  2  Sampling rate  2 data points/sec.  Temperature  20°C (approximately)  46  3.4  Data Analysis  3.4.1  A n a l y s i s o f sensory d a t a  3.4.1.1 E x p l o r a t o r y  analysis  Prior to performing complex statistical analyses o f the sensory data, some basic statistics were calculated.  For example, for each panellist, the number o f observations, the averages and standard  deviations were tallied for the six treatments for each attribute.  I n addition, the data were also  represented graphically in a series o f boxplots. Boxplots were also constructed for the reference in the same manner. T h r o u g h this exploratory data analysis, portions o f the data were found t o be unacceptable due t o an excessive number o f inconsistencies.  These included the first replicate (first sensory panel  session) as w e l l as all the data from panellist 6.  These unreliable portions o f the sensory data were  subsequently removed prior to further analysis, leaving 5 panellists and 9 taste panel days (replicates).  3.4.1.2 P r i n c i p a l c o m p o n e n t analysis ( P C A ) P C A was performed o n the pooled data for the sensory aroma attributes, as well as the pooled flavour and texture data using Systat software. For each set o f P C A data, a series o f graphs o f P C I vs. P C 2 were produced. Separate graphs were also produced by labelling data points w i t h the treatment number, the panel day number, and the panellist number.  47  3.4.1.3 ANOVA Several sets o f A N O V A s , were performed o n the sensory data. These included a t w o factor A N O V A o n reference data that examined the effect o f panellists, the day effect, and the interaction o f these t w o factors. F o r the treated samples, all the sensory attributes were subjected t o a three factor A N O V A , i n w h i c h the main effects o f ration level ( R L ) , swimming speed (SS), and panellists ( P A N ) were assessed.  3.4.1.4  Z-transformation of significant sensory attribute scores When A N O V A s were performed o n the r a w sensory data (treated samples), the panellist effect  was consistently found t o be highly significant.  Additionally, f o r several o f the attributes where a  significant treatment effect had been uncovered, the panellist X treatment interactions were also significant. T o remove the variation i n the sensory data due t o the panellist t o panellist variation, a ztransformation was performed o n all the sensory attributes that had been significantly affected b y either R L o r SS. W i t h respect t o this, z-scores f o r a given sensory attribute were calculated in several steps. First, the data were sorted, so that the responses o f each panellist could be identified. Following this, the averages and standard deviations o f the responses o f the individual judges were calculated from the r a w data. The z-score f o r each response was then calculated b y first subtracting the panellists average score from each response that she/he gave f o r that attribute, and then dividing the product b y her/his standard deviation (Reid and Durance, 1992).  48  Subsequently, the transformed data were examined using A N O V A .  Since the panellist to  panellist variation had been eliminated, only a t w o w a y A N O V A examining the effects o f the R L and SS was necessary.  3.4.2  Calculation o f Instron T P A parameters  3.4.2.1 C a l i b r a t i o n o f results Prior to using the Instron, the instrument was calibrated daily by using a k n o w n weight.  This  was accomplished by taking measurements w i t h the empty load cell for thirty seconds to establish a baseline, f o l l o w e d by placing a 1 k g weight o n the inverted load cell f o r 30 seconds. This procedure was repeated three times in successioa The calibration factor was then calculated by taking an average o f the scores recorded w h e n the 1 k g weight was applied, and then subtracting the baseline score. The test sample's data were calibrated by first subtracting the baseline value from the data and then dividing it by the calibration factor.  These measurements were then converted into Newtons.  This was accomplished by multiplying the scores by 9.8 m/s . The data were then used to measure or 2  calculate Instron T P A measurements as outlined in Table 6.  3.4.3  Calculation o f T P A "Firmness" Firmness, the m a x i m u m slope o f the compression cycle, was determined by measuring the  m a x i m u m slope o f the force curve. The slope was determined by calculating the distance the curve had risen o n the y-axis divided by the distance it had covered o n the x-axis. The slope was calculated for every 1 second interval o n the curve.  49  3.4.4  Calculation of peak area I n order t o measure cohesiveness and gumminess, it was necessary t o first calculate the area  under the curve. T o accomplish this, the area o f the "bites" was measured between the start o f the curve and the peak force (the highest measured force).  T o calculate the area under the curve, the  distance travelled o n the chart f o r each reading was first determined. This was accomplished b y first converting the chart speed t o mm/s and then dividing it by the sampling rate, giving the distance travelled o n the chart during each measurement.  The area was then determined by the sum o f this  value multiplied by each point o n the curve.  3.4.5  A N O V A of Instron T P A and p H data A t w o w a y A N O V A looking at the effect o f swimming speed and ration level o n various p H  and T P A measurements was performed.  W h e n a significant result was f o u n d in swimming speed,  having more then t w o levels, a T u k e y test was also conducted t o determine w h i c h levels were responsible f o r the significant differences. The data were analysed w i t h the aid o f Systat statistical software.  3.4.6  Principal Component Similarity (PCS) analysis of Sensory, and G C headspace volatile data P C A using Systat software, was performed separately o n the sensory and G C headspace  volatile data.  The sensory data included only those sensory attributes that had been significantly  affected b y either R L o r SS. These results had subsequently undergone a z-transformation t o eliminate  50  Table 6  Calculation o f Instron T P A parameters  Texture parameters  Definition  Reference  Hardness 1  Peak force during the first compression cycle  (Bourne, 1978)  Hardness 2  Peak force during the second compression cycle  (Bourne, 1978)  Firmness 1  M a x i m u m slope o f the first compression cycle  (Durance and Collins,  Firmness 2  M a x i m u m slope o f the second compression cycle  Cohesiveness  Ratio o f positive force area during the second  1991) (Durance and Collins, 1991) compression Gumminess  (Bourne, 1978)  cycle t o that o f the first ( A / A i ) 2  Product o f hardness X cohesiveness  51  (Bourne, 1978)  the panellist effect. B y averaging the responses o f all the judges f o r each replicate, a 5 f o l d reduction in the size o f the data set was achieved, leaving 9 replicates for each treatment combinatioa For the analysis o f the G C data, only those peaks that consistently appeared and were significantly affected by either SS or R L were used. This resulted i n 27 out o f a possible 71 peaks being included i n this analysis. P C A was performed o n these t w o sets o f data, and this resulted i n a print out for each, and the scores were saved. Starting w i t h the first P C , the percent o f total variance explained by the PC were added together until more than 90 % o f the variance was accounted f o r ; these are the P C that were used in the PCS. The scores from these PCs were copied into a data file to be imported into the PCS program.  PCS produces the slope and coefficient o f determination for each case, w h i c h were  subsequently saved in a data file. A f t e r importing this file into a computer spreadsheet, the slope was graphed against the coefficient o f determination.  52  4.  Results a n d Discussion  4.1  Sensory analysis o f cooked s a l m o n samples  4.1.1  Sensory p a n e l reference samples  4.1.1.1 Purpose o f reference sample A t both the morning and afternoon sensory panel sessions, a reference sample was presented t o the panellists along w i t h the test samples.  The reference samples were composite samples o f small,  randomly selected slices o f fish from several 0.9-1.5 k g farmed chinook salmon. Using this method, a large number o f fairly u n i f o r m samples was produced.  Since an individual fillet from each treatment  was used along w i t h a reference sample for each o f the nine panel days, it was essential t o include reference  samples.  W i t h o u t a reference it w o u l d have been difficult t o distinguish from the test  samples whether a statistical difference i n the day was due t o a true difference between the sessions o r simply stemmed from fish t o fish variation. Consistency between reference samples was also important as panellists have a tendency t o grade the test samples relative to the reference (Giovanni and Pangborn, 1983). A summary o f the sensory reference data is found i n Table 7. The reference was also used to help the panellists calibrate their responses.  These reference  samples were prepared for the panellists o n several panel training sessions. A t these training sessions, the panellists were able to interact w i t h each other, discussing h o w and w h y they w o u l d give these samples a particular score, eventually reaching a consensus o n the appropriate grade.  D u r i n g the  sensory panels, these reference samples were rated prior t o any o f the treated samples. This allowed the panellists t o calibrate their responses, between each other, as w e l l as from session t o session (Johnsen and Kelly, 1990). However, despite the use o f the reference samples and training, there  53  7  Range, mean, and standard deviation (St. D e v . ) o f sensory attributes o f the cooked, farmed chinook salmon reference samples (5 panellists, 9 panel days)  Attribute  Range  Mean&St.Dev  Seaweedy  0.7-7.0  3.75 ± 1 . 4 9  Boiled m i l k  0-4.0  1.29 ± 0 . 8 9  Boiled potato  0-5.8  2.51 ± 1.44  Lemony  0-5.8  1.62 ± 1 . 2 5  Sour  0-4.8  1.06 ± 0 . 9 3  Fishy  0.1-8.9  3.59 ± 1 . 7 7  Chickeny  0-7.0  1.76 ± 1 . 7 8  Oily  0-5.2  1.28 ± 0 . 9 6  " 1.7-9.6  5.72 ± 1 . 4 4  Flavour  1.0-9.1  5.43 ± 2 . 1 4  Fishy  0.2-8.8  3.49 ± 2 . 0 6  Earthy  0-5.5  1.30 ± 1 . 3 3  Papery  0-7.4  2.01 ± 1 . 4 8  Bitter  0-6.7  1.22±1.16  Sour  0-4.4  1.06 ± 0 . 8 4  Lemony  0-7.7  1.65 ± 1 . 5 4  Salty  0-4.1  1.53 ± 1 . 0 6  Spicy  0-7.2  2.13 ± 1 . 5 4  Brothy  0-7.7  2.15±2.17  Moistness  0.5-9.0  4.89 ± 1 . 9 3  Powderiness  0-8.9  2.55 ± 1 . 9 3  Flakiness  0.1-8.3  3.55 ± 2 . 2 5  Firmness  0.6-8.6  5.30 ±1.71  Chewiness  0.1-8.5  4.13 ± 2 . 1 8  Aroma  Fresh Flavour  Texture  Cohesiveness  0.4-7.7  4.17 ± 1 . 8 3  Adhesiveness  0-8.0  2.91 ± 1.85  Mushiness  0-7.3  1.94 ± 1.58  Overall  2.1-8.7  5.58 ± 1 . 3 7  54  w o u l d often be a wide range o f responses received from the panellists f o r a given sample.  The  panellists, each w i t h their o w n personal style, were consistent from session t o session.  4.1.1.2 Reference sample observations Significance f o r the day X panellist t e r m interaction that appears i n Table 8 is likely largely due to some o f the unavoidable differences between panel sessions. I t was not always possible t o have the panellists start the panel at the same time. F r o m time t o time, a panellist was unavoidably detained and came t o the session late. Occasionally a panellist was absent f o r one o f the t w o sessions o n a panel day. The panellist t o o k a short break following the scheduled session he/she attended and then rated the samples from the missed session. T h e resulting differences i n temperature and taste acuity may partially be responsible f o r the day X panellist interaction (Meilgaard et al., 1 9 9 1 ; L a r m o n d , 1977). I t was also noted that o n occasion a panellist d i d not comply w i t h the request t o abstain from eating lunch or consuming coffee prior t o the panel session and this m a y have resulted i n confusion o r carry-over sensations w i t h the samples (Rutledge and Hudson, 1990).  4.1.2  T r e a t e d samples  4.1.2.1 E x p l o r a t o r y analysis The data set f o r this p o r t i o n o f the research was extremely large and required some exploratory data analysis. F o r each individual panellist, boxplots o f each attribute were constructed (examples i n appendix A ) . Tables giving each panellist's average, standard deviation, range and number o f observations were also prepared (data not shown).  55  4.1.2.1.1  Boxplots  I t proved very difficult t o ascertain from the boxplots whether the treatments were significantly different from each other. E v e n i n attributes where a significant difference between treatments existed, the wide variation i n panellist rating styles masked evidence o f the treatment differences. These boxplots, however, did clearly show that despite training there was a large amount o f judge to judge variation. The judges differed greatly in their style o f rating o f the attributes, varying widely i n the range o f values that they used (data not shown).  They, however, appeared quite  consistent w i t h their individual styles o f rating the attributes, using the same range and psychological distances between grading levels i.e. what one panellist w o u l d grade as a 0.3 c m , a second might score as 1.5 c m .  Fortunately, according t o Stone et al. (1974), it is not o f critical importance that the  individual panellists used different segment o f the scale, as long as their individual performances were constant.  4.1.2.1.2 Deletion o f unacceptable d a t a U p o n examination o f these results, it was decided that some o f the data collected w o u l d not be used in any further analysis.  A l l the data collected o n the first day, as w e l l as the contribution o f  panellist 6, were removed. T h e data set collected o n the first day was eliminated because it contained numerous panellist errors, mostly related t o panellist fatigue. I n the first panel session, it was w r o n g l y assumed that all the panellists w o u l d be capable o f rating each o f the 7 samples (6 treatments and one reference) f o r the 28 sensory attributes without becoming fatigued. I n subsequent sessions, the panel days were divided into morning and afternoon sessions. Three randomly chosen treatments and a reference were presented t o  56  Table 8  A N O V A results o fjudge and panel day effect o n reference samples f o r 28 sensory attributes o f cultured chinook salmon (5 panellists, 9 panel days)  F ratio Sensory attributes  Day  Panellist  M e a n square D a y X Pan  error  Aroma Seaweedy  1.748  30.992***  1.006  0.848  Boiled m i l k  1.196  18.450***  3.748***  0.244  Boiled potato Lemony  0.482  18.504***  1.454  1.069  0.682  27.416***  0.941  0.668  Sour  0.261  3.494*  1.383  0.698  Fishy  3.048**  5.303**  1.330  2.164  Chickeny  1.740  1.325  1.149  Oily  2.576*  38.885*** 11.447***  1.233  0.486  Fresh  2.109  18.584***  1.233  0.486  Flavour  0.597  35.396***  0.762  1.762  Fishy  0.501  19.857***  1.474  2.315  Earthy  2.536*  47.925***  2.859**  0.418  Papery  1.321  5.834**  1.233  1.449  Bitter  0.978  5.266**  1.337  1.099  Sour  1.145  0.681  0.972  0.704  Lemony  1.209  12.622***  0.982  1.617  Salty  1.805  42.830***  3.383***  0.308  Spicy  0.853  20.321***  1.005  1.092  Brothy  1.364  129.229***  0.931  0.715  Moistness  0.904  16.656***  0.579  2.361  Powderiness  1.091  2.301  1.775*  2.469 1.817  Flavour  Texture  Flakiness  0.713  39.043***  1.100  Firmness  2.331*  8.238***  1.206  1.842  Chewiness  2.500*  67.577***  1.683*  1.232  Cohesiveness  1.593  13.919***  0.884  2.039  Adhesiveness  1.589  28.128***  1.776*  1.232  Mushiness  1.857  1.554  1.376  Overall  1.567  1.503  0.728  3.148* 24.223***  * p<.05, * * p < . 0 1 , * * * p<.001  57  the panellists i n the morning w i t h the remaining three treatments and a second reference offered at an afternoon session. A large number o f missing data points, as w e l l as excessive variation i n replicates (data n o t shown), made it necessary to omit the contribution o f panellist 6 from the data set.  The data  from  panellist 6 were actually a combination o f data contributed by three people. Each o f these three people had a personal style o f rating the samples that varied greatly from one other.  Since consistency and  accuracy are so very important for panellists in this type o f sensory analysis (Stone et al. 1974), it was decided not t o include the data from panellist 6.  U p o n elimination o f this data set a second set o f  summary statistics was calculated (Tables 9 - 1 5 ) .  4.1.2.2 The use of replacement panellists A s this panel t o o k place over a period o f several weeks during the summer, it proved an impossible task t o find six willing panellists w h o were able to commit t o being present throughout the duration o f the experiment. I t was decided that back-up panellists, w h o had also completed the training sessions, w o u l d  substitute for absent panellists. Fortunately, back-up panellists were only necessary  for panellists 5 and 6. Evidence o f the substitution o f a back-up panellist for panellist 5 o n t w o panel days became very apparent during data analysis. I n Figures 2-4 a set o f data points, one from each treatment, was separated from the main cluster. This set was evident in the aroma and pooled texture variables (Fig. 2 and 4 ) ; no evidence o f this set was readily apparent i n the corresponding flavour graph (Fig. 3). These irregularities in the data were further examined by producing a second set o f these figures where the treatment number was replaced w i t h the panel day number (Fig. 5-7). F r o m these  58  Table 9  Range, mean, and standard deviation (St. Dev.) o f cooked, cultured chinook salmon sensory attributes (all treatments combined; 5 panellists, 9 panel days) Attribute  Range  M e a n & St. Dev.  Aroma Seaweedy  0-9.9  4.05 ± 2 . 1 7  Boiled m i l k  0-6.1  1.26 ± 1 . 0 0  Boiled potato  0-6.7  2.20 ± 1 . 4 7  Lemony  0-6.3  Sour  0-6.3  1.44 ± 1.21 1.37 ± 1 . 2 3  Fishy  0-8.9  3.58 ± 1 . 9 2  Chickeny  0-7.6  1.51 ± 1.47  Oily  0-5.6  1.36 ± 1 . 0 3  Fresh  0.6-9.6  5.26 ± 1 . 7 1  Flavour  0.3-9.4  5.63 ± 2 . 0 7  Fishy  0.1-8.8  3.42 ± 2.20  Earthy  0-7.5  1.23 ± 1 . 2 6  Papery  0-8.6  1.99 ± 1 . 6 4  Bitter  0-7.1  1.32 ± 1 . 2 5  Flavour  Sour  0-7.1  1.27±1.11  Lemony  0-7.7  1.30 ± 1 . 6 7  Salty  0-7.0  1.82 ± 1.28  Spicy  0-8.8  2.35 ± 1 . 7 2  Brothy  0-8.4  2.31 ± 2 . 1 3  Moistness  0.5-9.2  5.34 ± 1 . 9 2  Powderiness  0-8.9  2.45 ± 1 . 9 7  Flakiness  0-8.4  3.14 ± 2 . 0 4  Firmness  0.5-8.8  4.63 ± 1 . 8 9  Chewiness  0-9.0  4.13 ± 2 . 1 5  Cohesiveness  0.2-9.0  4.14 ± 1 . 8 4  Texture  Adhesiveness  0-8.3  2.76 ± 1 . 8 8  Mushiness  0-8.6  2.43 ± 2 . 0 4  Overall  1.9-8.9  5.48 ± 1 . 4 4  59  Table 10  M e a n sensory scores and standard deviation o f cultured chinook salmon aroma attributes for each ration level X swimming speed treatment ( 5 panellists; 9 panel days)  Ration  Level  75%  100% Swimming  Attribute  Speed (bl/s)  0.5  1.0  1.5  0.5  1.0  1.5  Seaweedy  3.95 ±2.22  4.17 ±2.23  3.50 ±2.07  4.38 ±2.23  4.34 ± 2 . 4 6  4.52 ±2.63  Boiled milk  1.37 ±1.19  1.31 ±1.05  1.23 ±1.14  1.17 ±0.88  1.28 ±1.05  1.16 ±0.80  Boiled potato  2.28 ±1.56  2.19 ±1.61  2.36 ±1.50  2.06 ±1.50  1.84 ±1.26  1.89 ±1.29  Lemony  1.44 ±1.17  1.51 ± 1 . 3 3  1.26 ±1.14  1.34 ±1.07  1.56 ±1.33  1.17±1.00  Sour  1.56 ±1.29  1.36 ±1.23  1.06 ± 0 . 9 3  1.56 ±1.44  1.76 ±1.05  1.52 ±1.23  Fishy  3.44 ±1.99  1.46 ±1.71  3.46 ± 1 . 7 4  3.27 ±1.85  3.63 ± 2.20  4.09 ±2.18  Chickeny  1.49 ±1.28  1.30 ±1.12  1.75 ± 1 . 7 5  1.28 ±1.15  1.45 ±1.34  1.34 ±1.30  Oily  1.29 ±1.02  1.37 ± 0 . 9 7  1.37 ± 0 . 9 4  1.27 ± 0 . 9 9  1.51 ±1.29  1.51 ±1.04  Fresh  5.23 ±1.67  5.15 ±1.62  5.26 ± 1 . 3 0  5.24 ±1.98  5.00 ±1.98  4.78 ±1.88  60  Table 11  Range o f sensory scores o f cultured chinook salmon aroma attributes for each ration level X swimming speed treatment (5 panellists; 9 panel days)  Ration  Level  75%  Attribute  100% Swimming  Speed (bl/s)  0.5  1.0  1.5  0.5  1.0  1.5  0.7-9.3  0.2-9.6  0.2-9.7  0.7-9.2  0.2-9.9  0-9.8  Boiled m i l k  0-5.8  0-4.6  0-6.1  0-4.3  0-3.8  0-3.4  Boiled potato  0-5.7  0-6.0  0.1-6.7  0-6.0  0-5.3  0-5.3  Lemony  0-6.3  0-6.3  0-6.1  0-4.9  0-5.6  0-4.1  Sour  0-5.3  0-5.7  0-4.0  0.1-6.3  0-5.6  0-6.3  Fishy  0-8.1  0.1-7.3  .04-6.6  0.2-8.0  0.2-7.9  0.1-8.6  Chickeny  0-5.9  0-4.6  0-7.6  0-5.7  0-7.4  0-6.7  Oily  0-3.5  0-5.4  0-4.1  0-4.5  0-5.6  0-4.6  1.9-8.6  1.5-8.9  2.3-9.6  1.2-9.1  1.2-9.5  0.6-8.9  Seaweedy  Fresh  61  Table 12  M e a n sensory scores and standard deviation o f cultured chinook salmon flavour attributes for each ration level X sv\Tmming speed treatment (5 panellists; 9 panel days)  Ration  Level  75%  100% Svvdmming  Attribute  Speed (bl/s)  0.5  1.0  1.5  0.5  1.0  1.5  Flavour  5.95 ±1.84  5.86 ±2.13  5.69 ±1.89  5.31 ±2.07  5.69 ±1.89  5.60 ±2.07  Fishy  3.41 ±2.08  3.31 ±2.25  3.33 ±2.40  3.49 ±2.15  3.33 ±2.40  3.50 ±2.26  Earthy  1.09 ±1.02  1.36 ±1.31  1.34 ±1.42  1.13 ±1.03  1.34 ±1.42  1.09 ±1.21  Papery  1.92 ±1.73  2.02 ±1.42  2.06 ±1.76  2.04 ±1.64  2.06 ±1.76  1.92 ±1.70  Bitter  1.46 ±1.39  1.52 ±0.94  1.52 ±1.19  1.13 ±1.16  1.52 ±1.19  1.49 ±1.66  Sour  1.47 ±1.54  1.16 ±1.00  1.25 ±0.96  1.25 ±1.00  1.25 ±0.96  1.46 ±1.22  Lemony  1.28 ±1.05  1.19 ±0.95  1.22 ±0.97  1.15 ±1.06  1.19 ±1.02  1.06 ±0.92  Salty  2.03 ±1.35  2.01 ±1.34  1.85 ±1.32  1.79 ±1.39  1.87 ±1.28  1.91 ±1.30  Spicy  2.48 ±1.72  2.38 ±1.69  2.64 ±1.68  2.42 ±1.88  2.22 ±1.81  2.40 ±1.78  Brothy  2.51 ±2.05  2.56 ±2.12  2.48 ±2.28  2.07 ±2.00  2.27 ±1.99  2.28 ±2.06  62  Table 13  Range o f sensory scores o f cultured chinook salmon flavour attributes for each ration level X swimming speed treatment (5 panellists; 9 panel days)  Ration  Level  75%  100% Swimming  Attribute  Speed (bl/s)  0.5  1.0  1.5  0.5  1.0  1.5  Flavour  0.6-9.4  1.2-9.1  0.4-8.8  0.9-9.0  0.4-8.8  0.4-9.1  Fishy  0.1-8.0  0.1-8.0  0.1-8.2  0.2-8.2  0.1-8.2  0.1-8.1  Earthy  0-4.3  0-5.1  0-7.3  0-4.7  0-7.3  0-4.6  Papery  0-7.5  0-6.2  0-7.0  0-6.7  0-7.0  0.2-8.5  Bitter  0-5.3  0-4.4  0-6.3  0-6.2  0-6.3  0-7.1  Sour  0-7.1  0-4.2  0-4.0  0-3.7  0-4.0  0-5.0  Lemony  0-3.5  0-4.0  0-3.9  0-5.1  0-4.0  0-3.3  Salty  0-5.6  0-5.8  0.1-5.2  0-7.0  0-4.6  0.1-5.4  Spicy  0.1-5.9  0-7.8  0.1-8.0  0-7.8  0-6.9  0.1-8.8  Brothy  0.2-7.4  0-7.8  0.2-7.3  0-7.1  0.1-8.4  0.1-7.7  63  Table 14  M e a n sensory scores and standard deviations o f cultured chinook salmon texture attributes for each ration level X swimming speed treatment (5 panellists; 9 panel days)  Ration  Level  75%  100% Swimming  Speed (bl/s)  Attribute  0.5  1.0  1.5  0.5  1.0  1.5  Moistness  5.27 ±1.90  5.52 ±1.76  5.04 ±1.93  5.48 ±1.67  5.98 ±2.02  5.66 ±1.94  Powderiness  2.50 ±1.93  2.33 ±1.88  2.45 ±1.94  2.42 ±2.02  2.26 ±1.99  2.49 ±2.11  Flakiness  2.92 ±2.12  3.21 ±2.05  3.04 ±1.79  3.07±1.77  2.95 ±2.08  2.88 ±1.83  Firmness  4.78 ±1.68  4.49 ±1.98  5.02 ±1.30  4.21 ±1.97  3.80 ±2.03  4.22 ±2.07  Chewiness  4.27 ±2.26  4.03 ±2.12  4.33 ±1.92  3.83 ±2.18  3.99 ±2.05  4.30 ±2.23  Cohesiveness  4.34 ±1.58  4.17 ±1.75  4.62 ±1.72  3.74 ±1.78  4.03 ±2.07  3.91±2.02  Adhesiveness  2.83 ±1.89  2.77 ±1.89  2.67 ±1.85  5.58 ±1.64  2.94 ±1.98  5.52 ±2.05  Mushiness  2.35 ±1.70  2.17 ±1.83  1.95 ±1.69  2.82 ±2.34  3.19 ±2.50  3.02 ±2.30  Overall  5.65 ±1.34  5.70 ±1.32  5.49 ±1.43  5.23 ±1.32  5.20 ±1.62  5.39 ±1.60  64  Table 15  Range o f sensory scores o f cultured chinook salmon texture attributes for each ration level X svrimming speed treatment (5 panellists; 9 panel days)  Ration  Level  75%  100% SvvTmming  Speed (bl/s)  Attribute  0.5  1.0  1.5  0.5  1.0  1.5  Moistness  1.1-9.1  1.5-9.1  0.8-8.8  1.9-9.1  0.6-9.2  0.7-9.0  0-6.8  0-7.6  0-8.5  0-8.7  0-8.4  0-7.9  Flakiness  0.2-8.3  0-7.2  0-7.9  0.1-8.2  0.1-8.4  0-7.2  Firmness  1.5-8.0  0.6-8.8  2.2-7.5  0.6-7.7  0.5-7.9  0.6-8.4  Chewiness  0.1-8.3  0-9.0  0.7-8.5  0.3-8.0  0.2-8.8  0.1-8.3  Cohesiveness  0.8-8.5  0.4-7.6  0.5-7.7  0.2-7.4  0.4-9.0  0.3-8.2  Adhesiveness  0-7.4  0.1-7.7  0.1-7.0  0.1-7.8  0-8.2  0.1-8.3  Mushiness  0.2-7.4  0-8.6  0.1-6.8  0-7.9  0-8.1  0.2-8.0  Overall  3.4-8.7  2.7-8.9  2.0-8.5  1.9-8.6  2.2-8.5  2.2-8.5  Powderiness  65  2.5 23'  4  1.5  4  J\J  il-  2  L 0.5«  3  n  6 4  c  .  Oh  Z  4  4  4.  3  -0.5  2  1 -1  \l 6^  -1.5 -2  -4  -3  -2  -1  0  PC 1 ( 4 4 % )  Figure 2  P C 1 versus P C 2 using sensory aroma attribute scores from panellist 5, data points labelled w i t h treatment numbers  66  0  1 PC 1 (33 %)  Figure 3  P C 1 versus P C 2 o f panellist 5 sensory flavour attribute scores, data points labelled w i t h treatment numbers  67  Figure 4  PC 1 versus PC 2 of panellist 5 sensory texture attribute scores, data points labelled with treatment numbers  68  Figure 5  P C 1 versus P C 2 o f panellist 5 sensory aroma attribute scores, data points labelled w i t h panel day number  69  3  7 7 7  ?  1  1  13  5  6 o OO w CM x  0  U  4  8  ;  *  4 M  %  8  7  8  8 8  7  2#  4  9  -1  -2 9 -3  -4  9  -2  1  0  PC 1 (33 %)  Figure 6  PC 1 versus PC 2 of panellist 5 sensory flavour aroma attribute scores, data points labelled with panel day numbers  70  3  8/7 8 cs O  0  4  2*4  -2  -2  0 PC  Figure 7  1  (35 % )  P C 1 versus P C 2 o f panellist 5 sensory texture attribute scores, data points labelled w i t h panel day number  71  figures it is apparent that the anomalies in the data are due to events on panel day 9. With the panel day hi-lighted a second cluster, this time in day 7, was also identifiable. Unlike thefirstset of graphs (Fig. 2-4), these irregularities were also evident in theflavourgraphs. When notes recorded during the course of the sensory panel sessions were reviewed, it became apparent that in these two sessions a substitute panellist was used in place of panellist 5 (the same person on both occasions). This irregularity in the judging attributed to one panellist, although unlikely to have a large effect on statistics such as the mean, standard deviation and range of responses, can lead to problems when testing for significant difference in ANOVA. For example, it could lead to panellist X day interactions. When attempting to standardize the data to remove panellist effect, the skewed average and standard deviation for that panellist will, in turn, skew the results.  4.1.2.3 Summary statistics of treated samples  The sensory data scores, generally, were quite low. Upon examination of the means (Tables 10, 12 and 14), it was apparent that most were at the lower end of the 10 cm scale; only four had averages over 5 cm, while 11 out of the 28 attributes had an average under 2 cm In Tables 9, 11 and 13, the lower end of the range was often zero. On one or more occasions, this attribute was too faint to be discernible by at least one of the panellists. All the salmon samples had a very delicate flavour. In informal discussions held with the panellists, they would often comment that theflavournotes were quite faint and difficult to quantify. In addition, they commented that they could not discern much of a difference among the six treatments. Many of the sensory attributes proved impossible for individual panellists to even detect in some samples, resulting in a score of zero. 72  4.1.2.4 T h r e e f a c t o r A N O V A o f sensory a t t r i b u t e d a t a A three factor A N O V A was performed o n all the attributes individually, t o evaluate the contribution o f SS, R L , and panellists as w e l l as all the interactions (Tables 16-18). The panellist effect was highly significant ( p < 0.001, or p<0.01) for all attributes. E v e n after training, it is quite c o m m o n f o r the panellist effect t o account for a large p o r t i o n o f the variation i n the data. This variation stems from the subjective nature o f this type o f sensory evaluation and the individual differences between panellists (Stone et al. 1974). There were a f e w attributes where the R L X P A N interaction was significant. acceptability, there was also a significant SS X P A N interaction (p<0.05).  I n overall  E v e n i n cases where  panellists are screened and w e l l trained, there is always a possibility that panellist by treatment interaction w i l l occur due t o differences i n motivation, sensitivity or psychophysical  response  behaviour. This is especially true w h e n the panel size is small (Lundahl and McDanieL 1990) as it was i n this experiment.  Some confusion in scoring is acceptable, particularly i n cases such as this where  there is not a large degree o f difference between samples (Stone et al. 1974). O ut o f the 28 sensory attributes tested, no attributes were significantly affected by SS but eight were significantly affected by varying the R L .  O f the aroma attributes tested, "Boiled potato," and  "Sour" were significant at the p<0.05 level, and "Seaweedy" at p O . O l . Only one taste t e r m , " B r o t h y " was found t o be significant ( p O . O l ) . Four texture attributes were significantly influenced: "Moistness" (p<0.05), "Firmness" ( p O . 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C O VO NO m Og O  O ON O  § 9  * CN m O oo © co  §-2  oo  v  * *  CN  © V  T3  De  *  vel  2  •^•rONOcNNOCNOOoN  • H ^ ^ t t ^ r H t ^ ^ f f f )  1& CH  c 3 /  =,•8  I  >  o  O <  76  * *  O V  •S  d C .2 * H 8 H CH Vj  2 II  * at3 CH  oo  oo II II OO OO PH o  4.1.2.5 Z - t r a n s f o r m a t i o n o f sensory a t t r i b u t e s s i g n i f i c a n t l y affected b y t h e t r e a t m e n t A z-transformation t o remove the panellist effect was performed o n the eight attributes that were significantly affected b y R L . W i t h the panellist effect removed, analysis o f variance revealed that "Boiled Potato" was no longer significantly affected by R L (Table 19). The F scores from several o f the  other  attributes,  including  "Seaweedy,"  "Sour"  (aroma),  "Brothy,"  "Cohesiveness"  and  "Mushiness" also decreased while those o f "Firmness" and "Moistness" were largely unchanged.  4.1.2.6 P C A a n d P C S o f sensory d a t a P C A and then PCS were performed o n the transformed sensory attributes. The factor score coefficients o f the first 6 P C and the percentage o f the total variance that they explain are listed i n Table 20. These six PCs, cumulatively accounting f o r approximately 9 2 % o f the variation i n the data, were fed into the PCS p r o g r a m t o calculate slope and coefficient o f determination ( r ) . 2  PCS proved to be an ideal analysis method. First, the use o f PCS is most effective w h e n the individual P C do not account for a large percentage o f the variation ( V o d v o t z et al., 1993) or the size o f the data matrix is fairly large (Furtula et al. 1994a). these criteria; the  first  The sensory data in this experiment fit b o t h o f  P C only accounted f o r 40 percent o f the variation and the data set was  extremely large. PCS also has the capacity t o represent a larger number o f P C scores for graphic illustration than P C A alone (Turtula et al. 1994a) as w e l l as giving better group resolution ( V o d o v o t z et al., 1993). W h e n graphs were produced from different pairs o f PCs (not included), n o clumping or trends were observed. I n these types o f graphs portions o f the data that may be important are overlooked  77  Table 19  Summarised A N O V A results o f ration level and swimming speed effect o n standardised data from significant sensory attributes o f cultured chinook salmon a  (5 panellists; 9 panel days)  Sensory  F ratio  attributes Seaweedy  d  Boiled P o t a t o  d  Sour" Brothy  Ration  Swimming  level"  speed  Mean RLXSS  square error  0  7.840**  0.548  0.833  3.681  0.684  0.071  1.013  5.823*  1.057  0.200  0.992  0.871  4.839*  0.187  0.836  0.998  Moisture  f  4.235*  2.344  0.987  Firmness  f  9.964**  2.334  0.329 0.121  4.731*  0.113  0.140  1.003  10.264**  0.728  2.077  0.965  6  Cohesiveness Mushiness  f  f  0.968  * p<.05, * * p < . 0 1 , * * * p<.001 a  b  0  Standardised data = panellist effect removed by z transformation o f all scores w i t h i n each panellist ration level = 7 5 % and 100% o f full ration swimming speed = 0 . 5 , 1 . 0 , and 1.5 bl/s  d  aroma t e r m  e  flavour t e r m  f  texture t e r m  78  Table 20  Factor score coefficients o f the first 6 principal components o f the z-transformed sensory attribute scores o f the R L X SS treated chinook salmon samples (5 panellists; 9 panel days)  Sensory attributes  Principal Component 1  2  3  0.161  0.341  Boiled potato Sour  -0.199  0.266  0.128  -0.222  Brothy  -0.069  0.551  Moistness  0.237  Firmness Cohesiveness  4  5  6  0.234  0.822  -0.252  0.524  0.251  -0.347  -0.748  0.307  0.851  0.049  0.146  -0.458  0.306  -0.372  0.585  -0.016  0.224  -0.270  -0.070  0.463  0.198  -0.273  0.099  0.014  0.152  0.239  -0.183  -0.202  -0.311  0.176  0.121  0.474  1.018  0.236  -0.103  0.118  -0.639  -0.152  0.609  Fraction  0.398  0.177  0.119  0.090  0.083  0.061  Cumulative  0.398  0.575  0.694  0.784  0.867  0.928  Seaweedy  Mushiness Variance explained  79  ( V o d o v o t z et al., 1993). I n flavour analysis this is critical because seemingly minor compounds often play critical roles in constituting characteristic flavour notes ( V o d o v o t z et al., 1993). The reference chosen for PCS was a slow SS, high R L (treatment 4) replicate from panellist 1. This treatment was singled out as the reference because, according to Kiessling et al. (1994b), this was the most cost effective o f the six treatments. The particular replicate used for Figures 8-11 gave the best group resolution o f those tested. The PCS results were imported into a spreadsheet, where a series o f graphs o f r versus slope 2  were constructed. The data points were labelled w i t h either the treatment number, ration level number, swimming speed number or the panel day that they represented (Figures 8-11 respectively).  These  results served t o reconfirm that ration level, and not swimming speed, was largely responsible for the effect o n the sensory properties o f the cooked s a l m o a I n Figure 9, although a small degree o f overlap is present, the t w o ration levels are largely i n t w o separate groups. The lower R L was i n the lower, left side o f the graph, while the higher R L was primarily i n the upper right p o r t i o n o f the graph. I n Figure 10, where the data points are labelled w i t h the SS level, the three swimming speeds appear randomly scattered indicating that SS did not affect the sensory properties o f the salmon. There was no appreciable evidence o f an interaction effect observable from the treatment number graphs (Figure 8). Within the groups o f treatments w i t h the same R L , data from the three SS appear randomly distributed. Some o f the overlapping o f the t w o R L i n Figure 9 may have been due t o fish t o fish variability. Individual fillets were used i n this experiment, and not samples compiled from several fish from the same treatment, as i n all the other portions o f this research. Fish t o fish variation may be partially  80  1.8|— 1.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10  L  1 6  1  4 5  4  4  6 4  2  *  J  2  5  3 1 31  5  6 1  2  1  13 4  16  3 J  0  I  !  I  I  0.1  0.2  0.3  0.4  L  0.5  0.6  0.7  0.8  i  i  0.9  1  Coefficient o f Determination  Figure 8  PCS graph o f significant sensory attributes, data points labelled w i t h treatment numbers  81  0  0.1  0.2  0.3  0.4  0.5  0,6  0.7  0.8  0.9  1  Coefficient of Detennination  Figure 9  PCS graph o f significant sensory attributes, data points labelled w i t h ration level numbers (1 = 7 5 % ; 2 = 1 0 0 % ration level)  82  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1  Coefficient of Determination  F i g u r e 10  PCS graph o f significant sensory attributes, data points labelled w i t h swimming speed numbers ( 1 = 0 . 5 , 2 = 1 . 0 , 3 = 1 . 5 bl/s)  83  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1  Coefficient of Determination  Figure 11  PCS graph o f significant sensory attributes, data points labelled w i t h panel day number  84  responsible for this lack o f clear separation that is evident i n the G C headspace data. Graphs using the panel day numbers (Fig. 11) showed no obvious day effect.  4.1.2.7 E f f e c t o f SS o n t h e sensory a t t r i b u t e s F r o m these results, it appears that changing swimming speed did not result in significant changes i n the aroma, taste and texture o f the cooked fish. This conclusion agrees w i t h the results o f Kiessling et al. (1994a,b).  Kiessling et al.(1994b), measuring the proximate composition o f the fish  from this study, did not find that SS had had any effect o n the composition o f the chinook fillets (Kiessling et al. 1994b). Furthermore, Kiessling et al. (1994a) did not find any evidence o f hypertrophy i n the white muscle o f the salmon due to exercise. Additionally, no evidence was found t o indicate that SS affected the total muscle area. Also, SS did not influence the fibre size distribution. Kiessling et al. (1994a), however, d i d find a significant increase i n the amount o f  fibre  hypertrophy i n the red muscle that occurred as a result o f the increase i n SS. H o w e v e r , since the red muscle is only a fraction o f the size o f the white muscle tissue, it is unlikely that the hypertrophy o f red muscle w o u l d have a large impact o n the texture o f the fish.  4.1.2.8 Effect o f R L o n t h e sensory a t t r i b u t e s R L significantly affected some aspects o f the taste, aroma and texture o f the treated fish samples. This is likely attributable t o the significant difference i n fat content between the t w o R L noted by Kiessling et al. (1994b). Fat content can affect the mouthfeeL aroma and taste o f a f o o d system. V a n Gemert et al. (1987), w o r k i n g w i t h smoked sausages, found that there was a strong positive linear relationship between the percentage o f fat i n the sausages and their odour intensity. 85  Fat can affect the mouthfeel qualities o f f o o d systems i n several ways. First, it lubricates the f o o d while it is being chewed. Additionally, fat also imparts an oily sensation i n the m o u t h ; this affects the surface tension and can cause changes in the viscosity o f the product (Szczesniak, 1963).  4.1.3  I n s t r u m e n t a l analysis  4.1.4 G C headspace analysis A s w i t h the sensory data analyses, A N O V A , P C A and PCS were used t o determine what, i f any, effect the treatment had had o n the headspace gases.  O f the 71 G C peaks that consistently  appeared, 27 were significant for either SS or R L (Table 2 1 , Figure 12). Significant peaks were found throughout the graph, and included b o t h large and small peaks. P C A was performed using the areas o f the significant peaks. The factor score coefficients from the first seven P C and the percentage o f total variance explained by each o f these is presented i n Table 22. The graphs o f the PCS results, once again using a slow SS, h i g h R L treatment for the refernce and labelled w i t h treatment number, ration level number and swimming speed number, (Figures 13-15 respectively) were similar t o those produced from the sensory data (Figures 8-11). Again, there is a very clear group resolution o f the data due to ration level (Figure 14). The ration level PCS graphs o f b o t h the G C and sensory results showed very similar cluster patterns. I n both, the data points fell into the same pattern; the l o w e r ration level having a coefficient o f determination between 0 and 0.8, w i t h a range i n slopes between 0 and 1. The higher ration level fell in the range o f 0.7-1 coefficient o f determination w i t h a slope range o f 0.8-2.0.  86  Table 21  Peak labels, retention times and level o f significance for ration level and svvimming speed o f G C peaks that consistently appeared i n purge and trap G C headspace analysis o f cultured, cooked chinook salmon  Retention  Peak  time (minutes)  labels  RT/  Significance  6.69  a  *  7.08  b  7.21  c  7.82  d  8.94  e  **  9.1  f  9.51 9.84  g h  10.25  i  10.4 10.93  j k  10.97  1  12.19  m  12.52  n  12.74  o  13.04  P  13.19 13.49  q r  13.68  s  13.87  t  14.1  u  14.5  V  15.04  w  15.28  X  15.49  y z  15.57  aa  15.96  ab  16.05  ac  16.21  ad  16.68  ae  17.19  af  17.4 17.81  ag ah  18.27  al  18.45 19.18  aj ak  19.42  al  b  RLXSS  * Me He  15.29  SS  *  * * ** * * * * ** * *  *  87  Retention  Peak  time (minutes)  labels  20.12  am  20.51  an  20.88  ao  21.27  ap  21.48  aq  21.72  ar  21.97 22.32  as at  22.42  au  22.62  av  22.71  aw  22.81  ax  23.12  ay  23.27  az  25.21  ba  26.41  bb  26.68  be  27.27  bd  27.61  be  28.07  bf  29.16 30.94  bg bh  31.42  bi  31.51 31.75  bj bk  31.91  bl  33.14  bm  35.03  bn  35.37  bo  36.23  bp  36.58 36.84  bq br  38.28  bs  a  R L = ration level ( 7 5 % and 1 0 0 % o f full ration)  b  SS = swimming speed ( 0 . 5 , 1 . 0 , and 1.5 bl/s)  c  Significance RL  a  SS  *** *  **  **  *** * * *  *  *  *  *  b  RLXSS  * *  *  *  L e v e l o f significance * p<.05, * * p < . 0 1 , * * * p<.001  88  89  Table 22  Factor score coefficients o f the first 7 principal components from G C headspace peaks significantly affected by either ration level o r s w i m m i n g speed 3  Peak  b  Principal Components  Label 1  2  3  4  5  6  7  A  0.741  -0.547  -0.102  0.205  0.087  -0.173  -0.187  C  0.735  0.582  -0.147  0.008  -0.225  0.134  0.131  D  0.841  0.364  -0.011  0.107  0.054  -0.238  0.240  E  0.932  0.090  -0.286  -0.045  0.038  -0.029  0.016  K  0.734  0.591  -0.001  0.007  -0.200  -0.199  0.175  Q  0.468  0.587  -0.057  0.386  0.418  -0.181  0.011  s u  0.847  -0.329  0.067  -0.117  0.119  -0.039  -0.253  0.774  0.183  -0.441  0.059  0.086  0.223  -0.140  w  0.748  -0.599  0.075  -0.157  0.055  -0.042  0.136  X  0.529  -0.489  0.115  -0.547  0.250  0.020  0.286  Y  0.682  -0.427  0.301  0.275  -0.188  0.033  0.288  z  0.796  -0.423  0.047  0.215  0.163  -0.234  -0.196  AA  0.676  -0.543  0.139  0.416  0.031  0.024  0.079  AB  0.596  0.292  -0.681  0.016  0.131  0.247  0.054  AD  0.673  0.524  0.135  0.184  0.297  0.150  -0.030  AG  0.768  0.484  0.189  0.094  -0.276  -0.082  -0.116  AN  0.713  -0.257  -0.477  -0.232  -0.077  0.141  -0.087  AO  0.710  0.134  -0.291  -0.358  -0.178  -0.151  -0.221  AQ  0.845  -0.183  0.271  -0.026  -0.121  -0.155  -0.207  AR  0.922  0.042  0.095  -0.082  -0.113  0.016  0.004  AZ  0.782  0.421  0.163  -0.210  -0.099  -0.239  0.048  BB  0.861  0.021  -0.077  -0.123  0.125  0.391  -0.055  BC  0.644  -0.344  0.448  0.160  0.059  0.278  0.097  BD  0.841  -0.056  0.127  -0.238  0.113  -0.107  0.161  BE  0.412  -0.539  -0.463  0.264  -0.388  0.003  0.068  BM  0.161  0.296  0.863  -0.228  0.070  0.011  -0.108  BS  0.433  0.134  0.722  0.081  -0.202  0.355  -0.149  Variance explained Fraction  0.517  0.155  0.114  0.050  0.034  0.031  0.024  Cumulative  0.517  0.672  0.786  0.836  0.087  0.901  0.925  3  ration level = 7 5 % and 1 0 0 % o f f u l l ration  b  s w i m m i n g speed = 0 . 5 , 1 . 0 , and 1.5 bl/s  90  2.30  1.90  1.50 CL O  l . io  4-  0.70  0.30  0.70  -t-  0 • 76  0 .82 Coefficient  F i g u r e 13  0.88 of  —i 0.94  1.00  Determination  PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h treatment numbers  91  2.30  1.90  1.50  1.10  0.70  0.30  0.70  — I  0.76  1  Coefficient  F i g u r e 14  1  0.B2  0.88 of  —i 0.94  1.00  Determination  PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h ration level number (1 = 7 5 % , 2 = 1 0 0 % ration level)  92  2.30  1.90  1 .50  1 . 10  4-  0.70  0.30  0.70  0.76  0.82  0.88  Coefficient  Figure 15  of  —i 0.94  1.00  Determination  PCS graph o f significant G C peaks produced from a purge and trap headspace extraction o f cooked, cultured chinook salmon, data points labelled w i t h swimming speed number (1 = 0 . 5 , 2 = 1.0,3 = 1.5 bl/s)  93  4.1.5 Instron TPA analysis Six T P A parameters were determined from the curves produced b y the Instron Universal testing machine (Figures 1 6 - 2 1 ) .  T w o factor A N O V A s , looking at the effect o f R L , SS and the  interaction o f these treatments, were performed o n all six parameters. The results o f these analyses are presented i n Table 23. Unlike the sensory texture results, SS and not R L was significant. Hardness 1, Firmness 1 and 2 were all significantly affected b y SS (p<0.05). Dunajski (1979) d i d not feel that devices used for rheological testing o f f o o d were suitable t o measure the texture o f fish muscle.  She felt that at best they may be applicable t o r a w , but not t o  cooked fish. She pointed out that w h e n fish is eaten, the majority o f the energy is used f o r mastication A s a result, the mechanical measurement should measure the resistance o f the fibres t o mechanical disintegration (Dunajski, 1979). I n this experiment, only compression forces were measured.  Since  the force required t o shear the fibres was not measured, this is likely not a true representation o f the texture experienced b y the panellists. I t is also possible that the reason f o r the disagreement between the sensory and Instron results may be due t o the w a y i n w h i c h the samples were prepared. Samples f o r the sensory tests composed o f slices o f the fish placed side b y side, gelled together into a composite sample u p o n cooking. For the Instron samples, once cooked and cooled t o r o o m temperature, the fish was flaked and formed into a cylinder.  I t is possible that these differences i n sample temperature and structure contributed t o the  discrepancy i n results. There were many difficulties producing consistent samples. The cylinders o f fish w o u l d often fall apart and/or distort, prior t o , o r in the process o f transportation t o the Instron platform for testing. Additionally, the production o f samples o f consistent height proved t o be nearly impossible. Following  94  60  52  44  36  28  20 3 .  4  Treatment Number  F i g u r e 16  B o x p l o t o f the Instron T P A parameter Hardness 1 f o r cooked, cultured chinook salmon samples, results by treatment number  95  35 30  « c o  25 L  (D 2  20  15 10  I  3  4  Treatment Number  Figure 17  B o x p l o t o f the Instron T P A parameter Hardness 2 for cooked, cultured chinook salmon samples, results by treatment number  96  10.00  8.40  6.80  5.20  3.60  2.00  3  4  Treatment Number  F i g u r e 18  B o x p l o t o f the Instron T P A parameter Firmness 1 f o r cooked, cultured chinook salmon samples, results by treatment number  97  10.00  8.40 h  «2 -S  Z  f  6.80 f-  5.20  H  3.60  -  2.00  L-  1 1  J2  :  L  I  !_  3  4  5  Treatment Number  Figure 19  B o x p l o t o f the Instron T P A parameter Firmness 2 f o r cooked, cultured chinook salmon samples, results by treatment number  98  0.60  0.48  h  0.37  h  0.25  h  0.14  h  I  0.02 1  2  3  4  5  6  Treatment Number  Figure 20  B o x p l o t o f the Instron T P A parameter Cohesiveness f o r cooked, cultured chinook salmon samples, results by treatment number  99  25  s  20  h  1 5  h  10  h  5  h  1  2  3  4  5  6  Treatment Number  Figure 21  B o x p l o t o f the Instron T P A parameter Gumminess for cooked, cultured chinook salmon samples, results by treatment number  100  Table 23  Summarised A N O V A results o f ration level and swimming speed effect o n Instron T P A parameters o f cooked cultured chinook salmon  Sensory attributes  F ratio  Mean  Ration  Swimming  Ration level X  square  level  speed  Swimming speed  error  Hardness 1  0.533  3.888*  0.229  48.823  Hardness2  0.000  2.343  0.140  17.912  Firmness 1  0.173  5.077*  0.483  4.775  Firmness2  0.226  6.063*  0.512  3.571  Cohesiveness  0.434  2.115  1.222  0.005  Gumminess  0.010  0.507  0.615  0.120  a  * p<.05, * * p < . 0 1 , * * * p<.001 a  T u k e y test results: swimming speeds 0.5 bl/s significantly different from 1.0 bl/s (p<0.05)  101  compression o f the sample to the desired height and release o f the plunger o n the syringe, the sample w o u l d spring back.  4.1.6  p H analysis The post-mortem p H o f fish, as w i t h most other animals, is largely due to the degradation o f  glycogen t o lactic acid v i a the Emden-Meyerhof-Parnas pathway.  T h e ultimate post-mortem p H o f  most fish species usually falls i n the range o f 6.5 - 6.2 (Dunajski, 1979).  The p H o f most o f the fish  samples i n this experiment was above this; the majority ranging between 6.5 - 6.7 (Figure 22). A l t h o u g h the p H did not vary widely between treatments, significant differences were found (Table 24). H i g h l y significant statistical differences (p<.001) i n the p H were f o u n d t o have resulted from b o t h R L and SS. The interaction between R L and SS also proved to be highly significant. F r o m the b o x plot showing the effect o f the 6 treatment combinations o n p H (Figure 2 2 ) , some general trends become apparent. First, the fish that received the higher R L (treatments 4-6) generally had a l o w e r p H w h e n compared w i t h the salmon fed the l o w e r ration level. A small increase i n the p H also occurred w h e n the swimming speed was increased. I t is unfortunate that no data is available as to the glycogen content o f the salmon used i n this study. Kiessling et al. (1989b), i n a study where fish were fed different ration levels, found an increase i n the glycogen content o f fish muscle w h e n the fish ration level was increased i n the range between 5 0 % - 1 0 0 % R L . This appears to have also occurred i n this experiment; there was a decrease i n the post m o r t e m p H o f the fish at the higher ration levels that w o u l d be consistent w i t h an increased glycogen content in the live fish prior t o their being sacrificed.  102  6.85  6.76  6.67 32 CL  6.58  6.49  r-  6.40 3  4  Treatment Number  Figure 22  B o x p l o t b y treatment number o f the p H o f t h e cooked chinook salmon samples  103  Table 24  A N O V A table o f ration level and swimming speed effect o n the p H o f cultured cooked chinook salmon  Source  DF  SS  MS  F  P  SS  2  0.054  0.027  49.189  0.000  RL  1  0.015  0.015  27.273  0.000  SS*RL Error  2  0.097 0.010  0.048 0.001  87.977  0.000  18  a  T u k e y test results: swimming speeds 0.5 bl/s significantly different from 1.0 bl/s ( p O . 0 0 1 )  104  A slight increase in the p H that corresponds to an increase in the SS o f the fish is also seen in Figure 22.  There is no readily apparent explanation for this small increase in p H w i t h the  corresponding increase in swimming speed. For an u n k n o w n reason, the replicates from treatment number 4 (SS = 0.5 bl/s, R L = 100%) were significantly higher than all but treatment number 3 (SS = 1.5 bl/s, R L = 7 5 % ) . W h e n the effects o f SS and R L were looked at individually, the data from this treatment combination skewed the results. I t is w o r t h noting that no similar deviations in the results are observed for this treatment in either the sensory, Instron o r G C results.  4.1.6.1  Comparison of pH and sensory analysis results According to Dunajski (1979) and L o v e (1988) the p H o f the fish muscle is likely the most  important factor affecting its rheological properties. W i t h a drop in the p H , theoretically there should be an increase in the toughness o f the fish. Thus, the drop in the p H o f the cooked fish muscle that resulted w h e n the R L was increased should have caused these well-fed fish to have a slightly  firmer  eating texture. I n the sensory texture testing, however, although there was a significant difference in the texture o f the fish due to R L , the opposite trend was observed. The fish reared o n the 7 5 % R L were significantly more firm and less mushy, despite their higher p H , than the fish reared o n the 1 0 0 % R L . Similarly, w i t h a lower p H , it is expected that one w o u l d find the fish to be drier (Dunajski, 1979). Here t o o , the results were contrary to theory; in the sensory testing, the higher ration level fish samples were significantly more moist than those from their lower ration counterparts. There are t w o factors that may have contributed t o this contradiction o f the popular theory regarding the relationship between p H and the texture o f the fish. First, ignoring the treatment number  105  4 ( R L = 1 0 0 % X . SS=0.5 bl/s) data, the change i n p H is small, approximately 0.2 p H units. Second, the fish reared w i t h the higher R L had a significantly higher fat content than those at the lower R L (Kiessling et al. 1994b). This higher fat content w o u l d have caused a higher degree o f lubrication and an oily sensation in the m o u t h (Szczesniak, 1963). more moist, less t o u g h or firm sample.  106  This w o u l d result i n the sensory perception o f a  5. Conclusions 5.1 Sensory Analyses R L and panellist effect while SS d i d not.  significantly affected the sensory analysis p o r t i o n o f this experiment,  Eight sensory attributes (three aroma, one flavour, and four texture) were  significantly affected by ration level; no attributes were affected b y swimming speed. T h e PCS graphs (Figures 8 and 9 ) , using the P C from the seven significant sensory attributes, graphically depicted this phenomenon. The panellist effect was highly significant f o r all the attributes. After completing a ztransformation t o remove the panellist effect, and a second A N O V A was performed, one aroma t e r m "Boiled Potato" was no longer significant. These results are consistent w i t h those o f Kiessling et. al. (1994b). Kiessling et al. (1994b) d i d not find that SS had any effect o n the proximate composition o f the fish. They, however, d i d observe that the fat content o f the R L 1 0 0 samples were significantly higher than those o f the R L 7 5 samples. This difference i n fat content o f the fish samples was likely responsible f o r the significant R L effect observed i n the sensory analyses results. flavour  volatiles, affecting the  I t could explain b o t h changes i n b o t h the concentrations o f  aroma attributes "Seaweedy" and " S o u r , " and flavour attribute  " B r o t h y , " and the mouthfeel o f the samples affecting the perception o f the texture  attributes  " M o i s t u r e , " "Firmness," "Cohesiveness" and "Mushiness." The statistical analyses o f the sensory data also clearly demonstrated that the use o f replacement panellists i n Q D A sensory analysis should be avoided.  I n this type o f sensory analysis,  despite training towards uniformity, each panellist develops their o w n unique style o f grading the samples.  A s long as the panellist is consistent it is possible t o remove the panellist effect during  statistical analysis. Figures 2-7 illustrate that w h e n the replacement panellist was used o n panel days 7 107  and 9, that irregularities due t o the substitution were evident in the data. When a replacement panellist is used o n one or more panel sessions, as it was in this experiment, the removal o f panellist effect is compromised.  5.2  G C headspace analyses Out o f the 71 G C peaks that consistently appeared, 27 were found to be significant for either  SS and/or R L (Table 21). A n appreciable number o f peaks were significantly affected by R L , while comparatively fewer were significant for SS. The PCS graph o f these significant peaks clearly shows clear group resolution o n the basis o f R L (Figure 14), while no trend was observable w h e n the PCS graph data points were labelled w i t h either SS levels (Figure 15) o r treatment numbers (Figure 13). These results compare favourably w i t h b o t h the sensory analysis p o r t i o n o f this experiment and the findings o f Kiessling et. al (1994b).  5.3  Instron  TPA  The Instron T P A results did not f o l l o w the trend established i n the other areas o f this experiment where the R L  significantly affected the sensory properties o f cooked, cultured chinook  salmon, and SS did not. Rather, the Instron T P A results showed a significant difference due t o SS, while R L had no effect. I t is possible that the compression method o f texture measurement, employed i n this experiment, measured changes i n the cooked muscle that were not discernible by the panellists, and was unable t o account for the textural changes due t o the differences i n fat content between the t w o ration levels. I n the mastication o f cooked fish, the majority o f the energy is used f o r mastication. A s a result, to get an accurate instrumental measurement o f the texture o f the fish, the mechanical 108  measurement should measure the resistance o f the fibres to mechanical disintegration (Dunajski, 1979), w h i c h was not the case i n this experiment.  Other possible reasons for the discrepancy i n results  include: differences i n method o f sample preparation and extreme difficulty in sample preparation.  pH  5.4  The p H p o r t i o n o f this experiment was sensitive to b o t h the changes i n R L and SS, showing clear trends i n both. There was a highly significant statistical difference found in SS, R L and the R L X . SS interaction. I n this experiment, the drop i n muscle p H did not result in a firmer texture, but rather, the opposite trend was observed. I n the sensory analysis p o r t i o n o f this study, as the p H dropped, the fish became more mushy and they were rated lower in  firmness.  This trend may be attributable to a  corresponding increase in fat content. The unexplainably high results o f treatment 4 badly skewed the p H data. F r o m the T u k e y test results as w e l l as the highly significant R L X . SS interaction, it became apparent that the significant difference due t o SS was because o f this aberration in the data and was not evidence for a true SS effect.  5.5  O v e r a l l Conclusions This experiment demonstrated that although changing the r a t i o n level w i l l have an effect o n the  sensory attributes o f cooked chinook salmon muscle, the swimming speed o f the fish does not. A s a result, increasing the swimming speed o f chinook salmon i n a fish farming operation above that w h i c h is necessary for proper schooling, while decreasing the f o o d conversion rate, is unlikely t o result i n any 109  appreciable difference in consumer acceptability. 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I n , Flavour Science and Technology: Proceedings o f the 5th Weurman Flavor Research S y m p o s i u m held at Sara H o t e l Volsenasea Oslo. 23rd - 25th M a r c h . 1987. M . Martens, G.A. Dalen, and H . R u s s w u r m Jr (Ed).  John Wiley & Sons L t d . Chichenster, N e w Y o r k . pp. 439-452.  Vijayan, M . M . , and Leatherland, J.F. 1989. Cortisol-induced changes i n plasma glucose, protein, and thyroid hormone levels, and liver glycogen content o f coho salmon (Oncorhynchus kisutch Walbaum). Can. J. Z o o l . 67: 2746-2750. V o d o v o t z , Y . , Arteaga, G.E., and Nakai, S. 1993. Principal component similarity analysis for classification and its application to G C data o f mango. F o o d Res. Int. 26: 355-363. v o n Sydow, E., Andersson, J., A n j o u , BC, Karlsson, G., L a n d , D . and Griffiths, N . 1970. The aroma o f bilberries (Vaccinium myrtillus L.). 2. Evaluation o f the press juice by sensory methods and by gas chromatography and mass spectrometry. Lebensm-Wiss. u. Technol.  3: 11-17.  Weatherley, A H . and G i l l H.S. 1981. Characteristics o f mosaic muscle g r o w t h i n rainbow trout Salmo gairdneri. Experimentia 37: 1102-1103. Weatherley A H . and G i l l H.S. 1984. G r o w t h dynamics o f white m y o t o m a l muscle fibres i n the bluenose m i n n o w Pinephales notatus Rafinesque and comparison w i t h rainbow trout Salmo gairdneri Richardson. J. Fish B i o l . 25: 13-24. White, J.R. and L I H . W . 1985. Determination o f the energetic cost o f swimming from the analysis o f g r o w t h rate and body composition i n juvenile chinook salmon (Oncorhynchus tshawytschd). Comp. Biochem. P h y s i o l 81 A : 116  25-33.  Williams, A . A . and Tucknot, O. 1977. Misleading information from G C effluents i n aroma analysis. Chem. & I n d . 3 : 1 2 5 - 1 2 9 . W o o d w a r d , J.J., and Smith, L.S. 1985. Exercise training and the stress response i n rainbow trout, Salmo gairdneri Richardson. J. Fish B i o l . 26: 435-447.  117  Appendix A: Samples of sensory exploratory analysis boxplots  118  IT)  1 «  OO OO  CO 4>  >H  VO  OH  OJ  a £  11  •8.8 S  OH OH © O 1)  lis  o •  tfa vo *  J<  •a  ^  .S J$ *§ 3  ©  00 00  •8 J f 8 O OO co »H  eg  O PH  a 1 CO  ©'  oo  o o  CO  B  OH EH  CO  00 O  o o « ? ssaiunos %SL TH  ssaumos %00l TM  •  s  OX)  119  r--  1  i l  oo oo  g  OH  1.8 O  OJ  60  OH  "53  -2  a? t/3  co  o  oo oo  Is .a 1 OH  I 0 JH  42  s § JH  i o  1 O  M  e 8  s  £ & « 2 " s  ©  00 00  o 5  6 %SZ. TH  %00l TH S3  120  »i  I £ a 5  g  £  II  |  

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